Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ENZYMES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of enzymes and
to the use of
these sequences in the diagnosis, treatment, and prevention of
autoimmune/inflammatory disorders,
infectious disorders, immune deficiencies, disorders of metabolism,
reproductive disorders,
neurological disorders, cardiovascular disorders, eye disorders, and cell
proliferative disorders,
including cancer, and in the assessment of the effects of exogenous compounds
on the expression of
nucleic acid and amino acid sequences of enzymes.
BACKGROUND OF THE INVENTION
The cellular processes of biogenesis and biodegradation involve a number of
key enzyme classes
including oxidoreductases, transferases, hydrolases, lyases, isomerases,
ligases, and others. Each-
class of enzyme comprises many substrate-specific enzymes having precise and
well regulated
functions. Enzymes facilitate metabolic processes such as glycolysis, the
tricarboxylic cycle, and
fatty acid metabolism; synthesis or degradation of amino acids, steroids,
phospholipids, and alcohols;
regulation of cell signaling, proliferation, inflamation, and apoptosis; and
through catalyzing critical
steps in DNA replication and repair and the process of translation.
Oxidoreductases
Many pathways of biogenesis and biodegradation require oxidoreductase
(dehydrogenase or
reductase) activity, coupled to reduction or oxidation of a cofactor.
Potential cofactors include
cytochromes, oxygen, disulfide, iron-sulfur proteins, flavin adenine
dinucleotide (FAD), and the
nicotinamide adenine dinucleotides NAD and NADP (Newsholme, E.A. and Leech,
A.R. (1983)
Biochemistry for the Medical Sciences, John Wiley and Sons, Chichester, U. K.
pp. 779-793).
Reductase activity catalyzes transfer of electrons between substrates) and
cofactors) with concurrent
oxidation of the cofactor. Reverse dehydrogenase activity catalyzes the
reduction of a cofactor and
consequent oxidation of the substrate. Oxidoreductase enzymes are a broad
superfamily that catalyze
reactions in all cells of organisms, including metabolism of sugar, certain
detoxification reactions,
and synthesis or degradation of fatty acids, amino acids, glucocorticoids,
estrogens, androgens, and
prostaglandins. Different family members may be referred to as
oxidoreductases, oxidases,
reductases, or dehydrogenases, and they often have distinct cellular locations
such as the cytosol, the
plasma membrane, mitochondrial inner or outer membrane, and peroxisomes.
Short-chain alcohol dehydrogenases (SCADS) are a family of dehydrogenases that
share only
15% to 30% sequence identity, with similarity predominantly in the coenzyme
binding domain and
the substrate binding domain. In addition to their role in detoxification of
ethanol, SCADS are
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involved in synthesis and degradation of fatty acids, steroids, and some
prostaglandins, and are
therefore implicated in a variety of disorders such as lipid storage disease,
myopathy, SCAD
deficiency, and certain genetic disorders. For example, retinol dehydrogenase
is a SCAD-family
member (Simon, A. et al. (1995) J. Biol. Chem. 270:1107-1112) that converts
retinol to retinal, the
precursor of retinoic acid. Retinoic acid, a regulator of differentiation and
apoptosis, has been shown
to down-regulate genes involved in cell proliferation and inflammation (Chaff,
X. et al. (1995) J. Biol.
Chem. 270:3900-3904). In addition, retinol dehydrogenase has been linked to
hereditary eye diseases
such as autosomal recessive childhood-onset severe retinal dystrophy (Simon,
A. et al. (1996)
Genomics 36:424-430).
Membrane-bound succinate dehydrogenases (succinate:quinone reductases, SQR)
and
fumarate reductases (quinol:fumarate reductases, QFR) couple the oxidation of
succinate to fumarate
with the reduction of quinone to quinol, and also catalyze the reverse
reaction. QFR and SQR
complexes are collectively known as succinate:quinone oxidoreductases (EC
1.3.5.1) and have
similar compositions. The complexes consist of two hydrophilic and one or two
hydrophobic,
membrane-integrated subunits. The larger hydrophilic subunit A carries
covalently bound flavin
adenine dinucleotide; subunit B contains three iron-sulphur centers
(Lancaster, C.R. and Kroger, A.
(2000) Biochim. Biophys. Acta 1459:422-431). The full-length cDNA sequence for
the flavoprotein
subunit of human heart succinate dehydrogenase (succinate: (acceptor)
oxidoreductase; EC 1.3.99.1)
is similar to the bovine succinate dehydrogenase in that it contains a
cysteine triplet and in that the
active site contains an additional cysteine that is not present in yeast or
prokaryotic SQRs (Morris, A.
A. et al. (1994) Biochim. Biophys. Acta 29:125-128).
Propagation of nerve impulses, modulation of cell proliferation and
differentiation, induction
of the immune response, and tissue homeostasis involve neurotransmitter
metabolism (Weiss, B.
(1991) Neurotoxicology 12:379-386; Collins, S.M. et al. (1992) Ann. N.Y. Acad.
Sci. 664:415-424;
Brown, J.K. and Imam, H. (1991) J. Inherit. Metab. Dis. 14:436-458). Many
pathways of
neurotransmitter metabolism require oxidoreductase activity, coupled to
reduction or oxidation of a
cofactor, such as NAD+/NADH (Newsholme, E.A. and Leech, A.R. (1983)
Biochemistry for the
Medical Sciences, John Wiley and Sons, Chichester, U.K. pp. 779-793).
Degradation of
catecholamines (epinephrine or norepinephrine) requires alcohol dehydrogenase
(in the brain) or
aldehyde dehydrogenase (in peripheral tissue). NAD+-dependent aldehyde
dehydrogenase oxidizes
5-hydroxyindole-3-acetate (the product of S-hydroxytryptamine (serotonin)
metabolism) in the brain,
blood platelets, liver and pulmonary endothelium (Newsholme, E.A. and Leech,
A.R. supra, p. 786).,
Other neurotransmitter degradation pathways that utilize NAD+/NADH-dependent
oxidoreductase
activity include those of L-DOPA (precursor of dopamine, a neuronal excitatory
compound), glycine
(an inhibitory neurotransmitter in the brain and spinal cord), histamine
(liberated from mast cells
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during the inflammatory response), and taurine (an inhibitory neurotransmitter
of the brain stem,
spinal cord and retina) (Newsholme, E.A. and Leech, A.R. supra, pp. 790, 792).
Epigenetic or
genetic defects in neurotransmitter metabolic pathways can result in diseases
including Parkinson
disease and inherited myoclonus (McCance, K.L. and Huether, S.E. (1994)
Pathophysiology, Mosby-
Year Book, Inc., St. Louis, MO pp. 402-404; Gundlach, A.L. ( 1990) FASEB J.
4:2761-2766).
Tetrahydrofolate is a derivatized glutamate molecule that acts as a carrier,
providing
activated one-carbon units to a wide variety of biosynthetic reactions,
including synthesis of purines,
pyrimidines, and the amino acid methionine. Tetrahydrofolate is generated by
the activity of a
holoenzyme complex called tetrahydrofolate synthase, which includes three
enzyme activities:
tetrahydrofolate dehydrogenase, tetrahydrofolate cyclohydrolase, and
tetrahydrofolate synthetase.
Thus, tetrahydrofolate dehydrogenase plays an important role in generating
building blocks for
nucleic and amino acids, crucial to proliferating cells.
3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty acid metabolism.
It
catalyzes the reduction of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA, with
concomitant oxidation of
NAD to NADH, in the mitochondria and peroxisomes of eukaryotic cells. In
peroxisomes, 3HACD
and enoyl-CoA hydratase form an enzyme complex called bifunctional enzyme,
defects in which are
associated with peroxisomal bifunctional enzyme deficiency. This interruption
in fatty acid
metabolism produces accumulation of very-long chain fatty acids, disrupting
development of the
brain, bone, and adrenal glands. Infants born with this deficiency typically
die within 6 months
(Watkins, P. et al. (1989) J. Clin. Invest. 83:771-1277; Online Mendelian
Inheritance in Man
(OMIM), #261515). The neurodegeneration characteristic of Alzheimer's disease
involves
development of extracellular plaques in certain brain regions. A major protein
component of these
plaques is the peptide amyloid-~i (A~3), which is one of several cleavage
products of amyloid
precursor protein (APP). 3HACD has been shown to bind the A~i peptide, and is
overexpressed in
neurons affected in Alzheimer's disease. In addition, an antibody against
3HACD can block the toxic
effects of A(3 in a cell culture model of Alzheimer's disease (Yan, S. et al.
(1997) Nature 389:689-
695; OMIM, #602057).
Steroids such as estrogen, testosterone, and corticosterone are generated from
a common
precursor, cholesterol, and interconverted. Enzymes acting upon cholesterol
include dehydrogenases.
Steroid dehydrogenases, such as the hydroxysteroid dehydrogenases, are
involved in hypertension,
fertility, and cancer (Duax, W.L. and Ghosh, D. (1997) Steroids 62:95-100).
One such
dehydrogenase is 3-oxo-5-a-steroid dehydrogenase (OASD), a microsomal membrane
protein highly
expressed in prostate and other androgen-responsive tissues. OASD catalyzes
the conversion of
testosterone into dihydrotestosterone, which is the most potent androgen.
Dihydrotestosterone is
essential for the formation of the male phenotype during embryogenesis, as
well as for proper
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androgen-mediated growth of tissues such as the prostate and male genitalia. A
defect in DASD
leads to defective formation of the external genitalia (Andersson, S. et al.
(1991) Nature 354:159-161;
Labrie, F. et al. (1992) Endocrinology 131:1571-1573; OMIM #264600).
17/3-hydroxysteroid dehydrogenase (17(3HSD6) plays an important role in the
regulation of
the male reproductive hormone, dihydrotestosterone (DHTT). 17 ~iHSD6 acts to
reduce levels of
DHTT by oxidizing a precursor of DHTT, 3a-diol, to androsterone which is
readily glucuronidated
and removed. 17~3HSD6 is active with both androgen and estrogen substrates in
embryonic kidney
293 cells. Isozymes of l7~iHSD catalyze oxidation and/or reduction reactions
in various tissues with
preferences for different steroid substrates (Biswas, M.G. and Russell, D.W.
(1997) J. Biol. Chem.
272:15959-15966). For example, l7~iHSDl preferentially reduces estradiol and
is abundant in the
ovary and placenta. 17~3HSD2 catalyzes oxidation of androgens and is present
in the endometrium
and placenta. 17(3HSD3 is exclusively a reductive enzyme in the testis
(Geissler, W.M. et al. (1994)
Nature Genet. 7:34-39). An excess of androgens such as DHTT can contribute to
diseases such as
benign prostatic hyperplasia and prostate cancer.
The oxidoreductase isocitrate dehydrogenase catalyzes the conversion of
isocitrate to a-
ketoglutarate, a substrate of the citric acid cycle. Isocitrate dehydrogenase
can be either NAD or
1VADP dependent, and is found in the cytosol, mitochondria, and peroxisomes.
Activity of isocitrate
dehydrogenase is regulated developmentally, and by hormones,
neurotransmitters, and growth
factors.
Hydroxypyruvate reductase (HPR), a peroxisomal 2-hydroxyacid dehydrogenase in
the
glycolate pathway, catalyzes the conversion of hydroxypyruvate to glycerate
with the oxidation of
both NADH and NADPH. The reverse dehydrogenase reaction reduces NAD+ and
NADP+. HPR
recycles nucleotides and bases back into pathways leading to the synthesis of
ATP and GTP, which
are used to produce DNA and RNA and to control various aspects of signal
transduction and energy
metabolism. Purine nucleotide biosynthesis inhibitors are used as
antiproliferative agents to treat
cancer and viral diseases. HPR also regulates biochemical synthesis of serine
and cellular serine
levels available for protein synthesis.
The mitochondria) electron transport (or respiratory) chain is the series of
oxidoreductase-
type enzyme complexes in the mitochondria) membrane that is responsible for
the transport of
electrons from NADH to oxygen and the coupling of this oxidation to the
synthesis of ATP (oxidative
phosphorylation). ATP provides energy to drive energy-requiring reactions. The
key respiratory
chain complexes are NADH:ubiquinone oxidoreductase (complex I),
succinate:ubiquinone
oxidoreductase (complex II), cytochrome c,-b oxidoreductase (complex III),
cytochrome c oxidase
(complex IV), and ATP synthase (complex V) (Alberts, B. et al. (1994)
Molecular Biolo~y of the
Cell, Garland Publishing, Inc., New York, NY, pp. 677-678). All of these
complexes are located on
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the inner matrix side of the mitochondrial membrane except complex II, which
is on the cytosolic
side where it transports electrons generated in the citric acid cycle to the
respiratory chain. Electrons
released in oxidation of succinate to fumarate in the citric acid cycle are
transferred through electron
carriers in complex II to membrane bound ubiquinone (Q). Transcriptional
regulation of these
nuclear-encoded genes controls the biogenesis of respiratory enzymes. Defects
and altered
expression of enzymes in the respiratory chain are associated with a variety
of disease conditions.
Other dehydrogenase activities using NAD as a cofactor include 3-
hydroxyisobutyrate
dehydrogenase (3HBD), which catalyzes the NAD-dependent oxidation of 3-
hydroxyisobutyrate to
methylmalonate semialdehyde within mitochondria. 3-hydroxyisobutyrate levels
are elevated in
ketoacidosis, methylmalonic acidemia, and other disorders (Rougraff, P.M. et
al. (1989) J. Biol.
Chem. 264:5899-5903). Another mitochondrial dehydrogenase important in amino
acid metabolism
is the enzyme isovaleryl-CoA-dehydrogenase (IVD). IVD is involved in leucine
metabolism and
catalyzes the oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA. Human IVD
is a tetrameric
flavoprotein synthesized in the cytosol with a mitochondrial import signal
sequence. A mutation in
the gene encoding IVD results in isovaleric acidemia (Vockley, J. et al.
(1992) J. Biol. Chem.
267:2494-2501).
The family of glutathione peroxidases encompass tetrameric glutathione
peroxidases (GPxl-
3) and the monomeric phospholipid hydroperoxide glutathione peroxidase
(PHGPx/GPx4). Although
the overall homology between the tetrameric enzymes and GPx4 is less than 30%,
a pronounced
similarity has been detected in clusters involved in the active site and a
common catalytic triad has
been defined by structural and kinetic data (Epp, O. et al. (1983) Eur. J.
Biochem. 133:51-69). GPxl
is ubiquitously expressed in cells, whereas GPx2 is present in the liver and
colon, and GPx3 is
present in plasma. GPx4 is found at low levels in all tissues but is expressed
at high levels in the
testis (Ursini, F. et al (1995) Meth. Enzymol. 252:38-53). GPx4 is the only
monomeric glutathione
peroxidase found in mammals and the only mammalian glutathione peroxidase to
show high affinity
for and reactivity with phospholipid hydroperoxides, and to be membrane
associated. A tandem
mechanism for the antioxidant activities of GPx4 and vitamin E has been
suggested. GPx4 has
alternative transcription and translation start sites which determine its
subcellular localization
(Esworthy, R.S. et al. (1994) Gene 144:317-318; and Maiorino, M. et al. (1990)
Meth. Enzymol.
186:448-450).
The glutathione S-transferases (GST) are a ubiquitous family of enzymes with
dual substrate
specificities that perform important biochemical functions of xenobiotic
biotransformation and
detoxification, drug metabolism, and protection of tissues against
peroxidative damage. They
catalyze the conjugation of an electrophile with reduced glutathione (GSH)
which results in either
activation or deactivation/detoxification. The absolute requirement for
binding reduced GSH to a
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variety of chemicals necessitates a diversity in GST structures in various
organisms and cell types.
GSTs are homodimeric or heterodimeric proteins localized in the cytosol. The
major isozymes share
common structural and catalytic properties and include four major classes,
Alpha, Mu, Pi, and Theta.
Each GST possesses a common binding site for GSH, and a variable hydrophobic
binding site
S specific for its particular electrophilic substrates. Specific amino acid
residues within GSTs have
been identified as important for these binding sites and for catalytic
activity. Residues Q67, T68,
D101, E104, and 8131 are important for the binding of GSH (Lee, H.-C. et al.
(1995) J. Biol. Chem.
270:99-109). Residues R13, R20, and R69 are important for the catalytic
activity of GST (Stenberg,
G. et al. (1991) Biochem. J. 274:549-555).
GSTs normally deactivate and detoxify potentially mutagenic and carcinogenic
chemicals.
Some forms of rat and human GSTs are reliable preneoplastic markers of
carcinogenesis.
Dihalomethanes, which produce liver tumors in mice, are believed to be
activated by GST (Thier, R.
et al. (1993) Proc. Natl. Acad. Sci. USA 90:8567-8580). The mutagenicity of
ethylene dibromide and
ethylene dichloride is increased in bacterial cells expressing the human Alpha
GST, A1-l, while the
mutagenicity of aflatoxin B 1 is substantially reduced by enhancing the
expression of GST (Simula,
T.P. et al. (1993) Carcinogenesis 14:1371-1376). Thus, control of GST activity
may be useful in the
control of mutagenesis and carcinogenesis.
GST has been implicated in the acquired resistance of many cancers to drug
treatment, the
phenomenon known as multi-drug resistance (MDR). MDR occurs when a cancer
patient is treated
with a cytotoxic drug such as cyclophosphamide and subsequently becomes
resistant to this drug and
to a variety of other cytotoxic agents as well. Increased GST levels are
associated with some drug
resistant cancers, and it is believed that this increase occurs in response to
the drug agent which is
then deactivated by the GST catalyzed GSH conjugation reaction. The increased
GST levels then
protect the cancer cells from other cytotoxic agents for which GST has
affinity. Increased levels of
A1-1 in tumors has been linked to drug resistance induced by cyclophosphamide
treatment (Dirven,
H.A. et al. ( 1994) Cancer Res. 54:6215-6220). Thus control of GST activity in
cancerous tissues
may be useful in treating MDR in cancer patients.
The reduction of ribonucleotides to the corresponding deoxyribonucleotides,
needed for DNA
synthesis during cell proliferation, is catalyzed by the enzyme ribonucleotide
diphosphate reductase.
Glutaredoxin is a glutathione (GSH)-dependent hydrogen donor for
ribonucleotide diphosphate
reductase and contains the active site consensus sequence -C-P-Y-C-. This
sequence is conserved in
glutaredoxins from such different organisms as E. coli, vaccinia virus, yeast,
plants, and mammalian
cells. Glutaredoxin has inherent GSH-disulfide oxidoreductase
(thioltransferase) activity in a
coupled system with GSH, NADPH, and GSH-reductase, catalyzing the reduction of
low molecular
weight disulfides as well as proteins. Glutaredoxin has been proposed to exert
a general thiol redox
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control of protein activity by acting both as an effective protein disulfide
reductase, similar to
thioredoxin, and as a specific GSH-mixed disulfide reductase (Padilla, C.A. et
al. (1996) FEBS Lett.
378:69-73).
In addition to their important role in DNA synthesis and cell division,
glutaredoxin and other
thioproteins provide effective antioxidant defense against oxygen radicals and
hydrogen peroxide
(Schallreuter, K.U. and J.M. Wood (1991) Melanoma Res. 1:159-167).
Glutaredoxin is the principal
agent responsible for protein dethiolation in vivo and reduces dehydroascorbic
acid in normal human
neutrophils (Jung, C.H. and J.A. Thomas (1996) Arch. Biochem. Biophys. 335:61-
122; Park, J.B. and
M. Levine (1996) Biochem. J. 315:931-938).
The thioredoxin system serves as a hydrogen donor for ribonucleotide reductase
and as a
regulator of enzymes by redox control. It also modulates the activity of
transcription factors such as
NF-KB, AP-1, and steroid receptors. Several cytokines or secreted cytokine-
like factors such as adult
T-cell leukemia-derived factor, 3B6-interleukin-1, T-hybridoma-derived (MP-6)
B cell stimulatory
factor, and early pregnancy factor have been reported to be identical to
thioredoxin (Holmgren, A.
(1985) Annu. Rev. Biochem. 54:237-271; Abate, C. et al. (1990) Science
249:1157-1161; Tagaya, Y.
et al. ( 1989) EMBO J. 8:757-764; Wakasugi, H. ( 1987) Proc. Natl. Acad. Sci.
USA 84:804-808;
Rosen, A. et al. (1995) Int. Immunol. 7:625-633). Thus thioredoxin secreted by
stimulated
lymphocytes (Yodoi, J. and T. Tursz (1991) Adv. Cancer Res. 57:381-411;
Tagaya, N. et al. (1990)
Proc. Natl. Acad. Sci. USA 87:8282-8286) has extracellular activities
including a role as a regulator
of cell growth and a mediator in the immune system (Miranda-Vizuete, A. et al.
(1996) J. Biol.
Chem. 271:19099-19103; Yamauchi, A. et al. (1992) Mol. Immunol. 29:263-270).
Thioredoxin and
thioredoxin reductase protect against cytotoxicity mediated by reactive oxygen
species in disorders
such as Alzheimer's disease (Lovely M.A. (2000) Free Radic. Biol. Med. 28:418-
427).
The selenoprotein thioredoxin reductase is secreted by both normal and
neoplastic cells and
has been implicated as both a growth factor and as a polypeptide involved in
apoptosis (Soderberg, A.
et al. (2000) Cancer Res. 60:2281-2289). An extracellular plasmin reductase
secreted by hamster
ovary cells (HT-1080) has been shown to participate in the generation of
angiostatin from plasmin.
In this case, the reduction of the plasmin disulfide bonds triggers the
proteolytic cleavage of plasmin
which yields the angiogenesis inhibitor, angiostatin (Stathakis, P. et al.
(1997) J. Biol. Chem.
272:20641-20645). Low levels of reduced sulfhydryl groups in plasma has been
associated with
rheumatoid arthritis. The failure of these sulfhydryl groups to scavenge
active oxygen species (e.g.,
hydrogen peroxide produced by activated neutrophils) results in oxidative
damage to surrounding
tissues and the resulting inflammation (Hall, N.D. et al. (1994) Rheumatol.
Int. 4:35-38).
Another example of the importance of redox reactions in cell metabolism is the
degradation
of saturated and unsaturated fatty acids by mitochondrial and peroxisomal beta-
oxidation enzymes
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which sequentially remove two-carbon units from Coenzyme A (CoA)-activated
fatty acids. The
main beta-oxidation pathway degrades both saturated and unsaturated fatty
acids while the auxiliary
pathway performs additional steps required for the degradation of unsaturated
fatty acids.
The pathways of mitchondrial and peroxisomal beta-oxidation use similar
enzymes, but have
different substrate specificities and functions. Mitochondria oxidize short-,
medium-, and long-chain
fatty acids to produce energy for cells. Mitochondria) beta-oxidation is a
major energy source for
cardiac and skeletal muscle. In liver, it provides ketone bodies to the
peripheral circulation when
glucose levels are low as in starvation, endurance exercise, and diabetes
(Eaton, S. et al. (1996)
Biochem. J. 320:345-357). Peroxisomes oxidize medium-, long-, and very-long-
chain fatty acids,
dicarboxylic fatty acids, branched fatty acids, prostaglandins, xenobiotics,
and bile acid
intermediates. The chief roles of peroxisomal beta-oxidation are to shorten
toxic lipophilic
carboxylic acids to facilitate their excretion and to shorten very-long-chain
fatty acids prior to
mitochondria) beta-oxidation (Mannaerts, G.P. and P.P. Van Veldhoven (1993)
Biochimie 75:147-
158).
The auxiliary beta-oxidation enzyme 2,4-dienoyl-CoA reductase catalyzes the
following
reaction:
trans-2, cis/trans-4-dienoyl-CoA + NADPH + H+---> traps-3-enoyl-CoA + NADP+
This reaction removes even-numbered double bonds from unsaturated fatty acids
prior to their entry
into the main beta-oxidation pathway (Koivuranta, K.T. et al. (1994) Biochem.
J. 304:787-792). The
enzyme may also remove odd-numbered double bonds from unsaturated fatty acids
(Smeland, T.E. et
al. ( 1992) Proc. Nat). Acad. Sci. USA 89:6673-6677).
Rat 2,4-dienoyl-CoA reductase is located in both mitochondria and peroxisomes
(Dommes,
V. et al. (1981) J. Biol. Chem. 256:8259-8262). Two immunologically different
forms of rat
mitochondria) enzyme exist with molecular masses of 60 kDa and 120 kDa
(Hakkola, E.H. and J.K.
Hiltunen (1993) Eur. J. Biochem. 215:199-204). The 120 kDa mitochondria) rat
enzyme is
synthesized as a 335 amino acid precursor with a 29 amino acid N-terminal
leader peptide which is
cleaved to form the mature enzyme (Hirose, A. et al. (1990) Biochim. Biophys.
Acta 1049:346-349).
A human mitochondria) enzyme 83% similar to rat enzyme is synthesized as a 335
amino acid
residue precursor with a 19 amino acid N-terminal leader peptide (Koivuranta,
s-u~ra). These cloned
human and rat mitochondria) enzymes function as homotetramers (Koivuranta,
supra). A
Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is 295 amino
acids long, contains
a C-terminal peroxisomal targeting signal, and functions as a homodimer (Coe,
J.G.S. et al. (1994)
Mol. Gen. Genet. 244:661-672; and Gurvitz, A. et al. (1997) J. Biol. Chem.
272:22140-22147). All
2,4-dienoyl-CoA reductases have a fairly well conserved NADPH binding site
motif (Koivuranta,
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supra).
The main pathway beta-oxidation enzyme enoyl-CoA hydratase catalyzes the
reaction:
2-trans-enoyl-CoA + HZO <---> 3-hydroxyacyl-CoA
This reaction hydrates the double bond between C-2 and C-3 of 2-trans-enoyl-
CoA, which is
generated from saturated and unsaturated fatty acids (Engel, C.K. et al.
(1996) EMBO J. 15:5135-
5145). This step is downstream from the step catalyzed by 2,4-dienoyl-
reductase. Different enoyl-
CoA hydratases act on short-, medium-, and long-chain fatty acids (Eaton,
supra). Mitochondrial
and peroxisomal enoyl-CoA hydratases occur as both mono-functional enzymes and
as part of multi-
functional enzyme complexes. Human liver mitochondrial short-chain enoyl-CoA
hydratase is
synthesized as a 290 amino acid precursor with a 29 amino acid N-terminal
leader peptide
(Kanazawa, M. et al. (1993) Enzyme Protein 47:9-13; and Janssen, U. et al.
(1997) Genomics 40:470-
475). Rat short-chain enoyl-CoA hydratase is 87% identical to the human
sequence in the mature
region of the protein and functions as a homohexamer (Kanazawa, supra; and
Engel, supra). A
mitochondrial trifunctional protein exists that has long-chain enoyl-CoA
hydratase, 3-hydroxyacyl-
CoA dehydrogenase, and long-chain 3-oxothiolase activities (Eaton, supra). In
human peroxisomes,
enoyl-CoA hydratase activity is found in both a 327 amino acid residue mono-
functional enzyme and
as part of a mufti-functional enzyme, also known as bifunctional enzyme, which
possesses enoyl-
CoA hydratase, enoyl-CoA isomerase, and 3-hydroxyacyl-CoA hydrogenase
activities (FitzPatrick,
D.R. et al. (1995) Genomics 27:457-466; and Hoefler, G. et al. (1994) Genomics
19:60-67). A 339
amino acid residue human protein with short-chain enoyl-CoA hydratase activity
also acts as an AU-
specific RNA binding protein (Nakagawa, J. et al. (1995) Proc. Natl. Acad.
Sci. USA 92:2051-2055).
All enoyl-CoA hydratases share homology near two active site glutamic acid
residues, with 17 amino
acid residues that are highly conserved (Wu, W.-J. et al. (1997) Biochemistry
36:2211-2220).
Inherited deficiencies in mitochondrial and peroxisomal beta-oxidation enzymes
are
associated with severe diseases, some of which manifest soon after birth and
lead to death within a
few years. Mitochondrial beta-oxidation associated deficiencies include, e.g.,
carnitine palmitoyl
transferase and carnitine deficiency, very-long-chain acyl-CoA dehydrogenase
deficiency, medium-
chain acyl-CoA dehydrogenase deficiency, short-chain acyl-CoA dehydrogenase
deficiency, electron
transport flavoprotein and electron transport flavoprotein:ubiquinone
oxidoreductase deficiency,
trifunctional protein deficiency, and short-chain 3-hydroxyacyl-CoA
dehydrogenase deficiency
(Eaton, supra). Mitochondrial trifunctional protein (including enoyl-CoA
hydratase) deficient
patients have reduced long-chain enoyl-CoA hydratase activities and suffer
from non-ketotic
hypoglycemia, sudden infant death syndrome, cardiomyopathy, hepatic
dysfunction, and muscle
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weakness, and may die at an early age (Eaton, supra).
Defects in mitochondria) beta-oxidation are associated with Reye's syndrome, a
disease
characterized by hepatic dysfunction and encephalopathy that sometimes follows
viral infection in
children. Reye's syndrome patients may have elevated serum levels of free
fatty acids (Cotran, R.S.
et al. (1994) Robbins Pathologic Basis of Disease, W.B. Saunders Co.,
Philadelphia PA, p.866).
Patients with mitochondria) short-chain 3-hydroxyacyl-CoA dehydrogenase
deficiency and medium-
chain 3-hydroxyacyl-CoA dehydrogenase deficiency also exhibit Reye-like
illnesses (Eaton, supra;
and Egidio, R.J. et al. (1989) Am. Fam. Physician 39:221-226).
Inherited conditions associated with peroxisomal beta-oxidation include
Zellweger
syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, acyl-CoA
oxidase deficiency,
peroxisomal thiolase deficiency, and bifunctional protein deficiency (Suzuki,
Y. et al. (1994) Am. J.
Hum. Genet. 54:36-43; Hoefler, supra). Patients with peroxisomal bifunctional
enzyme deficiency,
including that of enoyl-CoA hydratase, suffer from hypotonia, seizures,
psychomotor defects, and
defective neuronal migration; accumulate very-long-chain fatty acids; and
typically die within a few
years of birth (Watkins, P.A. et al. (1989) J. Clin. Invest. 83:771-1277).
Peroxisomal beta-oxidation is impaired in cancerous tissue. Although
neoplastic human
breast epithelial cells have the same number of peroxisomes as do normal
cells, fatty acyl-CoA
oxidase activity is lower than in control tissue (e1 Bouhtoury, F. et al.
(1992) J. Pathol. 166:27-35).
Human colon carcinomas have fewer peroxisomes than normal colon tissue and
have lower fatty-
acyl-CoA oxidase and bifunctional enzyme (including enoyl-CoA hydratase)
activities than normal
tissue (Cable, S. et al. (1992) Virchows Arch. B Cell Pathol. Incl. Mol.
Pathol. 62:221-226).
6-phosphogluconate dehydrogenase (6-PGDH) catalyses the NADP+-dependent
oxidative
decarboxylation of 6-phosphogluconate to ribulose 5-phosphate with the
production of NADPH. The
absence or inhibition of 6-PGDH results in the accumulation of 6-
phosphogluconate to toxic levels in
eukaryotic cells. 6-PGDH is the third enzyme of the pentose phosphate pathway
(PPP) and is
ubiquitous in nature. In some heterofermentatative species, NAD+ is used as a
cofactor with the
subsequent production of NADH.
The reaction proceeds through a 3-keto intermediate which is decarboxylated to
give the enol
of ribulose 5-phosphate, then converted to the keto product following
tautomerization of the enol
(Berdis A.J. and P.F. Cook (1993) Biochemistry 32:2041-2046). 6-PGDH activity
is regulated by the
inhibitory effect of NADPH, and the activating effect of 6-phosphogluconate
(Rippa, M. et al. (1998)
Biochim. Biophys. Acta 1429:83-92). Deficiencies in 6-PGDH activity have been
linked to chronic
hemolytic anemia.
The targeting of specific forms of 6-PGDH (e.g., enzymes found in
trypanosomes) has been
suggested as a means for controlling parasitic infections (Tetaud, E. et al.
(1999) Biochem. J.
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338:55-60). For example, the T. brucei enzyme is markedly more sensitive to
inhibition by the
substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue
adenosine
2',5'-bisphosphate, compared to the mammalian enzyme (Hanau, S. et al. (1996)
Eur. J. Biochem.
240:592-599).
Ribonucleotide diphosphate reductase catalyzes the reduction of ribonucleotide
diphosphates
(i.e., ADP, GDP, CDP, and UDP) to their corresponding deoxyribonucleotide
diphosphates (i.e.,
dADP, dGDP, dCDP, and dUDP) which are used for the synthesis of DNA.
Ribonucleotide
diphosphate reductase thereby performs a crucial role in the de novo synthesis
of deoxynucleotide
precursors. Deoxynucleotides are also produced from deoxynucleosides by
nucleoside kinases via
the salvage pathway.
Mammalian ribonucleotide diphosphate reductase comprises two components, an
effector-
binding component (E) and a non-heme iron component (F). Component E binds the
nucleoside
triphosphate effectors while component F contains the iron radical necessary
for catalysis. Molecular
weight determinations of the E and F components, as well as the holoenzyme,
vary according to the
methods used in purification of the proteins and the particular laboratory.
Component E is
approximately 90-100 kDa, component F is approximately 100-120 kDa, and the
holoenzyme is 200-
250 kDa.
Ribonucleotide diphosphate reductase activity is adversely effected by iron
chelators, such as
thiosemicarbazones, as well as EDTA. Deoxyribonucleotide diphosphates also
appear to be negative
allosteric effectors of ribonucleotide diphosphate reductase. Nucleotide
triphosphates (both ribo- and
deoxyribo-) appear to stimulate the activity of the enzyme. 3-methyl-4-
nitrophenol, a metabolite of
widely used organophosphate pesticides, is a potent inhibitor of
ribonucleotide diphosphate reductase
in mammalian cells. Some evidence suggests that ribonucleotide diphosphate
reductase activity in
DNA virus (e.g., herpes virus) -infected cells and in cancer cells is less
sensitive to regulation by
allosteric regulators and a correlation exists between high ribonucleotide
diphosphate reductase
activity levels and high rates of cell proliferation (e.g., in hepatomas).
This observation suggests that
virus-encoded ribonucleotide diphosphate reductases, and those present in
cancer cells, are capable of
maintaining an increased supply deoxyribonucleotide pool for the production of
virus genomes or for
the increased DNA synthesis which characterizes cancers cells. Ribonucleotide
diphosphate
reductase is thus a target for therapeutic intervention (Nutter, L.M. and Y.-
C. Cheng (1984) Pharmac.
Ther. 26:191-207; and Wright, J.A. (1983) Pharmac. Ther. 22:81-102).
Dihydrodiol dehydrogenases (DD) are monomeric, NAD(P)+-dependent, 34-37 kDa
enzymes
responsible for the detoxification of traps-dihydrodiol and anti-diol epoxide
metabolites of
polycyclic aromatic hydrocarbons (PAH) such as benzo[a]yrene,
benz[a]anthracene, 7-methyl-
benz[a]anthracene, 7,12-dimethyl-Benz[a]anthracene, chrysene, and 5-methyl-
chrysene. In
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mammalian cells, an environmental PAH toxin such as benzo[a]yrene is initially
epoxidated~by a
microsomal cytochrome P450 to yield 7R,8R-arene-oxide and subsequently (-)-
7R,8R-dihydrodiol ((-
-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene or (-)-trans-B[a]P-diol) This
latter compound is
further transformed to the anti-diol epoxide of benzo[a]pyrene (i.e., (~)-anti-
7,~3,8a dihydroxy-
9a,10a epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene), by the same enzyme or a
different enzyme,
depending on the species. This resulting anti-diol epoxide of benzo[a]yrene,
or the corresponding
derivative from another PAH compound, is highly mutagenic.
DD efficiently oxidizes the precursor of the anti-diol epoxide (i.e., trans-
dihydrodiol) to
transient catechols which auto-oxidize to quinones, also producing hydrogen
peroxide and
semiquinone radicals. This reaction prevents the formation of the highly
carcinogenic anti-diol.
Anti-diols are not themselves substrates for DD yet the addition of DD to a
sample comprising an
anti-diol compound results in a significant decrease in the induced mutation
rate observed in the
Ames test. In this instance, DD is able to bind to and sequester the anti-
diol, even though it is not
oxidized. Whether through oxidation or sequestration, DD plays an important
role in the
detoxification of metabolites of xenobiotic polycyclic compounds (Penning,
T.M. (1993) Chemico-
Biological Interactions 89:1-34).
15-oxoprostaglandin 13-reductase (PGR) and 15-hydroxyprostaglandin
dehydrogenase ( 15-
PGDH) are enzymes present in the lung that are responsible for degrading
circulating prostaglandins.
Oxidative catabolism via passage through the pulmonary system is a common
means of reducing the
concentration of circulating prostaglandins. 15-PGDH oxidizes the 15-hydroxyl
group of a variety of
prostaglandins to produce the corresponding 15-oxo compounds. The 15-oxo
derivatives usually
have reduced biological activity compared to the 15-hydroxyl molecule. PGR
further reduces the
13,14 double bond of the 15-oxo compound which typically leads to a further
decrease in biological
activity. PGR is a monomer with a molecular weight of approximately 36 kDa.
The enzyme
requires NADH or NADPH as a cofactor with a preference for NADH. The 15-oxo
derivatives of
prostaglandins PGE,, PGE2, and PGEZa are all substrates for PGR; however, the
non-derivatized
prostaglandins (i.e., PGE,, PGE2, and PGEZa) are not substrates (Ensor, C.M.
et al. (1998) Biochem. J.
330:103-108).
15-PGDH and PGR also catalyze the metabolism of lipoxin A4 (LXA4). Lipoxins
(LX) are
autacoids, lipids produced at the sites of localized inflammation, which down-
regulate
polymorphonuclear leukocyte (PMN) function and promote resolution of localized
trauma. Lipoxin
production is stimulated by the administration of aspirin in that cells
displaying cyclooxygenase II
(COX II) that has been acetylated by aspirin and cells that possess 5-
lipoxygenase (5-LO) interact
and produce lipoxin. 15-PGDH generates 15-oxo-LXA4 with PGR further converting
the 15-oxo
compound to 13,14-dihydro-15-oxo-LXA4 (Clish, C.B. et al. (2000) J. Biol.
Chem.
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275:25372-25380). This finding suggests a broad substrate specificity of the
prostaglandin
dehydrogenases and has implications for these enzymes in drug metabolism and
as targets for
therapeutic intervention to regulate inflammation.
The GMC (glucose-methanol-choline) oxidoreductase family of enzymes was
defined based
on sequence alignments of Drosophila melano aster glucose dehydrogenase,
Escherichia coli choline
dehydrogenase, Asper illus niger glucose oxidase, and Hansenula polymorpha
methanol oxidase.
Despite their different sources and substrate specificities, these four
flavoproteins are homologous,
being characterized by the presence of several distinctive sequence and
structural features. Each
molecule contains a canonical ADP-binding, beta-alpha-beta mononucleotide-
binding motif close to
the amino terminus. This fold comprises a four-stranded parallel beta-sheet
sandwiched between a
three-stranded antiparallel beta-sheet and alpha-helices. Nucleotides bind in
similar positions relative
to this chain fold (Cavener, D.R. (1992) J. Mol. Biol. 223:811-814; Wierenga,
R.K. et al. (1986) J.
Mol. Biol. 187:101-107). Members of the GMC oxidoreductase family also share a
consensus
sequence near the central region of the polypeptide. Additional members of the
GMC oxidoreductase
family include cholesterol oxidases from Brevibacterium sterolicum and
Stre~omyces; and an
alcohol dehydrogenase from Pseudomonas oleovorans (Cavener, D.R., supra;
Henikoff, S. and J.G.
Henikoff (1994) Genomics 19:97-107; van Beilen, J.B. et al. (1992) Mol.
Microbiol. 6:3121-3136).
IMP dehydrogenase and GMP reductase are two oxidoreductases which share many
regions
of sequence similarity. IMP dehydrogenase (EC 1.1.1.205) catalyes the NAD-
dependent reduction of
IMP (inosine monophosphate) into XMP (xanthine monophosphate) as part of de
novo GTP
biosynthesis (Collart, F.R. and E. Huberman (1988) J. Biol. Chem. 263:15769-
15772). GMP
reductase catalyzes the NADPH-dependent reductive deamination of GMP into IMP,
helping to
maintain the intracellular balance of adenine and guanine nucleotides
(Andrews, S.C. and J.R. Guest
(1988) Biochem. J. 255:35-43).
Pyridine nucleotide-disulphide oxidoreductases are FAD flavoproteins involved
in the
transfer of reducing equivalents from FAD to a substrate. These flavoproteins
contain a pair of
redox-active cysteines contained within a consensus sequence which is
characteristic of this protein
family (Kurlyan, J. et al. (1991) Nature 352:172-174). Members of this family
of oxidoreductases
include glutathione reductase (EC 1.6.4.2); thioredoxin reductase of higher
eukaryotes (EC 1.6.4.5);
trypanothione reductase (EC 1.6.4.8); lipoamide dehydrogenase (EC 1.8.1.4),
the E3 component of
alpha-ketoacid dehydrogenase complexes; and mercuric reductase (EC 1.16.1.1).
Transferases
Transferases are enzymes that catalyze the transfer of molecular groups. The
reaction may
involve an oxidation, reduction, or cleavage of covalent bonds, and is often
specific to a substrate or
to particular sites on a type of substrate. Transferases participate in
reactions essential to such
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functions as synthesis and degradation of cell components, and regulation of
cell functions including
cell signaling, cell proliferation, inflammation, apoptosis, secretion and
excretion. Transferases are
involved in key steps in disease processes involving these functions.
Transferases are frequently
classified according to the type of group transferred. For example, methyl
transferases transfer one-
s carbon methyl groups, amino transferases transfer nitrogenous amino groups,
and similarly
denominated enzymes transfer aldehyde or ketone, acyl, glycosyl, alkyl or
aryl, isoprenyl, saccharyl,
phosphorous-containing, sulfur-containing, or selenium-containing groups, as
well as small
enzymatic groups such as Coenzyme A.
Acyl transferases include peroxisomal carnitine octanoyl transferase, which is
involved in the
fatty acid beta-oxidation pathway, and mitochondria) carnitine palmitoyl
transferases, involved in
fatty acid metabolism and transport. Choline O-acetyl transferase catalyzes
the biosynthesis of the
neurotransmitter acetylcholine. N-acyltransferase enzymes catalyze the
transfer of an amino acid
conjugate to an activated carboxylic group. Endogenous compounds and
xenobiotics are activated by
acyl-CoA synthetases in the cytosol, microsomes, and mitochondria. The acyl-
CoA intermediates are
then conjugated with an amino acid (typically glycine, glutamine, or taurine,
but also ornithine,
arginine, histidine, serine, aspartic acid, and several dipeptides) by N-
acyltransferases in the cytosol
or mitochondria to form a metabolite with an amide bond. One well-
characterized enzyme of this
class is the bile acid-CoA:amino acid N-acyltransferase (BAT) responsible for
generating the bile
acid conjugates which serve as detergents in the gastrointestinal tract
(Falany, C. N. et al. (1994) J.
Biol. Chem. 269:19375-9; Johnson, M. R. et al. (1991) J. Biol. Chem. 266:10227-
33). BAT is also
useful as a predictive indicator for prognosis of hepatocellular carcinoma
patients after partial
hepatectomy (Furutani, M.~et al. (1996) Hepatology 24:1441-5).
Acetyltransferases
Acetyltransferases have been extensively studied for their role in histone
acetylation.
Histone
acetylation results in the relaxing of the chromatin structure in eukaryotic
cells, allowing transcription
factors to gain access to promoter elements of the DNA templates in the
affected region of the
genome (or the genome in general). In contrast, histone deacetylation results
in a reduction in
transcription by closing the chromatin structure and limiting access of
transcription factors. To this
end, a common means of stimulating cell transcription is the use of chemical
agents that inhibit the
deacetylation of histones (e.g., sodium butyrate), resulting in a global
(albeit artifactual) increase in
gene expression. The modulation of gene expression by acetylation also results
from the acetylation
of other proteins, including but not limited to, p53, GATA-1, MyoD, ACTR,
TFIIE, TFIIF and the
high mobility group proteins (HMG). In the case of p53, acetylation results in
increased DNA
binding, leading to the stimulation of transcription of genes regulated by
p53. The prototypic histone
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acetylase (HAT) is GcnS from Saccharomyces cerevisiae. GcnS is a member of a
family of
acetylases that includes Tetrahymena p55, human GcnS, and human p300/CBP.
Histone acetylation
is reviewed in (Cheung, W.L. et al. (2000) Curr. Opin. Cell Biol. 12:326-333
and Berger, S.L (1999)
Curr. Opin. Cell Biol. 11:336-341). Some acetyltransferase enzymes possess the
alpha/beta
hydrolase fold (Center of Applied Molecular Engineering Inst. of Chemistry and
Biochemistry -
University of Salzburg, http://predict.Banger.ac.uk/irbm-course97/Docs/ms/)
common to several
other major classes of enzymes, including but not limited to,
acetylcholinesterases and
carboxylesterases (Structural Classification of Proteins,
http://scop.mrc-lmb.cam.ac.uk/scop/index.html).
N-acetyltransferases are cytosolic enzymes which utilize the cofactor acetyl-
coenzyme A
(acetyl-CoA) to transfer the acetyl group to aromatic amines and hydrazine
containing compounds.
In humans, there are two highly similar N-acetyltransferase enzymes, NAT1 and
NAT2; mice appear
to have a third form of the enzyme, NAT3. The human forms of N-
acetyltransferase have
independent regulation (NAT1 is widely-expressed, whereas NAT2 is in liver and
gut only) and
overlapping substrate preferences. Both enzymes appear to accept most
substrates to some extent,
but NAT1 does prefer some substrates (para-aminobenzoic acid, para-
aminosalicylic acid,
sulfamethoxazole, and sulfanilamide), while NAT2 prefers others (isoniazid,
hydralazine,
procainamide, dapsone, aminoglutethimide, and sulfamethazine). A recently
isolated human gene,
tubedown-1, is homologous to the yeast NAT-1 N-acetyltransferases and encodes
a protein associated
with acetyltransferase activity. The expression patterns of tubedown-1 suggest
that it may be
involved in regulating vascular and hematopoietic development (Gendron, R.L.
et al. (2000) Dev.
Dyn. 218:300-31 S).
Amino transferases comprise a family of pyridoxal 5'-phosphate (PLP) -
dependent enzymes
that catalyze transformations of amino acids. Amino transferases play key
roles in protein synthesis
and degradation, and they contribute to other processes as well. For example,
GABA
aminotransferase (GABA-T) catalyzes the degradation of GABA, the major
inhibitory amino acid
neurotransmitter. The activity of GABA-T is correlated to neuropsychiatric
disorders such as
alcoholism, epilepsy, and Alzheimer's disease (Sherif, F.M. and Ahmed, S.S.
(1995) Clin. Biochem.
28:145-154). Other members of the family include pyruvate aminotransferase,
branched-chain amino
acid aminotransferase, tyrosine aminotransferase, aromatic aminotransferase,
alanine:glyoxylate
aminotransferase (AGT), and kynurenine aminotransferase (Vacca, R.A. et al.
(1997) J. Biol. Chem.
272:21932-21937). Kynurenine aminotransferase catalyzes the irreversible
transamination of the
L-tryptophan metabolite L-kynurenine to form kynurenic acid. The enzyme may
also catalyzes the
reversible transamination reaction between L-2-aminoadipate and 2-oxoglutarate
to produce
2-oxoadipate and L-glutamate. Kynurenic acid is a putative modulator of
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neurotransmission, thus a deficiency in kynurenine aminotransferase may be
associated with
pleiotropic effects (Buchli, R. et al. (1995) J. Biol. Chem. 270:29330-29335).
Glycosyl transferases include the mammalian UDP-glucouronosyl transferases, a
family of
membrane-bound microsomal enzymes catalyzing the transfer of glucouronic acid
to lipophilic
substrates in reactions that play important roles in detoxification and
excretion of drugs, carcinogens,
and other foreign substances. Another mammalian glycosyl transferase,
mammalian UDP-galactose-
ceramide galactosyl transferase, catalyzes the transfer of galactose to
ceramide in the synthesis of
galactocerebrosides in myelin membranes of the nervous system. The UDP-
glycosyl transferases
share a conserved signature domain of about 50 amino acid residues (PROSITE:
PDOC00359,
http://expasy.hcuge.ch/sprot/prosite.html).
Methyl transferases are involved in a variety of pharmacologically important
processes.
Nicotinamide N-methyl transferase catalyzes the N-methylation of nicotinamides
and other pyridines,
an important step in the cellular handling of drugs and other foreign
compounds.
Phenylethanolamine N-methyl transferase catalyzes the conversion of
noradrenalin to adrenalin. 6-
O-methylguanine-DNA methyl transferase reverses DNA methylation, an important
step in
carcinogenesis. Uroporphyrin-III C-methyl transferase, which catalyzes the
transfer of two methyl
groups from S-adenosyl-L-methionine to uroporphyrinogen III, is the first
specific enzyme in the
biosynthesis of cobalamin, a dietary enzyme whose uptake is deficient in
pernicious anemia. Protein-
arginine methyl transferases catalyze the posttranslational methylation of
arginine residues in
proteins, resulting in the mono- and dimethylation of arginine on the
guanidino group. Substrates
include histones, myelin basic protein, and heterogeneous nuclear
ribonucleoproteins involved in
mRNA processing, splicing, and transport. Protein-arginine methyl transferase
interacts with
proteins upregulated by mitogens, with proteins involved in chronic
lymphocytic leukemia, and with
interferon, suggesting an important role for methylation in cytokine receptor
signaling (Lin, W.-J. et
al. (1996) J. Biol. Chem. 271:15034-15044; Abramovich, C. et al. (1997) EMBO
J. 16:260-266; and
Scott, H. S. et al. (1998) Genomics 48:330-340).
Phospho transferases catalyze the transfer of high-energy phosphate groups and
are important
in energy-requiring and -releasing reactions. The metabolic enzyme creatine
kinase catalyzes the
reversible phosphate transfer between creatine/creatine phosphate and ATP/ADP.
Glycocyamine
kinase catalyzes phosphate transfer from ATP to guanidoacetate, and arginine
kinase catalyzes
phosphate transfer from ATP to arginine. A cysteine-containing active site is
conserved in this
family (PROSITE: PDOC00103).
Prenyl transferases are heterodimers, consisting of an alpha and a beta
subunit, that catalyze
the transfer of an isoprenyl group. The Ras farnesyltransferase (FTase) enzyme
transfers a farnesyl
moiety from cytosolic farnesylpyrophosphate to a cysteine residue at the
carboxyl terminus of the
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Ras oncogene protein. This modification is required to anchor Ras to the cell
membrane so that it can
perform its role in signal transduction. FTase inhibitors block Ras function
and demonstrate
antitumor activity-(Buolamwini, J.K. (1999) Curr. Opin. Chem. Biol. 3:500-
509). Ftase, which
shares structural similarity with geranylgeranyl transferase, or Rab GG
transferase, prenylates Rab
proteins, allowing them to perform their roles in regulating vesicle transport
(Seabra, M.C. (1996) J.
Biol. Chem. 271:143913-24404).
Saccharyl transferases are glycating enzymes involved in a variety of
metabolic processes.
Oligosaccharyl transferase-48, for example, is a receptor for advanced
glycation endproducts, which
accumulate in vascular complications of diabetes, macrovascular disease, renal
insufficiency, and
Alzheimer's disease (Thornalley, P. J. (1998) Cell Mol. Biol. (Noisy-Le-Grand)
44:1013-1023).
Coenzyme A (CoA) transferase catalyzes the transfer of CoA between two
carboxylic acids.
Succinyl CoA:3-oxoacid CoA transferase, for example, transfers CoA from
succinyl-CoA to a
recipient such as acetoacetate. Acetoacetate is essential to the metabolism of
ketone bodies, which
accumulate in tissues affected by metabolic disorders such as diabetes
(PROSITE: PDOC00980).
Transglutaminase transferases (Tgases) are Ca2+ dependent enzymes capable of
forming
isopeptide bonds by catalyzing the transfer of the y-carboxy group from
protein-bound glutamine to
the E-amino group of protein-bound lysine residues or other primary amines.
Tgases are the enzymes
responsible for the cross-linking of cornified envelope (CE), the highly
insoluble protein structure on
the surface of corneocytes, into a chemically and mechanically resistant
protein polymer. Seven
known human Tgases have been identified. Individual transglutaminase gene
products are
specialized in the cross-linking of specific proteins or tissue structures,
such as factor XIIIa which
stabilizes the fibrin clot in hemostasis, prostrate transglutaminase which
functions in semen
coagulation, and tissue transglutaminase which is involved in GTP-binding in
receptor signaling.
Four (Tgases l, 2, 3, and X) are expressed in terminally differentiating
epithelia such as the
epidermis. Tgases are critical for the proper cross-linking of the CE as seen
in the pathology of
patients suffering from one form of the skin diseases referred to as
congenital ichthyosis which has
been linked to mutations in the keratinocyte transglutaminase (TG K) gene
(Nemes, Z. et al., (1999)
Proc. Natl. Acad. Sci. U.S.A. 96:8402-8407, Aeschlimann, D. et al., (1998) J.
Biol. Chem. 273:3452-
3460.)
Hydrolases
Hydrolases are a class of enzymes that catalyze the cleavage of various
covalent bonds in a
substrate by the introduction of a molecule of water. The reaction involves a
nucleophilic attack by
the water molecule's oxygen atom on a target bond in the substrate. The water
molecule is split
across the target bond, breaking the bond and generating two product
molecules. Hydrolases
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participate in reactions essential to such functions as synthesis and
degradation of cell components,
and for regulation of cell functions including cell signaling, cell
proliferation, inflamation, apoptosis,
secretion and excretion. Hydrolases are involved in key steps in disease
processes involving these
functions. Hydrolytic enzymes, or hydrolases, may be grouped by substrate
specificity into classes
including phosphatases, peptidases, lysophospholipases, phosphodiesterases,
glycosidases,
glyoxalases, aminohydrolases, carboxylesterases, sulfatases,
phosphohydrolases, nucleotidases,
lysozymes, and many others.
Phosphatases hydrolytically remove phosphate groups from proteins, an energy-
providing
step that regulates many cellular processes, including intracellular signaling
pathways that in turn
control cell growth and differentiation, cell-cell contact, the cell cycle,
and oncogenesis.
Peptidases, also called proteases, cleave peptide bonds that form the backbone
of peptide or
protein chains. Proteolytic processing is essential to cell growth,
differentiation, remodeling, and
homeostasis as well as inflammation and the immune response. Since typical
protein half-lives range
from hours to a few days, peptidases are continually cleaving precursor
proteins to their active form,
removing signal sequences from targeted proteins, and degrading aged or
defective proteins.
Peptidases function in bacterial, parasitic, and viral invasion and
replication within a host. Examples
of peptidases include trypsin and chymotrypsin (components of the complement
cascade and the
blood-clotting cascade) lysosomal cathepsins, calpains, pepsin, renin, and
chymosin (Beynon, R.J.
and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford
University Press, New
York, NY, pp. 1-5).
Lysophospholipases (LPLs) regulate intracellular lipids by catalyzing the
hydrolysis of ester
bonds to remove an acyl group, a key step in lipid degradation. Small LPL
isoforms, approximately
15-30 kD, function as hydrolases; larger isoforms function both as hydrolases
and transacylases. A
particular substrate for LPLs, lysophosphatidylcholine, causes lysis of cell
membranes. LPL activity
is regulated by signaling molecules important in numerous pathways, including
the inflammatory
response.
The phosphodiesterases catalyze the hydrolysis of one of the two ester bonds
in a
phosphodiester compound. Phosphodiesterases are therefore crucial to a variety
of cellular processes.
Phosphodiesterases include DNA and RNA endo- and exo-nucleases, which are
essential to cell
growth and replication as well as protein synthesis. Endonuclease V
(deoxyinosine 3'-endonuclease)
is an example of a type II site-specific deoxyribonuclease, a putative DNA
repair enzyme that cleaves
DNAs containing hypoxanthine, uracil, or mismatched bases. Escherichia coli
endonuclease V has
been shown to cleave DNA containing deoxyxanthosine at the second
phosphodiester bond 3' to
deoxyxanthosine, generating a 3'-hydroxyl and a 5'-phosphoryl group at the
nick site (He, B. et al.
(2000) Mutat. Res. 459:109-114). It has been suggested that Escherichia coli
endonuclease V plays a
18
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role in the removal of deaminated guanine, i.e., xanthine, from DNA, thus
helping to protect the cell
against the mutagenic effects of nitrosative deamination (Schouten, K.A. and
Weiss, B. (1999) Mutat.
Res. 435:245-254). In eukaryotes, the process of tRNA splicing requires the
removal of small tRNA
introns that interrupt the anticodon loop 1 base 3' to the anticodon. This
process requires the
stepwise action of an endonuclease, a ligase, and a phosphotransferase (Hong,
L. et al. (1998) Science
280:279-284). Ribonuclease P (RNase P) is a ubiquitous RNA processing
endonuclease that is
required for generating the mature tRNA 5'-end during the tRNA splicing
process. This is
accomplished through the catalysis of the cleavage of P-3'O bonds to produce
5'-phosphate and 3'-
hydroxyl end groups at a specific site on pre-tRNA. Catalysis by RNase P is
absolutely dependent on
divalent canons such as Mgz+ or Mnz+ (Kurz, J.C. et al. (2000) Curr. Opin.
Chem. Biol. 4:553-558).
Substrate recognition mechanisms of RNase P are well conserved among
eukaryotes and bacteria
(FENZMi, S. et al. (1998) Science 280:284-286). In S. cerevisiae, POPl
('processing of precursor
RNAs') encodes a protein component of both RNase P and RNase MRP, another RNA
processing
protein. Mutations in yeast POP1 are lethal (Lygerou, Z. et al. (1994) Genes
Dev. 8:1423-1433).
Another phosphodiesterase, acid sphingomyelinase, hydrolyzes the membrane
phospholipid
sphingomyelin to ceramide and phosphorylcholine. Phosphorylcholine functions
in synthesis of
phosphatidylcholine, which is involved in intracellular signaling pathways.
Ceramide is an essential
precursor for the generation of gangliosides, membrane lipids found in high
concentration in neural
tissue. Defective acid sphingomyelinase phosphodiesterase leads to Niemann-
Pick disease.
Glycosidases catalyze the cleavage of hemiacetyl bonds of glycosides, which
are compounds
that contain one or more sugar. Mammalian lactase-phlorizin hydrolase, for
example, is an intestinal
enzyme that splits lactose. Mammalian beta-galactosidase removes the terminal
galactose from
gangliosides, glycoproteins, and glycosaminoglycans, and deficiency of this
enzyme is associated
with a gangliosidosis known as Morquio disease type B (PROSITE PCDOC00910).
Vertebrate
lysosomal alpha-glucosidase, which hydrolyzes glycogen, maltose, and
isomaltose, and vertebrate
intestinal sucrase-isomaltase, which hydrolyzes sucrose, maltose, and
isomaltose, are widely
distributed members of this family with highly conserved sequences at their
active sites.
The glyoxylase system is involved in gluconeogenesis, the production of
glucose from
storage compounds in the body. It consists of glyoxylase I, which catalyzes
the formation of S-D-
lactoylglutathione from methyglyoxal, a side product of triose-phosphate
energy metabolism, and
glyoxylase II, which hydrolyzes S-D-lactoylglutathione to D-lactic acid and
reduced glutathione.
Glyoxylases are involved in hyperglycemia, non-insulin-dependent diabetes
mellitus, the
detoxification of bacterial toxins, and in the control of cell proliferation
and microtubule assembly.
NG,NG-dimethylarginine dimethylaminohydrolase (DDAH) is an enzyme that
hydrolyzes the
endogenous nitric oxide synthase (NOS) inhibitors, NG-monomethyl-arginine and
NG,NG-dimethyl-
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L-arginine, to L-citrulline. Inhibiting DDAH can cause increased intracellular
concentration of NOS
inhibitors to levels sufficient to inhibit NOS. Therefore, DDAH inhibition may
provide a method of
NOS inhibition, and changes in the activity of DDAH could play a role in
pathophysiological
alterations in nitric oxide generation (MacAllister, R.J. et al. (1996) Br. J.
Pharmacol. 119:1533-
1540). DDAH was found in neurons displaying cytoskeletal abnormalities and
oxidative stress in
Alzheimer's disease. In age-matched control cases, DDAH was not found in
neurons. This suggests
that oxidative stress- and nitric oxide-mediated events play a role in the
pathogenesis of Alzheimer's
disease (Smith, M.A. et al. ( 1998) Free Rad. Biol. Med. 25:898-902).
Acyl-CoA thioesterase is another member of the carboxylesterase family
(Alexson, S.E. et al.
(1993) Eur. J. Biochem. 214:719-727). Evidence suggests that acyl-CoA
thioesterase has a regulatory
role in steroidogenic tissues (Finkielstein, C. et al. (1998) Eur. J. Biochem.
256:60-66).
The alpha/beta hydrolase protein fold is common to several hydrolases of
diverse
phylogenetic origin and catalytic function. Enzymes with the alpha/beta
hydrolase fold have a
common core structure consisting of eight beta-sheets connected by alpha-
helices. The most
conserved structural feature of this fold is the loops of the nucleophile-
histidine-acid catalytic triad.
The histidine in the catalytic triad is completely conserved, while the
nucleophile and acid loops
accommodate more than one type of amino acid (Ollis, D.L. et al. (1992)
Protein Eng. 5:197-211).
Sulfatases are members of a highly conserved gene family that share extensive
sequence
homology and a high degree of structural similarity. Sulfatases catalyze the
cleavage of sulfate esters.
To perform this function, sulfatases undergo a unique post-translational
modification in the
endoplasmic reticulum that involves the oxidation of a conserved cysteine
residue. A human disorder
called multiple sulfatase deficiency is due to a defect in this post-
translational modification step,
leading to inactive sulfatases (Recksiek, M. et al. (1998) J. Biol. Chem.
273:6096-6103).
Phosphohydrolases are enzymes that hydrolyze phosphate esters. Some
phosphohydrolases
contain a mutT domain signature sequence. MutT is a protein involved in the GO
system responsible
for removing an oxidatively damaged form of guanine from DNA. A region of
about 40 amino acid
residues, found in the N-terminus of mutT, is also found in other proteins,
including some
phosphohydrolases (PROSITE PDOC00695).
Serine hydrolases are a large functional class of hydrolytic enzymes that
contain a serine
residue in their active site. This class of enzymes contains proteinases,
esterases, and lipases which
hydrolyze a variety of substrates and, therefore, have different biological
roles. Proteins in this
superfamily can be further grouped into subfamilies based on substrate
specificity or amino acid
similarities (Puente, X.S. and Lopez-Otin, C. (1995) J. Biol. Chem. 270: 12926-
12932).
Neuropathy target esterase (NTE) is an integral membrane protein present in
all neurons and
in some non-neural-cell types of vertebrates. NTE is involved in a cell-
signaling pathway controlling
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interactions between neurons and accessory glial cells in the developing
nervous system. NTE has
serine esterase activity and efficiently catalyses the hydrolysis of phenyl
valerate (PV) in vitro, but its
physiological substrate is unknown. NTE is not related to either the major
serine esterase family,
which includes acetylcholinesterase, nor to any other known serine hydrolases.
NTE contains at least
two functional domains: an N-terminal putative regulatory domain and a C-
terminal effector domain
which contains the esterase activity and is, in part, conserved in proteins
found in bacteria, yeast,
nematodes and insects. NTE's effector domain contains three predicted
transmembrane segments,
and the active-site serine residue lies at the center of one of these
segments. The isolated
recombinant domain shows PV hydrolase activity only when incorporated into
phospholipid
liposomes. NTE's esterase activity is largely redundant in adult vertebrates,
but organophosphates
which react with NTE in vivo initiate unknown events which lead to a
neuropathy with degeneration
of long axons. These neuropathic organophosphates leave a negatively charged
group covalently
attached to the active-site serine residue, which causes a toxic gain of
function in NTE (Glynn, P.
(1999) Biochem. J. 344:625-631). Further, the Drosophila neurodegeneration
gene Swiss-cheese
encodes a neuronal protein involved in glia-neuron interaction and is
homologous to the above human
NTE (Moser, M. et al. (2000) Mech. Dev. 90:279-282).
Chitinases are chitin-degrading enzymes present in a variety of organisms and
participate in
processes including cell wall remodeling, defense and catabolism. Chitinase
activity has been found
in human serum, leukocytes, granulocytes, and in association with fertilized
oocytes in mammals
(Escott, G.M. (1995) Infect. Immunol. 63:4770-4773; DeSouza, M.M. (1995)
Endrocrinology
136:2485-2496). Glycolytic and proteolytic molecules in humans are associated
with tissue damage
in lung diseases and with increased tumorigenicity and metastatic potential of
cancers (Mulligan,
M.S. (1993) Proc. Natl. Acad. Sci. 90:11523-11527; Matrisian, L.M. (1991) Am.
J. Med. Sci.
302:157-162; Witty, J.P. (1994) Cancer Res. 54:4805-4812). The discovery of a
human enzyme with
chitinolytic activity is noteworthy given the lack of endogenous chitin in the
human body (Raghavan,
N. (1994) Infect. Immun. 62:1901-1908). However, there is a group of mammalian
proteins that
share homology with chitinases from various non-mammalian organisms, such as
bacteria, fungi,
plants, and insects. The members of this family differ in their ability to
hydrolyze chitin or chitin-like
substrates. Some of the mammalian members of the family, such as a bovine whey
chitotriosidase
and human cartilage proteins which do not demonstrate specific chitinolytic
activity, are expressed in
association with tissue remodeling events (Rejman, J.J. (1988) Biochem.
Biophys. Res. Commun.
150:329-334, Nyirkos, P. (1990) Biochem. J. 268:265-268). Elevated levels of
human cartilage
proteins have been reported in the synovial fluid and cartilage of patients
with rheumatoid arthritis, a
disease which produces a severe degradation of the cartilage and a
proliferation of the synovial
membrane in the affected joints (Hakala, B.E. (1993) J. Biol. Chem. 268:25803-
25810).
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A small subclass of hydrolases acting on ether bonds includes the thioether
hydrolases. S-
adenosyl-L-homocysteine hydrolase, also known as AdoHcyase or SAHH (PROSITE
PDOC00603;
EC 3.3.1.1), is a thioether hydrolase first described in rat liver extracts as
the activity responsible for
the reversible hydrolysis of S-adenosyl-L-homocysteine (AdoHcy) to adenosine
and homocysteine
(Sganga, M.W. et al. (1992) PNAS 89:6328-6332). SAHH is a cytosolic enzyme
that has been found
in all cells that have been tested, with the exception of Escherichia coli and
certain related bacteria
(Walker, R.D. et al. (1975) Can. J. Biochem. 53:312-319; Shimizu, S. et al.
(1988) FEMS Microbiol.
Lett. 51:177-180; Shimizu, S. et al. (1984) Eur. J. Biochem. 141:385-392).
SAHH activity is
dependent on NAD+ as a cofactor. Deficiency of SAHH is associated with
hypermethioninemia
(Online Mendelian Inheritance in Man (OMIM) #180960 Hypermethioninemia), a
pathologic
condition characterized by neonatal cholestasis, failure to thrive, mental and
motor retardation, facial
dysmorphism with abnormal hair and teeth, and myocaridopathy (Labrune, P. et
al. (1990) J. Pediat.
117:220-226).
Another subclass of hydrolases includes those enzymes which act on carbon-
nitrogen (C-N)
bonds other than peptide bonds. To this subclass belong those enzymes
hydrolyzing amides,
amidines, and other C-N bonds. This subclass is further subdivided on the
basis of substrate
specificity such as linear amides, cyclic amides, linear amidines, cyclic
amidines, nitriles and other
compounds. A hydrolase belonging to the sub-subclass of enzymes acting on the
cyclic amidines is
adenosine deaminase (ADA). ADA catalyzes the breakdown of adenosine to
inosine. ADA is
present in many mammalian tissues, including placenta, muscle, lung, stomach,
digestive
diverticulum, spleen, erythrocytes, thymus, seminal plasma, thyroid, T-cells,
bone marrow stem cells,
and liver. A subclass of ADAs, ADAR, act on RNA and are classified as RNA
editases. An ADAR
from Drosophila, dADAR, expressed in the developing nervous system, may act on
para voltage-
gated Na+ channel transcripts in the central nervous system (Palladino, M.J.
et al. (2000) RNA
6:1004-1018). ADA deficiency causes profound lymphopenia with severe combined
immunodeficiency (SLID). Cells from patients with ADA deficiency contain low,
sometimes
undetectable, amounts of ADA catalytic activity and ADA protein. ADA
deficiency stems from
genetic mutations in the ADA gene (Hershfield, M.S. (1998) Semin. Hematol.
4:291-298). Metabolic
consequences of ADA deficiency are associated with defects in alveogenesis,
pulmonary
inflammation, and airway obstruction (Blackburn, M.R. et al. (2000) J. Exp.
Med. 192:159-170).
Pancreatic ribonucleases (RNase) are pyrimidine-specific endonucleases found
in high
quantity in the pancreas of certain mammalian taxa and of some reptiles
(Beintema, J.J. et al (1988)
Prog. Biophys. Mol. Biol. 51:165-192). Proteins in the mammalian pancreatic
RNase superfamily are
noncytosolic endonucleases that degrade RNA through a two-step
transphosphorolytic-hydrolytic
reaction (Beintema, J.J. et al. (1986) Mol. Biol. Evol. 3:262-275).
Specifically, the enzymes are
22
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involved in endonucleolytic cleavage of 3'-phosphomononucleotides and 3'-
phosphooligonucleotides
ending in C-P or U-P with 2',3'-cyclic phosphate intermediates. Ribonucleases
can unwind the DNA
helix by complexing with single-stranded DNA; the complex arises by an
extended multi-site
canon-anion interaction between lysine and arginine residues of the enzyme and
phosphate groups of
the nucleotides. Some of the enzymes belonging to this family appear to play a
purely digestive role,
whereas others exhibit potent and unusual biological activities (D'Alessio, G.
(1993) Trends Cell
Biol. 3:106-109). Proteins belonging to the pancreatic RNase family include:
bovine seminal vesicle
and brain ribonucleases; kidney non-secretory ribonucleases (Beintema, J.J. et
al (1986) FEBS Lett.
194:338-343); liver-type ribonucleases (Rosenberg, H.F. et al. (1989) PNAS
U.S.A. 86:4460-4464);
angiogenin, which induces vascularisation of normal and malignant tissues;
eosinophil cationic
protein (Hofsteenge, J. et al. (1989) Biochemistry 28:9806-9813), a cytotoxin
and helminthotoxin
with ribonuclease activity; and frog liver ribonuclease and frog sialic acid-
binding lectin. The
sequences of pancreatic RNases contain 4 conserved disulfide bonds and 3 amino
acid residues
involved in the catalytic activity.
ADP-ribosylation is a reversible post-translational protein modification in
which an ADP-
ribose moiety is transferred from (3-NAD to a target amino acid such as
arginine or cysteine. ADP-
ribosylarginine hydrolases regenerate arginine by removing ADP-ribose from the
protein, completing
the ADP-ribosylation cycle (Moss, J. et al. (1997) Adv. Exp. Med. Biol. 419:25-
33). ADP-
ribosylation is a well-known reaction among bacterial toxins. Cholera toxin,
for example, disrupts the
adenylyl cyclase system by ADP-ribosylating the a-subunit of the stimulatory G-
protein, causing an
increase in intracellular cAMP (Moss, J. and Vaughan, M. (Eds) (1990) ADP-
ribosylatin~ Toxins and
G-Proteins: Insights into Signal Transduction, American Society for
Microbiology, Washington,
D.C.). ADP-ribosylation may also have a regulatory function in eukaryotes,
affecting such processes
as cytoskeletal assembly (Zhou, H. et al. (1996) Arch. Biochem. Biophys.
334:214-222) and cell ,
proliferation in cytotoxic T-cells (Wang, J. et al. (1996) J. Immunol.
156:2819-2827).
Nucleotidases catalyze the formation of free nucleosides from nucleotides. The
cytosolic
nucleotidase cN-I (5' nucleotidase-I) cloned from pigeon heart catalyzes the
formation of adenosine
from AMP generated during ATP hydrolysis (Sala-Newby, G.B. et al. (1999) J.
Biol. Chem.
274:17789-17793). Increased adenosine concentration is thought to be a signal
of metabolic stress,
and adenosine receptors mediate effects including vasodilation, decreased
stimulatory neuron firing
and ischemic preconditioning in the heart (Schrader, J. (1990) Circulation
81:389-391; Rubino, A. et
al. (1992) Eur. J. Pharmacol. 220:95-98; de Jong, J.W. et al. (2000)
Pharmacol. Ther. 87:141-149).
Deficiency of pyrimidine 5'-nucleotidase can result in hereditary hemolytic
anemia (OMIM Entry
266120).
The lysozyme c superfamily consists of conventional lysozymes c, calcium-
binding
23
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lysozymes c, and a-lactalbumin (Prager, E.M. and P. Jolles (1996) EXS 75:9-
31). The proteins in this
superfamily have 35-40% sequence homology and share a common three-dimensional
fold, but can
have different functions. Lysozymes c are ubiquitous in a variety of tissues
and secretions and can
lyse the cell walls of certain bacteria (McKenzie, H.A. ( 1996) EXS 75:365-
409). Alpha-lactalbumin
is a metallo-protein that binds calcium and participates in the synthesis of
lactose (Iyer, L.K. and P.K.
Qasba ( 1999) Protein Eng. 12:129-139). Alpha-lactalbumin occurs in mammalian
milk and colostrum
(McKenzie, su ra).
Lysozymes catalyze the hydrolysis of certain mucopolysaccharides of bacterial
cell walls,
specifically, the beta (1-4) glycosidic linkages between N-acetylmuramic acid
and N-
acetylglucosamine, and cause bacterial lysis. Lysozymes occur in diverse
organisms including
viruses, birds, and mammals. In humans, lysozymes are found in spleen, lung,
kidney, white blood
cells, plasma, saliva, milk, tears, and cartilage (Online Mendelian
Inheritance in Man (OMIM)
#153450 Lysozyme; Weaver, L.H. et al. (1985) J. Mol. Biol. 184:739-741).
Lysozyme c functions in
ruminants as a digestive enzyme, releasing proteins from ingested bacterial
cells, and may perform
the same function in human newborns (Braun, O.H. et al. (1995) Klin. Pediatr.
207:4-7).
The two known forms of lysozymes, chicken-type and goose-type, were originally
isolated
from chicken and goose egg white, respectively. Chicken-type and goose-type
lysozymes have
similar three-dimensional structures, but different amino acid sequences
(Nakano, T. and T. Graf
(1991) Biochim. Biophys. Acta 1090:273-276). In chickens, both forms of
lysozyme are found in
neutrophil granulocytes (heterophils), but only chicken-type lysozyme is found
in egg white.
Generally, chicken-type lysozyme mRNA is found in both adherent monocytes and
macrophages and
nonadherent promyelocytes and granulocytes as well as in cells of the bone
marrow, spleen, bursa,
and oviduct. Goose-type lysozyme mRNA is found in non-adherent cells of the
bone marrow and
lung. Several isozymes have been found in rabbits, including leukocytic,
gastrointestinal, and
possibly lymphoepithelial forms (OMIM #153450, supra; Nakano and Graf, supra;
and GenBank GI
1310929). A human lysozyme gene encoding a protein similar to chicken-type
lysozyme has been
cloned (Yoshimura, K. et al. (1988) Biochem. Biophys. Res. Commun. 150:794-
801). A consensus
motif featuring regularly spaced cysteine residues has been derived from the
lysozyme C enzymes of
various species (PROSITE PS00128). Lysozyme C shares about 40% amino acid
sequence identity
with a-lactalbumin.
Lysozymes have several disease associations. Lysozymuria is observed in
diabetic
nephropathy (Shima, M. et a1.(1986) Clin. Chem. 32:1818-1822), endemic
nephropathy (Bruckner, I.
et al. (1978) Med. Interne. 16:117-125), urinary tract infections (Heidegger,
H. (1990) Minerva
Ginecol. 42:243-250), and acute monocytic leukemia (Shaw, M.T. (1978) Am. J.
Hematol. 4:97-103).
Nakano su ra) suggested a role for lysozyme in host defense systems. Older
rabbits with an
24
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inherited lysozyme deficiency show increased susceptibility to infections,
such as subcutaneous
abscesses (OMIM #153450, supra). Human lysozyme gene mutations cause
hereditary systemic
amyloidosis, a rare autosomal dominant disease in which amyloid deposits form
in the viscera,
including the kidney, adrenal glands, spleen, and liver. This disease is
usually fatal by the fifth
decade. The amyloid deposits contain variant forms of lysozyme. Renal
amyloidosis is the most
common and potentially the most serious form of organ involvement (Pepys, M.B.
et al. (1993)
Nature 362:553-557; OMIM #105200 Familial Visceral Amyloidosis; Cotran, R.S.
et al. (1994)
Robbins Pathologic Basis of Disease, W.B. Saunders Company, Philadelphia PA,
pp. 231-238).
Increased levels of lysozyme and lactate have been observed in the
cerebrospinal fluid of patients
with bacterial meningitis (Ponka, A. et al. (1983) Infection 11:129-131).
Acute monocytic leukemia
is characterized by massive lysozymuria (Den Tandt, W.R. (1988) Int. J.
Biochem. 20:713-719).
L_yases
Lyases are a class of enzymes that catalyze the cleavage of C-C, C-O, C-N, C-
S, C-(halide),
P-O, or other bonds without hydrolysis or oxidation to form two molecules, at
least one of which
contains a double bond (Stryer, L. (1995) Biochemistry, W.H. Freeman and Co.,
New York NY,
p.620). Under the International Classification of Enzymes (Webb, E. C. (1992)
Enzyme
Nomenclature 1992: Recommendations of the Nomenclature Committee of the
International Union of
Biochemistry and Molecular Biology on the Nomenclature and Classification of
Enzymes , Academic
Press, San Diego CA), lyases form a distinct class designated by the numeral 4
in the first digit of the
enzyme number (i.e., EC 4.x.x.x).
Further classification of lyases reflects the type of bond cleaved as well as
the nature of the
cleaved group. The group of C-C lyases includes carboxyl-lyases
(decarboxylases), aldehyde-lyases
(aldolases), oxo-acid-lyases, and other lyases. The C-O lyase group includes
hydro-lyases, lyases
acting on polysaccharides, and other lyases. The C-N lyase group includes
ammonia-lyases, amidine-
lyases, amine-lyases (deaminases), and other lyases. Lyases are critical
components of cellular
biochemistry, with roles in metabolic energy production, including fatty acid
metabolism and the
tricarboxylic acid cycle, as well as other diverse enzymatic processes.
One important family of lyases are the carbonic anhydrases (CA), also called
carbonate
dehydratases, which catalyze the hydration of carbon dioxide in the reaction
H20 + COZ = HC03- +
H+. CA accelerates this reaction by a factor of over 106 by virtue of a zinc
ion located in a deep cleft
about 15A below the protein's surface and co-ordinated to the imidazole groups
of three His residues.
Water bound to the zinc ion is rapidly converted to HC03 .
Eight enzymatic and evolutionarily related forms of carbonic anhydrase are
currently known
to exist in humans: three cytosolic isozymes (CAI, CAII, and CAIII), two
membrane-bound forms
CA 02443244 2003-10-07
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(CAIV and CAVII), a mitochondria) form (CAV), a secreted salivary form (CAVI)
and a yet
uncharacterized isozyme (PROSITE PDOC00146 Eukaryotic-type carbonic anhydrases
signature).
Though the isoenzymes CAI, CAII, and bovine CAIII have similar secondary
structures and
polypeptide-chain folds, CAI has 6 tryptophans, CAII has 7 and CAIII has 8
(Boren, K. et al. (1996)
S Protein Sci. 5:2479-2484). CAII is the predominant CA isoenzyme in the brain
of mammals.
CAs participate in a variety of physiological processes that involve pH
regulation, COZ and
HC03 transport, ion transport, and water and electrolyte balance. For example,
CAII contributes to
H+ secretion by gastric parietal cells, by renal tubular cells, and by
osteoclasts that secrete H+ to
acidify the bone-resorting compartment. In addition, CAII promotes HC03
secretion by pancreatic
duct cells, chary body epithelium, choroid plexus, salivary gland acinar
cells, and distal colonal
epithelium, thus playing a role in the production of pancreatic juice, aqueous
humor, cerebrospinal
fluid, and saliva, and contributing to electrolyte and water balance. CAII
also promotes CO Z
exchange in proximal tubules in the kidney, in erythrocytes, and in lung. CAIV
has roles in several
tissues: it facilitates HC03 reabsorption in the kidney; promotes COZ flux in
tissues including brain,
skeletal muscle, and heart muscle; and promotes COZ exchange from the blood to
the alveoli in the
lung. CAVI probably plays a role in pH regulation in saliva, along with CAII,
and may have a
protective effect in the esophagus and stomach. Mitochondria) CAV appears to
play important roles
in gluconeogenesis and ureagenesis, based on the effects of CA inhibitors on
these pathways. (Sly,
W.S. and Hu, P.Y. (1995) Ann. Rev. Biochem. 64:375-401.)
A number of disease states are marked by variations in CA activity. Mutations
in CAII which
lead to CAII deficiency are the cause of osteopetrosis with renal tubular
acidosis (Online Medelian
Inheritance in Man 259730 Osteopetrosis with Renal Tubular Acidosis). The
concentration of CAII
in the cerebrospinal fluid (CSF) appears to mark disease activity in patients
with brain damage. High
CA concentrations have been observed in patients with brain infarction.
Patients with transient
ischemic attack, multiple sclerosis, or epilepsy usually have CAII
concentrations in the normal range,
but higher CAII levels have been observed in the CSF of those with central
nervous system infection,
dementia, or trigeminal neuralgia (Parkkila, A.K. et al. (1997) Eur. J. Clin.
Invest. 27:392-397).
Colonic adenomas and adenocarcinomas have been observed to fail to stain for
CA, whereas non-
neoplastic controls showed CAI and CAII in the cytoplasm of the columnar cells
lining the upper half
of colonic crypts. The neoplasms show staining patterns similar to less mature
cells lining the base of
normal crypts (Gramlich T.L. et al. (1990) Arch. Pathol. Lab. Med. 114:415-
419).
Therapeutic interventions in a number of diseases involve altering CA
activity. CA inhibitors
such as acetazolamide are used in the treatment of glaucoma (Stewart, W.C.
(1999) Curr. Opin.
Opthamol. 10:99-108), essential tremor and Parkinson's disease (Uitti, R.J.
(1998) Geriatrics 53:46-
48, 53-57), intermittent ataxia (Singhvi, J.P. et al. (2000) Neurology India
48:78-80), and altitude
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related illnesses (Klocke, D.L. et al. (1998) Mayo Clin. Proc. 73:988-992).
CA activity can be particularly useful as an indicator of long-term disease
conditions, since
the enzyme reacts relatively slowly to physiological changes. CAI and zinc
concentrations have been
observed to decrease in hyperthyroid Graves' disease (Yoshida, K. ( 1996)
Tohoku J. Exp. Med.
178:345-356) and glycosylated CAI is observed in diabetes mellitus (Kondo, T.
et al. (1987) Clin.
Chim. Acta 166:227-236). A positive correlation has been observed between CAI
and CAII
reactivity and endometriosis (Brinton, D.A. et al. (1996) Ann. Clin. Lab. Sci.
26:409-420; D'Cruz ,
O.J. et al. ( 1996) Fertil. Steril. 66:547-556).
Another important member of the lyase family is ornithine decarboxylase (ODC),
the initial
rate-limiting enzyme in polyamine biosynthesis. ODC catalyses the
transformation of ornithine into
putrescine in the reaction L-ornithine = putrescine + COZ. Polyamines, which
include putrescine and
the subsequent metabolic pathway products spermidine and spermine, are
ubiquitous cell components
essential for DNA synthesis, cell differentiation, and proliferation. Thus the
polyamines play a key
role in tumor proliferation (Medina, M.A. et al. (1999) Biochem. Pharmacol.
57:1341-1344).
ODC is a pyridoxal-5'-phosphate (PLP)-dependent enzyme which is active as a
homodimer.
Conserved residues include those at the PLP binding site and a stretch of
glycine residues thought to
be part of a substrate binding region (PROSITE PDOC00685 Orn/DAP/Arg
decarboxylase family 2
signatures). Mammalian ODCs also contain PEST regions, sequence fragments
enriched in proline,
glutamic acid, serine, and threonine residues that act as signals for
intracellular degradation (Medina,
supra).
Many chemical carcinogens and tumor promoters increase ODC levels and
activity. Several
known oncogenes may increase ODC levels by enhancing transcription of the ODC
gene, and ODC
itself may act as an oncogene when expressed at very high levels. A high level
of ODC is found in a
number of precancerous conditions, and elevation of ODC levels has been used
as part of a screen for
tumor-promoting compounds (Pegg, A.E. et al. (1995) J. Cell. Biochem. Suppl.
22:132-138).
Inhibitors of ODC have been used to treat tumors in animal models and human
clinical trials,
and have been shown to reduce development of tumors of the bladder, brain,
esophagus,
gastrointestinal tract, lung, oral cavity, mammary gland, stomach, skin and
trachea (Pegg, supra;
McCann, P.P. and A.E. Pegg (1992) Pharmac. Ther. 54:195-215). ODC also shows
promise as a
target for chemoprevention (Pegg, supra). ODC inhibitors have also been used
to treat infections by
African trypanosomes, malaria, and Pneumocystis carinii, and are potentially
useful for treatment of
autoimmune diseases such as lupus and rheumatoid arthritis (McCann, supra).
Another family of pyridoxal-dependent decarboxylases are the group II
decarboxylases. This
family includes glutamate decarboxylase (GAD) which catalyzes the
decarboxylation of glutamate
into the neurotransmitter GABA; histidine decarboxylase (HDC), which catalyzes
the
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decarboxylation of histidine to histamine; aromatic-L-amino-acid decarboxylase
(DDC), also known
as L-dopa decarboxylase or tryptophan decarboxylase, which catalyzes the
decarboxylation of
tryptophan to tryptamine and also acts on 5-hydroxy-tryptophan and
dihydroxyphenylalanine (L-
dopa); and cysteine sulfuric acid decarboxylase (CSD), the rate-limiting
enzyme in the synthesis of
taurine from cysteine (PROSITE PDOC00329 DDC/GAD/HDC/TyrDC pyridoxal-phosphate
attachment site). Taurine is an abundant sulfonic amino acid in brain and is
thought to act as an
osmoregulator in brain cells (Bitoun, M. and Tappaz, M. (2000) J. Neurochem.
75:919-924).
Isomerases
Isomerases are a class of enzymes that catalyze geometric or structural
changes within a
molecule to form a single product. This class includes racemases and
epimerases, cis-trans-
isomerases, intramolecular oxidoreductases, intramolecular transferases
(mutases) and intramolecular
lyases. Isomerases are critical components of cellular biochemistry with roles
in metabolic energy
production including glycolysis, as well as other diverse enzymatic processes
(Stryer, L. (1995)
Biochemistry W.H. Freeman and Co. New York, NY pp.483-507).
Racemases are a subset of isomerases that catalyze inversion of a molecule's
configuration
around the asymmetric carbon atom in a substrate having a single center of
asymmetry, thereby
interconverting two racemers. Epimerases are another subset of isomerases that
catalyze inversion of
configuration around an asymmetric carbon atom in a substrate with more than
one center of
symmetry, thereby interconverting two epimers. Racemases and epimerases can
act on amino acids
and derivatives, hydroxy acids and derivatives, and carbohydrates and
derivatives. The
interconversion of UDP-galactose and UDP-glucose is catalyzed by UDP-galactose-
4'-epimerase.
Proper regulation and function of this epimerase is essential to the synthesis
of glycoproteins and
glycolipids. Elevated blood galactose levels have been correlated with UDP-
galactose-4'-epimerase
deficiency in screening programs of infants (Gitzelmann, R. (1972) Helv.
Paediat. Acta 27:125-130).
Correct folding of newly synthesized proteins is assisted by molecular
chaperones and
folding catalysts, two unrelated groups of helper molecules. Chaperones
suppress non-productive
side reactions by stoichiometric binding to folding intermediates, whereas
folding enzymes catalyze
some of the multiple folding steps that enable proteins to attain their final
functional configurations
(Kern, G. et al. (1994) FEBS Lett. 348: 145-148). One class of folding
enzymes, the peptidyl prolyl
cis-traps isomerases (PPIases), isomerizes certain proline imidic bonds in
what is considered to be a
rate limiting step in protein maturation and export. PPIases catalyze the cis
to traps isomerization of
certain proline imidic bonds in proteins. There are three evolutionarily
unrelated families of PPIases:
the cyclophilins, the FK506 binding proteins, and the newly characterized
parvulin family (Rahfeld,
J.U. et al. (1994) FEBS Lett. 352: 180-184).
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The cyclophilins (CyP) were originally identified as major receptors for the
immunosuppressive drug cyclosporin A (CsA), an inhibitor of T-cell activation
(Handschumacher,
R.E. et al. (1984) Science 226: 544-547; Harding, M.W. et al. (1986) J. Biol.
Chem. 261: 8547-8555).
Thus, the peptidyl-prolyl isomerase activity of CyP may be part of the
signaling pathway that leads to
T-cell activation. Subsequent work demonstrated that CyP's isomerase activity
is essential for
correct protein folding and/or protein trafficking, and may also be involved
in assembly/disassembly
of protein complexes and regulation of protein activity. For example, in
Drosophila, the CyP NinaA
is required for correct localization of rhodopsins, while a mammalian CyP
(Cyp40) is part of the
Hsp90/Hsp70 complex that binds steroid receptors. The mammalian CyP (CypA) has
been shown to
bind the gag protein from human immunodeficiency virus 1 (HIV-1), an
interaction that can be
inhibited by cyclosporin. Since cyclosporin has potent anti-HIV-1 activity,
CypA may play an
essential function in HIV-1 replication. Finally, Cyp40 has been shown to bind
and inactivate the
transcription factor c-Myb, an effect that is reversed by cyclosporin. This
effect implicates CyP in
the regulation of transcription, transformation, and differentiation (Bergsma,
D.J. et al (1991) J. Biol.
Chem. 266:23204-23214; Hunter, T. (1998) Cell 92: 141-143; and Leverson, J.D.
and Ness, S.A.
(1998) Mol. Cell. 1:203-211).
One of the major rate limiting steps in protein folding is the thiol:disulfide
exchange that is
necessary for correct protein assembly. Although incubation of reduced,
unfolded proteins in buffers
with defined ratios of oxidized and reduced thiols can lead to native
conformation, the rate of folding
is slow and the attainment of native conformation decreases proportionately
with the size and number
of cysteines in the protein. Certain cellular compartments such as the
endoplasmic reticulum of
eukaryotes and the periplasmic space of prokaryotes are maintained in a more
oxidized state than the
surrounding cytosol. Correct disulfide formation can occur in these
compartments, but at a rate that
is insufficient for normal cell processes and inadequate for synthesizing
secreted proteins. The
protein disulfide isomerases, thioredoxins and glutaredoxins are able to
catalyze the formation of
disulfide bonds and regulate the redox environment in cells to enable the
necessary thiol:disulfide
exchanges (Loferer, H. (1995) J. Biol. Cherii. 270:26178-26183).
Each of these proteins has somewhat different functions, but all belong to a
group of
disulfide-containing redox proteins that contain a conserved active-site
sequence and are ubiquitously
distributed in eukaryotes and prokaryotes. Protein disulfide isomerases are
found in the endoplasmic
reticulum of eukaryotes and in the periplasmic space of prokaryotes. They
function by exchanging
their own disulfide for a thiol in a folding peptide chain. In contrast, the
reduced thioredoxins and
glutaredoxins are generally found in the cytoplasm and function by directly
reducing disulfides in the
substrate proteins. .
Oxidoreductases can be isomerases as well. Oxidoreductases catalyze the
reversible transfer
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of electrons from a substrate that becomes oxidized to a substrate that
becomes reduced. This class
of enzymes includes dehydrogenases, hydroxylases, oxidases, oxygenases,
peroxidases, and
reductases. Proper maintenance of oxidoreductase levels is physiologically
important. For example,
genetically-linked deficiencies in lipoamide dehydrogenase can result in
lactic acidosis (Robinson, B.
H. et. al. (1977) Pediat. Res. 11:1198-1202).
Another subgroup of isomerases are the transferases (or mutases). Transferases
transfer a
chemical group from one compound (the donor) to another compound (the
acceptor). The types of
groups transferred by these enzymes include acyl groups, amino groups,
phosphate groups
(phosphotransferases or phosphomutases), and others. The transferase carnitine
palmitoyltransferase
is an important component of fatty acid metabolism. Genetically-linked
deficiencies in this
transferase can lead to myopathy (Scriver C. . et. al. (1995) The Metabolic
and Molecular Basis of
Inherited Disease, McGraw-Hill New York NY pp.1501-1533).
Yet another subgroup of isomerases are the topoisomersases. Topoisomerases are
enzymes
that affect the topological state of DNA. For example, defects in
topoisomerases or their regulation
can affect normal physiology. Reduced levels of topoisomerase II have been
correlated with some of
the DNA processing defects associated with the disorder ataxia-telangiectasia
(Singly S.P. et. al.
(1988) Nucleic Acids Res. 16:3919-3929).
Ligases
Ligases catalyze the formation of a bond between two substrate molecules. The
process
involves the hydrolysis of a pyrophosphate bond in ATP or a similar energy
donor. Ligases are
classified based on the nature of the type of bond they form, which can
include carbon-oxygen,
carbon-sulfur, carbon-nitrogen, carbon-carbon and phosphoric ester bonds.
Ligases forming carbon-oxygen bonds include the aminoacyl-transfer RNA (tRNA)
synthetases which are important RNA-associated enzymes with roles in
translation. Protein
biosynthesis depends on each amino acid forming a linkage with the appropriate
tRNA. The
aminoacyl-tRNA synthetases are responsible for the activation and correct
attachment of an amino
acid with its cognate tRNA. The 20 aminoacyl-tRNA synthetase enzymes can be
divided into two
structural classes, and each class is characterized by a distinctive topology
of the catalytic domain.
Class I enzymes contain a catalytic domain based on the nucleotide-binding
"Rossman fold". Class II
enzymes contain a central catalytic domain, which consists of a seven-stranded
antiparallel f3-sheet
motif, as well as N- and C- terminal regulatory domains. Class II enzymes are
separated into two
groups based on the heterodimeric or homodimeric structure of the enzyme; the
latter group is further
subdivided by the structure of the N- and C-terminal regulatory domains
(Hartlein, M. and Cusack, S.
(1995) J. Mol. Evol. 40:519-530). Autoantibodies against aminoacyl-tRNAs are
generated by
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patients with dermatomyositis and polymyositis, and correlate strongly with
complicating interstitial
lung disease (ILD). These antibodies appear to be generated in response to
viral infection, and
coxsackie virus has been used to induce experimental viral myositis in
animals.
Ligases forming carbon-sulfur bonds (acid-thiol ligases) mediate a large
number of cellular
biosynthetic intermediary metabolism processes involving intermolecular
transfer of carbon
atom-containing substrates (carbon substrates). Examples of such reactions
include the tricarboxylic
acid cycle, synthesis of fatty acids and long-chain phospholipids, synthesis
of alcohols and aldehydes,
synthesis of intermediary metabolites, and reactions involved in the amino
acid degradation
pathways. Some of these reactions require input of energy, usually in the form
of conversion of ATP
to either ADP or AMP and pyrophosphate.
In many cases, a carbon substrate is derived from a small molecule containing
at least two
carbon atoms. The carbon substrate is often covalently bound to a larger
molecule which acts as a
carbon substrate carrier molecule within the cell. In the biosynthetic
mechanisms described above,
the carrier molecule is coenzyme A. Coenzyme A (CoA) is structurally related
to derivatives of the
nucleotide ADP and consists of 4'-phosphopantetheine linked via a
phosphodiester bond to the alpha
phosphate group of adenosine 3',5'-bisphosphate. The terminal thiol group of
4'-phosphopantetheine
acts as the site for carbon substrate bond formation. The predominant carbon
substrates which utilize
CoA as a carrier molecule during biosynthesis and intermediary metabolism in
the cell are acetyl,
succinyl, and propionyl moieties, collectively referred to as acyl groups.
Other carbon substrates
include enoyl lipid, which acts as a fatty acid oxidation intermediate, and
carnitine, which acts as an
acetyl-CoA flux regulator/mitochondrial acyl group transfer protein. Acyl-CoA
and acetyl-CoA are
synthesized in the cell by acyl-CoA synthetase and acetyl-CoA synthetase,
respectively.
Activation of fatty acids is mediated by at least three forms of acyl-CoA
synthetase activity:
i) acetyl-CoA synthetase, which activates acetate and several other low
molecular weight carboxylic
acids and is found in muscle mitochondria and the cytosol of other tissues;
ii) medium-chain
acyl-CoA synthetase, which activates fatty acids containing between four and
eleven carbon atoms
(predominantly from dietary sources), and is present only in liver
mitochondria; and iii) acyl CoA
synthetase, which is specific for long chain fatty acids with between six and
twenty carbon atoms,
and is found in microsomes and the mitochondria. Proteins associated with acyl-
CoA synthetase
activity have been identified from many sources including bacteria, yeast,
plants, mouse, and man.
The activity of acyl-CoA synthetase may be modulated by phosphorylation of the
enzyme by
CAMP-dependent protein kinase.
Ligases forming carbon-nitrogen bonds include amide synthases such as
glutamine
synthetase (glutamate-ammonia ligase) that catalyzes the amination of glutamic
acid to glutamine by
ammonia using the energy of ATP hydrolysis. Glutamine is the primary source
for the amino group
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in various amide transfer reactions involved in de novo pyrimidine nucleotide
synthesis and in purine
and pyrimidine ribonucleotide interconversions. Overexpression of glutamine
synthetase has been
observed in primary liver cancer (Christa, L. et al. (1994) Gastroent.
106:1312-1320).
Acid-amino-acid ligases (peptide synthases) are represented by the ubiquitin
conjugating
enzymes which are associated with the ubiquitin conjugation system (UCS), a
major pathway for the
degradation of cellular proteins in eukaryotic cells and some bacteria. The
UCS mediates the
elimination of abnormal proteins and regulates the half-lives of important
regulatory proteins that
control cellular processes such as gene transcription and cell cycle
progression. In the UCS pathway,
proteins targeted for degradation are conjugated to ubiquitin (Ub), a small
heat stable protein. Ub is
first activated by a ubiquitin-activating enzyme (E1), and then transferred to
one of several Ub-
conjugating enzymes (E2). E2 then links the Ub molecule through its C-terminal
glycine to an
internal lysine (acceptor lysine) of a target protein. The ubiquitinated
protein is then recognized and
degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and
ubiquitin is released
for reutilization by ubiquitin protease. The UCS is implicated in the
degradation of mitotic cyclic
kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins,
cell surface receptors
associated with signal transduction, transcriptional regulators, and mutated
or damaged proteins
(Ciechanover, A. (1994) Cell 79:13-21).
Cyclo-ligases and other carbon-nitrogen ligases comprise various enzymes and
enzyme
complexes that participate in the de novo pathways of purine and pyrimidine
biosynthesis. Because
these pathways are critical to the synthesis of nucleotides for replication of
both RNA and DNA,
many of these enzymes have been the targets of clinical agents for the
treatment of cell proliferative
disorders such as cancer and infectious diseases.
Purine biosynthesis occurs de novo from the amino acids glycine and glutamine,
and other
small molecules. Three of the key reactions in this process are catalyzed by a
trifunctional enzyme
composed of glycinamide-ribonucleotide synthetase (GARS), aminoimidazole
ribonucleotide
synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART).
Together these three
enzymes combine ribosylamine phosphate with glycine to yield phosphoribosyl
aminoimidazole, a
precursor to both adenylate and guanylate nucleotides. This trifunctional
protein has been implicated
in the pathology of Downs syndrome (Aimi, J. et al. (1990) Nucleic Acid Res.
18:6665-6672).
Adenylosuccinate synthetase catalyzes a later step in purine biosynthesis that
converts inosinic acid
to adenylosuccinate, a key step on the path to ATP synthesis. This enzyme is
also similar to another
carbon-nitrogen ligase, argininosuccinate synthetase, that catalyzes a similar
reaction in the urea
cycle (Powell, S.M. et al. (1992) FEBS Lett. 303:4-10).
Adenylosuccinate synthetase, adenylosuccinate lyase, and AMP deaminase may be
considered as a functional unit, the purine nucleotide cycle. This cycle
converts AMP to inosine
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monophosphate (IMP) and reconverts IMP to AMP via adenylosuccinate, thereby
producing NH3 and
forming fumarate from aspartate. In muscle, the purine nucleotide cycle
functions, during intense
exercise, in the regeneration of ATP by pulling the adenylate kinase reaction
in the direction of ATP
formation and by providing Krebs cycle intermediates. In kidney, the purine
nucleotide cycle
accounts for the release of NH3 under normal acid-base conditions. In brain,
the purine nucleotide
cycle may contribute to ATP recovery. Adenylosuccinate lyase deficiency
provokes psychomotor
retardation, often accompanied by autistic features (Van den Berghe, G. et al.
(1992) Prog
Neurobiol.: 39:547-561). A marked imbalance in the enzymic pattern of purine
metabolism is linked
with transformation and/or progression in cancer cells. In rat hepatomas the
specific activities of the
anabolic enzymes, IMP dehydrogenase, GMP synthetase, adenylosuccinate
synthetase,
adenylosuccinase, AMP deaminase and amidophosphoribosyltransferase, increased
to 13.5-, 3.7-,
3.1-, 1.8-, 5.5- and 2.8-fold, respectively, of those in normal liver (Weber,
G. (1983) Clin Biochem
1983 Feb;l6(1):57-63).
Like the de novo biosynthesis of purines, de novo synthesis of the pyrimidine
nucleotides
uridylate and cytidylate also arises from a common precursor, in this instance
the nucleotide
orotidylate derived from orotate and phosphoribosyl pyrophosphate (PPRP).
Again a trifunctional
enzyme comprising three carbon-nitrogen ligases plays a key role in the
process. In this case the
enzymes aspartate transcarbamylase (ATCase), carbamyl phosphate synthetase II,
and dihydroorotase
(DHOase) are encoded by a single gene called CAD. Together these three enzymes
combine the
initial reactants in pyrimidine biosynthesis, glutamine, CO2, and ATP to form
dihydroorotate, the
precursor to orotate and orotidylate (Iwahana, H. et al. (1996) Biochem.
Biophys. Res. Commun.
219:249-255). Further steps then lead to the synthesis of uridine nucleotides
from orotidylate.
Cytidine nucleotides are derived from uridine-5'-triphosphate (UTP) by the
amidation of UTP using
glutamine as the amino donor and the enzyme CTP synthetase. Regulatory
mutations in the human
CTP synthetase are believed to confer mufti-drug resistance to agents widely
used in cancer therapy
(Yamauchi, M. et al. (1990) EMBO J. 9:2095-2099).
Ligases forming carbon-carbon bonds include the carboxylases acetyl-CoA
carboxylase and
pyruvate carboxylase. Acetyl-CoA carboxylase catalyzes the carboxylation of
acetyl-CoA from CO Z
and H20 using the energy of ATP hydrolysis. Acetyl-CoA carboxylase is the rate-
limiting enzyme in
the biogenesis of long-chain fatty acids. Two isoforms of acetyl-CoA
carboxylase, types I and types
II, are expressed in human in a tissue-specific manner (Ha, J. et al. (1994)
Eur. J. Biochem. 219:297-
306). Pyruvate carboxylase is a nuclear-encoded mitochondria) enzyme that
catalyzes the conversion
of pyruvate to oxaloacetate, a key intermediate in the citric acid cycle.
Ligases forming phosphoric ester bonds include the DNA ligases involved in
both DNA
replication and repair. DNA ligases seal phosphodiester bonds between two
adjacent nucleotides in a
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DNA chain using the energy from ATP hydrolysis to first activate the free 5'-
phosphate of one
nucleotide and then react it with the 3'-OH group of the adjacent nucleotide.
This resealing reaction
is used in DNA replication to join small DNA fragments called "Okazaki"
fragments that are
transiently formed in the process of replicating new DNA, and in DNA repair.
DNA repair is the
process by which accidental base changes, such as those produced by oxidative
damage, hydrolytic
attack, or uncontrolled methylation of DNA, are corrected before replication
or transcription of the
DNA can occur. Bloom's syndrome is an inherited human disease in which
individuals are partially
deficient in DNA ligation and consequently have an increased incidence of
cancer (Alberts, B. et al.
(1994) The Molecular Biology of the Cell, Garland Publishing Inc., New York,
NY, p. 247).
Pantothenate synthetase (D-pantoate; beta-alanine ligase (AMP-forming); EC
6.3.2.1) is the
last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis. It
catalyzes the condensation of
pantoate with beta-alanine in an ATP-dependent reaction. The enzyme is
dimeric, with two
well-defined domains per protomer: the N-terminal domain, a Rossmann fold,
contains the active site
cavity, with the C-terminal domain forming a hinged lid. The N-terminal domain
is structurally very
similar to class I aminoacyl-tRNA synthetases and is thus a member of the
cytidylyltransferase
superfamily (von Delft, F. et al. (2000) Structure (Camb) 9:439-450).
Farnesyl diphosphate synthase (FPPS) is an essential enzyme that is required
both for
cholesterol synthesis and protein prenylation. The enzyme catalyzes the
formation of farnesyl
diphosphate from dimethylallyl diphosphate and isopentyl diphosphate. FPPS is
inhibited by
nitrogen-containing biphosphonates, which can lead to the inhibition of
osteoclast-mediated bone
resorption by preventing protein prenylation (Dunford, J.E. et al. (2001) J.
Pharmacol. Exp. Ther.
296:235-242).
5-aminolevulinate synthase (ALAS; delta-aminolevulinate synthase; EC 2.3.1.37)
catalyzes
the rate-limiting step in heme biosynthesis in both erythroid and non-
erythroid tissues. This enzyme
is unique in the heme biosynthetic pathway in being encoded by two genes, the
first encoding
ALAS1, the non-erythroid specific enzyme which is ubiquitously expressed, and
the second
encoding ALAS2, which is expressed exclusively in erythroid cells. The genes
for ALAS 1 and
ALAS2 are located, respectively, on chromosome 3 and on the X chromosome.
Defects in the gene
encoding ALAS2 result in X-linked sideroblastic anemia. Elevated levels of
ALAS are seen in acute
hepatic porphyrias and can be lowered by zinc mesoporphyrin.
Drub Metabolizin Enzymes (DMEs)
The metabolism of a drug and its movement through the body (pharmacokinetics)
are
important in determining its effects, toxicity, and interactions with other
drugs. The three processes
governing pharmacokinetics are the absorption of the drug, distribution to
various tissues, and
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elimination of drug metabolites. These processes are intimately coupled to
drug metabolism, since a
variety of metabolic modifications alter most of the physicochemical and
pharmacological properties
of drugs, including solubility, binding to receptors, and excretion rates. The
metabolic pathways
which modify drugs also accept a variety of naturally occurring substrates
such as steroids, fatty
acids, prostaglandins, leukotrienes, and vitamins. The enzymes in these
pathways are therefore
important sites of biochemical and pharmacological interaction between natural
compounds, drugs,
carcinogens, mutagens, and xenobiotics. It has long been appreciated that
inherited differences in
drug metabolism lead to drastically different levels of drug efficacy and
toxicity among individuals.
Advances in pharmacogenomics research, of which DMEs constitute an important
part, are promising
to expand the tools and information that can be brought to bear on questions
of drug efficacy and
toxicity (See Evans, W.E. and R.V. Relling (1999) Science 286:487-491). DMEs
have broad
substrate specificities, unlike antibodies, for example, which are diverse and
highly specific. Since
DMEs metabolize a wide variety of molecules, drug interactions may occur at
the level of
metabolism so that, for example, one compound may induce a DME that affects
the metabolism of
another compound.
Drug metabolic reactions are categorized as Phase I, which prepare the drug
molecule for
functioning and further metabolism, and Phase II, which are conjugative. In
general, Phase I reaction
products are partially or fully inactive, and Phase II reaction products are
the chief excreted species.
However, Phase I reaction products are sometimes more active than the original
administered drugs;
this metabolic activation principle is exploited by pro-drugs (e.g. L-dopa).
Additionally, some
nontoxic compounds (e.g. aflatoxin, benzo[a]pyrene) are metabolized to toxic
intermediates through
these pathways. Phase I reactions are usually rate-limiting in drug
metabolism. Prior exposure to the
compound, or other compounds, can induce the expression of Phase I enzymes
however, and thereby
increase substrate flux through the metabolic pathways. (See Klaassen, C.D. et
al. (1996) Casarett
and Doull's Toxicolo~y: The Basic Science of Poisons, McGraw-Hill, New York,
NY, pp. 113-186;
Katzung, B.G. (1995) Basic and Clinical Pharmacoloey, Appleton and Lange,
Norwalk, CT, pp. 48-
59; Gibson, G.G. and P. Skett (1994) Introduction to Drug Metabolism, Blackie
Academic and
Professional, London.).
The major classes of Phase I enzymes include, but are not limited to,
cytochrome P450 and
flavin-containing monooxygenase. Other enzyme classes involved in Phase I-type
catalytic cycles
and reactions include, but are not limited to, NADPH cytochrome P450 reductase
(CPR), the
microsomal cytochrome b5/NADH cytochrome b5 reductase system, the
ferredoxin/ferredoxin
reductase redox pair, aldo/keto reductases, and alcohol dehydrogenases. The
major classes of Phase
II enzymes include, but are not limited to, UDP glucuronyltransferase,
sulfotransferase, glutathione
S-transferase, N-acyltransferase, and N-acetyl transferase.
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Cytochrome P450 and P450 catalytic cycle-associated enzymes
Members of the cytochrome P450 superfamily of enzymes catalyze the oxidative
metabolism
of a variety of substrates, including natural compounds such as steroids,
fatty acids, prostaglandins,
leukotrienes, and vitamins, as well as drugs, carcinogens, mutagens, and
xenobiotics. Cytochromes
P450, also known as P450 heme-thiolate proteins, usually act as terminal
oxidases in
multi-component electron transfer chains, called P450-containing monooxygenase
systems. Specific
reactions catalyzed include hydroxylation, epoxidation, N-oxidation,
sulfooxidation, N-, S-, and O-
dealkylations, desulfation, deamination, and reduction of azo, nitro, and N-
oxide groups. These
reactions are involved in steroidogenesis of glucocorticoids, cortisols,
estrogens, and androgens in
animals; insecticide resistance in insects; herbicide resistance and flower
coloring in plants; and
environmental bioremediation by microorganisms. Cytochrome P450 actions on
drugs, carcinogens,
mutagens, and xenobiotics can result in detoxification or in conversion of the
substance to a more
toxic product. Cytochromes P450 are abundant in the liver, but also occur in
other tissues; the
enzymes are located in microsomes. (See ExPASY ENZYME EC 1.14.14.1; Prosite
PDOC00081
Cytochrome P450 cysteine heme-iron ligand signature; PRINTS EP450I E-Class
P450 Group I
signature; Graham-Lorence, S. and Peterson, J.A. (1996) FASEB J. 10:206-214.)
Four hundred cytochromes P450 have been identified in diverse organisms
including
bacteria, fungi, plants, and animals (Graham-Lorence, supra). The B-class is
found in prokaryotes
and fungi, while the E-class is found in bacteria, plants, insects,
vertebrates, and mammals. Five
subclasses or groups are found within the larger family of E-class cytochromes
P450 (PRINTS
EP450I E-Class P450 Group I signature).
All cytochromes P450 use a heme cofactor and share structural attributes. Most
cytochromes
P450 are 400 to 530 amino acids in length. The secondary structure of the
enzyme is about 70%
alpha-helical and about 22% beta-sheet. The region around the heme-binding
site in the C-terminal
part of the protein is conserved among cytochromes P450. A ten amino acid
signature sequence in
this heme-iron ligand region has been identified which includes a conserved
cysteine involved in
binding the heme iron in the fifth coordination site. In eukaryotic
cytochromes P450, a
membrane-spanning region is usually found in the first 15-20 amino acids of
the protein, generally
consisting of approximately 15 hydrophobic residues followed by a positively
charged residue. (See
Prosite PDOC00081, supra; Graham-Lorence, supra.)
Cytochrome P450 enzymes are involved in cell proliferation and development.
The enzymes
have roles in chemical mutagenesis and carcinogenesis by metabolizing
chemicals to reactive
intermediates that form adducts with DNA (Nebert, D.W. and Gonzalez, F.J.
(1987) Ann. Rev.
Biochem. 56:945-993). These adducts can cause nucleotide changes and DNA
rearrangements that
lead to oncogenesis. Cytochrome P450 expression in liver and other tissues is
induced by xenobiotics
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such as polycyclic aromatic hydrocarbons, peroxisomal proliferators,
phenobarbital, and the
glucocorticoid dexamethasone (Dogra, S.C. et al. (1998) Clin. Exp. Pharmacol.
Physiol. 25:1-9). A
cytochrome P450 protein may participate in eye development as mutations in the
P450 gene CYP1B1
cause primary congenital glaucoma (Online Mendelian Inheritance in Man (OMIM)
*601771
Cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1; CYP1B1).
Cytochromes P450 are associated with inflammation and infection. Hepatic
cytochrome
P450 activities are profoundly affected by various infections and inflammatory
stimuli, some of
which are suppressed and some induced (Morgan, E.T. (1997) Drug Metab. Rev.
29:1129-1188).
Effects observed in vivo can be mimicked by proinflammatory cytokines and
interferons.
Autoantibodies to two cytochrome P450 proteins were found in patients with
autoimmune
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), a polyglandular
autoimmune
syndrome (OMIM *240300 Autoimmune polyenodocrinopathy-candidiasis-ectodermal
dystrophy).
Mutations in cytochromes P450 have been linked to metabolic disorders,
including
congenital adrenal hyperplasia, the most common adrenal disorder of infancy
and childhood;
pseudovitamin D-deficiency rickets; cerebrotendinous xanthomatosis, a lipid
storage disease
characterized by progressive neurologic dysfunction, premature
atherosclerosis, and cataracts; and an
inherited resistance to the anticoagulant drugs coumarin and warfarin
(Isselbacher, K.J. et al. (1994)
Harrison's Principles of Internal Medicine, McGraw-Hill, Inc. New York, NY,
pp. 1968-1970;
Takeyama, K. et al. (1997) Science 277:1827-1830; Kitanaka, S. et al. (1998)
N. Engl. J. Med.
338:653-661; OMIM *213700 Cerebrotendinous xanthomatosis; and OMIM #122700
Coumarin
resistance). Extremely high levels of expression of the cytochrome P450
protein aromatase were
found in a fibrolamellar hepatocellular carcinoma from a boy with severe
gynecomastia
(feminization) (Agarwal, V.R. (1998) J. Clin. Endocrinol. Metab. 83:1797-
1800).
The cytochrome P450 catalytic cycle is completed through reduction of
cytochrome P450 by
NADPH cytochrome P450 reductase (CPR). Another microsomal electron transport
system
consisting of cytochrome b5 and NADPH cytochrome b5 reductase has been widely
viewed as a
minor contributor of electrons to the cytochrome P450 catalytic cycle.
However, a recent report by
Lamb, D.C. et al. (1999; FEBS Lett. 462:283-288) identifies a Candida albicans
cytochrome P450
(CYP51) which can be efficiently reduced and supported by the microsomal
cytochrome b5/NADPH
cytochrome b5 reductase system. Therefore, there are likely many cytochromes
P450 which are
supported by this alternative electron donor system.
Cytochrome b5 reductase is also responsible for the reduction of oxidized
hemoglobin
(methemoglobin, or fernhemoglobin, which is unable to carry oxygen) to the
active hemoglobin
(ferrohemoglobin) in red blood cells. Methemoglobinemia results when there is
a high level of
oxidant drugs or an abnormal hemoglobin (hemoglobin M) which is not
efficiently reduced.
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Methemoglobinemia can also result from a hereditary deficiency in red cell
cytochrome b5 reductase
(Reviewed in Mansour, A. and Lurie, A.A. (1993) Am. J. Hematol. 42:7-12).
Members of the cytochrome P450 family are also closely associated with vitamin
D synthesis
and catabolism. Vitamin D exists as two biologically equivalent prohormones,
ergocalciferol
(vitamin DZ), produced in plant tissues, and cholecalciferol (vitamin D3),
produced in animal tissues.
The latter form, cholecalciferol, is formed upon the exposure of 7-
dehydrocholesterol to near
ultraviolet light (i.e., 290-310 nm), normally resulting from even minimal
periods of skin exposure to
sunlight (reviewed in Miller, W.L. and Portale, A.A. (2000) Trends Endocrinol.
Metab. 11:315-319).
Both prohormone forms are further metabolized in the liver to 25-
hydroxyvitamin D
(25(OH)D) by the enzyme 25-hydroxylase. 25(OH)D is the most abundant precursor
form of vitamin
D which must be further metabolized in the kidney to the active form, 1 a,25-
dihydroxyvitamin D
(1a,25(OH)ZD), by the enzyme 25-hydroxyvitamin D la-hydroxylase (la-
hydroxylase). Regulation
of 1a,25(OH)zD production is primarily at this final step in the synthetic
pathway. The activity of
la-hydroxylase depends upon several physiological factors including the
circulating level of the
enzyme product (1a,25(OH)ZD) and the levels of parathyroid hormone (PTH),
calcitonin, insulin,
calcium, phosphorus, growth hormone, and prolactin. Furthermore, extrarenal 1
a-hydroxylase
activity has been reported, suggesting that tissue-specific, local regulation
of 1 a,25(OH)2D
production may also be biologically important. The catalysis of 1 a,25(OH)ZD
to
24,25-dihydroxyvitamin D (24,25(OH)ZD), involving the enzyme 25-hydroxyvitamin
D
24-hydroxylase (24-hydroxylase), also occurs in the kidney. 24-hydroxylase can
also use 25(OH)D
as a substrate (Shinki, T. et al. (1997) Proc. Natl. Acad. Sci. U.S.A.
94:12920-12925; Miller, W.L.
and Portale, A.A. supra; and references within).
Vitamin D 25-hydroxylase, la-hydroxylase, and 24-hydroxylase are all NADPH-
dependent,
type I (mitochondrial) cytochrome P450 enzymes that show a high degree of
homology with other
members of the family. Vitamin D 25-hydroxylase also shows a broad substrate
specificity and may
also perform 26-hydroxylation of bile acid intermediates and 25, 26, and 27-
hydroxylation of
cholesterol (Dilworth, F.J. et al. (1995) J. Biol. Chem. 270:16766-16774;
Miller, W.L. and Portale,
A.A. supra; and references within).
The active form of vitamin D (1 a,25(OH)zD) is involved in calcium and
phosphate
homeostasis and promotes the differentiation of myeloid and skin cells.
Vitamin D deficiency
resulting from deficiencies in the enzymes involved in vitamin D metabolism
(e.g., 1 a-hydroxylase)
causes hypocalcemia, hypophosphatemia, and vitamin D-dependent (sensitive)
rickets, a disease
characterized by loss of bone density and distinctive clinical features,
including bandy or bow
leggedness accompanied by a waddling gait. Deficiencies in vitamin D 25-
hydroxylase cause
cerebrotendinous xanthomatosis, a lipid-storage disease characterized by the
deposition of cholesterol
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and cholestanol in the Achilles' tendons, brain, lungs, and many other
tissues. The disease presents
with progressive neurologic dysfunction, including postpubescent cerebellar
ataxia, atherosclerosis,
and cataracts. Vitamin D 25-hydroxylase deficiency does not result in rickets,
suggesting the
existence of alternative pathways for the synthesis of 25(OH)D (Griffin, J.E.
and Zerwekh, J.E.
(1983) J. Clin. Invest. 72:1190-1199; Gamblin, G.T. et al. (1985) J. Clin.
Invest. 75:954-960; and
Miller, W.L. and Portale, A.A. supra).
Ferredoxin and ferredoxin reductase are electron transport accessory proteins
which support
at least one human cytochrome P450 species, cytochrome P450c27 encoded by the
CYP27 gene
(Dilworth, F.J. et al. (1996) Biochem. J. 320:267-71). A Streptomyces ng 'seus
cytochrome P450,
CYP104D1, was heterologously expressed in E. coli and found to be reduced by
the endogenous
ferredoxin and ferredoxin reductase enzymes (Taylor, M. et al. (1999) Biochem.
Biophys. Res.
Commun. 263:838-42), suggesting that many cytochrome P450 species may be
supported by the
ferredoxin/ferredoxin reductase pair. Ferredoxin reductase has also been found
in a model drug
metabolism system to reduce actinomycin D, an antitumor antibiotic, to a
reactive free radical species
(Flitter, W.D. and Mason, R.P. (1988) Arch. Biochem. Biophys. 267:632-639).
Flavin-containing monooxygenase (FMO)
Flavin-containing monooxygenases oxidize the nucleophilic nitrogen, sulfur,
and phosphorus
heteroatom of an exceptional range of substrates. Like cytochromes P450, FMOs
are microsomal and
use NADPH and OZ; there is also a great deal of substrate overlap with
cytochromes P450. The tissue
distribution of FMOs includes liver, kidney, and lung.
Isoforms of FMO in mammals include FMO1, FM02, FM03, FM04, and FMOS, which are
expressed in a tissue-specific manner. The isoforms differ in their substrate
specificities and
properties such as inhibition by various compounds and stereospecificity of
reaction. FMOs have a
13 amino acid signature sequence, the components of which span the N-terminal
two-thirds of the
sequences and include the FAD binding region and the FATGY motif found in many
N-
hydroxylating enzymes (Stehr, M. et al. (1998) Trends Biochem. Sci. 23:56-57;
PRINTS
FMOXYGENASE Flavin-containing monooxygenase signature). Specific reactions
include
oxidation of nucleophilic tertiary amines to N-oxides, secondary amines to
hydroxylamines and
nitrones, primary amines to hydroxylamines and oximes, and sulfur-containing
compounds and
phosphines to S- and P-oxides. Hydrazines, iodides, selenides, and boron-
containing compounds are
also substrates. FMOs are more heat labile and less detergent-sensitive than
cytochromes P450 in
vitro though FMO isoforms vary in thermal stability and detergent sensitivity.
FMOs play important roles in the metabolism of several drugs and xenobiotics.
FMO
(FM03 in liver) is predominantly responsible for metabolizing (S)-nicotine to
(S)-nicotine N-1'-
oxide, which is excreted in urine. FMO is also involved in S-oxygenation of
cimetidine, an Hz
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antagonist widely used for the treatment of gastric ulcers. Liver-expressed
forms of FMO are not
under the same regulatory control as cytochrome P450. In rats, for example,
phenobarbital treatment
leads to the induction of cytochrome P450, but the repression of FMO1.
LYSVI oxidase
Lysyl oxidase (lysine 6-oxidase, LO) is a copper-dependent amine oxidase
involved in the
formation of connective tissue matrices by crosslinking collagen and elastin.
LO is secreted as an N-
glycosylated precursor protein of approximately 50 kDa and cleaved to the
mature form of the
enzyme by a metalloprotease, although the precursor form is also active. The
copper atom in LO is
involved in the transport of electrons to and from oxygen to facilitate the
oxidative deamination of
lysine residues in these extracellular matrix proteins. While the coordination
of copper is essential to
LO activity, insufficient dietary intake of copper does not influence the
expression of the apoenzyme.
However, the absence of the functional LO is linked to the skeletal and
vascular tissue disorders that
are associated with dietary copper deficiency. LO is also inhibited by a
variety of semicarbazides,
hydrazines, and amino nitrites, as well as heparin. Beta-aminopropionitrile is
a commonly used
inhibitor. LO activity is increased in response to ozone, cadmium, and
elevated levels of hormones
released in response to local tissue trauma, such as transforming growth
factor-beta, platelet-derived
growth factor, angiotensin II, and fibroblast growth factor. Abnormalities in
LO activity have been
linked to Menkes syndrome and occipital horn syndrome. Cytosolic forms of the
enzyme have been
implicated in abnormal cell proliferation (reviewed in Rucker, R.B. et al.
(1998) Am. J. Clin. Nutr.
67:996S-10025 and Smith-Mungo, L.I. and Kagan, H.M. (1998) Matrix Biol. 16:387-
398).
Dihydrofolate reductases
Dihydrofolate reductases (DHFR) are ubiquitous enzymes that catalyze the
NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, an essential
step in the de novo
synthesis of glycine and purines as well as the conversion of deoxyuridine
monophosphate (BUMP)
to deoxythymidine monophosphate (dTMP). The basic reaction is as follows:
7,8-dihydrofolate + NADPH -~ 5,6,7,8-tetrahydrofolate + NADP+
The enzymes can be inhibited by a number of dihydrofolate analogs, including
trimethroprim and
methotrexate. Since an abundance of dTMP is required for DNA synthesis,
rapidly dividing cells
require the activity of DHFR. The replication of DNA viruses (i.e.,
herpesvirus) also requires high
levels of DHFR activity. As a result, drugs that target DHFR have been used
for cancer
chemotherapy and to inhibit DNA virus replication. (For similar reasons,
thymidylate synthetases are
also target enzymes.) Drugs that inhibit DHFR are preferentially cytotoxic for
rapidly dividing cells
(or DNA virus-infected cells) but have no specificity, resulting in the
indiscriminate destruction of
CA 02443244 2003-10-07
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dividing cells. Furthermore, cancer cells may become resistant to drugs such
as methotrexate as a
result of acquired transport defects or the duplication of one or more DHFR
genes (Stryer, L. (1988)
Biochemistry. W.H Freeman and Co., Inc. New York. pp. 511-5619).
Aldo/keto reductases
Aldo/keto reductases are monomeric NADPH-dependent oxidoreductases with broad
substrate specificities (Bohren, K.M. et al. (1989) J. Biol. Chem. 264:9547-
9551). These enzymes
catalyze the reduction of carbonyl-containing compounds, including carbonyl-
containing sugars and
aromatic compounds, to the corresponding alcohols. Therefore, a variety of
carbonyl-containing
drugs and xenobiotics are likely metabolized by enzymes of this class.
One known reaction catalyzed by a family member, aldose reductase, is the
reduction of
glucose to sorbitol, which is then further metabolized to fructose by sorbitol
dehydrogenase. Under
normal conditions, the reduction of glucose to sorbitol is a minor pathway. In
hyperglycemic states,
however, the accumulation of sorbitol is implicated in the development of
diabetic complications
(OMIM * 103880 Aldo-keto reductase family 1, member B 1). Members of this
enzyme family are
also highly expressed in some liver cancers (Cao, D. et al. ( 1998) J. Biol.
Chem. 273:11429-11435).
Alcohol dehydropenases
Alcohol dehydrogenases (ADHs) oxidize simple alcohols to the corresponding
aldehydes.
ADH is a cytosolic enzyme, prefers the cofactor NAD+, and also binds zinc ion.
Liver contains the
highest levels of ADH, with lower levels in kidney, lung, and the gastric
mucosa.
Known ADH isoforms are dimeric proteins composed of 40 kDa subunits. There are
five
known gene loci which encode these subunits (a, b, g, p, c), and some of the
loci have characterized
allelic variants (b,, b2, b3, g,, g2). The subunits can form homodimers and
heterodimers; the subunit
composition determines the specific properties of the active enzyme. The
holoenzymes have
therefore been categorized as Class I (subunit compositions aa, ab, ag, bg,
gg), Class II (pp), and
Class III (cc). Class I ADH isozymes oxidize ethanol and other small aliphatic
alcohols, and are
inhibited by pyrazole. Class II isozymes prefer longer chain aliphatic and
aromatic alcohols, are
unable to oxidize methanol, and are not inhibited by pyrazole. Class III
isozymes prefer even longer
chain aliphatic alcohols (five carbons and longer) and aromatic alcohols, and
are not inhibited by
pyrazole.
The short-chain alcohol dehydrogenases include a number of related enzymes
with a variety
of substrate specificities. Included in this group are the mammalian enzymes D-
beta-hydroxybutyrate
dehydrogenase, (R)-3-hydroxybutyrate dehydrogenase, 15-hydroxyprostaglandin
dehydrogenase,
NADPH-dependent carbonyl reductase, coi~ticosteroid 11-beta-dehydrogenase, and
estradiol 17-beta-
dehydrogenase, as well as the bacterial enzymes acetoacetyl-CoA reductase,
glucose 1-
dehydrogenase, 3-beta-hydroxysteroid dehydrogenase, 20-beta-hydroxysteroid
dehydrogenase, ribitol
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dehydrogenase, 3-oxoacyl reductase, 2,3-dihydro-2,3-dihydroxybenzoate
dehydrogenase, sorbitol-6-
phosphate 2-dehydrogenase, 7-alpha-hydroxysteroid dehydrogenase, cis-1,2-
dihydroxy-3,4-
cyclohexadiene-1-carboxylate dehydrogenase, cis-toluene dihydrodiol
dehydrogenase, cis-benzene
glycol dehydrogenase, biphenyl-2,3-dihydro-2,3-diol dehydrogenase, N-
acylmannosamine 1-
dehydrogenase, and 2-deoxy-D-gluconate 3-dehydrogenase (Krozowski, Z. (1994)
J. Steroid
Biochem. Mol. Biol. 51:125-130; Krozowski, Z. (1992) Mol. Cell Endocrinol.
84:C25-31; and Marks,
A.R. et al. (1992) J. Biol. Chem. 267:15459-15463).
Sulfotransferase
Sulfate conjugation occurs on many of the same substrates which undergo O-
glucuronidation
to produce a highly water-soluble sulfuric acid ester. Sulfotransferases (ST)
catalyze this reaction by
transferring SO 3- from the cofactor 3'-phosphoadenosine-5'-phosphosulfate
(PAPS) to the substrate.
ST substrates are predominantly phenols and aliphatic alcohols, but also
include aromatic amines and
aliphatic amines, which are conjugated to produce the corresponding
sulfamates. The products of
these reactions are excreted mainly in urine.
STs are found in a wide range of tissues, including liver, kidney, intestinal
tract, lung,
platelets, and brain. The enzymes are generally cytosolic, and multiple forms
are often co-expressed.
For example, there are more than a dozen forms of ST in rat liver cytosol.
These biochemically
characterized STs fall into five classes based on their substrate preference:
arylsulfotransferase,
alcohol sulfotransferase, estrogen sulfotransferase, tyrosine ester
sulfotransferase, and bile salt
sulfotransferase.
ST enzyme activity varies greatly with sex and age in rats. The combined
effects of
developmental cues and sex-related hormones are thought to lead to these
differences in ST
expression profiles, as well as the profiles of other DMEs such as cytochromes
P450. Notably, the
high expression of STs in cats partially compensates for their low level of
UDP glucuronyltransferase
activity.
Several forms of ST have been purified from human liver cytosol and cloned.
There are two
phenol sulfotransferases with different thermal stabilities and substrate
preferences. The
thermostable enzyme catalyzes the sulfation of phenols such as para-
nitrophenol, minoxidil, and
acetaminophen; the thermolabile enzyme prefers monoamine substrates such as
dopamine,
epinephrine, and levadopa. Other cloned STs include an estrogen
sulfotransferase and an N-
acetylglucosamine-6-O-sulfotransferase. This last enzyme is illustrative of
the other major role of
STs in cellular biochemistry, the modification of carbohydrate structures that
may be important in
cellular differentiation and maturation of proteoglycans. Indeed, an inherited
defect in a
sulfotransferase has been implicated in macular corneal dystrophy, a disorder
characterized by a
failure to synthesize mature keratan sulfate proteoglycans (Nakazawa, K. et
al. (1984) J. Biol. Chem.
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259:13751-13757; OMIM *217800 Macular dystrophy, corneal).
Galactosyltransferases
Galactosyltransferases are a subset of glycosyltransferases that transfer
galactose (Gal) to the
terminal N-acetylglucosamine (GIcNAc) oligosaccharide chains that are part of
glycoproteins or
glycolipids that are free in solution (Kolbinger, F. et al. (1998) J. Biol.
Chem. 273:433-440; Amado,
M. et al. (1999) Biochim. Biophys. Acta 1473:35-53). Galactosyltransferases
have been detected on
the cell surface and as soluble extracellular proteins, in addition to being
present in the Golgi. (31,3-
galactosyltransferases form Type I carbohydrate chains with Gal ( (31-3)GIcNAc
linkages. Known
human and mouse ~i 1,3-galactosyltransferases appear to have a short cytosolic
domain, a single
transmembrane domain, and a catalytic domain with eight conserved regions.
(Kolbinger, supra and
Rennet, T. et al. (1998) J. Biol. Chem. 273:58-65). In mouse UDP-galactose:~i-
N-acetylglucosamine
X31,3-galactosyltransferase-I region 1 is located at amino acid residues 78-
83, region 2 is located at
amino acid residues 93-102, region 3 is located at amino acid residues 116-
119, region 4 is located at
amino acid residues 147-158, region 5 is located at amino acid residues 172-
183, region 6 is located
at amino acid residues 203-206, region 7 is located at amino acid residues 236-
246, and region 8 is
located at amino acid residues 264-275. A variant of a sequence found within
mouse UDP-
galactose: (3-N-acetylglucosamine (31,3-galactosyltransferase-I region 8 is
also found in bacterial
galactosyltransferases, suggesting that this sequence defines a
galactosyltransferase sequence motif
(Rennet, supra). Recent work suggests that brainiac protein is a (31,3-
galactosyltransferase (Yuan, Y.
et al. (1997) Cell 88:9-11; and Rennet, supra).
UDP-Gal:GlcNAc-1,4-galactosyltransferase (-1,4-GaIT) (Sato, T. et al., (1997)
EMBO J.
16:1850-1857) catalyzes the formation of Type II carbohydrate chains with Gal
( (31-4)GIcNAc
linkages. As is the case with the ~i 1,3-galactosyltransferase, a soluble form
of the enzyme is formed
by cleavage of the membrane-bound form. Amino acids conserved among (31,4-
galactosyltransferases include two cysteines linked through a disulfide-bond
and a putative UDP-
galactose-binding site in the catalytic domain (Yadav, S. and Brew, K. (1990)
J. Biol. Chem.
265:14163-14169; Yadav, S.P. and Brew, K. (1991) J. Biol. Chem. 266:698-703;
and Shaper, N.L. et
al. (1997) J. Biol. Chem. 272:31389-31399). X31,4-galactosyltransferases have
several specialized
roles in addition to synthesizing carbohydrate chains on glycoproteins or
glycolipids. In mammals a
X31,4-galactosyltransferase, as part of a heterodimer with a-lactalbumin,
functions in lactating
mammary gland lactose production. A (31,4-galactosyltransferase on the surface
of sperm functions
as a receptor that specifically recognizes the egg. Cell surface (31,4-
galactosyltransferases also
function in cell adhesion, cell/basal lamina interaction, and normal and
metastatic cell migration.
(Shur, B. (1993) Curr. Opin. Cell Biol. 5:854-863; and Shaper, J. (1995) Adv.
Exp. Med. Biol.
376:95-104).
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Gamma- lug tamyl transpeptidase
Gamma-glutamyl transpeptidases are ubiquitously expressed enzymes that
initiate
extracellular glutathione (GSH) breakdown by cleaving gamma-glutamyl amide
bonds. The
breakdown of GSH provides cells with a regional cysteine pool for biosynthetic
pathways.
Gamma-glutamyl transpeptidases also contribute to cellular antioxidant
defenses and expression is
induced by oxidative stress. The cell surface-localized glycoproteins.are
expressed at high levels in
cancer cells. Studies have suggested that the high level of gamma-glutamyl
transpeptidase activity
present on the surface of cancer cells could be exploited to activate
precursor drugs, resulting in high
local concentrations of anti-cancer therapeutic agents (Hanigan, M.H. (1998)
Chem. Biol. Interact.
111-112:333-42; Taniguchi, N. and Ikeda, Y. (1998) Adv. Enzymol. Relat. Areas
Mol. Biol.
72:239-78; Chikhi, N. et al. (1999) Comp. Biochem. Physiol. B. Biochem. Mol.
Biol. 122:367-380).
Aminotransferases
Aminotransferases comprise a family of pyridoxal 5'-phosphate (PLP) -dependent
enzymes
that catalyze transformations of amino acids. Aspartate aminotransferase
(AspAT) is the most
extensively studied PLP-containing enzyme. It catalyzes the reversible
transamination of
dicarboxylic L-amino acids, aspartate and glutamate, and the corresponding 2-
oxo acids, oxalacetate
and 2-oxoglutarate. Other members of the family include pyruvate
aminotransferase, branched-chain
amino acid aminotransferase, tyrosine aminotransferase, aromatic
aminotransferase,
alanine:glyoxylate aminotransferase (AGT), and kynurenine aminotransferase
(Vacca, R.A. et al.
(1997) J. Biol. Chem. 272:21932-21937).
Primary hyperoxaluria type-1 is an autosomal recessive disorder resulting in a
deficiency in
the liver-specific peroxisomal enzyme, alanine:glyoxylate aminotransferase-1.
The phenotype of the
disorder is a deficiency in glyoxylate metabolism. In the absence of AGT,
glyoxylate is oxidized to
oxalate rather than being transaminated to glycine. The result is the
deposition of insoluble calcium
oxalate in the kidneys and urinary tract, ultimately causing renal failure
(Lumb, M.J. et al. (1999) J.
Biol. Chem. 274:20587-20596).
Kynurenine aminotransferase catalyzes the irreversible transamination of the L-
tryptophan
metabolite L-kynurenine to form kynurenic acid. The enzyme may also catalyze
the reversible
transamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce
2-oxoadipate and
L-glutamate. Kynurenic acid is a putative modulator of glutamatergic
neurotransmission; thus a
deficiency in kynurenine aminotransferase may be associated with pleotrophic
effects (Buchli, R. et
al. (1995) J. Biol. Chem. 270:29330-29335).
Catechol-O-methyltransferase
Catechol-O-methyltransferase (COMT) catalyzes the transfer of the methyl group
of S-
adenosyl-L-methionine (AdoMet; SAM) donor to one of the hydroxyl groups of the
catechol
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substrate (e.g., L-dopa, dopamine, or DBA). Methylation of the 3'-hydroxyl
group is favored over
methylation of the 4'-hydroxyl group and the membrane bound isoform of COMT is
more
regiospecific than the soluble form. Translation of the soluble form of the
enzyme results from
utilization of an internal start codon in a full-length mRNA ( 1.5 kb) or from
the translation of a
shorter mRNA (1.3 kb), transcribed from an internal promoter. The proposed SN2-
like methylation
reaction requires Mg++ and is inhibited by Ca++. The binding of the donor and
substrate to COMT
occurs sequentially. AdoMet first binds COMT in a Mg++-independent manner,
followed by the
binding of Mg++ and the binding of the catechol substrate.
The amount of COMT in tissues is relatively high compared to the amount of
activity
normally required, thus inhibition is problematic. Nonetheless, inhibitors
have been developed for in
vitro use (e.g., gallates, tropolone, U-0521, and 3',4'-dihydroxy-2-methyl-
propiophetropolone) and for
clinical use (e.g., nitrocatechol-based compounds and tolcapone).
Administration of these inhibitors
results in the increased half-life of L-dopa and the consequent formation of
dopamine. Inhibition of
COMT is also likely to increase the half-life of various other catechol-
structure compounds,
including but not limited to epinephrine/norepinephrine, isoprenaline,
rimiterol, dobutamine,
fenoldopam, apomorphine, and a-methyldopa. A deficiency in norepinephrine has
been linked to
clinical depression, hence the use of COMT inhibitors could be usefull in the
treatment of depression.
COMT inhibitors are generally well tolerated with minimal side effects and are
ultimately
metabolized in the liver with only minor accumulation of metabolites in the
body (Mannisto, P.T. and
Kaakkola, S. (1999) Pharmacol. Rev. 51:593-628).
Copper-zinc superoxide dismutases
Copper-zinc superoxide dismutases are compact homodimeric metalloenzymes
involved in
cellular defenses against oxidative damage. The enzymes contain one atom of
zinc and one atom of
copper per subunit and catalyze the dismutation of superoxide anions into OZ
and HzOz. The rate of
dismutation is diffusion-limited and consequently enhanced by the presence of
favorable electrostatic
interactions between the substrate and enzyme active site. Examples of this
class of enzyme have
been identified in the cytoplasm of all the eukaryotic cells as well as in the
periplasm of several
bacterial species. Copper-zinc superoxide dismutases are robust enzymes that
are highly resistant to
proteolytic digestion and denaturing by urea and SDS. In addition to the
compact structure of the
enzymes, the presence of the metal ions and intrasubunit disulfide bonds is
believed to be responsible
for enzyme stability. The enzymes undergo reversible denaturation at
temperatures as high as 70°C
(Battistoni, A. et al. (1998) J. Biol. Chem. 273:5655-5661).
Overexpression of superoxide dismutase has been implicated in enhancing
freezing tolerance
of transgenic alfalfa as well as providing resistance to environmental toxins
such as the diphenyl
ether herbicide, acifluorfen (McKersie, B.D. et al. ( 1993) Plant Physiol.
103:1155-1163). In addtion,
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yeast cells become more resistant to freeze-thaw damage following exposure to
hydrogen peroxide
which causes the yeast cells to adapt to further peroxide stress by
upregulating expression of
superoxide dismutases. In this study, mutations to yeast superoxide dismutase
genes had a more
detrimental effect on freeze-thaw resistance than mutations which affected the
regulation of
glutathione metabolism, long suspected of being important in determining an
organism's survival
through the process of cryopreservation (Jong-In Park, J.-I. et al. (1998) J.
Biol. Chem.
273:22921-22928).
Expression of superoxide dismutase is also associated with Mycobacterium
tuberculosis, the
organism that causes tuberculosis. Superoxide dismutase is one of the ten
major proteins secreted by
M. tuberculosis and its expression is upregulated approximately 5-fold in
response to oxidative stress.
M. tuberculosis expresses almost two orders of magnitude more superoxide
dismutase than the
nonpathogenic mycobacterium M. smegmatis, and secretes a much higher
proportion of the expressed
enzyme. The result is the secretion of 350-fold more enzyme by M. tuberculosis
than M.
smeg_matis, providing substantial resistance to oxidative stress (Harth, G.
and Horwitz, M.A. (1999) J.
Biol. Chem. 274:4281-4292).
The reduced expression of copper-zinc superoxide dismutases, as well as other
enzymes with
anti-oxidant capabilities, has been implicated in the early stages of cancer.
The expression of copper-
zinc superoxide dismutases is reduced in prostatic intraepithelial neoplasia
and prostate carcinomas,
(Bostwick, D.G. (2000) Cancer 89:123-134).
Phosphoesterases
Phosphotriesterases (PTE, paraoxonases) are enzymes that hydrolyze toxic
organophosphorus
compounds and have been isolated from a variety of tissues.
Phosphotriesterases play a central role
in the detoxification of insecticides by mammals. Birds and insects lack PTE,
and as a result have
reduced tolerance for organophosphorus compounds (Vilanova, E. and Sogorb,
M.A. (1999) Crit.
Rev. Toxicol. 29:21-57). Phosphotriesterase activity varies among individuals
and is lower in
infants than adults. PTE knockout mice are markedly more sensitive to the
organophosphate-based
toxins diazoxon and chlorpyrifos oxon (Furlong, C.E., et al. (2000)
Neurotoxicology 21:91-100).
Phosphotriesterase is also implicated in atherosclerosis and diseases
involving lipoprotein
metabolism.
Glycerophosphoryl diester phosphodiesterase (also known as
glycerophosphodiester
phosphodiesterase) is a phosphodiesterase which hydrolyzes deacetylated
phospholipid
glycerophosphodiesters to produce sn-glycerol-3-phosphate and an alcohol.
Glycerophosphocholine,
glycerophosphoethanolamine, glycerophosphoglycerol, and glycerophosphoinositol
are examples of
substrates for glycerophosphoryl diester phosphodiesterases. A
glycerophosphoryl diester
phosphodiesterase from E. coli has broad specificity for glycerophosphodiester
substrates (Larson,
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T.J. et al. (1983) J. Biol. Chem. 248:5428-5432).
Cyclic nucleotide phosphodiesterases (PDEs) are crucial enzymes in the
regulation of the
cyclic nucleotides cAMP and cGMP. cAMP and eGMP function as intracellular
second messengers
to transduce a variety of extracellular signals including hormones, light, and
neurotransmitters. PDEs
degrade cyclic nucleotides to their corresponding monophosphates, thereby
regulating the
intracellular concentrations of cyclic nucleotides and their effects on signal
transduction. Due to their
roles as regulators of signal transduction, PDEs have been extensively studied
as chemotherapeutic
targets (Perry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481;
Torphy, J.T. (1998)
Am. J. Resp. Crit. Care Med. 157:351-370).
Families of mammalian PDEs have been classified based on their substrate
specificity and
affinity, sensitivity to cofactors, and sensitivity to inhibitory agents
(Beavo, J.A. (1995) Physiol. Rev.
75:725-748; Conti, M. et al. (1995) Endocrine Rev. 16:370-389). Several of
these families contain
distinct genes, many of which are expressed in different tissues as splice
variants. Within PDE
families, there are multiple isozymes and multiple splice variants of these
isozymes (Conti, M. and
S.-L.C. Jin (1999) Prog. Nucleic Acid Res. Mol. Biol. 63:1-38). The existence
of multiple PDE
families, isozymes, and splice variants is an indication of the variety and
complexity of the regulatory
pathways involving cyclic nucleotides (Houslay, M.D. and G. Milligan (1997)
Trends Biochem. Sci.
22:217-224).
Type 1 PDEs (PDEls) are Caz+/calmodulin-dependent and appear to be encoded by
at least
three different genes, each having at least two different splice variants
(Kakkar, R. et al. (1999) Cell
Mol. Life Sci. 55:1164-1186). PDEls have been found in the lung, heart, and
brain. Some PDE1
isozymes are regulated in vitro by phosphorylation/dephosphorylation.
Phosphorylation of these
PDE1 isozymes decreases the affinity of the enzyme for calmodulin, decreases
PDE activity, and
increases steady state levels of cAMP (Kakkar, su ra). PDEls may provide
useful therapeutic targets
for disorders of the central nervous system and the cardiovascular and immune
systems, due to the
involvement of PDEls in both cyclic nucleotide and calcium signaling (Perry,
M.J. and G.A. Higgs
(1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE2s are cGMP-stimulated PDEs that have been found in the cerebellum,
neocortex, heart,
kidney, lung, pulmonary artery, and skeletal muscle (Sadhu, K. et al. ( 1999)
J. Histochem. Cytochem.
47:895-906). PDE2s are thought to mediate the effects of cAMP on catecholamine
secretion,
participate in the regulation of aldosterone (Beavo, supra), and play a role
in olfactory signal
transduction (Juilfs, D.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:3388-
3395).
PDE3s have high affinity for both cGMP and cAMP, and so these cyclic
nucleotides act as
competitive substrates for PDE3s. PDE3s play roles in stimulating myocardial
contractility,
inhibiting platelet aggregation, relaxing vascular and airway smooth muscle,
inhibiting proliferation
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of T-lymphocytes and cultured vascular smooth muscle cells, and regulating
catecholamine-induced
release of free fatty acids from adipose tissue. The PDE3 family of
phosphodiesterases are sensitive
to specific inhibitors such as cilostamide, enoximone, and lixazinone.
Isozymes of PDE3 can be
regulated by cAMP-dependent protein kinase, or by insulin-dependent kinases
(Degerman, E. et al.
(1997) J. Biol. Chem. 272:6823-6826).
PDE4s are specific for cAMP; are localized to airway smooth muscle, the
vascular
endothelium, and all inflammatory cells; and can be activated by cAMP-
dependent phosphorylation.
Since elevation of cAMP levels can lead to suppression of inflammatory cell
activation and to
relaxation of bronchial smooth muscle, PDE4s have been studied extensively as
possible targets for
novel anti-inflammatory agents, with special emphasis placed on the discovery
of asthma treatments.
PDE4 inhibitors are currently undergoing clinical trials as treatments for
asthma, chronic obstructive
pulmonary disease, and atopic eczema. All four known isozymes of PDE4 are
susceptible to the
inhibitor rolipram, a compound which has been shown to improve behavioral
memory in mice
(Barad, M. et al. (1998) Proc. Natl. Acad. Sci. USA 95:15020-15025). PDE4
inhibitors have also
been studied as possible therapeutic agents against acute lung injury,
endotoxemia, rheumatoid
arthritis, multiple sclerosis, and various neurological and gastrointestinal
indications (Doherty, A.M.
(1999) Curr. Opin. Chem. Biol. 3:466-473).
PDES is highly selective for cGMP as a substrate (Turko, LV. et al. (1998)
Biochemistry
37:4200-4205), and has two allosteric cGMP-specific binding sites (McAllister-
Lucas, L.M. et al.
(1995) J. Biol. Chem. 270:30671-30679). Binding of cGMP to these allosteric
binding sites seems to
be important for phosphorylation of PDES by cGMP-dependent protein kinase
rather than for direct
regulation of catalytic activity. High levels of PDES are found in vascular
smooth muscle, platelets,
lung, and kidney. The inhibitor zaprinast is effective against PDES and PDEls.
Modification of
zaprinast to provide specificity against PDES has resulted in sildenafil
(VIAGRA; Pfizer, Inc., New
York NY), a treatment for male erectile dysfunction (Terrett, N. et al. (1996)
Bioorg. Med. Chem.
Lett. 6:1819-1824). Inhibitors of PDES are currently being studied as agents
for cardiovascular
therapy (Perry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE6s, the photoreceptor cyclic nucleotide phosphodiesterases, are crucial
components of
the phototransduction cascade. In association with the G-protein transducin,
PDE6s hydrolyze cGMP
to regulate cGMP-gated cation channels in photoreceptor membranes. In addition
to the cGMP-
binding active site, PDE6s also have two high-affinity cGMP-binding sites
which are thought to play
a regulatory role in PDE6 function (Artemyev, N.O. et al. (1998) Methods 14:93-
104). Defects in
PDE6s have been associated with retinal disease. Retinal degeneration in the
rd mouse (Yan, W. et
al. (1998) Invest. Opthalmol. Vis. Sci. 39:2529-2536), autosomal recessive
retinitis pigmentosa in
humans (Danciger, M. et al. (1995) Genomics 30:1-12), and rod/cone dysplasia 1
in Irish Setter dogs
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(Suber, M.L. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3968-3972) have been
attributed to
mutations in the PDE6B gene.
The PDE7 family of PDEs consists of only one known member having multiple
splice
variants (Bloom, T.J. and J.A. Beavo (1996) Proc. Natl. Acad. Sci. USA
93:141813-24192). PDE7s
are cAMP specific, but little else is known about their physiological
function. Although mRNAs
encoding PDE7s are found in skeletal muscle, heart, brain, lung, kidney, and
pancreas, expression of
PDE7 proteins is restricted to specific tissue types (Han, P. et al. (1997) J.
Biol. Chem. 272:16152-
16157; Perry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE7s are very
closely related to the PDE4 family; however, PDE7s are not inhibited by
rolipram, a specific inhibitor
of PDE4s (Beavo, supra).
PDEBs are cAMP specific, and are closely related to the PDE4 family. PDEBs are
expressed
in thyroid gland, testis, eye, liver, skeletal muscle, heart, kidney, ovary,
and brain. The cAMP-
hydrolyzing activity of PDEBs is not inhibited by the PDE inhibitors rolipram,
vinpocetine,
milrinone, IBMX (3-isobutyl-1-methylxanthine), or zaprinast, but PDEBs are
inhibited by
dipyridamole (Fisher, D.A. et al. (1998) Biochem. Biophys. Res. Commun.
246:570-577; Hayashi,
M. et al. (1998) Biochem. Biophys. Res. Commun. 250:751-1256; Soderling, S.H.
et al. (1998) Proc.
Natl. Acad. Sci. USA 95:8991-8996).
PDE9s are cGMP specific and most closely resemble the PDE8 family of PDEs.
PDE9s are
expressed in kidney, liver, lung, brain, spleen, and small intestine. PDE9s
are not inhibited by
sildenafil (VIAGRA; Pfizer, Inc., New York NY), rolipram, vinpocetine,
dipyridamole, or IBMX (3-
isobutyl-1-methylxanthine), but they are sensitive to the PDES inhibitor
zaprinast (Fisher, D.A. et al.
(1998) J. Biol. Chem. 273:15559-15564; Soderling, S.H. et al. (1998) J. Biol.
Chem. 273:15553-
15558).
PDElOs are dual-substrate PDEs, hydrolyzing both cAMP and cGMP. PDElOs are
expressed
in brain, thyroid, and testis. (Soderling, S.H. et al. (1999) Proc. Natl.
Acad. Sci. USA 96:7071-
12076; Fujishige, K. et al. (1999) J. Biol. Chem. 274:18438-18445; Loughney,
K. et al (1999) Gene
234:109-117).
PDEs are composed of a catalytic domain of about 270-300 amino acids, an N-
terminal
regulatory domain responsible for binding cofactors, and, in some cases, a
hydrophilic C-terminal
domain of unknown function (Conti, M. and S.-L.C. Jin (1999) Prog. Nucleic
Acid Res. Mol. Biol.
63:1-38). A conserved, putative zinc-binding motif has been identified in the
catalytic domain of all
PDEs. N-terminal regulatory domains include non-catalytic cGMP-binding domains
in PDE2s,
PDESs, and PDE6s; calmodulin-binding domains in PDEls; and domains containing
phosphorylation
sites in PDE3s and PDE4s. In PDES, the N-terminal cGMP-binding domain spans
about 380 amino
acid residues and comprises tandem repeats of a conserved sequence motif
(McAllister-Lucas, L.M.
49
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et al. (1993) J. Biol. Chem. 268:22863-22873). The NKXnD motif has been shown
by mutagenesis
to be important for cGMP binding (Turko, LV. et al. (1996) J. Biol. Chem.
271:22240-22244). PDE
families display approximately 30% amino acid identity within the catalytic
domain; however,
isozymes within the same family typically display about 85-95% identity in
this region (e.g. PDE4A
vs PDE4B). Furthermore, within a family there is extensive similarity (>60%)
outside the catalytic
domain; while across families, there is little or no sequence similarity
outside this domain.
Many of the constituent functions of immune and inflammatory responses are
inhibited by
agents that increase intracellular levels of cAMP (Verghese, M.W. et al.
(1995) Mol. Pharmacol.
47:1164-1171). A variety of diseases have been attributed to increased PDE
activity and associated
with decreased levels of cyclic nucleotides. For example, a form of diabetes
insipidus in mice has
been associated with increased PDE4 activity, an increase in low-Km cAMP PDE
activity has been
reported in leukocytes of atopic patients, and PDE3 has been associated with
cardiac disease.
Many inhibitors of PDEs have undergone clinical evaluation (ferry, M.J. and
G.A. Higgs
(1998) Curr. Opin. Chem. Biol. 2:472-481; Torphy, T.J. (1998) Am. J. Respir.
Crit. Care Med.
157:351-370). PDE3 inhibitors are being developed as antithrombotic agents,
antihypertensive
agents, and as cardiotonic agents useful in the treatment of congestive heart
failure. Rolipram, a
PDE4 inhibitor, has been used in the treatment of depression, and other PDE4
inhibitors have an
anti-inflammatory effect. Rolipram may inhibit HIV-1 replication (Angel, J.B.
et al. (1995) AIDS
9:1137-1144). Additionally, rolipram suppresses the production of cytokines
such as TNF-a and ~
and interferon y, and thus is effective against encephalomyelitis. Rolipram
may also be effective in
treating tardive dyskinesia and multiple sclerosis (Sommer, N. et al. (1995)
Nat. Med. 1:244-248;
Sasaki, H. et al. (1995) Eur. J. Pharmacol. 282:71-76). Theophylline is a
nonspecific PDE inhibitor
used in treatment of bronchial asthma and other respiratory diseases.
Theophylline is believed to act
on airway smooth muscle function and in an anti-inflammatory or
immunomodulatory capacity
(Banner, K.H. and C.P. Page (1995) Eur. Respir. J. 8:996-1000). Pentoxifylline
is another
nonspecific PDE inhibitor used in the treatment of intermittent claudication
and diabetes-induced
peripheral vascular disease. Pentoxifylline is also known to block TNF-a
production and may inhibit
HIV-1 replication (Angel et al., supra).
PDEs have been reported to affect cellular proliferation of a variety of cell
types (Conti et al.
(1995) Endocrine Rev. 16:370-389) and have been implicated in various cancers.
Growth of prostate
carcinoma cell lines DU145 and LNCaP was inhibited by delivery of cAMP
derivatives and PDE
inhibitors (Bang, Y.J. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334).
These cells also
showed a permanent conversion in phenotype from epithelial to neuronal
morphology. It has also
been suggested that PDE inhibitors can regulate mesangial cell proliferation
(Matousovic, K. et al.
(1995) J. Clin. Invest. 96:401-410) and lymphocyte proliferation (Joulain, C.
et al. (1995) J. Lipid
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Mediat. Cell Signal. 11:63-79). One cancer treatment involves intracellular
delivery of PDEs to
particular cellular compartments of tumors, resulting in cell death
(Deonarain, M.P. and A.A.
Epenetos ( 1994) Br. J. Cancer 70:786-794).
Members of the UDP glucuronyltransferase family (UGTs) catalyze the transfer
of a
glucuronic acid group from the cofactor uridine diphosphate-glucuronic acid
(UDP-glucuronic acid)
to a substrate. The transfer is generally to a nucleophilic heteroatom (O, N,
or S). Substrates include
xenobiotics which have been functionalized by Phase I reactions, as well as
endogenous compounds
such as bilirubin, steroid hormones, and thyroid hormones. Products of
glucuronidation are excreted
in urine if the molecular weight of the substrate is less than about 250
g/mol, whereas larger
glucuronidated substrates are excreted in bile.
UGTs are located in the microsomes of liver, kidney, intestine, skin, brain,
spleen, and nasal
mucosa, where they are on the same side of the endoplasmic reticulum membrane
as cytochrome
P450 enzymes and flavin-containing monooxygenases. UGTs have a C-terminal
membrane-spanning
domain which anchors them in the endoplasmic reticulum membrane, and a
conserved signature
domain of about 50 amino acid residues in their C terminal section (PROSITE
PDOC00359 UDP-
glycosyltransferase signature).
UGTs involved in drug metabolism are encoded by two gene families, UGT1 and
UGT2.
Members of the UGT1 family result from alternative splicing of a single gene
locus, which has a
variable substrate binding domain and constant region involved in cofactor
binding and membrane
insertion. Members of the UGT2 family are encoded by separate gene loci, and
are divided into two
families, UGT2A and UGT2B. The 2A subfamily is expressed in olfactory
epithelium, and the 2B
subfamily is expressed in liver microsomes. Mutations in UGT genes are
associated with
hyperbilirubinemia (OMIM #143500 Hyperbilirubinemia I); Crigler-Najjar
syndrome, characterized
by intense hyperbilirubinemia from birth (OMIM #218800 Crigler-Najjar
syndrome); and a milder
form of hyperbilirubinemia termed Gilbert's disease (OMIM *191740 UGT1).
Thioesterases
Two soluble thioesterases involved in fatty acid biosynthesis have been
isolated from
mammalian tissues, one which is active only toward long-chain fatty-acyl
thioesters and one which is
active toward thioesters with a wide range of fatty-acyl chain-lengths. These
thioesterases catalyze
the chain-terminating step in the de novo biosynthesis of fatty acids. Chain
termination involves the
hydrolysis of the thioester bond which links the fatty acyl chain to the 4'-
phosphopantetheine
prosthetic group of the acyl carrier protein (ACP) subunit of the fatty acid
synthase (Smith, S.
(1981a) Methods Enzymol. 71:181-188; Smith, S. (1981b) Methods Enzymol. 71:188-
200).
E. coli contains two soluble thioesterases, thioesterase I which is active
only toward long-
chain acyl thioesters, and thioesterase II (TEII) which has a broad chain-
length specificity (Naggert,
S1
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J. et al. (1991) J. Biol. Chem. 266:11044-11050). E. coli TEII does not
exhibit sequence similarity
with either of the two types of mammalian thioesterases which function as
chain-terminating
enzymes in de novo fatty acid biosynthesis. Unlike the mammalian
thioesterases, E. coli TEII lacks
the characteristic serine active site gly-X-ser-X-gly sequence motif and is
not inactivated by the
serine modifying agent diisopropyl fluorophosphate. However, modification of
histidine 58 by
iodoacetamide and diethylpyrocarbonate abolished TEII activity. Overexpression
of TEII did not
alter fatty acid content in E. coli, which suggests that it does not function
as a chain-terminating
enzyme in fatty acid biosynthesis (Naggert et al., sue). For that reason,
Naggert et al. (su ra)
proposed that the physiological substrates for E. coli TEII may be coenzyme A
(CoA)-fatty acid
esters instead of ACP-phosphopanthetheine-fatty acid esters.
Carboxylesterases
Mammalian carboxylesterases are a multigene family expressed in a variety of
tissues and
cell types. Acetylcholinesterase, butyrylcholinesterase, and carboxylesterase
are grouped into the
serine superfamily of esterases (B-esterases). Other carboxylesterases include
thyroglobulin,
thrombin, Factor IX, gliotactin, and plasminogen. Carboxylesterases catalyze
the hydrolysis of ester-
and amide- groups from molecules and are involved in detoxification of drugs,
environmental toxins,
and carcinogens. Substrates for carboxylesterases include short- and long-
chain acyl-glycerols,
acylcarnitine, carbonates, dipivefrin hydrochloride, cocaine, salicylates,
capsaicin,
palmitoyl-coenzyme A, imidapril, haloperidol, pyrrolizidine alkaloids,
steroids, p-nitrophenyl acetate,
malathion, butanilicaine, and isocarboxazide. Carboxylesterases are also
important for the
conversion of prodrugs to free acids, which may be the active form of the drug
(e.g., lovastatin, used
to lower blood cholesterol) (reviewed in Satoh, T. and Hosokawa, M. (1998)
Annu. Rev. Pharmacol.
Toxico1.38:257-288). Neuroligins are a class of molecules that (i) have N-
terminal signal sequences,
(ii) resemble cell-surface receptors, (iii) contain carboxylesterase domains,
(iv) are highly expressed
in the brain, and (v) bind to neurexins in a calcium-dependent manner. Despite
the homology to
carboxylesterases, neuroligins lack the active site serine residue, implying a
role in substrate binding
rather than catalysis (Ichtchenko, K. et al. (1996) J. Biol. Chem. 271:2676-
2682).
Saualene epoxidase
Squalene epoxidase (squalene monooxygenase, SE) is a microsomal membrane-
bound, FAD-
dependent oxidoreductase that catalyzes the first oxygenation step in the
sterol biosynthetic pathway
of eukaryotic cells. Cholesterol is an essential structural component of
cytoplasmic membranes
acquired via the LDL receptor-mediated pathway or the biosynthetic pathway. SE
converts squalene
to 2,3(S)-oxidosqualene, which is then converted to lanosterol and then
cholesterol.
High serum cholesterol levels result in the formation of atherosclerotic
plaques in the arteries
of higher organisms. This deposition of highly insoluble lipid material onto
the walls of essential
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blood vessels results in decreased blood flow and potential necrosis. HMG-CoA
reductase is
responsible for the first committed step in cholesterol biosynthesis,
conversion of 3-hydroxyl-3-
methyl-glutaryl CoA (HMG-CoA) to mevalonate. HMG-CoA is the target of a number
of
pharmaceutical compounds designed to lower plasma cholesterol levels, but
inhibition of MHG-CoA
also results in the reduced synthesis of non-sterol intermediates required for
other biochemical
pathways. Since SE catalyzes a rate-limiting reaction that occurs later in the
sterol synthesis pathway
with cholesterol as the only end product, SE is a better ideal target for the
design of
anti-hyperlipidemic drugs (Nakamura, Y. et al. (1996) 271:8053-8056).
oxide hydrolases
Epoxide hydrolases catalyze the addition of water to epoxide-containing
compounds, thereby
hydrolyzing epoxides to their corresponding 1,2-diols. They are related to
bacterial haloalkane
dehalogenases and show sequence similarity to other members of the a/~i
hydrolase fold family of
enzymes. This family of enzymes is important for the detoxification of
xenobiotic epoxide
compounds which are often highly electrophilic and destructive when
introduced. Examples of
epoxide hydrolase reactions include the hydrolysis of some leukotoxin to
leukotoxin diol, and
isoleukotoxin to isoleukotoxin diol. Leukotoxins alter membrane permeability
and ion transport and
cause inflammatory responses. In addition, epoxide carcinogens are produced by
cytochrome P450
as intermediates in the detoxification of drugs and environmental toxins.
Epoxide hydrolases possess
a catalytic triad composed of Asp, Asp, and His (Arand, M. et al. (1996) J.
Biol. Chem.
271:4223-4229; Rink, R. et al. (1997) J. Biol. Chem. 272:14650-14657;
Argiriadi, M.A. et al. (2000)
J. Biol. Chem. 275:15265-15270).
Enzymes involved in tyrosine catalysis
The degradation of the amino acid tyrosine, to either succinate and pyruvate
or fumarate and
acetoacetate, requires a large number of enzymes and generates a large number
of intermediate
compounds. In addition, many xenobiotic compounds may be metabolized using one
or more
reactions that are part of the tyrosine catabolic pathway. Enzymes involved in
the degradation of
tyrosine to succinate and pyruvate (e.g., in Arthrobacter species) include 4-
hydroxyphenylpyruvate
oxidase, 4-hydroxyphenylacetate 3-hydroxylase, 3,4-dihydroxyphenylacetate 2,3-
dioxygenase,
5-carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenase, trans,cis-
5-carboxymethyl-2-hydroxymuconate isomerase, homoprotocatechuate
isomerase/decarboxylase,
cis-2-oxohept-3-ene-1,7-dioate hydratase, 2,4-dihydroxyhept-traps-2-ene-1,7-
dioate aldolase, and
succinic semialdehyde dehydrogenase. Enzymes involved in the degradation of
tyrosine to fumarate
and acetoacetate (e.g., in Pseudomonas species) include 4-
hydroxyphenylpyruvate dioxygenase,
homogentisate 1,2-dioxygenase, maleylacetoacetate isomerase,
fumarylacetoacetase and
4-hydroxyphenylacetate. Additional enzymes associated with tyrosine metabolism
in different
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organisms include 4-chlorophenylacetate-3,4-dioxygenase, aromatic
aminotransferase,
5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase, 2-oxo-kept-3-ene-1,7-
dioate hydratase, and
5-carboxymethyl-2-hydroxymuconate isomerase (Elks, L.B.M. et al. ( 1999)
Nucleic Acids Res.
27:373-376; Wackett, L.P. and Ellis, L.B.M. (1996) J. Microbiol. Meth. 25:91-
93; and Schmidt, M.
(1996) Amer. Soc. Microbiol. News 62:102).
In humans, acquired or inherited genetic defects in enzymes of the tyrosine
degradation
pathway may result in hereditary tyrosinemia. One form of this disease,
hereditary tyrosinemia 1
(HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase,
the last enzyme in the
pathway in organisms that metabolize tyrosine to fumarate and acetoacetate.
HT1 is characterized
by progressive liver damage beginning at infancy, and increased risk for liver
cancer (Endo, F. et al.
(1997) J. Biol. Chem. 272:24426-24432).
Expression profiling
Array technology can provide a simple way to explore the expression of a
single polymorphic
gene or the expression profile of a large number of related or unrelated
genes. When the expression
of a single gene is examined, arrays are employed to detect the expression of
a specific gene or its
variants. When an expression profile is examined, arrays provide a platform
for identifying genes
that are tissue specific, are affected by a substance being tested in a
toxicology assay, are part of a
signaling cascade, carry out housekeeping functions, or are specifically
related to a particular genetic
predisposition, condition, disease, or disorder.
The discovery of new enzymes, and the polynucleotides encoding them, satisfies
a need in the
art by providing new compositions which are useful in the diagnosis,
prevention, and treatment of
autoimmune/inflammatory disorders, infectious disorders, immune deficiencies,
disorders of
metabolism, reproductive disorders, neurological disorders, cardiovascular
disorders, eye disorders,
and cell proliferative disorders, including cancer, and in the assessment of
the effects of exogenous
compounds on the expression of nucleic acid and amino acid sequences of
enzymes.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, enzymes, referred to
collectively as "ENZM"
and individually as "ENZM-1," "ENZM-2," "ENZM-3," "ENZM-4," "ENZM-5," "ENZM-
6,"
"ENZM-7," "ENZM-8," "ENZM-9," "ENZM-10," "ENZM-11," and "ENZM-12." In one
aspect, the
invention provides an isolated polypeptide selected from the group consisting
of a) a polypeptide
comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:l-12, b) a
polypeptide comprising a naturally occurring amino acid sequence at least
90°lo identical to an amino
acid sequence selected from the group consisting of SEQ >Z7 NO:1-12, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
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m NO:1-12, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ >I7 NO:1-12. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ )D NO:1-12.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
S from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-12, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID NO: l-12, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ >D NO:1-12, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ >D NO:1-12.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
SEQ ID NO:1-12. In another alternative, the polynucleotide is selected from
the group consisting of
SEQ m N0:13-24.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ >D NO:1-12, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ >D NO: l-12, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-12, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ )D NO:1-12, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ )D NO:1-12, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
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sequence selected from the group consisting of SEQ )D NO:1-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ >D NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ >D
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )D NO:1-12.
The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ >D N0:13-24, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ >D
N0:13-24, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the
polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
selected from the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ )D N0:13-24, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence selected from the
group consisting of
SEQ )D N0:13-24, c) a polynucleotide complementary to the polynucleotide of
a), d) a
polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The
method comprises a) hybridizing the sample with a probe comprising at least 20
contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
hybridization complex is formed between said probe and said target
polynucleotide or fragments
thereof, and b) detecting the presence or absence of said hybridization
complex, and optionally, if
present, the amount thereof. In one alternative, the probe comprises at least
60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ )D N0:13-24, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ )D
N0:13-24, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
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reaction amplification, and b) detecting the presence or absence of said
amplified target
polynucleotide or fragment thereof, and, optionally, if present, the amount
thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ 1D NO:1-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical ~to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )D NO:1-12, and a pharmaceutically acceptable excipient. In
one embodiment, the
composition comprises an amino acid sequence selected from the group
consisting of SEQ 1D NO:1-
12. The invention additionally provides a method of treating a disease or
condition associated with
decreased expression of functional ENZM, comprising administering to a patient
in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:l-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )D NO:1-12. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional ENZM, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ 1D NO:1-12,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-12. The method comprises a) exposing a sample
comprising the
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polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
ENZM, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ >D NO:1-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ >D NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-12. The method comprises a) combining the
polypeptide with at least
one test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ >I7 NO:1-12. The method comprises a) combining the
polypeptide with at least
one test compound under conditions permissive for the activity of the
polypeptide, b) assessing the
activity of the polypeptide in the presence of the test compound, and c)
comparing the activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence
of the test compound, wherein a change in the activity of the polypeptide in
the presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
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The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:13-24, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
target polynucleotide
in the biological sample, said target polynucleotide selected from the group
consisting of i) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:13-24, ii) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:13-24,
iii) a polynucleotide complementary to the polynucleotide of i), iv) a
polynucleotide complementary
to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target
polynucleotide comprises a fragment of a polynucleotide sequence selected from
the group consisting
of i)-v) above; c) quantifying the amount of hybridization complex; and d)
comparing the amount of
hybridization complex in the treated biological sample with the amount of
hybridization complex in
an untreated biological sample, wherein a difference in the amount of
hybridization complex in the
treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability scores for the
matches between each
polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
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Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"ENZM" refers to the amino acid sequences of substantially purified ENZM
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
ENZM. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of ENZM either by
directly interacting with
ENZM or by acting on components of the biological pathway in which ENZM
participates.
An "allelic variant" is an alternative form of the gene encoding ENZM. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
CA 02443244 2003-10-07
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polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur~alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding ENZM include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as ENZM or a
polypeptide with at least one functional characteristic of ENZM. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding ENZM, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
ENZM. The encoded protein may also be "altered," and may contain deletions,
insertions, or
substitutions of amino acid residues which produce a silent change and result
in a functionally
equivalent ENZM. Deliberate amino acid substitutions may be made on the basis
of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the
residues, as long as the biological or immunological activity of ENZM is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of ENZM. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of ENZM either by
directly interacting with ENZM or by acting on components of the biological
pathway in which
ENZM participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
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thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind ENZM polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules.
The nucleotide components of an aptamer may have modified sugar groups (e.g.,
the 2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in
the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
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RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
S molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic ENZM, or of
any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding ENZM or fragments of
ENZM may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
Wl7 or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended and
assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
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amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which
retains at least one biological or immunological function of the natural
molecule. A derivative
polypeptide is one modified by glycosylation, pegylation, or any similar
process that retains at least
one biological or immunological function of the polypeptide from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
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exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of ENZM or the polynucleotide encoding ENZM
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ m N0:13-24 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ >D N0:13-24, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ JD N0:13-24 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ )D N0:13-24 from related polynucleotide sequences. The precise length of a
fragment of SEQ
>D N0:13-24 and the region of SEQ )D N0:13-24 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-12 is encoded by a fragment of SEQ m N0:13-24. A
fragment
of SEQ >D NO:1-12 comprises a region of unique amino acid sequence that
specifically identifies
SEQ >D NO:1-12. For example, a fragment of SEQ >D NO:1-12 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ >D NO:1-12.
The precise length of
a fragment of SEQ )D NO:1-12 and the region of SEQ >D NO:1-12 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
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to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.govBLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Cap: 5 and Extension Gap: 2 penalties
Gap x drop-off.' S0
Expect: l0
Word Size: Il
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
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as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: 1 D
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
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example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 ~,g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
S°C to 20°C lower than the thermal melting point (Tin) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
( 1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1 % SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1%.
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Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
~,g/ml. Organic
solvent, such as formamide at a concentration of about 35-50% v/v, may also be
used under particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of ENZM
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of ENZM which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of ENZM. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of ENZM.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
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"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an ENZM may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of ENZM.
"Probe" refers to nucleic acid sequences encoding ENZM, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
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MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
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A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing ENZM,
nucleic acids encoding ENZM, or fragments thereof may comprise a bodily fluid;
an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" refers to the collective pattern
of gene
expression by a particular cell type or tissue under given conditions at a
given time.
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"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed cells" includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. In one alternative, the nucleic acid can be introduced by
infection with a
recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002)
Science 295:868-872). The
term genetic manipulation does not include classical cross-breeding, or in
vitro fertilization, but
rather is directed to the introduction of a recombinant DNA molecule. The
transgenic organisms
contemplated in accordance with the present invention include bacteria,
cyanobacteria, fungi, plants
and animals. The isolated DNA of the present invention can be introduced into
the host by methods
known in the art, for example infection, transfection, transformation or
transconjugation. Techniques
for transferring the DNA of the present invention into such organisms are
widely known and provided
in references such as Sambrook et al. ( 1989), supra.
A "variant" of a-particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
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another. The resulting polypeptides will generally have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human enzymes (ENZM), the
polynucleotides
encoding ENZM, and the use of these compositions for the diagnosis, treatment,
or prevention of
autoimmune/inflammatory disorders, infectious disorders, immune deficiencies,
disorders of
metabolism, reproductive disorders, neurological disorders, cardiovascular
disorders, eye disorders,
and cell proliferative disorders, including cancer.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project >D). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide )D) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ >D NO:) and
an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide >D)
as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide )D) for polypeptides of the
invention. Column 3
shows the GenBank identification number (GenBank )D NO:) of the nearest
GenBank homolog.
Column 4 shows the probability scores for the matches between each polypeptide
and its homolog(s).
Column 5 shows the annotation of the GenBank homolog(s) along with relevant
citations where
applicable, all of which are expressly incorporated by reference herein.
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Table 3 shows various structural features of the polypeptides of the
invention. Columns 1
and 2 show the polypeptide sequence identification number (SEQ 117 NO:) and
the corresponding
Incyte polypeptide sequence number (Incyte Polypeptide )D) for each
polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI). Column 6 shows amino acid residues comprising signature
sequences, domains, and
motifs. Column 7 shows analytical methods for protein structure/function
analysis and in some cases,
searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are enzymes. For example,
SEQ ID NO:1 is 38%
identical, from residue L59 to residue I250, to human cytosolic epoxide
hydrolase (GenBank >D
g181395) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 3.4e-35, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID NO:1 also contains an
alphabeta hydrolase fold
domain as determined by searching for statistically significant matches in the
hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
BLIMPS and additional BLAST analyses provide further corroborative evidence
that SEQ )D NO:1 is
an epoxide hydrolase. In an alternative example, SEQ ID N0:2 is 64% identical,
from residue E275 to
residue I864, to mouse acetyltransferase Tubedown-1 (GenBank >D g8497318) as
determined by the
Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 4.4e-
210, which indicates the probability of obtaining the observed polypeptide
sequence alignment by
chance. SEQ ID N0:2 also contains TPR domains as determined by searching for
statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from additional BLAST analysis against the
PRODOM
database provides further corroborative evidence that SEQ ID N0:2 is an
acetyltransferase. In an
alternative example, SEQ ID N0:3 is 97% identical, from residue Ml to residue
K376, to human
trans-prenyltransferase (GenBank >D g4732024) as determined by the Basic Local
Alignment Search
Tool (BLAST). (See Table 2.) The BLAST probability score is 5.6e-194, which
indicates the
probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ )D N0:3 also
contains a polyprenyl synthetase domain as determined by searching for
statistically significant
matches in the hidden Markov model (HMM)-based PFAM database of conserved
protein family
domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses
provide
further corroborative evidence that SEQ )D N0:3 is a trans-prenyltransferase.
In an alternative
example, SEQ )D N0:6 is 75% identical, from residue M1 to residue K399, to rat
argininosuccinate
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synthetase (GenBank ID 855767) as determined by the Basic Local Alignment
Search Tool (BLAST).
(See Table 2.) The BLAST probability score is 6.3e-158, which indicates the
probability of obtaining
the observed polypeptide sequence alignment by chance. SEQ )D N0:6 also
contains an
arginosuccinate synthase domain as determined by searching for statistically
significant matches in
the hidden Markov model (HMM)-based PFAM database of conserved protein family
domains. (See
Table 3.) Data from BLIMPS and PROFILESCAN analyses provide further
corroborative evidence
that SEQ )D N0:6 is an argininosuccinate synthetase. In an alternative
example, SEQ ID N0:8 is
100% identical, from residue M1 to Y583, to human succinate dehydrogenase
flavoprotein subunit
(GenBank ID 8347134) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 0.0, which indicates the probability
of obtaining the
observed polypeptide sequence alignment by chance. SEQ >D N0:8 also contains a
FAD binding
domain and a fumarate reductase/succinate dehydrogenase flavoprotein C-
terminal domain as
determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database of conserved protein family domains. (See Table 3.) Data
from BLIMPS,
MOTIFS, PROFILESCAN and other BLAST analyses provide further corroborative
evidence that
SEQ >D N0:8 is a succinate dehydrogenase flavoprotein subunit. In an
alternative example, SEQ >D
NO:10 is 34% identical, from residue V23 to residue L380, to rat arylacetamide
deacetylase
(GenBank >D 85923874) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 2.0e-51, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. Data from BLIMPS analysis
provide further
corroborative evidence that SEQ ID NO:10 is an esterase. In an alternative
example, SEQ >D N0:12
is 65% identical, from residue M35 to residue L326, to human neuropathy target
esterase (GenBank
ID 82982501) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.)
The BLAST probability score is 2.4e-95, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ 1D N0:12 also contains a cyclic
nucleotide-binding
domain as determined by searching for statistically significant matches in the
hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
BLAST analysis provide further corroborative evidence that SEQ ID N0:12 is a
neuropathy target
esterase. SEQ )D N0:4-5, SEQ ID N0:7, SEQ m N0:9, and SEQ >D NO:11 were
analyzed and
annotated in a similar manner. The algorithms and parameters for the analysis
of SEQ ID NO:1-12
are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Column 1 lists the polynucleotide
sequence
identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte
polynucleotide
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consensus sequence number (Incyte ID) for each polynucleotide of the
invention, and the length of
each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start
(5') and stop (3')
positions of the cDNA and/or genomic sequences used to assemble the full
length polynucleotide
sequences of the invention, and of fragments of the polynucleotide sequences
which are useful, for
example, in hybridization or amplification technologies that identify SEQ ID
N0:13-24 or that
distinguish between SEQ ID N0:13-24 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotide sequences. In
addition, the polynucleotide fragments described in column 2 may identify
sequences derived from
the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences
including the
designation "ENST"). Alternatively, the polynucleotide fragments described in
column 2 may be
derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those
sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the polynucleotide
fragments described in
column 2 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, a polynucleotide sequence
identified as
FL XXXXXX_N~ Nz_YYYYY Nj N4 represents a "stitched" sequence in which XXXXXX
is the
identification number of the cluster of sequences to which the algorithm was
applied, and YYYYY is
the number of the prediction generated by the algorithm, and N,,2,3..,, if
present, represent specific
exons that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of exons brought
together by an
"exon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FLXXXXXX_gAAAAA~BBBBB_1 N is a "stretched" sequence, with XXXXXX being the
Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the
GenBank identification number or NCBI RefSeq identification number of the
nearest GenBank
protein homolog, and N referring to specific exons (See Example V). In
instances where a RefSeq
sequence was used as a protein homolog for the "exon-stretching" algorithm, a
RefSeq identifier
(denoted by "NM," "NP," or "NT") may be used in place of the GenBank
identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
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Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK).
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in Table
4 was obtained to confirm the final consensus polynucleotide sequence, but the
relevant Incyte cDNA
identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
The invention also encompasses ENZM variants. A preferred ENZM variant is one
which
has at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the ENZM amino acid sequence, and which contains at least
one functional or
structural characteristic of ENZM.
The invention also encompasses polynucleotides which encode ENZM. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:13-24, which encodes ENZM. The
polynucleotide
sequences of SEQ ID N0:13-24, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
ENZM. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding ENZM. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:13-24 which has at least about 70%, or alternatively at least about 85%, or
even at least about
95% polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting
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of SEQ )D N0:13-24. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of ENZM.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant
of a polynucleotide sequence encoding ENZM. A splice variant may have portions
which have
significant sequence identity to the polynucleotide sequence encoding ENZM,
but will generally have
a greater or lesser number of polynucleotides due to additions or deletions of
blocks of sequence
arising from alternate splicing of exons during mRNA processing. A splice
variant may have less
than about 70%, or alternatively less than about 60%, or alternatively less
than about 50%
polynucleotide sequence identity to the polynucleotide sequence encoding ENZM
over its entire
length; however, portions of the splice variant will have at least about 70%,
or alternatively at least
about 85%, or alternatively at least about 95%, or alternatively 100%
polynucleotide sequence
identity to portions of the polynucleotide sequence encoding ENZM. Any one of
the splice variants
described above can encode an amino acid sequence which contains at least one
functional or
structural characteristic of ENZM.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding ENZM, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring ENZM, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode ENZM and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring ENZM under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding ENZM or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding ENZM and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater
half-life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode ENZM
and
ENZM derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell
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systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding ENZM or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ 117
N0:13-24 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Applied
Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Bioloey, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding ENZM may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and ligations may be used to insert an engineered double-stranded
sequence into a region
of unknown sequence before performing PCR. Other methods which may be used to
retrieve
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unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need
to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 primer analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode ENZM may be cloned in recombinant DNA molecules that direct
expression of ENZM,
or fragments or functional equivalents thereof, in appropriate host cells. Due
to the inherent
degeneracy of the genetic code, other DNA sequences which encode substantially
the same or a
functionally equivalent amino acid sequence may be produced and used to
express ENZM.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter ENZM-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
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as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of ENZM, such as its biological or enzymatic
activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurnng genes in a
directed and controllable
manner.
In another embodiment, sequences encoding ENZM may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, ENZM itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Pro ep
roes, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of ENZM, or any part thereof, may be altered during direct
synthesis and/or
combined with sequences from other proteins, or any part thereof, to produce a
variant polypeptide or
a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active ENZM, the nucleotide sequences
encoding ENZM or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
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encoding ENZM. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
ENZM. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding ENZM and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals
may be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used. (See, e.g.,
Scharf, D. et al. (1994)
Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding ENZM and appropriate transcriptional and
translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989)
Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York
NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding ENZM. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, su ra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harnngton, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
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The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding ENZM. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding ENZM can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding ENZM into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of ENZM are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of ENZM may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of ENZM. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of ENZM. Transcription of
sequences
encoding ENZM may be driven by viral promoters, e.g., the 355 and 195
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and TechnoloQy
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding ENZM
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses ENZM in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
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virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of ENZM in cell lines is preferred. For example, sequences encoding ENZM can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using
tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), f3 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding ENZM is inserted within a marker gene sequence, transformed
cells containing
sequences encoding ENZM can be identified by the absence of marker gene
function. Alternatively,
CA 02443244 2003-10-07
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a marker gene can be placed in tandem with a sequence encoding ENZM under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding ENZM
and that express
ENZM may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of ENZM using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on ENZM is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding ENZM
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding ENZM, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding ENZM may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode ENZM may be designed to contain signal
sequences which
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direct secretion of ENZM through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
S phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding ENZM may be ligated to a heterologous sequence resulting in
translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric ENZM protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of ENZM activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the ENZM encoding sequence and the
heterologous protein
sequence, so that ENZM may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled ENZM may
be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
ENZM of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to ENZM. At least one and up to a plurality of test
compounds may be screened
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for specific binding to ENZM. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
ENZM, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e:g., Coligan, J.E. et al. (1991) Current
Protocols in ImmunoloQV 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which ENZM
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express ENZM,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing ENZM or cell membrane fractions which contain ENZM
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either ENZM or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
ENZM, either in
solution or affixed to a solid support, and detecting the binding of ENZM to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
ENZM of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of ENZM. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
ENZM activity, wherein ENZM is combined with at least one test compound, and
the activity of
ENZM in the presence of a test compound is compared with the activity of ENZM
in the absence of
the test compound. A change in the activity of ENZM in the presence of the
test compound is
indicative of a compound that modulates the activity of ENZM. Alternatively, a
test compound is
combined with an in vitro or cell-free system comprising ENZM under conditions
suitable for ENZM
activity, and the assay is performed. In either of these assays, a test
compound which modulates the
activity of ENZM may do so indirectly and need not come in direct contact with
the test compound.
At least one and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding ENZM or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
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models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi, M.R.
( 1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding ENZM may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding ENZM can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding ENZM is injected into animal ES cells, and
the injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the
blastulae are implanted as described above. Transgenic progeny or inbred lines
are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress ENZM, e.g., by secreting ENZM in
its milk, may also
serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of ENZM and enzymes. In addition, examples of tissues
expressing ENZM can be
found in Table 6. Therefore, ENZM appears to play a role in
autoimmune/inflammatory disorders,
infectious disorders, immune deficiencies, disorders of metabolism,
reproductive disorders,
neurological disorders, cardiovascular disorders, eye disorders, and cell
proliferative disorders,
including cancer. In the treatment of disorders associated with increased ENZM
expression or
activity, it is desirable to decrease the expression or activity of ENZM. In
the treatment of disorders
associated with decreased ENZM expression or activity, it is desirable to
increase the expression or
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activity of ENZM.
Therefore, in one embodiment, ENZM or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of ENZM. Examples of such disorders include, but are not limited to,
an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis,
autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, and trauma; an infectious disorder such as a viral
infection, e.g., caused by
an adenovirus (acute respiratory disease, pneumonia), an arenavirus
(lymphocytic choriomeningitis),
a bunyavirus (Hantavirus), a coronavirus (pneumonia, chronic bronchitis), a
hepadnavirus (hepatitis),
a herpesvirus (herpes simplex virus, varicella-zoster virus, Epstein-Barr
virus, cytomegalovirus), a
flavivirus (yellow fever), an orthomyxovirus (influenza), a papillomavirus
(cancer), a paramyxovirus
(measles, mumps), a picornovirus (rhinovirus, poliovirus, coxsackie-virus), a
polyomavirus (BK
virus, JC virus), a poxvirus (smallpox), a reovirus (Colorado tick fever), a
retrovirus (human
immunodeficiency virus, human T lymphotropic virus), a rhabdovirus (rabies), a
rotavirus
(gastroenteritis), and a togavirus (encephalitis, rubella), and a bacterial
infection, a fungal infection, a
parasitic infection, a protozoal infection, and a helminthic infection; an
immune deficiency, such as
acquired immunodeficiency syndrome (AlDS), X-linked agammaglobinemia of
Bruton, common
variable immunodeficiency (CV>7, DiGeorge's syndrome (thymic hypoplasia),
thymic dysplasia,
isolated IgA deficiency, severe combined immunodeficiency disease (SC)D),
immunodeficiency with
thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi
syndrome, chronic
granulomatous diseases, hereditary angioneurotic edema, and immunodeficiency
associated with
Cushing's disease; a disorder of metabolism such as Addison's disease,
cerebrotendinous
xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic
fibrosis, diabetes, fatty
hepatocirrhosis, fructose-I,6-diphosphatase deficiency, galactosemia, goiter,
glucagonoma, glycogen
storage diseases, hereditary fructose intolerance, hyperadrenalism,
hypoadrenalism,
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hyperparathyroidism, hypoparathyroidism, hypercholesterolemia,
hyperthyroidism, hypoglycemia,
hypothyroidism, hyperlipidemia, hyperlipemia, a lipid myopathy, a
lipodystrophy, a lysosomal
storage disease, mannosidosis, neuraminidase deficiency, obesity, pentosuria
phenylketonuria,
pseudovitamin D-deficiency rickets; a reproductive disorder such as a disorder
of prolactin
production, infertility, including tubal disease, ovulatory defects, and
endometriosis, a disruption of
the estrous cycle, a disruption of the menstrual cycle, polycystic ovary
syndrome, ovarian
hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids,
autoimmune disorders,
ectopic pregnancies, and teratogenesis, cancer of the breast, fibrocystic
breast disease, and
galactorrhea, disruptions of spermatogenesis, abnormal sperm physiology,
cancer of the testis, cancer
of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of
the male breast, and gynecomastia; a neurological disorder such as epilepsy,
ischemic
cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease,
Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating
diseases, bacterial and
viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease; prion diseases
including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome; fatal
familial insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system,
cerebral palsy,
neuroskeletal disorders, autonomic nervous system disorders, cranial nerve
disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders, peripheral
nervous system
disorders, dermatomyositis and polymyositis; inherited, metabolic, endocrine,
and toxic myopathies;
myasthenia gravis, periodic paralysis; mental disorders including mood,
anxiety, and schizophrenic
disorders; seasonal affective disorder (SAD); akathesia, amnesia, catatonia,
diabetic neuropathy,
tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder; a
cardiovascular disorder, such as arteriovenous fistula, atherosclerosis,
hypertension, vasculitis,
Raynaud's disease, aneurysms, arterial dissections, varicose veins,
thrombophlebitis and
phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon
angioplasty, vascular
replacement, and coronary artery bypass graft surgery, congestive heart
failure, ischemic heart
disease, angina pectoris, myocardial infarction, hypertensive heart disease,
degenerative valvular
heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic
valve, mural annular
calcification, mitral valve prolapse, rheumatic fever and rheumatic heart
disease, infective
endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic
lupus erythematosus,
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carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic
heart disease,
congenital heart disease, and complications of cardiac transplantation,
congenital lung anomalies,
atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary
hemorrhage,
pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive
pulmonary disease,
restrictive pulmonary, disease, chronic obstructive pulmonary disease,
emphysema, chronic
bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and
mycoplasmal pneumonia,
lung abscess, pulmonary tuberculosis, diffuse interstitial diseases,
pneumoconioses, sarcoidosis,
idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis,
hypersensitivity pneumonitis,
pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse
pulmonary
hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary
hemosiderosis, pulmonary
involvement in collagen-vascular disorders, pulmonary alveolar proteinosis,
lung tumors,
inflammatory and noninflammatory pleural effusions, pneumothorax, pleural
tumors, drug-induced
lung disease, radiation-induced lung disease, and complications of lung
transplantation; an eye
disorder such as ocular hypertension and glaucoma; a disorder of cell
proliferation such as actinic
keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue
disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis,
primary thrombocythemia; and a cancer, including adenocarcinoma, leukemia,
lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen,
testis, thymus, thyroid, and uterus.
In another embodiment, a vector capable of expressing ENZM or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of ENZM including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
ENZM in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent
a disorder associated with decreased expression or activity of ENZM including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of ENZM
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of ENZM including, but not limited to, those listed above.
In a further embodiment, an antagonist of ENZM may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of ENZM.
Examples of such
disorders include, but are not limited to, those autoimmune/inflammatory
disorders, infectious
disorders, immune deficiencies, disorders of metabolism, reproductive
disorders, neurological
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disorders, cardiovascular disorders, eye disorders, and cell proliferative
disorders, including cancer
described above. In one aspect, an antibody which specifically binds ENZM may
be used directly as
an antagonist or indirectly as a targeting or delivery mechanism for bringing
a pharmaceutical agent
to cells or tissues which express ENZM.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding ENZM may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of ENZM including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of ENZM may be produced using methods which are generally known
in the
art. In particular, purified ENZM may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind ENZM.
Antibodies to ENZM may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use. Single chain
antibodies (e.g., from
camels or llamas) may be potent enzyme inhibitors and may have advantages in
the design of peptide
mimetics, and in the development of immuno-adsorbents and biosensors
(Muyldermans, S. (2001) J.
Biotechnol. 74:277-302).
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, camels,
dromedaries, llamas, humans, and others may be immunized by injection with
ENZM or with any
fragment or oligopeptide thereof which has immunogenic properties. Depending
on the host species,
various adjuvants may be used to increase immunological response. Such
adjuvants include, but are
not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
ENZM have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
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fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of ENZM amino acids may be fused with those of another protein, such as KLH,
and antibodies to the
chimeric molecule may be produced.
Monoclonal antibodies to ENZM may be prepared using any technique which
provides for
the production of antibody molecules by continuous cell lines in culture.
These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
ENZM-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. ( 1989) Proc.
Natl. Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for ENZM may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
ENZM and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering ENZM epitopes is generally used, but a
competitive binding assay
may also be employed (Pound, supra).
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Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for ENZM. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of ENZM-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The K~ determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple ENZM epitopes, represents the average affinity, or
avidity, of the antibodies for
ENZM. The K~ determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular ENZM epitope, represents a true measure of affinity. High-affinity
antibody preparations
with Ka ranging from about 109 to 10'Z L/mole are preferred for use in
immunoassays in which the
ENZM-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with Ka ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of ENZM, preferably in active
form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of ENZM-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity, and
guidelines for antibody quality and usage in various applications, are
generally available. (See, e.g.,
Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding ENZM, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding ENZM. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding ENZM. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
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9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding ENZM may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in ENZM expression or regulation causes
disease, the expression of
ENZM from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
ENZM are treated by constructing mammalian expression vectors encoding ENZM
and introducing
these vectors by mechanical means into ENZM-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (
1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of ENZM include,
but are not
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limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
ENZM may
be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes),
(ii) an inducible
promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard
(1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and
H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in
the T-REX plasmid
(Invitrogen)); the ecdysone-inducible promoter (available in the plasmids
PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone
inducible promoter
(Rossi, F.M.V. and H.M. Blau, supra)), or (iii) a tissue-specific promoter or
the native promoter of the
endogenous gene encoding ENZM from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIP>D
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to ENZM expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding ENZM under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
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cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding ENZM to cells which have one or more genetic
abnormalities with respect
to the expression of ENZM. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding ENZM to target cells which have one or more genetic
abnormalities with
respect to the expression of ENZM. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing ENZM to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,.804,413 teaches the use of
recombinant HSV d92 which
consists of a genome containing at least one exogenous gene to be transferred
to a cell under the
control of the appropriate promoter for purposes including human gene therapy.
Also taught by this
patent are the construction and use of recombinant HSV strains deleted for
ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and
Xu, H. et al. (1994)
Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of
cloned herpesvirus
sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
herpesvirus, and the infection of cells with herpesvirus are techniques well
known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
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deliver polynucleotides encoding ENZM to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
ENZM into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
ENZM-coding RNAs and the synthesis of high levels of ENZM in vector transduced
cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of ENZM into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding ENZM.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
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corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding ENZM. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding ENZM.
Compounds which may be effective in altering expression of a specific
polynucleotide may include,
but are not limited to, oligonucleotides, antisense oligonucleotides, triple
helix-forming
oligonucleotides, transcription factors and other polypeptide transcriptional
regulators, and non-
macromolecular chemical entities which are capable of interacting with
specific polynucleotide
sequences. Effective compounds may alter polynucleotide expression by acting
as either inhibitors or
promoters of polynucleotide expression. Thus, in the treatment of disorders
associated with increased
ENZM expression or activity, a compound which specifically inhibits expression
of the
polynucleotide encoding ENZM may be therapeutically useful, and in the
treatment of disorders
associated with decreased ENZM expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding ENZM may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
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altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding ENZM is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
ENZM are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding ENZM. The amount of hybridization may be
quantified, thus
forming the basis for a comparison of the expression of the polynucleotide
both with and without
exposure to one or more test compounds. Detection of a change in the
expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in
altering the expression of
the polynucleotide. A screen for a compound effective in altering expression
of a specific
polynucleotide can be carried out, for example, using a Schizosaccharom~pombe
gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. ( 1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
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Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of ENZM, antibodies to ENZM, and mimetics, agonists, antagonists, or
inhibitors of ENZM.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton,
J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage
of administration
without needle injection, and obviates the need for potentially toxic
penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising ENZM or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, ENZM or a fragment thereof may be joined to
a short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be .used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
ENZM or fragments thereof, antibodies of ENZM, and agonists, antagonists or
inhibitors of ENZM, .
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
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therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 ~cg, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind ENZM may be used for
the
diagnosis of disorders characterized by expression of ENZM, or in assays to
monitor patients being
treated with ENZM or agonists, antagonists, or inhibitors of ENZM. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for ENZM include methods which utilize the antibody and a label to detect ENZM
in human body
fluids or in extracts of cells or tissues. The antibodies may be used with or
without modification, and
may be labeled by covalent or non-covalent attachment of a reporter molecule.
A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring ENZM, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
ENZM expression. Normal
or standard values for ENZM expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to ENZM
under conditions suitable for complex formation. The amount of standard
complex formation may be
quantitated by various methods, such as photometric means. Quantities of ENZM
expressed in
subject, control, and disease samples from biopsied tissues are compared with
the standard values.
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Deviation between standard and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding ENZM may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of ENZM
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of ENZM, and to monitor regulation of ENZM levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding ENZM or closely related
molecules may be used
to identify nucleic acid sequences which encode ENZM. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding ENZM, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the ENZM encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:13-24 or from
genomic sequences including promoters, enhancers, and introns of the ENZM
gene.
Means for producing specific hybridization probes for DNAs encoding ENZM
include the
cloning of polynucleotide sequences encoding ENZM or ENZM derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding ENZM may be used for the diagnosis of
disorders
associated with expression of ENZM. Examples of such disorders include, but
are not limited to, an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis,
autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
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myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, and trauma; an infectious disorder such as a viral
infection, e.g., caused by
an adenovirus (acute respiratory disease, pneumonia), an arenavirus
(lymphocytic choriomeningitis),
a bunyavirus (Hantavirus), a coronavirus (pneumonia, chronic bronchitis), a
hepadnavirus (hepatitis),
a herpesvirus (herpes simplex virus, varicella-zoster virus, Epstein-Ban
virus, cytomegalovirus), a
flavivirus (yellow fever), an orthomyxovirus (influenza), a papillomavirus
(cancer), a paramyxovirus
(measles, mumps), a picornovirus (rhinovirus, poliovirus, coxsackie-virus), a
polyomavirus (BK
virus, JC virus), a poxvirus (smallpox), a reovirus (Colorado tick fever), a
retrovirus (human
immunodeficiency virus, human T lymphotropic virus), a rhabdovirus (rabies), a
rotavirus
(gastroenteritis), and a togavirus (encephalitis, rubella), and a bacterial
infection, a fungal infection, a
parasitic infection, a protozoal infection, and a helminthic infection; an
immune deficiency, such as
acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of
Breton, common
variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia),
thymic dysplasia,
isolated IgA deficiency, severe combined immunodeficiency disease (SCID),
immunodeficiency with
thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi
syndrome, chronic
granulomatous diseases, hereditary angioneurotic edema, and immunodeficiency
associated with
Cushing's disease; a disorder of metabolism such as Addison's disease,
cerebrotendinous
xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic
fibrosis, diabetes, fatty
hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter,
glucagonoma, glycogen
storage diseases, hereditary fructose intolerance, hyperadrenalism,
hypoadrenalism,
hyperparathyroidism, hypoparathyroidism, hypercholesterolemia,
hyperthyroidism, hypoglycemia,
hypothyroidism, hyperlipidemia, hyperlipemia, a lipid myopathy, a
lipodystrophy, a lysosomal
storage disease, mannosidosis, neuraminidase deficiency, obesity, pentosuria
phenylketonuria,
pseudovitamin D-deficiency rickets; a reproductive disorder such as a disorder
of prolactin
production, infertility, including tubal disease, ovulatory defects, and
endometriosis, a disruption of
the estrous cycle, a disruption of the menstrual cycle, polycystic ovary
syndrome, ovarian
hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids,
autoimmune disorders,
ectopic pregnancies, and teratogenesis, cancer of the breast, fibrocystic
breast disease, and
galactorrhea, disruptions of spermatogenesis, abnormal sperm physiology,
cancer of the testis, cancer
of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of
the male breast, and gynecomastia; a neurological disorder such as epilepsy,
ischemic
cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease,
Pick's disease,
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Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating
diseases, bacterial and
viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease; prion diseases
including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome; fatal
familial insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system,
cerebral palsy,
neuroskeletal disorders, autonomic nervous system disorders, cranial nerve
disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders, peripheral
nervous system
disorders, dermatomyositis and polymyositis; inherited, metabolic, endocrine,
and toxic myopathies;
myasthenia gravis, periodic paralysis; mental disorders including mood,
anxiety, and schizophrenic
disorders; seasonal affective disorder (SAD); akathesia, amnesia, catatonia,
diabetic neuropathy,
tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder; a
cardiovascular disorder, such as arteriovenous fistula, atherosclerosis,
hypertension, vasculitis,
Raynaud's disease, aneurysms, arterial dissections, varicose veins,
thrombophlebitis and
phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon
angioplasty, vascular
replacement, and coronary artery bypass graft surgery, congestive heart
failure, ischemic heart
disease, angina pectoris, myocardial infarction, hypertensive heart disease,
degenerative valvular
heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic
valve, mural annular
calcification, mitral valve prolapse, rheumatic fever and rheumatic heart
disease, infective
endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic
lupus erythematosus,
carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic
heart disease,
congenital heart disease, and complications of cardiac transplantation,
congenital lung anomalies,
atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary
hemorrhage,
pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive
pulmonary disease,
restrictive pulmonary disease, chronic obstructive pulmonary disease,
emphysema, chronic
bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and
mycoplasmal pneumonia,
lung abscess, pulmonary tuberculosis, diffuse interstitial diseases,
pneumoconioses, sarcoidosis,
idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis,
hypersensitivity pneumonitis,
pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse
pulmonary
hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary
hemosiderosis, pulmonary
involvement in collagen-vascular disorders, pulmonary alveolar proteinosis,
lung tumors,
inflammatory and noninflammatory pleural effusions, pneumothorax, pleural
tumors, drug-induced
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lung disease, radiation-induced lung disease, and complications of lung
transplantation; an eye
disorder such as ocular hypertension and glaucoma; a disorder of cell
proliferation such as actinic
keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue
disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis,
primary thrombocythemia; and a cancer, including adenocarcinoma, leukemia,
lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen,
testis, thymus, thyroid, and uterus. The polynucleotide sequences encoding
ENZM may be used in
Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies;
in dipstick, pin, and multiformat ELISA-like assays; and in microarrays
utilizing fluids or tissues
from patients to detect altered ENZM expression. Such qualitative or
quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding ENZM may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding ENZM may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding ENZM in the
sample indicates the presence of the associated disorder. Such assays may also
.be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
ENZM, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding ENZM, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
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successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding ENZM
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding ENZM, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
ENZM, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding ENZM may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are not limited to,
single-stranded
conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In
SSCP,
oligonucleotide primers derived from the polynucleotide sequences encoding
ENZM are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
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useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibrosis,
sickle cell anemia, or chronic granulomatous disease. For example, variants in
the mannose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl transferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the core promoter of
the ALOXS gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations. (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu
(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641.)
Methods which may also be used to quantify the expression of ENZM include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, ENZM, fragments of ENZM, or antibodies specific for
ENZM may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
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generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expression of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
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treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
supra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for ENZM
to quantify
the levels of ENZM expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed
by a variety of methods known in the art, for example, by reacting the
proteins in the sample with a
thiol- or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at
each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
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should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
' cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are
well known and thoroughly described in DNA Microarrays: A Practical Approach,
M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding ENZM
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a mufti-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
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region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial
chromosomes (HACs), yeast artificial chromosomes (PACs), bacterial artificial
chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See,
e.g., Harrington, J.J.
et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134;
and Trask, B.J.
(1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of
the invention may be
used to develop genetic linkage maps, for example, which correlate the
inheritance of a disease state
with the inheritance of a particular chromosome region or restriction fragment
length polymorphism
(RFLP). (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl.
Acad. Sci. USA 83:7353-
7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding ENZM on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to l 1q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, ENZM, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between ENZM and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with ENZM, or
fragments thereof,
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and washed. Bound ENZM is then detected by methods well known in the art.
Purified ENZM can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding ENZM specifically compete with a test compound
for binding ENZM.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with ENZM.
In additional embodiments, the nucleotide sequences which encode ENZM may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No.60/322,181, U.S. Ser. No.60/315,874, U.S. Ser.
No.60/311,447, U.S. Ser.
No.60/308,182, U.S. Ser. No.60/293,572, U.S. Ser. No.60/291,544, and U.S. Ser.
No.60/283,793 are
expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in
guanidinium
isothiocyanate, while others were homogenized and lysed in phenol or in a
suitable mixture of
denaturants, such as TRIZOL (Life Technologies), a monophasic solution of
phenol and guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or
extracted with
chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
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purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV, plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte
Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant
plasmids were
transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF, or
SOLR from
Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96
plasmid purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
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cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo
Sapiens,
Rattus norve-icus, Mus musculus, Caenorhabditis elegans, Saccharomyces
cerevisiae,
Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); hidden
Markov model (HMM)-based protein family databases such as PFAM, INCY, and
TIGRFAM (Haft,
D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain
databases such as
SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic,
I. et al. (2002)
Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which
analyzes consensus
primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr.
Opin. Struct. Biol.
6:361-365.) The queries were performed using programs based on BLAST, FASTA,
BLIMPS, and
HMMER. The Incyte cDNA sequences were assembled to produce full length
polynucleotide
sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,
stretched sequences,
or Genscan-predicted coding sequences (see Examples IV and V) were used to
extend Incyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention
may begin at any of the methionine residues of the full length translated
polypeptide. Full length
polypeptide sequences were subsequently analyzed by querying against databases
such as the
GenBank protein databases (genpept), SwissProt, the PROTEOME databases,
BLOCKS, PRINTS,
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DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family
databases such as
PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART.
Full
length polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi
Software Engineering, South San Francisco CA) and LASERGENE software
(DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default
parameters specified
by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence
alignment program
(DNASTAR), which also calculates the percent identity between aligned
sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
references, all of which are incorporated by reference herein in their
entirety, and the fourth column
presents, where applicable, the scores, probability values, and other
parameters used to evaluate the
strength of a match between two sequences (the higher the score or the lower
the probability value,
the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
)D N0:13-24. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization
and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative enzymes were initially identified by running the Genscan gene
identification
program against public genomic sequence databases (e.g., gbpri and gbhtg).
Genscan is a general-
purpose gene identification program which analyzes genomic DNA sequences from
a variety of
organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and
Burge, C. and S. Karlin
(1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates
predicted exons to form an
assembled cDNA sequence extending from a methionine to a stop codon. The
output of Genscan is a
FASTA database of polynucleotide and polypeptide sequences. The maximum range
of sequence for
Genscan to analyze at once was set to 30 kb. To determine which of these
Genscan predicted cDNA
sequences encode enzymes, the encoded polypeptides were analyzed by querying
against PFAM
models for enzymes. Potential enzymes were also identified by homology to
Incyte cDNA sequences
that had been annotated as enzymes. These selected Genscan-predicted sequences
were then
compared by BLAST analysis to the genpept and gbpri public databases. Where
necessary, the
Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to
correct errors in the sequence predicted by Genscan, such as extra or omitted
exons. BLAST analysis
was also used to fmd any Incyte cDNA or public cDNA coverage of the Genscan-
predicted
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sequences, thus providing evidence for transcription. When Incyte cDNA
coverage was available,
this information was used to correct or confirm the Genscan predicted
sequence. Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
Incyte cDNA sequences and/or public cDNA sequences using the assembly process
described in
Example III. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Se9uences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Seguences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
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may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of ENZM Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:13-24 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:13-24 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) su~a, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
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BLAST Score x Percent Identity
x minimum {length(Seq. 1), length(Seq. 2)}
5 The product score takes into account both the degree of similarity between
two sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding ENZM are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; heroic and immune system; liver; musculoskeletal
system; nervous
system; pancreas; respiratory system; sense organs; skin; stomatognathic
system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and
divided by the total number
of libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding ENZM. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of ENZM Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
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synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz+, (NH4)zS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE-enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p.1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~.1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x carb liquid media.
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The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, S min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in ENZM Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ ID N0:13-24 using the LIF'ESEQ database (Incyte Genomics).
Sequences from the
same gene were clustered together and assembled as described in Example III,
allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of ,
basecall errors by requiring a minimum Phred quality score of 15, and removed
sequence alignment
errors and errors resulting from improper trimming of vector sequences,
chimeras, and splice
variants. An automated procedure of advanced chromosome analysis analysed the
original
chromatogram files in the vicinity of the putative SNP. Clone error filters
used statistically generated
algorithms to identify errors introduced during laboratory processing, such as
those caused by reverse
transcriptase, polymerase, or somatic mutation. Clustering error filters used
statistically generated
algorithms to identify errors resulting from clustering of close homologs or
pseudogenes, or due to
contamination by non-human sequences. A final set of filters removed
duplicates and SNPs found in
immunoglobulins or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
Hispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele
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frequencies were first analyzed in the Caucasian population; in some cases
those SNPs which showed
no allelic variance in this population were not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ~ N0:13-24 are employed to screen cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ,uCi of
[y 32P~ adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher.& Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
XI. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena ( 1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
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array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/p,l oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/pl RNase inhibitor, 500 p,M dATP, 500 ~,M
dGTP, 500 ~,M dTTP, 40
p,M dCTP, 40 ~.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium
hydroxide and
incubated for 20 minutes at 85°C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~l SX SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
pg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
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and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 p.1 of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°C
followed by washes in
0.2% SDS and distilled water as before. _
Hybridization
Hybridization reactions contain 9 p,1 of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 p1 of 5X SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60° C. The arrays are washed for 10
min at 45° C in a first wash
buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45°C in a
second wash buffer (0.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
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although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
XII. Complementary Polynucleotides
Sequences complementary to the ENZM-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring ENZM. Although
use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of ENZM. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5'sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a
complementary oligonucleotide is designed to prevent ribosomal binding to the
ENZM-encoding
transcript.
XIII. Expression of ENZM
Expression and purification of ENZM is achieved using bacterial or virus-based
expression
systems. For expression of ENZM in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
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promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express ENZM upon induction with isopropyl beta-
D-
thiogalactopyranoside (1PTG). Expression of ENZM in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant AutoQra~hica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding ENZM by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, ENZM is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26
kilodalton enzyme from Schistosoma ~ponicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
ENZM at specifically engineered sites. FLAG, an 8-amino. acid peptide, enables
immunoaffmity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel (1995,
supra, ch. 10 and 16). Purified ENZM obtained by these methods can be used
directly in the assays
shown in Examples XVII, XVIII, and XIX where applicable.
XIV. Functional Assays
ENZM function is assessed by expressing the sequences encoding ENZM at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 ~cg of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
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recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. ( 1994) Flow Cytometry, Oxford, New York NY.
The influence of ENZM on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding ENZM and either CD64 or CD64-GFP.
CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding ENZM and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XV. Production of ENZM Specific Antibodies
ENZM substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using
standard protocols.
Alternatively, the ENZM amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-ENZM activity by, for example, binding the peptide or
ENZM to a substrate,
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blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XVI. Purification of Naturally Occurring ENZM Using Specific Antibodies
Naturally occurring or recombinant ENZM is substantially purified by
immunoaffinity
chromatography using antibodies specific for ENZM. An immunoaffinity column is
constructed by
covalently coupling anti-ENZM antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing ENZM are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of ENZM (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/ENZM binding (e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope, such
as urea or thiocyanate ion), and ENZM is collected.
XVII. Identification of Molecules Which Interact with ENZM
ENZM, or biologically active fragments thereof, are labeled with '25I Bolton-
Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a mufti-well plate are incubated with the
labeled ENZM, washed,
and any wells with labeled ENZM complex are assayed. Data obtained using
different concentrations
of ENZM are used to calculate values for the number, affinity, and association
of ENZM with the
candidate molecules.
Alternatively, molecules interacting with ENZM are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
ENZM may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVIII. Demonstration of ENZM Activity
ENZM activity is demonstrated through a variety of specific enzyme assays;
some of which
are outlined below.
ENZM oxidoreductase activity is measured by the increase in extinction
coefficient of
NAD(P)H coenzyme at 340 nmfor the measurement of oxidation activity, or the
decrease in
extinction coefficient of NAD(P)H coenzyme at 340 nmfor the measurement of
reduction activity
(Dalziel, K. (1963) J. Biol. Chem. 238:2850-2858). One of three substrates may
be used: Asn-(3Gal,
biocytidine, or ubiquinone-10. The respective subunits of the enzyme reaction,
for example,
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cytochrome c,-b oxidoreductase and cytochrome c, are reconstituted. The
reaction mixture contains
a)1-2 mg/ml ENZM; and b) 15 mM substrate, 2.4 mM NAD(P)+ in 0.1 M phosphate
buffer, pH 7.1
(oxidation reaction), or 2.0 mM NAD(P)H, in 0.1 M NazHP04 buffer, pH 7.4 (
reduction reaction); in
a total volume of 0.1 ml. Changes in absorbance at 340 nm (A3ao) are measured
at 23.5 °C using a
recording spectrophotometer (Shimadzu Scientific Instruments, Inc.,
Pleasanton, CA). The amount of
NAD(P)H is stoichiometrically equivalent to the amount of substrate initially
present, and the change
in A3ao is a direct measure of the amount of NAD(P)H produced; DA340 =
6620[NADH]. ENZM
activity is proportional to the amount of NAD(P)H present in the assay.
Aldo/keto reductase activity of ENZM is proportional to the decrease in
absorbance at 340
nm as NADPH is consumed (or increased absorbance if NADPH is produced, i.e.,
if the reverse
reaction is monitored). A standard reaction mixture is 135 mM sodium phosphate
buffer (pH 6.2-7.2
depending on enzyme), 0.2 mM NADPH, 0.3 M lithium sulfate, 0.5-2.5 mg ENZM and
an
appropriate level of substrate. The reaction is incubated at 30 °C and
the reaction is monitored
continuously with a spectrophotometer. ENZM activity is calculated as mol
NADPH consumed / mg
of ENZM.
Acyl-CoA dehydrogenase activity of ENZM is measured using an anaerobic
electron
transferring flavoprotein (ETF) assay. The reaction mixture comprises 50 mM
Tris-HCI (pH 8.0),
0.5% glucose, and 50 p,M acyl-CoA substrate (i.e., isovaleryl-CoA) that is pre-
warmed to 32 °C. The
mixture is depleted of oxygen by repeated exposure to vacuum followed by
layering with argon.
Trace amounts of oxygen are removed by the addition of glucose oxidase and
catalase followed by
the addition of ETF to a final concentration of 1 ~,M. The reaction is
initiated by addition of purified
ENZM or a sample containing ENZM and exciting the reaction at 342 nm.
Quenching of
fluorescence caused by the transfer of electrons from the substrate to ETF is
monitored at 496 nm. 1
unit of acyl-CoA dehydrogenase activity is defined as the amount of ENZM
required to reduce 1
~mol of ETF per minute (Reinard, T. et al. (2000) J. Biol. Chem. 275:33738-
33743).
Alcohol dehydrogenase activity of ENZM is measured by following the conversion
of NAD+
to NADH at 340 nm (E3ao = 6.22 mM-' cm') at 25°C in 0.1 M potassium
phosphate (pH 7.5), 0.1 M
glycine (pH 10.0), and 2.4 mM NAD+. Substrate (e.g., ethanol) and ENZM are
then added to the
reaction. The production of NADH results in an increase in absorbance at 340
nm and correlates with
the oxidation of the alcohol substrate and the amount of alcohol dehydrogenase
activity in the ENZM
sample (Svensson, S. (1999) J. Biol. Chem. 274:29712-29719).
Aldehyde dehydrogenase activity of ENZM is measured by determining the total
hydrolase +
dehydrogenase activity of ENZM and subtracting the hydrolase activity.
Hydrolase activity is first
determined in a reaction mixture containing 0.05 M Tris-HCl (pH 7.8), 100 mM 2-
mercaptoethanol,
and 0.5-18 ~M substrate, e.g., 10-HCO-HPteGlu (10-formyltetrahydrofolate;
HPteGlu,
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tetrahydrofolate) or 10-FDDF (10-formyl-5,8-dideazafolate). Approximately lp,g
of ENZM is added
in a final volume of 1.0 ml. The reaction is monitored and read against a
blank cuvette, containing all
components except enzyme. The appearance of product is measured at either 295
nm for
5,8-dideazafolate or 300 nm for HPteGlu using molar extinction coefficients of
1.89x104 and
2.17x104 for 5,8-dideazafolate and HPteGlu, respectively. The addition of
NADP+ to~the reaction
mixture allows the measurement of both dehydrogenase and hydrolase activity
(assays are performed
as before). Based on the production of product in the presence of NADP+ and
the production of
product in the absence of the cofactor, aldehyde dehydrogenase activity is
calculated for ENZM. In
the alternative, aldehyde dehydrogenase activity is assayed using propanal as
substrate. The reaction
mixture contains 60 mM sodium pyrophosphate buffer (pH 8.5), 5 mM propanal, 1
mM NADP+, and
ENZM in a total volume of 1 ml. Activity is determined by the increase in
absorbance at 340 nm,
resulting from the generation of NADPH, and is proportional to the aldehyde
dehydrogenase activity
in the sample (Krupenko, S.A. et al. (1995) J. Biol. Chem. 270:519-522).
6-phosphogluconate dehydrogenase activity of ENZM is measured by incubating
purified
ENZM, or a composition comprising ENZM, in 120 mM triethanolamine (pH 7.5),
0.1 mM EDTA,
0.5 mM NADP+, and 10-150 ~.M 6-phosphogluconate as substrate at 20-
25°C. The production of
NADPH is measured fluorimetrically (340 nm excitation, 450 nm emission) and is
indicative of
6-phosphogluconate dehydrogenase activity. Alternatively, the production of
NADPH is measured
photometrically, based on absorbance at 340 nm. The molar amount of NADPH
produced in the
reaction is proportional to the 6-phosphogluconate dehy.drogenase activity in
the sample (Tetaud, E.
et al. (1999) Biochem. J. 338:55-60).
Ribonucleotide diphosphate reductase activity of ENZM is determined by
incubating purified
ENZM, or a composition comprising ENZM, along with dithiothreitol, Mg++, and
ADP, GDP, CDP,
or UDP substrate. The product of the reaction, the corresponding
deoxyribonucleotide, is separated
from the substrate by thin-layer chromatography. The reaction products can be
distinguished from
the reactants based on rates of migration. The use of radiolabeled substrates
is an alternative for
increasing the sensitivity of the assay. The amount of deoxyribonucleotides
produced in the reaction
is proportional to the amount of ribonucleotide diphosphate reductase activity
in the sample (note that
this is true only for pre-steady state kinetic analysis of ribonucleotide
diphosphate reductase activity,
as the enzyme is subject to negative feedback inhibition by products) (Nutter,
L.M. and Y.-C. Cheng
(1984) Pharmac. Ther. 26:191-207).
Dihydrodiol dehydrogenase activity of ENZM is measured by incubating purified
ENZM, or
a composition comprising ENZM, in a reaction mixture comprising 50 mM glycine
(pH 9.0), 2.3 mM
NADP+, 8% DMSO, and a trans-dihydrodiol substrate, selected from the group
including but not
limited to, (~)-trans-naphthalene-1,2-dihydrodiol, (~)-trans-phenanthrene-1,2-
dihydrodiol, and (~)-
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trans-chrysene-1,2-dihydrodiol. The oxidation reaction is monitored at 340 nm
to detect the
formation of NADPH, which is indicative of the oxidation of the substrate. The
reaction mixture can
also be analyzed before and after the addition of ENZM by circular dichroism
to determine the
stereochemistry of the reaction components and determine which enantiomers of
a racemic substrate
S composition are oxidized by the ENZM (Penning, T.M. (1993) Chemico-
Biological Interactions
89:1-34).
Glutathione S-transferase (GST) activity of ENZM is determined by measuring
the ENZM
catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB), a common
substrate for
most GSTs. ENZM is incubated with 1 mM CDNB and 2.S mM GSH together in O.1M
potassium
phosphate buffer, pH 6.5, at 2S °C. The conjugation reaction is
measured by the change in
absorbance at 340 nm using an ultraviolet spectrophometer. ENZM activity is
proportional to the
change in absorbance at 340 nm.
1S-oxoprostaglandin 13-reductase (PGR) activity of ENZM is measured following
the
separation of contaminating 1S-hydroxyprostaglandin dehydrogenase (1S-PGDH)
activity by DEAF
chromatography. Following isolation of PGR containing fractions (or using the
purified ENZM),
activity is assayed in a reaction comprising 0.1 M sodium phosphate (pH 7.4),
1 mM 2-
mercaptoethanol, 20 p,g substrate (e.g., 1S-oxo derivatives of prostaglandins
PGE,, PGE2, and PGEZa),
and 1 mM NADH (or a higher concentration of NADPH). ENZM is added to the
reaction which is
then incubated for 10 min at 37 °C before termination by the addition
of 0.25 ml 2 N NaOH. The
amount of 1S-oxo compound remaining in the sample is determined by measuring
the maximum
absorption at S00 nm of the terminated reaction and comparing this value to
that of a terminated
control reaction that received no ENZM. 1 unit of enzyme is defined as the
amount required to
catalyze the oxidation of 1 pmol substrate per minute and is proportional to
the amount of PGR
activity in the sample.
2S Choline dehydrogenase activity of ENZM is identified by the ability of E.
coli, transformed
with an ENZM expression vector, to grow on media containing choline as the
sole carbon and
nitrogen source. The ability of the transformed bacteria to thrive is
indicative of choline
dehydrogenase activity (Magne ~steras, M. (1998) Proc. Natl. Acad. Sci. USA
95:11394-11399).
ENZM thioredoxin activity is assayed as described (Luthman, M. (1982)
Biochemistry
21:6628-6633). Thioredoxins catalyze the formation of disulfide bonds and
regulate the redox
environment in cells to enable the necessary thiol:disulfide exchanges. One
way to measure the
thiol:disulfide exchange is by measuring the reduction of insulin in a mixture
containing 0.1 M
potassium phosphate, pH 7.0, 2 mM EDTA, 0.16 ~M insulin, 0.33 mM DTT, and 0.48
mM NADPH.
Different concentrations of ENZM are added to the mixture, and the reaction
rate is followed by
monitoring the oxidation of NADPH at 340 nM.
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ENZM transferase activity is measured through assays such as a methyl
transferase assay in
which the transfer of radiolabeled methyl groups between a donor substrate and
an acceptor substrate
is measured (Bokar, J.A. et al. (1994) J. Biol. Chem. 269:17697-17704).
Reaction mixtures (50 ~,l
final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgClz, 10 mM dithiothreitol,
3%
polyvinylalcohol, 1.5 ~.Ci [methyl-3H]AdoMet (0.375 ~.M AdoMet) (DuPont-NEN),
0.6 ~.g ENZM,
and acceptor substrate (0.4 p,g [35S]RNA or 6-mercaptopurine (6-MP) to 1 mM
final concentration).
Reaction mixtures are incubated at 30 °C for 30 minutes, then at 65
°C for 5 minutes. The products
are separated by chromatography or electrophoresis and the level of methyl
transferase activity is
determined by quantification of methyl-3H recovery.
Aminotransferase activity of ENZM is assayed by incubating samples containing
ENZM for
1 hour at 37°C in the presence of 1 mM L-kynurenine and 1 mM 2-
oxoglutarate in a final volume of
200 ~,1 of 150 mM Tris acetate buffer (pH 8.0) containing 70 ~M PLP. The
formation of kynurenic
acid is quantified by HPLC with spectrophotometric detection at 330 nm using
the appropriate
standards and controls well known to those skilled in the art. In the
alternative,
L-3-hydroxykynurenine is used as substrate and the production of xanthurenic
acid is determined by
HPLC analysis of the products with UV detection at 340 nm. The production of
kynurenic acid and
xanthurenic acid, respectively, is indicative of aminotransferase activity
(Buchli, R. et al. (1995) J.
Biol. Chem. 270:29330-29335).
In another alternative, aminotransferase activity of ENZM is measured by
determining the
activity of purified ENZM or crude samples containing ENZM toward various
amino and oxo acid
substrates under single turnover conditions by monitoring the changes in the
UV/VIS absorption
spectrum of the enzyme-bound cofactor, pyridoxal 5'-phosphate (PLP). The
reactions are performed
at 25°C in 50 mM 4-methylmorpholine (pH 7.5) containing 9 ~,M purified
ENZM or ENZM
containing samples and substrate to be tested (amino and oxo acid substrates).
The half-reaction
from amino acid to oxo acid is followed by measuring the decrease in
absorbance at 360 nm and the
increase in absorbance at 330 nm due to the conversion of enzyme-bound PLP to
pyridoxamine 5'
phosphate (PMP). The specificity and relative activity of ENZM is determined
by the activity of the
enzyme preparation against specific substrates (Vacca, R. A. et al. (1997) J.
Biol. Chem.
272:21932-21937).
ENZM chitinase activity is determined with the fluorogenic substrates 4-
methylumbelliferyl
chitotriose, methylumbelliferyl chitobiose, or methylumbelliferyl N-
acetylglucosamine. Purified
ENZM is incubated with O.SuM substrate at pH 4.0 (0.1M citrate buffer), pH 5.0
(0.1M phosphate
buffer), or pH 6.0 (0.1M Tris-HCL). After various times of incubation, the
reaction is stopped by the
addition of O.1M glycine buffer, pH 10.4, and the concentration of free
methylumbelliferone is
determined fluorometrically. Chitinase B from Serratia marcescens may be used
as a positive control
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(Hakala, B. E. (1993) J. Biol. Chem. 268 (34):25803-25810).
ENZM isomerase activity is determined by measuring 2-hydroxyhepta-2,4-
diene,l,7 dioate
isomerase (HHDD isomerase) activity, as described by Garrido-Peritierra, A.
and Cooper, R.A. (Eur.
J. Biochem. (1981)17:581-584). The sample is combined with 5-carboxymethyl-2-
oxo-hex-3-ene-
1,5, dioate (CMHD), which is the substrate for HHDD isomerase. CMHD
concentration is monitored
by measuring its absorbance at 246 nm. Decrease in absorbance at 246 nm is
proportional to HHDD
isomerase activity of ENZM.
ENZM isomerase activity such as peptidyl prolyl cisltrans isomerase activity
can be assayed
by an enzyme assay described by Rahfeld, J. U., et al. (1994) (FEBS Lett. 352:
180-184). The assay
is performed at 10°C in 35 mM HEPES buffer, pH 7.8, containing
chymotrypsin (0.5 mg/ml) and
ENZM at a variety of concentrations. Under these assay conditions, the
substrate, Suc-Ala-Xaa-Pro-
Phe-4-NA, is in equilibrium with respect to the prolyl bond, with 80-95% in
trans and 5-20% in cis
conformation. An aliquot (2 p.1) of the substrate dissolved in dimethyl
sulfoxide (10 mg/ml) is added
to the reaction mixture described above. Only the cis isomer is a substrate
for cleavage by
chymotrypsin. Thus, as the substrate is isomerized by ENZM, the product is
cleaved by
chymotrypsin to produce 4-nitroanilide, which is detected by its absorbance at
390 nm. 4-
Nitroanilide appears in a time-dependent and a ENZM concentration-dependent
manner.
Alternatively, peptidyl prolyl cis-trans isomerase activity of ENZM can be
assayed using a
chromogenic peptide in a coupled assay with chymotrypsin (Fischer, G. et al.
(1984) Biomed.
Biochim. Acta 43:1101-1111).
UDP glucuronyltransferase activity of ENZM is measured using a colorimetric
determination
of free amine groups (Gibson, G.G. and P. Skett (1994) Introduction to Drug
Metabolism, Blackie
Academic and Professional, London). An amine-containing substrate, such as 2-
aminophenol, is
incubated at 37 °C with an aliquot of the enzyme in a reaction buffer
containing the necessary
cofactors (40 mM Tris pH 8.0, 7.5 mM MgClz, 0.025% Triton X-100, 1 mM ascorbic
acid, 0.75 mM
UDP-glucuronic acid). After sufficient time, the reaction is stopped by
addition of ice-cold 20%
trichloroacetic acid in 0.1 M phosphate buffer pH 2.7, incubated on ice, and
centrifuged to clarify the
supernatant. Any unreacted 2-aminophenol is destroyed in this step. Sufficient
freshly-prepared
sodium nitrite is then added; this step allows formation of the diazonium salt
of the glucuronidated
product. Excess nitrite is removed by addition of sufficient ammonium
sulfamate, and the diazonium
salt is reacted with an aromatic amine (for example, N-naphthylethylene
diamine) to produce a
colored azo compound which can be assayed spectrophotometrically (at 540 nm,
for example). A
standard curve can be constructed using known concentrations of aniline, which
will form a
chromophore with similar properties to 2-aminophenol glucuronide.
Adenylosuccinate synthetase activity of ENZM is measured by synthesis of AMP
from IMP.
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The sample is combined with AMP. IMP concentration is monitored
spectrophotometrically at 248
nm at 23°C (Wang, W. et al. (1995) J. Biol. Chem. 270:13160-13163). The
increase in IMP
concentration is proportional to ENZM activity.
Alternatively, AMP binding activity of ENZM is measured by combining the
sample with
32P-labeled AMP. The reaction is incubated at 37°C and terminated by
addition of trichloroacetic
acid. The acid extract is neutralized and subjected to gel electrophoresis to
remove unbound label.
The radioactivity retained in the gel is proportional to ENZM activity.
In another alternative, xenobiotic carboxylic acid:CoA ligase activity of ENZM
is measured
by combining the sample with y-33P-ATP and measuring the formation of y-33P-
pyrophosphate with
time (Vessey, D.A. et al. (1998) J. Biochem. Mol. Toxicol. 12:151-155).
Protein phosphatase (PP) activity can be measured by the hydrolysis of P-
nitrophenyl
phosphate (PNPP). ENZM is incubated together with PNPP in HEPES buffer pH 7.5,
in the presence
of 0.1% p-mercaptoethanol at 37°C for 60 min. The reaction is stopped
by the addition of 6 ml of 10
N NaOH (Diamond, R.H. et al. (1994) Mol. Cell. Biol. 14:3752-62).
Alternatively, acid phosphatase activity of ENZM is demonstrated by incubating
ENZM
containing extract with 100 ~,l of 10 mM PNPP in 0.1 M sodium citrate, pH 4.5,
and 50 ~,l of 40 mM
NaCI at 37°C for 20 min. The reaction is stopped by the addition of 0.5
ml of 0.4 M glycine/NaOH,
pH 10.4 (Saftig, P. et al. (1997) J. Biol. Chem. 272:18628-18635). The
increase in light absorbance
at 410 nm resulting from the hydrolysis of PNPP is measured using a
spectrophotometer. The
increase in light absorbance is proportional to the activity of ENZM in the
assay.
In the alternative, ENZM activity is determined by measuring the amount of
phosphate
removed from a phosphorylated protein substrate. Reactions are performed with
2 or 4 nM ENZM in
a final volume of 30 ~.1 containing 60 mM Tris, pH 7.6, 1 mM EDTA, 1 mM EGTA,
0.1%
2-mercaptoethanol and 10 p.M substrate, 32P-labeled on serine/threonine or
tyrosine, as appropriate.
Reactions are initiated with substrate and incubated at 30° C for 10-15
min. Reactions are quenched
with 450 ~,1 of 4% (w/v) activated charcoal in 0.6 M HCI, 90 mM Na4P20,, and 2
mM NaHZP04, then
centrifuged at 12,000 x g for 5 min. Acid-soluble 32Pi is quantified by liquid
scintillation counting
(Sinclair, C. et al. (1999) J. Biol. Chem. 274:23666-23672).
The adenosine deaminase activity of ENZM is determined by measuring the rate
of
deamination that occurs when adenosine substrate is incubated with ENZM.
Reactions are performed
with a predetermined amount of ENZM in a final volume of 3.0 ml containing
53.3 mM potassium
phosphate and 0.045 mM adenosine. Assay reagents excluding ENZM are mixed in a
quartz cuvette
and equilibrated to 25° C. Reactions are initiated by the addition of
ENZM and are mixed
immediately by inversion. The decrease in light absorbance at 265 nm resulting
from the hydrolysis
of adenosine to inosine is measured using a spectrophotometer. The decrease in
the Albs nm is
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recorded for approximately 5 minutes. The decrease in light absorbance is
proportional to the activity
of ENZM in the assay.
ENZM hydrolase activity is measured by the hydrolysis of appropriate synthetic
peptide
substrates conjugated with various chromogenic molecules in which the degree
of hydrolysis is
quantified by spectrophotometric (or fluorometric) absorption of the released
chromophore (Beynon,
R.J. and J.S. Bond (1994) Proteol~ic Enzymes: A Practical Approach, Oxford
University Press, New
York, NY, pp.25-55). Peptide substrates are designed according to the category
of protease activity
as endopeptidase (serine, cysteine, aspartic proteases), aminopeptidase
(leucine aminopeptidase), or
carboxypeptidase (Carboxypeptidase A and B, procollagen C-proteinase).
An assay for carbonic anhydrase activity of ENZM uses the fluorescent pH
indicator 8-
hydroxypyrene-1,3,6-trisulfonate (pyranine) in combination with stopped-flow
fluorometry to
measure carbonic anhydrase activity (Shingles, et al. 1997, Anal. Biochem.
252: 190-197). A pH 6.0
solution is mixed with a pH 8.0 solution and the initial rate of bicarbonate
dehydration is measured.
Addition of carbonic anhydrase to the pH 6.0 solution enables the measurement
of the initial rate of
activity at physiological temperatures with resolution times of 2 ms. Shingles
et al. used this assay to
resolve differences in activity and sensitivity to sulfonamides by comparing
mammalian carbonic
anhydrase isoforms. The fluorescent technique's sensitivity allows the
determination of initial rates
with a protein concentration as little as 65 ng/ml.
Decarboxylase activity of ENZM is measured as the release of COz from labeled
substrate.
For example, ornithine decarboxylase activity of ENZM is assayed by measuring
the release of CO Z
from L-[1-'°C]-ornithine (Reddy, S.G et al. (1996) J. Biol. Chem.
271:24945-24953). Activity is
measured in 200 p1 assay buffer (50 mM Tris/HCI, pH 7.5, 0.1 mM EDTA, 2 mM
dithiothreitol, 5
mM NaF, 0.1% Brij35, 1 mM PMSF, 60 pM pyridoxal-5-phosphate) containing 0.5 mM
L-ornithine
plus 0.5 pCi L-[1-'4C]ornithine. The reactions are stopped after 15-30 minutes
by addition of 1 M
citric acid, and the '4C02 evolved is trapped on a paper disk filter saturated
with 20 p1 of 2 N NaOH.
The radioactivity on the disks is determined by liquid scintillation
spectography. The amount of
'4C02 released is proportional to ornithine decarboxylase activity of ENZM.
AdoHCYase activity of ENZM in the hydrolytic direction is performed
spectroscopically by
measuring the rate of the product (homocysteine) formed by reaction with 5,5'-
Dithiobis(2-
nitrobenzoic acid) (DTNB). To 800 p.1 of an enzyme solution containing 4.7 ~g
of ENZM and 4 units
of adenosine deaminase in 50 mM potassium phosphate buffer, pH 7.2, containing
1 mM EDTA
(buffer A), is added 200 ~1 of S-Adenosyl-L-homocysteine (500 ~M) containing
250 ~.M DTNB in
buffer A. The reaction mixture is incubated at 37 °C for 2 minutes.
Hydrolytic activity is monitored
at 412 nm continuously using a diode array UV spectrophotometer. Enzyme
activity is defined as the
amount of enzyme that can hydrolyze 1 ~mol of S-Adenosyl-L-homocysteine/minute
(Yuan, C-S et
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al. (1996) J. Biol. Chem. 271:28009-28015).
AdoHCYase activity of ENZM can be measured in the synthetic direction as the
production
of S-adenosyl homocysteine using 3-deazaadenosine as a substrate (Sganga, M.W.
et al. suvra).
Briefly, ENZM is incubated in a 100 p,1 volume containing 0.1 mM 3-
deazaadenosine, 5 mM
homocysteine, 20 mM HEPES (pH 7.2). The assay mixture is incubated at 37
°C for 15 minutes. The
reaction is terminated by the addition of 10 ~l of 3 M perchloric acid. After
incubation on ice for 15
minutes, the mixture is centrifuged for S minutes at 18,000 x g in a
microcentrifuge at 4 °C. The
supernatant is removed, neutralized by the addition of 1 M potassium
carbonate, and centrifuged
again. A 50 ~.l aliquot of supernatant is then chromatographed on an Altex
Ultrasphere ODS column
(5 ~m particles, 4.6 x 250 mm) by isocratic elution with 0.2 M ammonium
dihydrogen phosphate
(Aldrich) at a flow rate of 1 ml/min. Protein is determined by the
bicinchoninic acid assay (Pierce).
Alternatively, AdoHCYase activity of ENZM can be measured in the synthetic
direction by a
TLC method (Hershfield, M.S. et al. (1979) J. Biol. Chem. 254:22-25). In a
preincubation step, 50
p,M [8-'4C]adenosine is incubated with 5 molar equivalents of NAD+ for 15
minutes at 22°C. Assay
samples containing ENZM in a 50 ~.1 final volume of 50 mM potassium phosphate
buffer, pH 7.4, 1
mM DTT, and 5 mM homocysteine, are mixed with the preincubated [8-
'4C]adenosine/NAD+ to
initiate the reaction. The reaction is incubated at 37 °C, and 1 ~,l
samples are spotted on TLC plates at
5 minute intervals for 30 minutes. The chromatograms are developed in butanol-
1/glacial acetic
acid/water (12:3:5, v/v) and dried. Standards are used to identify substrate
and products under
ultraviolet light. The complete spots containing ['4C]adenosine and ['4C]SAH
are then detected by
exposing x-ray film to the TLC plate. The radiolabeled substrate and product
are then cut from the
chromatograms and counted by liquid scintillation spectrometry. Specific
activity of the enzyme is
determined from the linear least squares slopes of the product vs time plots
and the milligrams of
protein in the sample (Bethin, K.E. et al. (1995) J. Biol. Chem. 270:20698-
20702).
Asparaginase activity of ENZM can be measured in the hydrolytic direction by
determining
the amount of radiolabeled L-aspartate released from 0.6 mM N4-Vii'-N-
acetylglucosaminyl-L-
asparagine substrate when it is incubated at 25 °C with ENZM in 50 mM
phosphate buffer, pH 7.5
(Kaartinen, V. et al. (1991) J. Biol. Chem. 266:5860-5869).
Measurement of acyl CoA acid hydrolase activity of ENZM in the hydrolytic
direction is
performed spectroscopically by monitoring the appearance of the product
(CoASH) formed by
reaction of substrate (acyl-CoA) and ENZM with 5,5'-Dithiobis(2-nitrobenzoic
acid) (DTNB). The
final reaction volume is 1 ml of 0.05 M potassium phosphate buffer, pH 8,
containing 0.1 mM
DTNB, 20 ~.g/ml bovine serum albumin, 10 ~.M of acyl-CoA of different lengths
(C6-CoA, C10-CoA,
C14-CoA and C18-CoA, Sigma), and ENZM. The reaction mixture is incubated at 22
°C for 7
minutes. Hydrolytic activity is monitored spectrophotometrically by measuring
absorbance at 412
137
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
nm (Poupon, V. et al. supra).
RNase activity of ENZM can be measured spectrophotometrically by determining
the amount
of solubilized RNA that is produced as a result of incubation of RNA substrate
with ENZM. 5 ~.1 (20
fig) of a 4 mg/ml solution of yeast tRNA (Sigma) is added to 0.8 ml of 40 mM
sodium phosphate, pH
7.5, containing ENZM. The reaction is incubated at 25 °C for 15
minutes. The reaction is stopped by
addition of 0.5 ml of an ice-cold fresh solution of 20 mM lanthanum nitrate
plus 3% perchloric acid.
The stopped reaction is incubated on ice for at least 15 min, and the
insoluble tRNA is removed by
centrifugation for 5 min at 10,000 g. Solubilized tRNA is determined as UV
absorbance (260 nm) of
the remaining supernatant, with A2~ of 1.0 corresponding to 40 ~,g of
solubilized RNA (Rosenberg,
H.F. et al. (1996) Nucleic Acids Research 24:3507-3513).
RNase P or tRNA splicing endonuclease activity can be determined as the
ability of ENZM
to cleave 32P internally labeled T. thermophila pre-tRNA~'". RNase P or tRNA
splicing endonuclease
and substrate are added to reaction vessels and reactions are carried out in
MBB buffer (50 mM Tris-
HCl (pH 7.5), 10 mM MgClz) for 1 hour at 37 °C. Reactions are
terminated with the addition of an
equal volume of sample loading buffer (SLB: 40 mM EDTA, 8 M urea, 0.2% xylene
cyanol, and
0.2% bromophenol blue). The reaction products are separated by electrophoresis
on 8 M urea, 6%
polyacrylamide gels and analyzed using detection instruments and software
capable of quantification
of the products. One unit of RNase P or tRNA splicing endonuclease activity is
defined as the
amount of enzyme required to cleave 10% of 28 fmol of T. thermophila pre-
tRNA°'" to mature
products in 1 hour at 37°C (True, H.L. et al. (1996) J. Biol. Chem.
271:16559-16566).
Alternatively, cleavage of 32P internally labeled substrate tRNA by RNase P or
tRNA
splicing endonuclease can be determined in a 20 ~1 reaction mixture containing
30 mM HEPES-KOH
(pH 7.6), 6 mM MgCl2, 30 mM KCI, 2 mM DTT, 25 ~g/ml bovine serum albumin, 1
unit/~1 rRNasin,
and 5,000-50,000 cpm of gel-purified substrate RNA. 3.0 /.d of RNase P or tRNA
splicing
endonuclease is added to the reaction mixture, which is then incubated at 37
°C for 30 minutes. The
reaction is stopped by guanidinium/phenol extraction, precipitated with
ethanol in the presence of
glycogen, and subjected to denaturing polyacrylamide gel electrophoresis (6 or
8% polyacrylamide, 7
M urea) and autoradiography (Rossmanith, W. et al. (1995) J. Biol. Chem.
270:12885-12891). The
RNase P or tRNA splicing endonuclease activity is proportional to the amount
of cleavage products
detected.
ENZM activity can be measured by determining the amount of free adenosine
produced by
the hydrolysis of AMP, as described by Sala-Newby et al. supra. Briefly, ENZM
is incubated with
AMP in a suitable buffer for 10 minutes at 37°C. Free adenosine is
separated from AMP and
measured by reverse phase HPLC.
Alternatively, ENZM activity is measured by the hydrolysis of ADP-
ribosylarginine
138
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
(Konczalik, P. and J. Moss (1999) J. Biol. Chem. 274:16736-16740). 50 ng of
ENZM is incubated
with 100 ~,M ADP-ribosyl-['4C]arginine (78,000 cpm) in 50 mM potassium
phosphate, pH 7.5, 5 mM
dithiothreitol, 10 mM MgCl2 in a final volume of 100 p,1. After 1 h at
37° C, 90 ~,1 of the sample is
applied to a column (0.5 x 4 cm) of Affi-Gel 601 (boronate) equilibrated and
eluted with five 1-ml
portions of 0.1 M glycine, pH 9.0, 0.1 M NaCI, and 10 mM MgClz. Free '4C-Arg
in the total eluate is
measured by liquid scintillation counting.
Epoxide hydrolase activity of ENZM can be determined with a radiometric assay
utilizing
[H3]-labeled traps-stilbene oxide (TSO) as substrate. Briefly, ENZM is
preincubated in Tris-HCl pH
7.4 buffer in a total volume of 100 ,u1 for 1 minute at 37 ° C. 1 ~cl
of [H3]-labeled TSO (0.5 ~M in
EtOH) is added and the reaction mixture is incubated at 37°C for 10
minutes. The reaction mixture is
extracted with 200 ~l n-dodecane. 50 ,u1 of the aqueous phase is removed for
quantification of diol
product in a liquid scintillation counter (LSC). ENZM activity is calculated
as nmol diol
producbmin/mg protein (Gill, S.S. et al. (1983) Analytical Biochemistry
131:273-282).
Lysophosphatidic acid acyltransferase activity of ENZM is measured by
incubating samples
containing ENZM with 1 mM of the thiol reagent S,5'-dithiobis(2-nitrobenzoic
acid) (DTNB), 50 ~m
LPA, and 50 pm acyl-CoA in 100 mM Tris-HCI, pH 7.4. The reaction is initiated
by addition of acyl-
CoA, and allowed to reach equilibrium. Transfer of the acyl group from acyl-
CoA to LPA releases
free CoA, which reacts with DTNB. The product of the reaction between DTNB and
free CoA
absorbs at 413 nm. The change in absorbance at 413 nm is measured using a
spectrophotometer, and
is proportional to the lysophosphatidic acid acyltransferase activity of ENZM
in the sample.
N-acyltransferase activity of ENZM is measured using radiolabeled amino acid
substrates
and measuring radiolabel incorporation into conjugated products. ENZM is
incubated in a reaction
buffer containing an unlabeled acyl-CoA compound and radiolabeled amino acid,
and the
radiolabeled acyl-conjugates are separated from the unreacted amino acid by
extraction into n-
butanol or other appropriate organic solvent. For example, Johnson, M. R. et
al. (1990; J. Biol.
Chem. 266:10227-10233) measured bile acid-CoA:amino acid N-acyltransferase
activity by
incubating the enzyme with cholyl-CoA and 3H-glycine or 3H-taurine, separating
the tritiated cholate
conjugate by extraction into n-butanol, and measuring the radioactivity in the
extracted product by
scintillation. Alternatively, N-acyltransferase activity is measured using the
spectrophotometric
determination of reduced CoA (CoASH) described below.
N-acetyltransferase activity of ENZM is measured using the transfer of
radiolabel from
['4C]acetyl-CoA to a substrate molecule (for example, see Deguchi, T. ( 1975)
J. Neurochem.
24:1083-5). Alternatively, a newer spectrophotometric assay based on DTNB
reaction with CoASH
may be used. Free thiol-containing CoASH is formed during N-acetyltransferase
catalyzed transfer of
an acetyl group to a substrate. CoASH is detected using the absorbance of DTNB
conjugate at 412
139
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
nm (De Angelis, J. et al. (1997) J. Biol. Chem. 273:3045-3050). ENZM activity
is proportional to the
rate of radioactivity incorporation into substrate, or the rate of absorbance
increase in the
spectrophotometric assay.
Galactosyltransferase activity of ENZM is determined by measuring the transfer
of galactose
from UDP-galactose to a GIcNAc-terminated oligosaccharide chain in a
radioactive assay.
(Kolbinger, F. et al. (1998) J. Biol. Chem. 273:58-65.) The ENZM sample is
incubated with 14 p,1 of
assay stock solution ( 180 mM sodium cacodylate, pH 6.5, 1 mg/ml bovine serum
albumin, 0.26 mM
UDP-galactose, 2 ~,1 of UDP-['H]galactose), 1 p1 of MnCl2 (500 mM), and 2.5
p,1 of GIcNAc~iO-
(CHZ)$ COZMe (37 mg/ml in dimethyl sulfoxide) for 60 minutes at 37°C.
The reaction is quenched by
the addition of 1 ml of water and loaded on a C18 Sep-Pak cartridge (Waters),
and the column is
washed twice with 5 ml of water to remove unreacted UDP-['H]galactose. The
['H]galactosylated
GIcNAc(30-(CHz)e-COZMe remains bound to the column during the water washes and
is eluted with 5
ml of methanol. Radioactivity in the eluted material is measured by liquid
scintillation counting and
is proportional to galactosyltransferase activity of ENZM in the starting
sample.
Phosphoribosyltransferase activity of ENZM is measured as the transfer of a
phosphoribosyl
group from phosphoribosylpyrophosphate (PRPP) to a purine or pyrimidine base.
Assay mixture (20
~1) containing 50 mM Tris acetate, pH 9.0, 20 mM 2-mercaptoethanol, 12.5 mM
MgCI Z, and 0.1 mM
labeled substrate, for example, ['4C]uracil, is mixed with 20 p1 of ENZM
diluted in 0.1 M Tris
acetate, pH 9.7, and 1 mg/ml bovine serum albumin. Reactions are preheated for
1 min at 37 °C,
initiated with 10 ~1 of 6 mM PRPP, and incubated for 5 min at 37 °C.
The reaction is stopped by
heating at 100°C for 1 min. The product ['4C]UMP is separated from
['4C]uracil on DEAF-cellulose
paper (Turner, R.J. et al. (1998) J. Biol. Chem. 273:5932-5938). The amount of
['4C]UMP produced
is proportional to the phosphoribosyltransferase activity of ENZM.
ADP-ribosyltransferase activity of ENZM is measured as the transfer of
radiolabel from
adenine-NAD to agmatine (Weng, B. et al. (1999) J. Biol. Chem. 274:31797-
31803). Purified ENZM
is incubated at 30°C for 1 hr in a total volume of 300 ~,1 containing
50 mM potassium phosphate (pH.
7.5), 20 mM agmatine, and 0.1 mM [adenine-U-'4C]NAD (0.05 mCi). Samples (100
~1) are applied
to Dowex columns and ["C]ADP-ribosylagmatine eluted with 5 ml of water for
liquid scintillation
counting. The amount of radioactivity recovered is proportional to ADP-
ribosyltransferase activity of
ENZM.
An ENZM activity assay measures aminoacylation of tRNA in the presence of a
radiolabeled
substrate. SYNT is incubated with ['°C]-labeled amino acid and the
appropriate cognate tRNA (for
example, ['4C]alanine and tRNAa'a) in a buffered solution. '4C-labeled product
is separated from free
['aC]amino acid by chromatography, and the incorporated'"C is quantified using
a scintillation
counter. The amount of '4C-labeled product detected is proportional to the
activity of ENZM in this
140
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
assay (Ibba, M. et al. ( 1997) Science 278:1119-1122).
Alternatively, agininosuccinate synthase activity of ENZM is measured based on
the
conversion of [3H]aspartate to [3H]argininosuccinate. ENZM is incubated with a
mixture of
[3H]aspartate, citrulline, Tris-HCl (pH 7.5), ATP, MgClz, KCI,
phosphoenolpyruvate, pyruvate ,
kinase, myokinase, and pyrophosphatase, and allowed to proceed for 60 minutes
at 37 °C. Enzyme
activity was terminated with addition of acetic acid and heating for 30
minutes at 90 °C.
[3H]argininosuccinate is separated from un-catalyzed [3H]aspartate by
chromatography and quantified
by liquid scintillation spectrometry. The amount of [3H]argininosuccinate
detected is proportional to
the activity of ENZM in this assay (O'Brien, W. E. (1979) Biochemistry 18:5353-
5356).
Alternatively, the esterase activity of ENZM is assayed by the hydrolysis of p-
nitrophenylacetate (NPA). ENZM is incubated together with 0.1 ~M NPA in 0.1 M
potassium
phosphate buffer (pH 7.25) containing 150 mM NaCI. The hydrolysis of NPA is
measured by the
increase of absorbance at 400 nm with a spectrophotometer. The increase in
light absorbance is
proportional to the activity of ENZM (Probst, M.R. et al. (1994) J. Biol.
Chem. 269:21650-21656).
XIX. Identification of ENZM Agonists and Antagonists
Agonists or antagonists of ENZM activation or inhibition may be tested using
the assays
described in section XV)TI. Agonists cause an increase in ENZM activity and
antagonists cause a
decrease in ENZM activity.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
141
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
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CA 02443244 2003-10-07
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CA 02443244 2003-10-07
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CA 02443244 2003-10-07
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CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
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153
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Table 4
PolynucleotideSequence Fragments
SEQ ID NO:/
Incyte ID/
Sequence
Length
13/2425607CB1-278, 85-563, 247-563, 264-316, 304-927, 304-935,
1/ 768-944, 769-944, 770-
1437 944,796-1161,797-1042,817-944,868-1112,868-1356,871-1409,904-1319,
967-1426,1041-1428,1043-1428,1071-1356,1074-1428,1263-1412,1263-
1437,1268-1347,1334-1426
14/2786919CB1-590, 1-648, 1-817, 3-470, 25-491, 299-2302,
1/ 338-610, 343-938, 382-948, 404-
3269 666, 424-895, 435-695, 537-817, 537-1063, 573-850,
588-927, 595-1132, 796-
1411,
891-1467,1054-1502,1098-1351,1271-1526,1275-1502,1294-1502,1305-
1502, 1332-1502, 1349-2075, 1433-1958, 1488-1727,
1488-1786, 1784-1929,
1794-2101,
1840-2061, 1990-2089, 1997-2101, 2021-2296,
2081-2313, 2090-2728, 2103-
2774, 2123-2774, 2142-2395, 2165-2400, 2180-2830,
2234-2491, 2247-2774,
2488-2773,
2518-2787, 2522-2774, 2540-2774, 2540-2775,
2540-2783, 2540-3049, 2654-
2898, 2655-3269, 2681-3253, 2708-2918, 2725-3179,
2770-3042, 2777-3269,
2781-3028, 2781-3054, 2781-3064, 2781-3067,
2781-3203, 2781-3269
15/1801130CB1/1-732, 372-625, 378-478, 378-568, 378-583,
378-615, 378-617, 378-657, 378-
1534 666, 378-686, 378-718, 378-732, 379-732, 405-732,
411-649, 411-732, 441-618,
447-1092,
474-695,474-732,499-682,499-732,505-732,525-732,554-732,594-1115,
689-1234,696-1023,712-996,727-1117,727-1469,739-1354,757-1035,772-
1290,
822-1376,823-1082,858-1118,858-1365,858-1420,880-1187,886-1354,912-
1151,913-1170,913-1172,936-1534,957-1470
16/3535146CB1/1-783, 220-567, 223-789, 223-843, 239-693,
241-409, 241-588, 242-588, 252-
3400 386, 263-722, 282-692, 285-1142, 291-461, 291-466,
292-796, 299-670, 303-
529, 303-706, 303-794, 308-553, 314-590, 316-601,
433-992, 463-636, 626-
1230, 626-1283, 818-1331, 851-1473, 957-1444,
1044-1312, 1072-1313, 1072-
1426, 1122-1529, 1142-1757, 1168-1375, 1169-1304,
1201-1608, 1254-1494,
1322-2090, 1465-1700, 1483-2061, 1483-2064,
1486-2064, 1495-2064, 1524-
2109,1538-2185,1550-1767,1556-2047,1577-2243,1587-2128,1591-2362,
1746-2199, 1748-1869, 1756-1995, 1756-2296,
1779-2369, 1780-2044, 1790-
2249, 1790-2272, 1790-2299, 1790-2343, 1790-2390,
1790-2403, 1790-2405,
1790-2406, 1793-2408, 1808-2657, 1827-2464,
1832-2083, 1832-2456, 1832-
2512,1834-3400,1854-2506,1926-2170,1930-2572,1978-2251,1978-2262,
1978-2485, 2066-2312, 2084-2190, 2090-2748,
2191-2353, 2365-3059, 2369-
3079, 2373-3194, 2376-3050, 2383-3018, 2395-2835,
2457-3082, 2527-2933,
2527-3020,2527-3035,2527-3083,2527-3139,2527-3249,2527-3316,2531-
3083, 2559-3033, 2575-3276, 2596-2857, 2606-2860,
2624-2912, 2641-2907,
2649-3371, 2674-2897, 2677-3350, 2679-3194,
2688-2943, 2702-2932,
2712-3374, 2737-3001, 2744-3116, 2749-3386,
2760-3383, 2766-3029, 2766-
3048, 2767-3087, 2779-3400, 2842-2991, 2872-2962,
2913-2933
154
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Table 4
Po(ynucleotideSequence Fragments
SEQ ID NO:/
Incyte ID/
Sequence
Length
17/1436543CB1/1-207, 1-450, 15-219, 35-663, 153-313, 153-543,
153-637, 154-650, 157-429,
3395 162-393,162-530,162-849,173-589,174-306,174-312,174-543,179-628,192-
496,204-892,208-421,208-435,208-510,241-613,471-1017,491-865,525-
933,525-1025,578-839,584-652,622-923,688-944,696-1301,738-1361,746-
1183,798-1077,836-1409,886-1380,887-1380,947-1263,972-1259,972-
1477,989-1590,1048-1321,1050-1395,1071-1726,1280-1483,1317-1770,
1324-1571,1324-1858,1373-1609,1535-1801,1623-1884,1623-2197,1672-
1817, 1731-1982, 1777-2328, 2003-2218, 2003-2476,
2010-2480, 2064-2339,
2089-2334, 2106-2586, 2149-2386, 2156-2677,
2227-2614, 2254-2535, 2254-
2538, 2255-2526, 2286-2527, 2286-2883, 2288-2440,
2350-2638, 2431-2914,
2433-2725, 2457-2640, 2506-2696, 2516-2780,
2540-2795, 2567-3169, 2569-
2827, 2616-2835, 2616-3286, 2637-2884, 2656-2919,
2672-3350, 2676-3352,
2682-3362,2721-3357,2728-3004,2760-2910,2772-3358,2809-3085,2817-
3386, 2857-3395, 2862-3390, 2862-3395, 2871-3153,
2875-3361, 2876-3343,
2876-3347,2876-3355,2889-3382,2892-3366,2914-3365,2915-3369,2915-
3389, 2918-3366, 2919-3366, 2919-3369, 2922-3375,
2923-3171, 2923-3345, 2923-3367, 2923-3380,
2925-3366, 2927-3367, 2935-
3198, 2937-3366, 2953-3350, 2955-3392, 2962-3361,
2966-3196, 2968-3366,
2969-3381, 2976-3361, 2977-3366, 2977-3386,
2983-3367, 2984-3366, 3015-
3249, 3015-3364, 3031-3369, 3032-3367, 3043-3370,
3063-3384, 3065-3341,
3080-3324,
3081-3369, 3111-3371, 3114-3366, 3120-3382,
3131-3364, 3138-3361, 3153-
3366, 3176-3388, 3244-3366, 3244-3393, 3248-3375
18/7491063CB1/1-1132, 477-701, 991-1843, 1062-1657, 1284-1880,
1384-2017, 1385-1606,
2283 1385-1792,1385-1879,1385-1884,1385-1888,1385-1891,1385-1907,1385-
1916,1385-1974,1385-2036,1387-2283
19/7625645CB1/1-256, 1-279, 1-646, 5-621, 62-776, 135-442,
157-442, 430-1187, 430-1193, 521-
1193 1193,587-1193,598-1193,601-1193,611-1193
20/5730123CB1/1-654, 33-129, 33-288, 33-302, 33-318, 33-320,
33-330, 33-364, 33-400, 33-
2092 409, 33-451, 33-454, 33-481, 33-551, 33-562,
33-635, 35-259, 35-469, 37-291,
39-279, 39-297, 39-318, 39-345, 40-656, 42-251,
43-186, 43-248, 43-315, 44-
296,44-310,44-315,45-164,45-274,45-279,45-297,45-306,45-339,45-453,
45-471, 45-621, 45-722, 46-207, 46-224, 46-228,
46-246, 46-280, 46-308, 46-
337, 46-350, 46-489, 49-296, 49-377, 50-201,
50-330, 52-256, 52-329, 57-316,
58-147, 58-257, 58-325, 58-329, 58-332, 59-201,
60-733, 86-698, 262-885, 348-
770,350-617,373-621,387-645,397-834,397-844,405-614,405-705,406-897,
408-688,409-711,427-677,427-995,428-726,430-693,436-885,447-1151,
447-1223, 458-909, 474-982, 478-756, 481-976,
492-753, 492-1041, 504-771,
505-1074,506-828,521-784,522-774,524-1017,525-970,530-837,536-806,
536-856, 537-794, 546-1057, 558-924, 561-858,
570-895, 582-863, 583-795,
583-895,610-766,610-818,610-844,610-855;610-894,610-905,613-1295,
614-722,614-911,618-867,618-905,618-1029,624-963,627-969,634-1073,
640-968,
155
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Table 4
PolynucleotideSequence Fragments
SEQ ID NO:/
Incyte ID/
Sequence
Length
641-884,659-899,669-929,672-925,679-905,688-961,688-974,688-988,688-
993, 690-914, 691-978, 704-945, 714-959, 714-1236,
721-939, 722-1095, 724-
967, 724-968,
734-981, 736-1028, 740-995, 740-1018, 749-988,
755-1045, 758-1003, 760-
1007,767-987,767-1013,983-1061,1058-1744,1058-1748,1058-1776,1058-
1784,
1058-1790,1058-1818,1058-1840,1058-1842,1058-1856,1063-1731,1072-
1818,1106-1838,1226-2083,1304-2087,1318-1976,1431-2087,1588-2092
21/7481031CB1/1-581, 105-130, 105-132, 105-133, 105-163,
105-164, 105-165, 105-194, 105-
4213 204, 105-227, 105-234, 105-236, 105-242, 105-265,
105-278, 105-301, 105-349,
105-356,105-360,105-361,106-336,109-360,111-359,113-360,117-360,140-
360, 163-355, 186-360,
194-360, 199-360, 202-360, 221-360, 232-360,
261-360, 292-357, 330-360, 491-
1286, 637-3078, 830-1350, 835-1447, 837-1404,
838-1512, 842-1451, 881-
1163,899-1026,982-1286,994-1419,1000-1439,1000-1440,1000-1450,1000-
1488, 1000-1514,
1000-1521, 1000-1551, 1000-1552, 1000-1592,
1000-1615, 1000-1622, 1000-
1689,1000-1785,1035-1624,1041-1630,1087-1653,1094-1858,1114-1419,
1140-1803,1204-1520,1258-1973,1274-1513,1275-1518,1277-1518,1278-
1518, 1278-1975,
1285-1553,1319-1582,1319-1841,1319-1936,1349-2051,1373-2070,1441-
2209,1441-2389,1442-1666,1442-1737,1442-1937,1442-1967,1442-1983,
1442-1999,1442-2013,1442-2036,1442-2039,1442-2059,1442-2069,1442-
2072 1442-2082
1442-2088,1442-2091,1442-2109,1442-2120,1442-2201,1442-2224,1442-
2346, 1442-2355, 1442-2356, 1443-2042, 1456-2188,
1480-2085, 1493-1748,
1499-1790,1501-2003,1505-2070,1528-2178,1551-1743,1574-1845,1582-
2082,1587-2567,
1590-2082,1609-2298,1620-2079,1649-2515,1680-2463,1715-2444,1718-
2261, 1730-2410, 1751-2196, 1762-2812, 1780-2247,
1799-2336, 1799-2488,
1800-2636,1824-2525,1831-2685,1855-2512,1867-2682,1870-2082,1874-
2708,1883-2082,
1890-2082, 1899-2642, 1912-2463, 1925-2709,
2027-2746, 2028-2709, 2035-
2726, 2050-2511, 2053-2574, 2060-2932, 2071-2569,
2074-2908, 2078-2913,
2102-2696, 2106-2830, 2115-2804, 2123-2793,
2144-2885, 2145-2851, 2146-
2200, 2146-2315,
2146-2515, 2146-2555, 2146-2704, 2146-2873,
2161-2889, 2164-2819, 2167-
2874, 2185-2749, 2186-3027, 2191-2425, 2206-2798,
2206-2850, 2224-2722,
2238-2814, 2253-2823, 2261-2819, 2267-2821,
2271-3076, 2273-3078, 2274-
2961, 2275-2891,
2281-2883,2282-3101,2282-3250,2290-3107,2297-3143,2310-2939,2326-
3140, 2329-3050, 2330-3090, 2330-3097, 2350-3015,
2361-3045, 2376-2794,
2378-3228,2386-3090,2394-3037,2402-3020,2416-3157,2422-2973,2422-
3034,
156
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Table 4
PolynucleotideSequence Fragments
SEQ ID NO:/
Incyte ID/
Sequence
Length
2430-3252, 2438-3096, 2443-3051, 2445-2902,
2445-3009, 2456-3311, 2460-
3153, 2468-3061, 2474-3179, 2490-3061, 2497-3078,
2501-3409, 2503-3307,
2504-3234, 2506-3024, 2508-3250, 2511-3208,
2511-3337, 2512-3112,
2533-3144, 2533-3169, 2537-3223, 2537-3358,
2540-3156, 2541-3338, 2544-
3069, 2544-3225, 2553-3222, 2553-3240, 2570-3086,
2591-3418,
2596-3129, 2600-3240, 2603-3316, 2608-3451,
2625-3141, 2627-3281, 2627-
3425, 2630-3298, 2630-3459, 2631-3524, 2640-3178,
2641-3180, 2646-3157,
2650-3069, 2650-3458, 2653-3288, 2658-3230,
2660-3330, 2660-3496, 2667-
3361, 2668-3070, 2668-3075, 2668-3101, 2668-3102,
2668-3193, 2668-3243,
2668-3252, 2668-3253, 2668-3260, 2668-3265,
2668-3277, 2668-3281, 2668-
3297, 2668-3302, 2668-3337, 2668-3367, 2668-3369,
2668-3395, 2668-3411,
2668-3417, 2668-3511, 2668-3523, 2668-3529,
2668-3585, 2668-3590, 2668-
3592, 2668-3597, 2668-3614, 2670-3558, 2671-3297,
2681-3522, 2682-3554,
2710-3083,
2712-3058, 2723-3606, 2724-3499, 2728-3547,
2730-3540, 2735-3674, 2741-
3580, 2745-3521, 2759-3577, 2788-3572, 2811-3565,
2825-3730, 2828-3544,
2835-3619,
2843-3242,2844-3532,2844-3685,2845-3545,2845-3651,2847-3554,2848-
3562, 2853-3731, 2856-3565, 2869-3598, 2871-3733,
2881-3601, 2908-3546,
2926-3578,
2927-3560, 2938-3553, 2942-3858, 2946-3824,
2947-3519, 2948-3867, 2958-
3598, 2983-3499, 2996-3525, 2996-3526, 2997-3635,
2999-3797, 3003-3884,
3007-3691
3013-3553, 3014-3813, 3035-3921, 3037-3497,
3046-3556, 3050-3698, 3051-
3959, 3070-3813, 3071-3562, 3072-3752, 3078-3574,
3080-3522, 3088-3522,
3090-3521,3099-3604,3105-3833,3111-3783,3121-4035,3127-3544,3129-
3613,3129-3670,3136-3845,3141-3976,3145-3761,3145-3888,3147-3574,
3153-4002, 3155-3774, 3156-4037, 3160-3591,
3161-3625, 3164-3888, 3169-
3712,3174-3712,3176-3771,3178-3733,3181-3679,3184-3866,3190-3871,
3195-3766,3195-3978,3198-3994,3199-3712,3208-3683,3208-3770,3216-
3509, 3218-3815, 3220-3688, 3223-3913, 3228-3647,
3228-3908, 3235-4154,
3239-3519,3239-3772,3239-3776,3242-3761,3245-3534,3263-4110,3267-
3997, 3268-3823, 3274-3968, 3276-3841, 3285-3870,
3285-3998, 3292-3631,
3293-3812,3294-3819,3299-4019,3302-3920,3310-4213,3327-3882,3333-
3611, 3335-3800, 3338-3748, 3339-4041, 3349-4012,
3361-3906, 3367-4213,
3374-4160, 3381-3763, 3381-4058, 3382-4061,
3382-4156, 3394-3490, 3399-
4028,3410-3835,3417-4213,3418-3702,3422-4035,3424-4018,3426-4213,
3436-4004, 3441-3882, 3451-4154, 3460-3671,
3465-4095, 3470-4065, 3472-
3596, 3473-3729, 3480-3730, 3480-3731, 3480-3741,
3486-4213,
3493-4133,3502-3766,3515-4074,3517-4208,3526-4031,3542-4096,3546-
4131, 3946-3993, 3946-4033, 3958-4016, 3958-4032,
3968-3999,
3968-4033,4007-4109,4014-4056,4014-4057,4014-4068,4014-4074,4014-
4087,4014-4094,4014-4097,4014-4109,4063-4109,4063-4145,4063-4149
22/7491216CB1/1-788,1-1784,345-1179,487-1167,819-1005,819-1007,953-1167,1130-
1781
1784
157
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Table 4
PolynucleotideSequence Fragments
SEQ ID NO:/
Incyte ID/
Sequence
Length
23/71624817CB1-639, 1-2125, 138-647, 138-737, 254-731, 366-490,
1/ 368-612, 386-2141, 612-
2141 1004, 699-737, 848-897, 848-1102
24/6945964CB1/1-780, 102-917, 109-1154, 141-766, 142-673,
142-678, 142-679, 142-699, 142-
2518 702, 142-721, 142-725, 142-736, 142-755, 142-756,
142-761, 142-769,
142-772, 142-786, 142-790, 142-799, 142-828,
142-838, 142-853, 142-877, 142-
881,142-888,142-889,142-890,142-977,142-982,142-987,143-801,146-678,
146-861, 146-914, 146-929, 146-1056, 160-814,
160-858, 166-827, 182-914,
183-713, 184-1095, 188-912, 211-777, 220-965,
233-1037, 235-1192, 236-785,
260-1028,271-1196,272-890,282-1034,308-1040,319-842,321-1047,326-
1089,343-984,348-1056,397-957,400-1223,409-1139,412-875,418-1029,
422-1040, 433-898, 436-943, 438-800, 446-999,
470-1107, 472-978, 473-945,
483-1103, 510-773, 519-942, 523-674, 530-1028,
532-1084, 535-1028,
543-673, 545-1040, 563-1062, 572-998, 575-1026,
584-1077, 663-1064, 686-
1104, 714-914, 714-1040, 714-1058, 714-1070,
714-1071, 728-975,
796-1035, 1051-1666, 1178-1841, 1410-1468,
1414-1444, 1416-1478, 1444-
1498,1450-1498 1494-1968,1537-1975 1651-1980,1908-2518
158
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Table 5
PolynucleotideIncyte ProjectRepresentative Library
SEQ ID:
ID NO:
13 2425607CB BRAYDIN03
1
14 2786919CB EPIPUNA01
1
15 1801130CB TLYMNOT08
1
16 3535146CB PITUDIRO1
1
17 1436543CB SINTFER02
1
18 7491063CB BRAVTXT03
1
19 7625645CB1 KIDNFEE02
20 5730123CB1 ADRENOT11
21 7481031CB1 BRAWTDR02
23 71624817CB LIVRTUN04
1
24 6945964CB THYRDIE01
1
159
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
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CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
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CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
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WO 02/083873 PCT/US02/15253
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CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
<110> INCYTE GENOMICS, INC.
TANG, Y. Tom
YUE, Henry
SANJANWALA, Madhusudan M.
RAMKUMAR, Jayalaxini
YAO, Monique G.
SWARNAKAR, Anita
DING, Li
ELLIOTT, Vicki S.
GRIFFIN, Jennifer A.
LI, Joana X.
LAL, Preeti G.
LU, Dyung Aina M.
LU, Yan
GORVAD, Ann E.
FORSYTHE, Ian J.
DUGGAN, Brendan M.
THANGAVELU, Kavitha
EMERLING, Brooke M.
HAFALIA, April J.A.
BAUGHN, Mariah R.
BECHA, Shanya D.
SPRAGUE, William W.
<120> ENZYMES
<130> PI-0403 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/283,793; 60/291,544; 60/293,572; 60/308,182; 60/311,447;
60/315,874; 60/322,181
<151> 2001-04-13; 2001-05-16; 2001-05-25; 2001-07-27; 2001-08-09
2001-OS-29; 2001-09-14
<160> 24
<170> PERL Program
<210> 1
<211> 363
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2425607CD1
<400> 1
Met Ala Arg Leu Arg Asp Cys Leu Pro Arg Leu Met Leu Thr Leu
1 5 10 15
Arg Ser Leu Leu Phe Trp Ser Leu Val Tyr Cys Tyr Cys Gly Leu
20 25 30
Cys Ala Ser Ile His Leu Leu Lys Leu Leu Trp Ser Leu Gly Lys
35 40 45
Gly Pro Arg Arg Pro Ser Gly Gly Pro Pro Gly Ser Thr Leu Pro
50 55 60
Ala Cys Leu Ser Asp Pro Ser Leu Gly Thr His Cys Tyr Val Arg
65 70 75
Ile Lys Asp Ser Gly Leu Arg Phe His Tyr Val Ala Ala Gly Glu
80 85 90
Arg Gly Lys Pro Leu Met Leu Leu Leu His Gly Phe Pro Glu Phe
1/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
95 100 105
Trp Tyr Ile Ser Gly Gly Asn Gln Leu Arg Glu Phe Lys Ser Glu
110 115 120
Tyr Arg Val Val Ala Leu Asp Leu Arg Gly Tyr Gly Glu Thr Asp
125 130 135
Ala Pro Ile His Arg Gln Asn Tyr Lys Leu Asp Cys Leu Ile Thr
140 145 150
Asp Ile Lys Asp Ile Leu Asp Ser Leu Gly Tyr Ser Lys Cys Val
155 160 165
Leu Ile Gly His Asp Trp Gly Gly Met Ile Ala Trp Leu Ile Ala
170 175 180
Ile Cys Tyr Pro Glu Met Val Met Lys Leu Ile Val Ile Asn Phe
185 190 195
Pro His Pro Asn Val Phe Thr Glu Tyr Ile Leu Arg His Pro Ala
200 205 210
Gln Leu Leu Lys Ser Ser Tyr Tyr Tyr Phe Phe Gln Ile Pro Trp
215 220 225
Phe Pro Glu Phe Met Phe Ser Ile Asn Asp Phe Lys Val Leu Lys
230 235 240
His Leu Phe Thr Ser His Ser Thr Gly Ile Gly Arg Lys Gly Cys
245 250 255
Gln Leu Thr Thr Glu Asp Leu Glu Ala Tyr Ile Tyr Val Phe Ser
260 265 270
Gln Pro Gly Ala Leu Ser Gly Pro Ile Asn His Tyr Arg Asn Ile
275 280 285
Phe Ser Cys Leu Pro Leu Lys His His Met Val Thr Thr Pro Thr
290 295 300
Leu Leu Leu Trp Gly Glu Asn Asp Ala Phe Met Glu Val Glu Met
305 310 315
Ala Glu Val Thr Lys Ile Tyr Val Lys~Asn Tyr Phe Arg Leu Thr
320 325 330
Ile Leu Ser Glu Ala Ser His Trp Leu Gln Gln Asp Gln Pro Asp
335 340 345
Ile Val Asn Lys Leu Ile Trp Thr Phe Leu Lys Glu Glu Thr Arg
350 355 360
Lys Lys Asp
<210> 2
<211> 864
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2786919CD1
<400> 2
Met Pro Asn Val Leu Leu Pro Pro Lys Glu Ser Asn Leu Phe Lys
1 5 10 15
Arg Ile Leu Lys Cys Tyr Glu Gln Lys Gln Tyr Lys Asn Gly Leu
20 25 30
Lys Phe Cys Lys Met Ile Leu Ser Asn Pro Lys Phe Ala Glu His
35 40 45
Gly Glu Thr Leu Ala Met Lys Gly Leu Thr Leu Asn Cys Leu Gly
50 55 60
Lys Lys Glu Glu Ala Tyr Glu Phe Val Arg Lys Gly Leu Arg Asn
65 70 75
Asp Val Lys Ser His Val Cys Trp His Val Tyr Gly Leu Leu Gln
80 85 90
Arg Ser Asp Lys Lys Tyr Asp Glu Ala Ile Lys Cys Tyr Arg Asn
95 100 105
Ala Leu Lys Leu Asp Lys Asp Asn Leu Gln Ile Leu Arg Asp Leu
2/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
110 115 120
Ser Leu Leu Gln Ile Gln Met Arg Asp Leu Glu Gly Tyr Arg Glu
125 130 135
Thr Arg Tyr Gln Leu Leu Gln Leu Arg Pro Thr Gln Arg Ala Ser
140 145 150
Trp Ile Gly Tyr Ala Ile Ala Tyr His Leu Leu Lys Asp Tyr Asp
155 160 165
Met Ala Leu Lys Leu Leu Glu Glu Phe Arg Gln Thr Gln Gln Val
170 175 180
Pro Pro Asn Lys Ile Asp Tyr Glu Tyr Ser Glu Leu Ile Leu Tyr
185 190 195
Gln Asn Gln Val Met Arg Glu Ala Asp Leu Leu Gln Glu Ser Leu
200 205 210
Glu His Ile Glu Met Tyr Glu Lys Gln Ile Cys Asp Lys Leu Leu
215 220 225
Val Glu Glu Ile Lys Gly Glu Ile Leu Leu Lys Leu Gly Arg Leu
230 235 240
Lys Glu Ala Ser Glu Val Phe Lys Asn Leu Ile Asp Arg Asn Ala
245 250 255
Glu Asn Trp Cys Tyr Tyr Glu Gly Leu Glu Lys Ala Leu Gln Ile
260 265 270
Ser Thr Leu Glu Glu Arg Leu Gln Ile Tyr Glu Glu Ile Ser Lys
275 280 285
Gln His Pro Lys Ala Ile Thr Pro Arg Arg Leu Pro Leu Thr Leu
290 295 300
Val Pro Gly Glu Arg Phe Arg Glu Leu Met Asp Lys Phe Leu Arg
305 310 315
Val Asn Phe Ser Lys Gly Cys Pro Pro Leu Phe Thr Thr Leu Lys
320 325 330
Ser Leu Tyr Tyr Asn Thr Glu Lys Val Ser Ile Ile Gln Glu Leu
335 340 345
Val Thr Asn Tyr Glu Ala Ser Leu Lys Thr Cys Asp Phe Phe Ser
350 355 360
Pro Tyr Glu Asn Gly Glu Lys Glu Pro Pro Thr Thr Leu Leu Trp
365 370 375
Val Gln Tyr Phe Leu Ala Gln His Phe Asp Lys Leu Gly Gln Tyr
380 385 390
Ser Leu Ala Leu Asp Tyr Ile Asn Ala Ala Ile Ala Ser Thr Pro
395 400 405
Thr Leu Ile Glu Leu Phe Tyr Met Lys Ala Lys Ile Tyr Lys His
410 415 420
Ile Gly Asn Leu Lys Glu Ala Ala Lys Trp Met Asp Glu Ala Gln
425 430 435
Ser Leu Asp Thr Ala Asp Arg Phe Ile Asn Ser Lys Cys Ala Lys
440 445 450
Tyr Met Leu Arg Ala Asn Met Ile Lys Glu Ala Glu Glu Met Cys
455 460 465
Ser Lys Phe Thr Arg Glu Gly Thr Ser Ala Met Glu Asn Leu Asn
470 475 480
Glu Met Gln Cys Met Trp Phe Gln Thr Glu Cys Ile Ser Ala Tyr
485 490 495
Gln Arg Leu Gly Arg Tyr Gly Asp Ala Leu Lys Lys Cys His Glu
500 505 510
Val Glu Arg His Phe Phe Glu Ile Thr Asp Asp Gln Phe Asp Phe
515 520 525
His Thr Tyr Cys Met Arg Lys Met Thr Leu Arg Ala Tyr Val Asp
530 535 540
Leu Leu Arg Leu Glu Asp Ile Leu Arg Arg His Ala Phe Tyr Phe
545 550 555
Lys Ala Ala Arg Ser Ala Ile Glu Ile Tyr Leu Lys Leu Tyr Asp
560 565 570
Asn Pro Leu Thr Asn Glu Ser Lys Gln Gln Glu Ile Asn Ser Glu
575 580 585
3/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Asn Leu Ser Ala Lys Glu Leu Lys Lys Met Leu Ser Lys Gln Arg
590 595 600
Arg Ala Gln Lys Lys Ala Lys Leu Glu Glu Glu Arg Lys His Ala
605 610 615
Glu Arg Glu Arg Gln Gln Lys Asn Gln Lys Lys Lys Arg Asp Glu
620 625 630
Glu Glu Glu Glu Ala Ser Gly Leu Lys Glu Glu Leu Ile Pro Glu
635 640 645
Lys Leu Glu Arg Val Glu Asn Pro Leu Glu Glu Ala Val Lys Phe
650 655 660
Leu Ile Pro Leu Lys Asn Leu Val Ala Asp Asn Ile Asp Thr His
665 670 675
Leu Leu Ala Phe Glu Ile Tyr Phe Arg Lys Gly Lys Phe Leu Leu
680 685 690
Met Leu Gln Ser Val Lys Arg Ala Phe Ala Ile Asn Ser Asn Asn
695 700 705
Pro Trp Leu His Glu Cys Leu Ile Arg Phe Ser Lys Ser Val Ser
710 715 720
Asn His Ser Asn Leu Pro Asp Ile Val Ser Lys Val Leu Ser Gln
725 730 735
Glu Met Gln Lys Ile Phe Val Lys Lys Asp Leu Glu Ser Phe Asn
740 745 750
Glu Asp Phe Leu Lys Arg Asn Ala Thr Ser Leu Gln His Leu Leu
755 760 765
Ser Gly Ala Lys Met Met Tyr Phe Leu Asp Lys Ser Arg Gln Glu
770 775 780
Lys Ala Ile Ala Ile Ala Thr Arg Leu Asp Glu Thr Ile Lys Asp
785 790 795
Lys Asp Val Lys Thr Leu Ile Lys Val Ser Glu Ala Leu Leu Asp
800 805 810
Gly Ser Phe Gly Asn Cys Ser Ser Gln Tyr Glu Glu Tyr Arg Met
815 820 825
Ala Cys His Asn Leu Leu Pro Phe Thr Ser Ala Phe Leu Pro Ala
830 835 840
Val Asn Glu Val Asp Asn Pro Asn Val Ala Leu Asn His Thr Ala
845 850 855
Asn Tyr Asp Val Leu Ala Asn Glu Ile
860
<210> 3
<211> 376
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1801130CD1
<400> 3
Met Ser Ala Gln Val His Arg Gln Lys Gly Leu Asp Leu Ser Gln
1 5 10 15
Ile Pro Tyr Phe Asn Leu Val Lys His Leu Thr Pro Ala Cys Pro
20 25 30
Asn Val Tyr Ser Ile Ser Gln Phe His His Thr Thr Pro Asp Ser
35 40 45
Lys Thr His Ser Gly Glu Lys Tyr Thr Asp Pro Phe Lys Leu Gly
50 55 60
Trp Arg Asp Leu Lys Gly Leu Tyr Glu Asp Ile Arg Lys Glu Leu
65 70 75
Leu Ile Ser Thr Ser Glu Leu Lys Glu Met Ser Glu Tyr Tyr Phe
80 85 90
Asp Gly Lys Gly Lys Ala Phe Arg Pro Ile Ile Val Ala Leu Met
95 100 105
4/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Ala Arg Ala Cys Asn Ile His His Asn Asn Ser Arg His Val Gln
110 115 120
Ala Ser Gln Arg Ala Ile Ala Leu Ile Ala Glu Met Ile His Thr
125 130 135
Ala Ser Leu Val His Asp Asp Val Ile Asp Asp Ala Ser Ser Arg
140 145 150
Arg Gly Lys His Thr Val Asn Lys Ile Trp Gly Glu Lys Lys Ala
155 160 165
Val Leu Ala Gly Asp Leu Ile Leu Ser Ala Ala Ser Ile Ala Leu
170 175 180
Ala Arg Ile Gly Asn Thr Thr Val Ile Ser Ile Leu Thr Gln Val
185 190 195
Ile Glu Asp Leu Val Arg Gly Glu Phe Leu Gln Leu Gly Ser Lys
200 205 210
Glu Asn Glu Asn Glu Arg Phe Ala His Tyr Leu Glu Lys Thr Phe
215 220 225
Lys Lys Thr Ala Ser Leu Ile Ala Asn Ser Cys Lys Ala Val Ser
230 235 240
Val Leu Gly Cys Pro Asp Pro Val Val His Glu Ile Ala Tyr Gln
245 250 255
Tyr Gly Lys Asn Val Gly Ile Ala Phe Gln Leu Ile Asp Asp Val
260 265 270
Leu Asp Phe Thr Ser Cys Ser Asp Gln Met Gly Lys Pro Thr Ser
275 280 285
Ala Asp Leu Lys Leu Gly Leu Ala Thr Gly Pro Val Leu Phe Ala
290 295 300
Cys Gln Gln Phe Pro Glu Met Asn Ala Met Ile Met Arg Arg Phe
305 310 315
Ser Leu Pro Gly Asp Val Asp Arg Ala Arg Gln Tyr Val Leu Gln
320 325 330
Ser Asp Gly Val Gln Gln Thr Thr Tyr Leu Ala Gln Gln Tyr Cys
335 340 345
His Glu Ala Ile Arg Glu Ile Ser Lys Leu Arg Pro Ser Pro Glu
350 355 360
Arg Asp Ala Leu Ile Gln Leu Ser Glu Ile Val Leu Thr Arg Asp
365 370 375
Lys
<210> 4
<211> 978
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3535146CD1
<400> 4
Met Gly Thr Arg Leu Pro Leu Val Leu Arg Gln Leu Arg Arg Pro
1 5 10 15
Pro Gln Pro Pro Gly Pro Pro Arg Arg Leu Arg Val Pro Cys Arg
20 25 30
Ala Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Arg Glu Gly
35 40 45
Leu Leu Gly Gln Arg Arg Pro Gln Asp Gly Gln Ala Arg Ser Ser
50 55 60
Cys Ser Pro Gly Gly Arg Thr Pro Ala Ala Arg Asp Ser Ile Val
65 70 75
Arg Glu Val Ile Gln Asn Ser Lys Glu Val Leu Ser Leu Leu Gln
80 85 90
Glu Lys Asn Pro Ala Phe Lys Pro Val Leu Ala Ile Ile Gln Ala
95 100 105
5/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Gly Asp Asp Asn Leu Met Gln Glu Ile Asn Gln Asn Leu Ala Glu
110 115 120
Glu Ala Gly Leu Asn Ile Thr His Ile Cys Leu Pro Pro Asp Ser
125 130 135
Ser Glu Ala Glu Ile Ile Asp Glu Ile Leu Lys Ile Asn Glu Asp
140 145 150
Thr Arg Val His Gly Leu Ala Leu Gln Ile Ser Glu Asn Leu Phe
155 160 165
Ser Asn Lys Val Leu Asn Ala Leu Lys Pro Glu Lys Asp Val Asp
170 175 180
Gly Val Thr Asp Ile Asn Leu Gly Lys Leu Val Arg Gly Asp Ala
185 190 195
His Glu Cys Phe Val Ser Pro Val Ala Lys Ala Val Ile Glu Leu
200 205 210
Leu Glu Lys Ser Gly Val Asn Leu Asp Gly Lys Lys Ile Leu Val
215 220 225
Val Gly Ala His Gly Ser Leu Glu Ala Ala Leu Gln Cys Leu Phe
230 235 240
Gln Arg Lys Gly Ser Met Thr Met Ser Ile Gln Trp Lys Thr Arg
245 250 255
Gln Leu Gln Ser Lys Leu His Glu Ala Asp Ile Val Val Leu Gly
260 265 270
Ser Pro Lys Pro Glu Glu Ile Pro Leu Thr Trp Ile Gln Pro Gly
275 280 285
Thr Thr Val Leu Asn Cys Ser His Asp Phe Leu Ser Gly Lys Val
290 295 300
Gly Cys Gly Ser Pro Arg Ile His Phe Gly Gly Leu Ile Glu Glu
305 310 315
Asp Asp Val Ile Leu Leu Ala Ala Ala Leu Arg Ile Gln Asn Met
320 325 330
Val Ser Ser Gly Arg Arg Trp Leu Arg Glu Gln Gln His Arg Arg
335 340 345
Trp Arg Leu His Cys Leu Lys Leu Gln Pro Leu Ser Pro Val Pro
350 355 360
Ser Asp Ile Glu Ile Ser Arg Gly Gln Thr Pro Lys Ala Val Asp
365 370 375
Val Leu Ala Lys Glu Ile Gly Leu Leu Ala Asp Glu Ile Glu Ile
380 385 390
Tyr Gly Lys Ser Lys Ala Lys Val Arg Leu Ser Val Leu Glu Arg
395 400 405
Leu Lys Asp Gln Ala Asp Gly Lys Tyr Val Leu Val Ala Gly Ile
410 415 420
Thr Pro Thr Pro Leu Gly Glu Gly Lys Ser Thr Val Thr Ile Gly
425 430 435
Leu Val Gln Ala Leu Thr Ala His Leu Asn Val Asn Ser Phe Ala
440 445 450
Cys Leu Arg Gln Pro Ser Gln Gly Pro Thr Phe Gly Val Lys Gly
455 460 465
Gly Ala Ala Gly Gly Gly Tyr Ala Gln Val Ile Pro Met Glu Glu
470 475 480
Phe Asn Leu His Leu Thr Gly Asp Ile His Ala Ile Thr Ala Ala
485 490 495
Asn Asn Leu Leu Ala Ala Ala Ile Asp Thr Arg Ile Leu His Glu
500 505 510
Asn Thr Gln Thr Asp Lys Ala Leu Tyr Asn Arg Leu Val Pro Leu
515 520 525
Val Asn Gly Val Arg Glu Phe Ser Glu Ile Gln Leu Ala Arg Leu
530 535 540
Lys Lys Leu Gly Ile Asn Lys Thr Asp Pro Ser Thr Leu Thr Glu
545 550 555
Glu Glu Val Ser Lys Phe Ala Arg Leu Asp Ile Asp Pro Ser Thr
560 565 570
Ile Thr Trp Gln Arg Val Leu Asp Thr Asn Asp Arg Phe Leu Arg
6/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
575 580 585
Lys Ile Thr Ile Gly Gln Gly Asn Thr Glu Lys Gly His Tyr Arg
590 595 600
Gln Ala Gln Phe Asp Ile Ala Val Ala Ser Glu Ile Met Ala Val
605 610 615
Leu Ala Leu Thr Asp Ser Leu Ala Asp Met Lys Ala Arg Leu Gly
620 625 630
Arg Met Val Val Ala Ser Asp Lys Ser Gly.Gln Pro Val Thr Ala
635 640 645
Asp Asp Leu Gly Val Thr Gly Ala Leu Thr Val Leu Met Lys Asp
650 655 660
Ala Ile Lys Pro Asn Leu Met Gln Thr Leu Glu Gly Thr Pro Val
665 670 675
Phe Val His Ala Gly Pro Phe Ala Asn Ile Ala His Gly Asn Ser
680 685 690
Ser Val Leu Ala Asp Lys Ile Ala Leu Lys Leu Val Gly Glu Glu
695 700 705
Gly Phe Val Val Thr Glu Ala Gly Phe Gly Ala Asp Ile Gly Met
710 715 720
Glu Lys Phe Phe Asn Ile Lys Cys Arg Ala Ser Gly Leu Val Pro
725 730 735
Asn Val Val Val Leu Val Ala Thr Val Arg Ala Leu Lys Met His
740 745 750
Gly Gly Gly Pro Ser Val Thr Ala Gly Val Pro Leu Lys Lys Glu
755 760 765
Tyr Thr Glu Glu Asn Ile Gln Leu Val Ala Asp Gly Cys Cys Asn
770 775 780
Leu Gln Lys Gln Ile Gln Ile Thr Gln Leu Phe Gly Val Pro Val
785 790 795
Val Val Ala Leu Asn Val Phe Lys Thr Asp Thr Arg Ala Glu Ile
800 805 810
Asp Leu Val Cys Glu Leu Ala Lys Arg Ala Gly Ala Phe Asp Ala
815 820 825
Val Pro Cys Tyr His Trp Ser Val Gly Gly Lys Gly Ser Val Asp
830 835 840
Leu Ala Arg Ala Val Arg Glu Ala Ala Ser Lys Arg Ser Arg Phe
845 850 855
Gln Phe Leu Tyr Asp Val Gln Val Pro Ile Val Asp Lys Ile Arg
860 865 870
Thr Ile Ala Gln Ala Val Tyr Gly Ala Lys Asp Ile Glu Leu Ser
875 880 885
Pro Glu Ala Gln Ala Lys Ile Asp Arg Tyr Thr Gln Gln Gly Phe
890 895 900
Gly Asn Leu Pro Ile Cys Met Ala Lys Thr His Leu Ser Leu Ser
905 910 915
His Gln Pro Asp Lys Lys Gly Val Pro Arg Asp Phe Ile Leu Pro
920 925 930
Ile Ser Asp Val Arg Ala Ser Ile Gly Ala Gly Phe Ile Tyr Pro
935 940 945
Leu Val Gly Thr Met Ser Thr Met Pro Gly Leu Pro Thr Arg Pro
950 955 960
Cys Phe Tyr Asp Ile Asp Leu Asp Thr Glu Thr Glu Gln Val Lys
965 970 975
Gly Leu Phe
<210> 5
<211> 349
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
7/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
<223> Incyte ID No: 1436543CD1
<400> 5
Met Ala Ala Ser Glu Ala Ala Val Val Ser Ser Pro Ser Leu Lys
1 5 10 15
Thr Asp Thr Ser Pro Val Leu Glu Thr Ala Gly Thr Val Ala Ala
20 25 30
Met.Ala Ala Thr Pro Ser Ala Arg Ala Ala Ala Ala Val Val Ala
35 40 45
Ala Ala Ala Arg Thr Gly Ser Glu Ala Arg Val Ser Lys Ala Ala
50 55 60
Leu Ala Thr Lys Leu Leu Ser Leu Ser Gly Val Phe Ala Val His
65 70 75
Lys Pro Lys Gly Pro Thr Ser Ala Glu Leu Leu Asn Arg Leu Lys
80 85 90
Glu Lys Leu Leu Ala Glu Ala Gly Met Pro Ser Pro Glu Trp Thr
95 100 105
Lys Arg Lys Lys Gln Thr Leu Lys Ile Gly His Gly Gly Thr Leu
110 115 120
Asp Ser Ala Ala Arg Gly Val Leu Val Val Gly Ile Gly Ser Gly
125 130 135
Thr Lys Met Leu Thr Ser Met Leu Ser Gly Ser Lys Arg Tyr Thr
140 145 150
Ala Ile Gly Glu Leu Gly Lys Ala Thr Asp Thr Leu Asp Ser Thr
155 160 165
Gly Arg Val Thr Glu Glu Lys Pro Tyr Asp Lys Ile Thr Gln Glu
170 175 180
Asp Ile Glu Gly Ile Leu Gln Lys Phe Thr Gly Asn Ile Met Gln
185 190 195
Val Pro Pro Leu Tyr Ser Ala Leu Lys Lys Asp Gly Gln Arg Leu
200 205 210
Ser Thr Leu Met Lys Arg Gly Glu Val Val Glu Ala Lys Pro Ala
215 220 225
Arg Pro Val Thr Val Tyr Ser Ile Ser Leu Gln Lys Phe Gln Pro
230 235 240
Pro Phe Phe Thr Leu Asp Val Glu Cys Gly Gly Gly Phe Tyr Ile
245 250 255
Arg Ser Leu Val Ser Asp Ile Gly Lys Glu Leu Ser Ser Cys Ala
260 265 270
Asn Val Leu Glu Leu Thr Arg Thr Lys Gln Gly Pro Phe Thr Leu
275 280 285
Glu Glu His Ala Leu Pro Glu Asp Lys Trp Thr Ile Asp Asp Ile
290 295 300
Ala Gln Ser Leu Glu His Cys Ser Ser Leu Phe Pro Ala Glu Leu
305 310 315
Ala Leu Lys Lys Ser Lys Pro Glu Ser Asn Glu Gln Val Leu Ser
320 325 330
Cys Glu Tyr Ile Thr Leu Asn Glu Pro Lys Arg Glu Asp Asp Val
335 340 345
Ile Lys Thr Cys
<210> 6
<211> 399
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7491063CD1
<400> 6
Met Phe Ser Lys Gly Ser Met Val Leu Ala Tyr Ser Ala Gly Leu
8/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
1 5 10 15
Asp Thr Ser Cys Ile Leu Val Trp Leu Lys Glu Gln Gly Tyr Asp
20 25 30
Ile Ile Ala Tyr Leu Ala Asn Val Gly Gln Lys Glu Asp Phe Lys
35 40 45
Glu Ala Arg Lys Lys Ala Leu Asn Leu Gly Ser Lys Lys Val Phe
50 55 60
Ile Glu Asp Val Ser Lys Glu Phe Val Glu Glu Phe Ile Trp Pro
65 70 75
Tyr Glu Asp Cys Tyr Leu Leu Gly Pro Ser Leu Pro Arg Pro Tyr
80 85 90
Ile Thr Arg Lys Gln Val Glu Ile Ala Gln Trp Glu Gly Ala Lys
95 100 105
Tyr Val Ser His Ser Ala Met Gly Lys Gly Asn Asp Gln Val Trp
110 115 120
Phe Glu Leu Ala Cys Tyr Ser Leu Ala Pro Gln Ile Lys Val Ile
125 130 135
Ala Pro Gly Arg Ile Pro Glu Phe Tyr Asn Gln Ser Lys Gly Arg
140 145 150
Ser Asp Leu Met Glu Tyr Ala Glu Lys His Gly Ile Pro Ile Pro
155 160 165
Val Thr Leu Lys His Pro Trp Asn Met Asp Glu Asn Leu Met His
170 175 180
Ile Ser His Glu Gly Trp Asn Leu Gly Glu Pro Gln Glu Pro Glu
185 190 195
Ala Pro Ser Gly Leu Tyr Met Lys Ile Gln Asp Leu Ala Lys Ala
200 205 210
Pro Asn Thr Pro Asn Ile Phe Lys Thr Glu Gly Val Pro Val Lys
215 220 225
Val Thr Ser Ile Lys Asp Gly Thr Thr His Gln Thr Ser Leu Val
230 235 240
Leu Leu His Val Thr Trp Asn Glu Val Ala Gly Lys His Ser Val
245 250 255
Gly His Ile Asp Ile Val Glu Asn Arg Phe Ile Glu Met Asn Ile
260 265 270
Cys Lys Thr Pro Ala Gly Thr Ile Leu Tyr His Pro His Leu Asp
275 280 285
Ile Glu Gly Phe Ala Met Glu Gln Glu Val Arg Lys Ile Lys Gln
290 295 300
Gly Leu Gly Leu Lys Phe Ala Glu Leu Val Tyr Thr Gly Phe Trp
305 310 315
His Asn Pro Gln Cys Asp Phe Ala His His Cys Ile Ala Lys Ser
320 325 330
Gln Asp Arg Val Glu Gly Lys Val Gln Val Ser Ile Phe Lys Gly
335 340 345
Gln Val Tyr Ile Leu Cys Gln Glu Pro Pro Leu Ser Leu Tyr Ser
350 355 360
Gly Glu Gln Val Ser Met Asn Val Glu Gly Asn Asp Glu Pro Ala
365 370 375
Ser Arg Leu Ile Asn Ile Asn Ser Leu Arg Met Lys Glu Tyr His
380 385 390
His Leu Gln Ser Lys Val Thr Ala Lys
395
<210> 7
<211> 278
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7625645CD1
9/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
<400> 7
Met Glu Ile Ile Arg Thr Ile Ala Glu Leu Arg Val Arg Leu Ala
1 5 10 15
Arg Glu Ser Asn Ile Ala Leu Val Pro Thr Met Gly Asn Ile His
20 25 30
Asp Gly His Leu Ser Leu Val Arg Ile Gly Leu Lys Arg Ala Pro
35 40 45
Phe Thr Val Thr Ser Ile Phe Val Asn Arg Leu Gln Phe Ile Gln
50 55 60
Gly Glu Asp Phe Asp Lys Tyr Pro Arg Thr Phe Glu Glu Asp Cys
65 70 75
Glu Lys Leu Glu Ala Ala Gly Asn Ser Val Val Phe Ala Pro Asp
80 85 90
Glu Thr Gln Met Tyr Pro Glu Arg Gln Val Phe Ile Val Glu Pro
95 100 105
Pro Pro Ile Ala Asn Lys Leu Glu Gly Arg Phe Arg Pro Gly His
110 115 120
Phe Arg Gly Val Ser Thr Val Val Leu Lys Leu Phe Asn Met Val
125 130 135
Gln Pro Gln Val Ala Val Phe Gly Lys Lys Asp Tyr Gln Gln Leu
140 145 150
Ser Ile Val Arg His Met Val Asp Gln Leu Ala Leu Pro Leu Ser
155 160 165
Ile Ile Pro Ala Glu Thr Val Arg Ala Glu Asp Gly Leu Ala Leu
170 175 180
Ser Ser Arg Asn Arg Tyr Leu Ser Pro Glu Glu Arg Ser Glu Ala
185 190 195
Pro Arg Leu Asn Glu Ala Leu Arg Gln Val Lys Gln Ala Val Glu
200 205 210
Ser Gly Asp Arg Asn Phe Glu Ala Leu Glu Phe Ala Ala Asp Ala
215 220 225
Leu Leu Ala Arg His Arg Trp His Val Asp Tyr Val Val Val Arg
230 235 240
Ser Arg Arg Thr Leu Met Gln Pro Asp Pro Asp Glu Arg Glu Leu
245 250 255
Val Val Leu Gly Ala Ala Arg Leu Gly Gln Thr Arg Leu Ile Asp
260 265 270
Asn Leu Glu Ile Leu Ala Pro Ala
275
<210> 8
<211> 583
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5730123CD1
<400> 8
Met Ser Gly Val Arg Gly Leu Ser Arg Leu Leu Ser Ala Arg Arg
1 5 10 15
Leu Ala Leu Ala Lys Ala Trp Pro Thr Val Leu Gln Thr Gly Thr
20 25 30
Arg Gly Phe His Phe Thr Val Asp Gly Asn Lys Arg Ala Ser Ala
35 40 45
Lys Val Ser Asp Ser Ile Ser Ala Gln Tyr Pro Val Val Asp His
50 55 60
Glu Phe Asp Ala Val Val Val Gly Ala Gly Gly Ala Gly Leu Arg
65 70 75
Ala Ala Phe Gly Leu Ser Glu Ala Gly Phe Asn Thr Ala Cys Val
80 85 90
Thr Lys Leu Phe Pro Thr Arg Ser His Thr Val Ala Ala Gln Gly
10/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
95 100 105
Gly Ile Asn Ala Ala Leu Gly Asn Met Glu Glu Asp Asn Trp Arg
110 115 120
Trp His Phe Tyr Asp Thr Val Lys Gly Ser Asp Trp Leu Gly Asp
125 130 135
Gln Asp Ala Ile His Tyr Met Thr Glu Gln Ala Pro Ala Ala Val
140 145 150
Val Glu Leu Glu Asn Tyr Gly Met Pro Phe Ser Arg Thr Glu Asp
155 160 165
Gly Lys Ile Tyr Gln Arg Ala Phe Gly Gly Gln Ser Leu Lys Phe
170 175 180
Gly Lys Gly Gly Gln Ala His Arg Cys Cys Cys Val Ala Asp Arg
185 190 195
Thr Gly His Ser Leu Leu His Thr Leu Tyr Gly Arg Ser Leu Arg
200 205 210
Tyr Asp Thr Ser Tyr Phe Val Glu Tyr Phe Ala Leu Asp Leu Leu
215 220 225
Met Glu Asn Gly Glu Cys Arg Gly Val Ile Ala Leu Cys Ile Glu
230 235 240
Asp Gly Ser Ile His Arg Ile Arg Ala Lys Asn Thr Val Val Ala
245 250 255
Thr Gly Gly Tyr Gly Arg Thr Tyr Phe Ser Cys Thr Ser Ala His
260 265 270
Thr Ser Thr Gly Asp Gly Thr Ala Met Ile Thr Arg Ala Gly Leu
275 280 285
Pro Cys Gln Asp Leu Glu Phe Val Gln Phe His Pro Thr Gly Ile
290 295 300
Tyr Gly Ala Gly Cys Leu Ile Thr Glu Gly Cys Arg Gly Glu Gly
305 310 315
Gly Ile Leu Ile Asn Ser Gln Gly Glu Arg Phe Met Glu Arg Tyr
320 325 330
Ala Pro Val Ala Lys Asp Leu Ala Ser Arg Asp Val Val Ser Arg
335 340 345
Ser Met Thr Leu Glu Ile Arg Glu Gly Arg Gly Cys Gly Pro Glu
350 355 360
Lys Asp His Val Tyr Leu Gln Leu His His Leu Pro Pro Glu Gln
365 370 375
Leu Ala Thr Arg Leu Pro Gly Ile Ser Glu Thr Ala Met Ile Phe
380 385 390
Ala Gly Val Asp Val Thr Lys Glu Pro Ile Pro Val Leu Pro Thr
395 400 405
Val His Tyr Asn Met Gly Gly Ile Pro Thr Asn Tyr Lys Gly Gln
410 415 420
Val Leu Arg His Val Asn Gly Gln Asp Gln Ile Val Pro Gly Leu
425 430 435
Tyr Ala Cys Gly Glu Ala Ala Cys Ala Ser Val His Gly Ala Asn
440 445 450
Arg Leu Gly Ala Asn Ser Leu Leu Asp Leu Val Val Phe Gly Arg
455 460 465
Ala Cys Ala Leu Ser Ile Glu Glu Ser Cys Arg Pro Gly Asp Lys
470 475 480
Val Pro Pro Ile Lys Pro Asn Ala Gly Glu Glu Ser Val Met Asn
485 490 495
Leu Asp Lys Leu Arg Phe Ala Asp Gly Ser Ile Arg Thr Ser Glu
500 505 510
Leu Arg Leu Ser Met Gln Lys Val Arg Ile Asp Glu Tyr Asp Tyr
515 520 525
Ser Lys Pro Ile Gln Gly Gln Gln Lys Lys Pro Phe Glu Glu His
530 535 540
Trp Arg Lys His Thr Leu Ser Phe Val Asp Val Gly Thr Gly Lys
545 550 555
Val Thr Leu Glu Tyr Arg Pro Val Ile Asp Lys Thr Leu Asn Glu
560 565 570
11/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Ala Asp Cys Ala Thr Ile Pro Pro Ala Ile Arg Ser Tyr
575 580
<210> 9
<211> 1032
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7481031CD1
<400> 9
Met Gln Arg Pro Gly Gly Arg Trp Ala Gly Pro Glu Arg Val Gln
1 5 10 15
Val Arg Arg Ala Leu Trp Gly Pro Cys Ser Leu Arg Gly Pro Glu
20 25 30
Trp Ala Ala Pro Asp Thr Arg Glu Cys Val Gln Thr Arg Leu Leu
35 40 45
Thr Ala Ala Ser Pro Ala Asp His Gly Gly Gly Arg Pro Ala Ala
50 55 60
Ala Gly Pro Leu Leu Lys Arg Ser His Ser Val Pro Ala Pro Ser
65 70 75
Ile Arg Lys Gln Ile Leu Glu Glu Leu Glu Lys Pro Gly Ala Gly
80 85 90
Asp Pro Asp Pro Ser Ala Pro Gln Gly Gly Pro Gly Ser Ala Thr
95 100 105
Ser Asp Leu Gly Met Ala Cys Asp Arg Ala Arg Val Phe Leu His
110 115 120
Ser Asp Glu His Pro Gly Ser Ser Val Ala Ser Lys Ser Arg Lys
125 130 135
Ser Val Met Val Ala Glu Ile Pro Ser Thr Val Ser Gln His Ser
140 145 150
Glu Ser His Thr Asp Glu Thr Leu Ala Ser Arg Lys Ser Asp Ala
155 160 165
Ile Phe Arg Ala Ala Lys Lys Asp Leu Leu Thr Leu Met Lys Leu
170 175 180
Glu Asp Ser Ser Leu Leu Asp Gly Arg Val Ala Leu Leu His Val
185 190 195
Pro Ala Gly Thr Val Val Ser Arg Gln Gly Asp Gln Asp Ala Ser
200 205 210
Ile Leu Phe Val Val Ser Gly Leu Leu His Val Tyr Gln Arg Lys
215 220 225
Ile Gly Ser Gln Glu Asp Thr Cys Leu Phe Leu Thr Arg Pro Gly
230 235 240
Glu Met Val Gly Gln Leu Ala Val Leu Thr Gly Glu Pro Leu Ile
245 250 255
Phe Thr Val Lys Ala Asn Arg Asp Cys Ser Phe Leu Ser Ile Ser
260 265 270
Lys Ala His Phe Tyr Glu Ile Met Arg Lys Gln Pro Thr Val Val
275 280 285
Leu Gly Val Ala His Thr Val Val Lys Arg Met Ser Ser Phe Val
290 295 300
Arg Gln Ile Asp Phe Ala Leu Asp Trp Val Glu Val Glu Ala Gly
305 310 315
Arg Ala Ile Tyr Arg Gln Gly Asp Lys Ser Asp Cys Thr Tyr Ile
320 325 330
Met Leu Ser Gly Arg Leu Arg Ser Val Ile Arg Lys Asp Asp Gly
335 340 345
Lys Lys Arg Leu Ala Gly Glu Tyr Gly Arg Gly Asp Leu Val Gly
350 355 360
Val Val Glu Thr Leu Thr His Gln Ala Arg Ala Thr Thr Val His
365 370 375
12/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
Ala Val Arg Asp Ser Glu Leu Ala Lys Leu Pro Ala Gly Ala Leu
380 385 390
Thr Ser Ile Lys Arg Arg Tyr Pro Gln Val Val Thr, Arg Leu Ile
395 400 405
His Leu Leu Gly Glu Lys Ile Leu Gly Ser Leu Gln Gln Gly Pro
410 415 420
Val Thr Gly His Gln Leu Gly Leu Pro Thr Glu Gly Ser Lys Trp
425 430 435
Asp Leu Gly Asn Pro Ala Val Asn Leu Ser Thr Val Ala Val Met
440 445 450
Pro Val Ser Glu Glu Val Pro Leu Thr Ala Phe Ala Leu Glu Leu
455 460 465
Glu His Ala Leu Ser Ala Ile Gly Pro Thr Leu Leu Leu Thr Ser
470 475 480
Asp Asn Ile Lys Arg Arg Leu Gly Ser Ala Ala Leu Asp Ser Val
485 490 495
His Glu Tyr Arg Leu Ser Ser Trp Leu Gly Gln Gln Glu Asp Thr
500 505 510
His Arg Ile Val Leu Tyr Gln Ala Asp Gly Thr Leu Thr Pro Trp
515 520 525
Thr Gln Arg Cys Val Arg Gln Ala Asp Cys Ile Leu Ile Val Gly
530 535 540
Leu Gly Asp Gln Glu Pro Thr Val Gly Glu Leu Glu Arg Met Leu
545 550 555
Glu Ser Thr Ala Val Arg Ala Gln Lys Gln Leu Ile Leu Leu His
560 565 570
Arg Glu Glu Gly Pro Ala Pro Ala Arg Thr Val Glu Trp Leu Asn
575 580 585
Met Arg Ser Trp Cys Ser Gly His Leu His Leu Cys Cys Pro Arg
590 595 600
Arg Val Phe Ser Arg Arg Ser Leu Pro Lys Leu Val Glu Met Tyr
605 610 615
Lys His Val Phe Gln Arg Pro Pro Asp Arg His Ser Asp Phe Ser
620 625 630
Arg Leu Ala Arg Val Leu Thr Gly Asn Ala Ile Ala Leu Val Leu
635 640 645
Gly Gly Gly Gly Ala Arg Gly Cys Ala Gln Val Gly Val Leu Lys
650 655 660
Ala Leu Ala Glu Cys Gly Ile Pro Val Asp Met Val Gly Gly Thr
665 670 675
Ser Ile Gly Ala Phe Val Gly Ala Leu Tyr Ser Glu Glu Arg Asn
680 685 690
Tyr Ser Gln Met Arg Ile Arg Ala Lys Gln Trp Ala Glu Gly Met
695 700 705
Thr Ser Leu Met Lys Ala Ala Leu Asp Leu Thr Tyr Pro Ile Thr
710 715 720
Ser Met Phe Ser Gly Ala Gly Phe Asn Ser Ser Ile Phe Ser Val
725 730 735
Phe Lys Asp Gln Gln Ile Glu Asp Leu Trp Ile Pro Tyr Phe Ala
740 745 750
Ile Thr Thr Asp Ile Thr Ala Ser Ala Met Arg Val His Thr Asp
755 760 765
Gly Ser Leu Trp Arg Tyr Val Arg Ala Ser Met Ser Leu Ser Gly
770 775 780
Tyr Met Pro Pro Leu Cys Asp Pro Lys Asp Gly His Leu Leu Met
785 790 795
Asp Gly Gly Tyr Ile Asn Asn Leu Pro Ala Asp Val Ala Arg Ser
800 805 810
Met Gly Ala Lys Val Val Ile Ala Ile Asp Val Gly Ser Arg Asp
815 820 825
Glu Thr Asp Leu Thr Asn Tyr Gly Asp Ala Leu Ser Gly Trp Trp
830 835 840
Leu Leu Trp Lys Arg Trp Asn Pro Leu Ala Thr Lys Val Lys Val
13/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
845 850 855
Leu Asn Met Ala Glu Ile Gln Thr Arg Leu Ala Tyr Val Cys Cys
860 865 870
Val Arg Gln Leu Glu Val Val Lys Ser Ser Asp Tyr Cys Glu Tyr
875 880 885
Leu Arg Pro Pro Ile Asp Ser Tyr Ser Thr Leu Asp Phe Gly Lys
890 895 900
Phe Asn Glu Ile Cys Glu Val Gly Tyr Gln His Gly Arg Thr Val
905 910 915
Phe Asp Ile Trp Gly Arg Ser Gly Val Leu Glu Lys Met Leu Arg
920 925 930
Asp Gln Gln Gly Pro Ser Lys Lys Pro Ala Ser Ala Val Leu Thr
935 940 945
Cys Pro Asn Ala Ser Phe Thr Asp Leu Ala Glu Ile Val Ser Arg
950 955 960
Ile Glu Pro Ala Lys Pro Ala Met Val Asp Asp Glu Ser Asp Tyr
965 970 975
Gln Thr Glu Tyr Glu Glu Glu Leu Leu Asp Val Pro Arg Asp Ala
980 985 990
Tyr Ala Asp Phe Gln Ser Thr Ser Ala Gln Gln Gly Ser Asp Leu
995 1000 1005
Glu Asp Glu Ser Ser Leu Arg His Arg His Pro Ser Leu Ala Phe
1010 1015 1020
Pro Lys Leu Ser Glu Gly Ser Ser Asp Gln Asp Gly
1025 1030
<210> 10
<211> 407
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7491216CD1
<400> 10
Met Trp Asp Leu Ala Leu Ile Phe Leu Ala Ala Ala Cys Val Phe
1 5 10 15
Ser Leu Gly Val Thr Leu Trp Val Ile Cys Ser His Phe Phe Thr
20 25 30
Val His Ile Pro Ala Ala Val Gly His Pro Val Lys Leu Arg Val
35 40 45
Leu His Cys Ile Phe Gln Leu Leu Leu Thr Trp Gly Met Ile Phe
50 55 60
Glu Lys Leu Arg Ile Cys Ser Met Pro Gln Phe Phe Cys Phe Met
65 70 75
Gln Asp Leu Pro Pro Leu Lys Tyr Asp Pro Asp Val Val Val Thr
80 85 90
Asp Phe Arg Phe Gly Thr Ile Pro Val Lys Leu Tyr Gln Pro Lys
95 100 105
Ala Ser Thr Cys Thr Leu Lys Pro Gly Ile Val Tyr Tyr His Gly
110 115 120
Gly Gly Gly Val Met Gly Ser Leu Lys Thr His His Gly Ile Cys
125 130 135
Ser Arg Leu Cys Lys Glu Ser Asp Ser Val Val Leu Ala Val Gly
140 145 150
Tyr Arg Lys Leu Pro Lys His Lys Phe Pro Val Pro Val Arg Asp
155 160 165
Cys Leu Val Ala Thr Ile His Phe Leu Lys Ser Leu Asp Ala Tyr
170 175 180
Gly Val Asp Pro Ala Arg Val Val Val Cys Gly Asp Ser Phe Gly
185 190 195
Gly Ala Ile Ala Ala Val Val Cys Gln Gln Leu Val Asp Arg Pro
14/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
200 205 210
Asp Leu Pro Arg Ile Arg Ala Gln Ile Leu Ile Tyr Ala Ile Leu
215 220 225
Gln Ala Leu Asp Leu Gln Thr Pro Ser Phe Gln Gln Arg Lys Asn
230 235 240
Ile Pro Leu Leu Thr Trp Ser Phe Ile Cys Tyr Cys Phe Phe Gln
245 250 255
Asn Leu Asp Phe Ser Ser Ser Trp Gln Glu Val Ile Met Lys Gly
260 265 270
Ala His Leu Pro Ala Glu Val Trp Glu Lys Tyr Arg Lys Trp Leu
275 280 285
Gly Pro Glu Asn Ile Pro Glu Arg Phe Lys Glu Arg Gly Tyr Gln
290 295 300
Leu Lys Pro His Glu Pro Met Asn Glu Ala Ala Tyr Leu Glu Val
305 310 315
Ser Val Val Leu Asp Val Met Cys Ser Pro Leu Ile Ala Glu Asp
320 325 330
Asp Ile Val Ser Gln Leu Pro Glu Thr Cys Ile Val Ser Cys Glu
335 340 345
Tyr Asp Ala Leu Arg Asp Asn Ser Leu Leu Tyr Lys Lys Arg Leu
350 355 360
Glu Asp Leu Gly Val Pro Val Thr Trp His His Met Glu Asp Gly
365 370 375
Phe His Gly Val Leu Arg Thr Ile Asp Met Ser Phe Leu His Phe
380 385 390
Pro Cys Ser Met Arg Ile Leu Ser Ala Leu Val Gln Phe Val Lys
395 400 405
Gly Leu
<210> 11
<211> 352
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71624817CD1
<400> 11
Met Ser Leu Tyr Cys Gly Ile Ala Cys Arg Arg Lys Phe Phe Trp
1 5 10 15
Cys Tyr Arg Leu Leu Ser Thr Tyr Val Thr Lys Thr Arg Tyr Leu
20 25 30
Phe Glu Leu Lys Glu Asp Asp Asp Ala Cys Lys Lys Ala Gln Gln
35 40 45
Thr Gly Ala Phe Tyr Leu Phe His Ser Leu Ala Pro Leu Leu Gln
50 55 60
Thr Ser Ala His Gln Tyr Leu Ala Pro Arg His Ser Leu Leu Glu
65 70 75
Leu Glu Arg Leu Leu Gly Lys Phe Gly Gln Asp Ala Gln Arg Ile
80 85 90
Glu Asp Ser Val Leu Ile Gly Cys Ser Glu Gln Gln Glu Ala Trp
95 100 105
Phe Ala Leu Asp Leu Gly Leu Asp Ser Ser Phe Ser Ile Ser Ala
110 115 120
Ser Leu His Lys Pro Glu Met Glu Thr Glu Leu Lys Gly Ser Phe
125 130 135
Ile Glu Leu Arg Lys Ala Leu Phe Gln Leu Asn Ala Arg Asp Ala
140 145 150
Ser Leu Leu Ser Thr Ala Gln Ala Leu Leu Arg Trp His Asp Ala
155 160 165
His Gln Phe Cys Ser Arg Ser Gly Gln Pro Thr Lys Lys Asn Val
15/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
170 175 180
Ala Gly Ser Lys Arg Val Cys Pro Ser Asn Asn Ile Ile Tyr Tyr
185 190 195
Pro Gln Met Ala Pro Val Ala Ile Thr Leu Val Ser Asp Gly Thr
200 205 210
Arg Cys Leu Leu Ala Arg Gln Ser Ser Phe Pro Lys Gly Met Tyr
215 220 225
Ser Ala Leu Ala Gly Phe Cys Asp Ile Gly Glu Ser Val Glu Glu
230 235 240
Thr Ile Arg Arg Glu Val Ala Glu Glu Val Gly Leu Glu Val Glu
245 250 255
Ser Leu Gln Tyr Tyr Ala Ser Gln His Trp Pro Phe Pro Ser Gly
260 265 270
Ser Leu Met Ile Ala Cys His Ala Thr Val Lys Pro Gly Gln Thr
275 280 285
Glu Ile Gln Val Asn Leu Arg Glu Leu Glu Thr Ala Ala Trp Phe
290 295 300
Ser His Asp Glu Val Ala Thr Ala Leu Lys Arg Lys Gly Pro Tyr
305 310 315
Thr Gln Gln Gln Asn Gly Thr Phe Pro Phe Trp Leu Pro Pro Lys
320 325 330
Leu Ala Ile Ser His Gln Leu Ile Lys Glu Trp Val Glu Lys Gln
335 340 345
Thr Cys Ser Ser Leu Pro Ala
350
<210> 12
<211> 385
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6945964CD1
<400> 12
Met Glu Glu Glu Lys Asp Asp Ser Pro Gln Ala Asp Phe Cys Leu
1 5 10 15
Gly Thr Ala Leu His Ser Trp Gly Leu Trp Phe Thr Glu Glu Gly
20 25 30
Ser Pro Ser Thr Met Leu Thr Gly Ile Ala Val Gly Ala Leu Leu
35 40 45
Ala Leu Ala Leu Val Gly Val Leu Ile Leu Phe Met Phe Arg Arg
50 55 60
Leu Arg Gln Phe Arg Gln Ala Gln Pro Thr Pro Gln Tyr Arg Phe
65 70 75
Arg Lys Arg Asp Lys Val Met Phe Tyr Gly Arg Lys Ile Met Arg
80 85 90
Lys Val Thr Thr Leu Pro Asn Thr Leu Val Glu Asn Thr Ala Leu
95 100 105
Pro Arg Gln Arg Ala Arg Lys Arg Thr Lys Val Leu Ser Leu Ala
110 115 120
Lys Arg Ile Leu Arg Phe Lys Lys Glu Tyr Pro Ala Leu Gln Pro
125 130 135
Lys Glu Pro Pro Pro Ser Leu Leu Glu Ala Asp Leu Thr Glu Phe
140 145 150
Asp Val Lys Asn Ser His Leu Pro Ser Glu Val Leu Tyr Met Leu
155 160 165
Lys Asn Val Arg Val Leu Gly His Phe Glu Lys Pro Leu Phe Leu
170 175 180
Glu Leu Cys Lys His Ile Val Phe Val Gln Leu Gln Glu Gly Glu
185 190 195
His Val Phe Gln Pro Arg Glu Pro Asp Pro Ser Ile Cys Val Val
16/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
200 205 210
Gln Asp Gly Arg Leu Glu Val Cys Ile Gln Asp Thr Asp Gly Thr
215 220 225
Glu Val Val Val Lys Glu Val Leu Ala Gly Asp Ser Val His Ser
230 235 240
Leu Leu Ser Ile Leu Asp Ile Ile Thr Gly His Ala Ala Pro Tyr
245 250 255
Lys Thr Val Ser Val Arg Ala Ala Ile Pro Ser Thr Ile Leu Arg
260 265 270
Leu Pro Ala Ala Ala Phe His Gly Val Phe Glu Lys Tyr Pro Glu
275 280 285
Thr Leu Val Arg Val Val Gln Ile Ile Met Val Arg Leu Gln Arg
290 295 300
Val Thr Phe Leu Ala Leu His Asn Tyr Leu Gly Leu Thr Thr Glu
305 310 315
Leu Phe Asn Ala Glu Ser Gln Ala Ile Pro Leu Val Ser Val Ala
320 325 330
Ser Val Ala Ala Gly Lys Ala Lys Lys Gln Val Phe Tyr Gly Glu
335 340 345
Glu Glu Arg Leu Lys Lys Pro Pro Arg Leu Gln Glu Ser Cys Asp
350 355 360
Ser Gly Thr Val Leu His Gln Gly Gly Gln Cys Pro Ala Pro Glu
365 370 375
Trp Gly Met Glu Trp Thr Glu Trp Ser Glu
380 385
<210> 13
<211> 1437
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2425607CB1
<400> 13
tgcgacggcg ggctggcctg gcgcgctgcg gacgtcgctc acccgctccc gaggaagggc 60
agtgggcccc gccgccgcct cccaatggcg aggctgcggg attgcctgcc ccgcctgatg 120
ctcacgctcc ggtccctgct cttctggtcc ctggtctact gctactgcgg gctctgcgcc 180
tccatccacc tgctcaaact tttgtggagc ctcggcaagg ggccgcgcag accttccggc 240
ggcccgcccg ggagcaccct ccccgcgtgc ctgagcgacc cctccttggg cacccactgc 300
tacgtgcgga tcaaggattc agggttaaga tttcactatg ttgctgctgg agaaagaggc 360
aaaccactta tgctgctgct tcatggattt cccgaattct ggtatatctc tggcggtaac 420
caactgagag aatttaaaag tgaatatcga gttgtagcac tggatttgag aggttatgga 480
gaaacagatg ctcccattca tcgacagaat tataaattgg attgtctaat tacagatata 540
aaggatattt tagattcttt agggtatagc aaatgtgttc ttattggcca tgactggggg 600
ggcatgattg cttggctaat tgccatctgt tatcctgaaa tggtgatgaa gcttattgtt 660
attaacttcc ctcatccaaa tgtatttaca gaatatattt tacgacaccc tgctcagctg 720
ttgaaatcca gttattatta cttcttccaa ataccatggt tcccagaatt tatgttctca 780
ataaatgatt tcaaggtttt gaaacatctg tttaccagtc acagcactgg cattggaaga 840
aaaggatgcc aattaacaac agaggatctt gaagcttata tttatgtctt ttctcagcct 900
ggagcattaa gtggcccaat taaccattac cgaaatatct tcagctgcct gcctctcaaa 960
catcacatgg tgaccactcc aacactacta ctgtggggag agaatgacgc attcatggag 1020
gttgagatgg ctgaagtcac aaagatttat gttaaaaact atttcaggct aactattttg 1080
tcagaagcca gtcattggct tcagcaagac caacctgaca tagtgaacaa attgatatgg 1140
acatttctaa aagaagaaac aagaaaaaaa gattgacttt tctttatctt ctatgaaggg 1200
tctgtaatga aatctctaaa taatttttaa aaattgttca tcaacttctt tatgttttat 1260
tagaaaaaaa ctgttttaat gtgctttatc ataaataaat atcctgacaa atggtattga 1320
aaaaaatctg agatcatgtg aacatataat acaatgttga ttttcttctg gtgtattttg 1380
cacagacaag catctgcctt aaaatatata cacttgtaca aaaaggaaaa aaaaaaa 1437
<210> 14
<211> 3269
17/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2786919CB1
<400> 14
cgccgccgcc gccgctggca aaaagcggag cccagggaag cgtgtcctgc tcagaccgcc 60
ttccttctcc attgccaccc gtgccgaaca gccaggctgc ccaattgcaa ctgtagacca 120
atgaactaat ccatcgcccg cagcccgact ctcagcagcg gttcgtcccg gtgcccaccc 180
ccgcgaagcg gagcgcccgg gcacctagcc tccctgccgg ccacctagcc tccctgccgg 240
ccacgatgcc gaacgtgctg ctgccgccca aggagagcaa cctcttcaaa cgcatcttga 300
aatgttatga acagaagcag tacaaaaatg gcctcaagtt ttgcaagatg attctgtcga 360
acccaaaatt tgctgaacat ggagagactt tggctatgaa aggattaaca ctgaactgtt 420
taggaaaaaa agaagaagct tatgagtttg ttcgtaaagg acttcgtaat gatgtcaaga 480
gtcatgtctg ttggcatgta tatggactct tgcagcgttc tgataaaaaa tatgatgaag 540
ctataaaatg ttaccgaaat gccctcaaat tagataaaga taacctgcaa attttgaggg 600
atctctcact gttgcagatc caaatgagag accttgaagg ttaccgagag acaagatacc 660
agcttcttca gttgcgcccc acacagcgtg cctcctggat tggatatgct attgcatacc 720
atttgctgaa agattatgat atggccctaa aactgttgga agaatttaga caaactcagc 780
aagttcctcc aaacaaaata gattatgaat atagtgaatt gatattatac cagaatcaag 840
tgatgagaga ggcagatctg ttgcaggaat ctttggaaca tatagaaatg tatgagaaac 900
aaatatgtga taaacttttg gtggaagaaa ttaaagggga aatactgttg aaattgggaa 960
gattaaaaga agccagtgaa gtgttcaaaa acttgattga tcgaaatgca gaaaactggt 1020
gttattatga aggcttggaa aaagctctac aaattagcac tttagaagag aggcttcaaa 1080
tttatgaaga aattagtaag cagcacccca aagcaattac acccagaaga ttacctttga 1140
ctcttgtccc aggtgaaaga tttagagaac taatggataa gttcctgagg gttaacttca 1200
gtaaaggctg cccacccttg tttactactt tgaaatcttt atattacaat acagaaaagg 1260
tttctataat ccaggaactt gttactaatt atgaagcctc tcttaaaacg tgtgactttt 1320
ttagcccata tgagaatggg gagaaggaac ccccgacaac actactctgg gttcagtatt 1380
tcctggcaca gcactttgat aaacttggac agtattcttt ggctttggat tatattaatg 1440
ctgcaattgc tagtactcca actctaatag aattattcta tatgaaagca aaaatttaca 1500
agcatatagg taatctcaaa gaagctgcaa agtggatgga tgaagcacag tctttggaca 1560
cagctgatag attcatcaat tccaaatgtg caaaatacat gcttcgagca aatatgataa 1620
aagaagcaga ggaaatgtgc tccaagttca caagggaagg aacatctgcc atggaaaatc 1680
taaatgaaat gcagtgtatg tggtttcaga cagaatgcat ttcagcttat cagcgtctgg 1740
ggagatacgg ggatgccttg aaaaaatgtc atgaagtaga aaggcatttt tttgagataa 1800
ctgatgacca attcgacttc catacatact gcatgagaaa gatgaccctt cgtgcctatg 1860
ttgacctttt gagattagaa gatatactca gaagacatgc cttttatttc aaggctgcta 1920
gatcagcgat tgaaatatac ttgaaattgt atgataatcc cttaaccaat gaaagcaaac 1980
aacaagaaat aaactcagaa aacttgtcag ccaaagaatt gaagaaaatg cttagcaagc 2040
agagaagagc tcagaaaaag gctaaactag aagaagaaag aaagcatgca gaaagagaac 2100
gtcaacagaa aaatcaaaag aaaaaaagag atgaagaaga agaagaagcc agtggcctta 2160
aggaagaact tatacctgaa aaattagaaa gggtagaaaa tccattagag gaagccgtta 2220
agttccttat acctcttaag aaccttgttg ctgataacat tgacactcat ctgttagcat 2280
ttgaaatata ttttagaaaa ggaaagtttc tgttaatgct gcagtctgtc aaacgagctt 2340
ttgccattaa cagtaataac ccatggttac atgaatgttt aattagattt tctaaatctg 2400
tgtctaatca tagtaatctt ccagacattg tgagcaaagt tctatctcaa gaaatgcaga 2460
aaatatttgt caaaaaggat ttggaaagtt ttaatgagga ttttctgaaa cgtaacgcta 2520
cctctcttca gcatctactt tcaggtgcta aaatgatgta ttttctggac aagtcaaggc 2580
aggagaaagc aattgctata gccactagac tagatgaaac tataaaagat aaagatgtaa 2640
agacattaat aaaggtttct gaagcactgc ttgatggcag ctttgggaac tgtagttccc 2700
aatatgaaga atataggatg gcctgtcata acctgcttcc ttttacatct gccttcttgc 2760
ctgctgtgaa tgaagtcgac aatcctaatg tggcactgaa ccatacagct aattatgatg 2820
tcttggcaaa tgaaatttga aatcttctag aaagtagact cctatagact caaagctgaa 2880
ttgagatcag ggtttctttt ccaggttgca ttttaatata cgtatgaaat gaaatatttg 2940
gttaggattt ttaaatggca tattctgtaa gcttattttg ttctttaccc gacctgccaa 3000
tttacataac catctgttaa aattacctgt ttattcttac acagttttgt ggtagctccg 3060
atcgcttctg tataattata atttcttact aagctaagtt gtatacaacc ccactgtatt 3120
attttcttta gattctgatt tcaggatctg tgctttgatt ccttgtctgt tttatcgtat 3180
atttgcttgc tttttaatta aagcacatca gttttaaagt gttaactata tgttaacaat 3240
atggaggctg ggcgtggtgg ctcacacct 3269
18/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
<210> 15
<211> 1534
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1801130CB1
<220>
<221> unsure
<222> 91
<223> a, t, c, g, or other
<400> 15
atggaggaaa aaggaataaa gaggatttca aaaccgctag gagtgaagcg agaaggtaaa 60
ggatataaag aatataagag gtttcaggct ncaggtcctg gcaggaggat cgcgggccag 120
aggcggagcc accatcctac cgcgactttc agactccgac cgtggcctcg cgttggtgac 180
gggggcggct cggctgttgc tggagaccgg cggcgtgaga ccgctccccg agccttgcgg 240
ggccgccggg tccgcgcgcc gctgccgatg tccgcgcagg ttcataggca gaagggactt 300
gacttgtctc agatacccta ttttaatctt gtgaagcatt taacccctgc ctgtccaaat 360
gtatatagta tatcacagtt tcatcacaca accccagaca gtaaaacaca cagtggtgaa 420
aaatacaccg atcctttcaa actcggttgg agagacttga aaggtctgta tgaggacatt 480
agaaaggaac tgcttatatc aacatcagaa cttaaggaaa tgtctgagta ctactttgat 540
gggaaaggga aagcctttcg accaattatt gtggcgctaa tggcccgagc atgcaatatt 600
catcataaca actcccgaca tgtgcaagcc agccagcgcg ccatagcctt aattgcagaa 660
atgatccaca ctgctagtct ggttcacgat gacgttattg acgatgcaag ttctcgaaga 720
ggaaaacaca cagttaataa gatctggggt gaaaagaagg ctgttcttgc tggagattta 780
attctttctg cagcatctat agctctggca cgaattggaa atacaactgt tatatctatt 840
ttaacccaag ttattgaaga tttggtgcgt ggtgaatttc ttcagctcgg gtcaaaagaa 900
aatgagaatg aaagatttgc acactacctt gagaagacat tcaagaagac cgccagcctg 960
atagccaaca gttgtaaagc agtctctgtt ctaggatgtc ccgacccagt ggtgcatgag 1020
atcgcctatc agtacggaaa aaatgtagga atagcttttc agctaataga tgatgtattg 1080
gacttcacct cgtgttctga ccagatgggc aaaccaacat cagctgatct gaagctcggg 1140
ttagccactg gtcctgtcct gtttgcctgt cagcagttcc cagaaatgaa tgctatgatc 1200
atgcgacggt tcagtttgcc tggagatgta gacagagctc gacagtatgt actacagagt 1260
gatggtgtgc aacaaacaac ctacctcgcc cagcagtact gccatgaagc aataagagag 1320
atcagtaaac ttcgaccatc cccagaaaga gatgccctca ttcagctttc agaaattgta 1380
ctcacaagag ataaatgaca actctttctg ttctttctgg cagctattct taccagactg 1440
tgcctaaaga attttgtgga atacactttg tttgcttcat gtgcagatga ccaaaaatca 1500
ttttaaagat atcaaactta ttgatgggca ttta 1534
<210> 16
<211> 3400
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3535146CB1
<400> 16
ggcacgcgcc ccgccgccct cggcagccgc agctccgtgt ccctgagaac cagccgtccc 60
gcgccatggg cacgcgtctg ccgctcgtcc tgcgccagct ccgccgcccg ccccagcccc 120
cgggccctcc gcgccgcctc cgtgtgccct gtcgcgctag cagcggcggc ggcggaggcg 180
gcggcggtgg ccgggagggc ctgcttggac agcggcggcc gcaggatggc caggcccgga 240
gcagctgcag ccccggcggc cgaacgcccg cggcgcggga ctccatcgtc agagaagtca 300
ttcagaattc aaaagaagtt ctaagtttat tgcaagaaaa aaaccctgcc ttcaagccgg 360
ttcttgcaat tatccaggca ggtgacgaca acttgatgca ggaaatcaac cagaatttgg 420
ctgaggaggc tggtctgaac atcactcaca tttgcctccc tccagatagc agtgaagccg 480
agattataga tgaaatctta aagatcaatg aagataccag agtacatggc cttgcccttc 540
agatctctga gaacttgttt agcaacaaag tcctcaatgc cttgaaacca gaaaaagatg 600
tggatggagt aacagacata aacctgggga agctggtgcg aggggatgcc catgaatgtt 660
19/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
ttgtttcacc tgttgccaaa gctgtaattg aacttcttga aaaatcaggt gtcaacctag 720
atggaaagaa gattttggta gtgggggccc atgggtcttt ggaagctgct ctacaatgcc 780
tgttccagag aaaagggtcc atgacaatga gcatccagtg gaaaacacgc cagcttcaaa 840
gcaagcttca cgaggctgac attgtggtcc taggctcacc taagccagaa gagattcccc 900
ttacttggat acaaccagga actactgttc tcaactgctc ccatgacttc ctgtcaggga 960
aggttgggtg tggctctcca agaatacatt ttggtggact cattgaggaa gatgatgtga 1020
ttctccttgc tgcagctctg cgaattcaga acatggtcag tagtggaagg agatggcttc 1080
gtgaacagca gcacaggcgg tggagacttc actgcttgaa acttcagcct ctctcccctg 1140
tgccaagtga cattgagatt tcaagaggac aaactccaaa agctgtggat gtccttgcca 1200
aggagattgg attgcttgca gatgaaattg aaatctatgg caaaagcaaa gccaaagtac 1260
gtttgtccgt gctagaaagg ttaaaggatc aagcagatgg aaaatacgtc ttagttgctg 1320
ggatcacacc cacccctctt ggagaaggga agagcacagt caccatcggg cttgtgcagg 1380
ctctgaccgc acacctgaat gtcaactcct ttgcctgctt gaggcagcct tcccaaggac 1440
cgacgtttgg agtgaaagga ggagccgcgg gtggtggata tgcccaggtc atccccatgg 1500
aggagttcaa ccttcacttg actggagaca tccacgccat caccgctgcc aataacttgc 1560
tggctgccgc catcgacacg aggattcttc atgaaaacac gcaaacagat aaggctctgt 1620
ataatcggct ggttccttta gtgaatggtg tcagagaatt ttcagaaatt cagcttgctc 1680
ggctaaaaaa actgggaata aataagactg atccgagcac actgacagaa gaggaagtga 1740
gtaaatttgc ccgtctcgac atcgacccat ctaccatcac gtggcagaga gtattggata 1800
caaatgaccg atttctacga aaaataacca tcgggcaggg aaacacagag aagggccatt 1860
accggcaggc gcagtttgac atcgcagtgg ccagcgagat catggcggtg ctggccctga 1920
cggacagcct cgcagacatg aaggcacggc tgggaaggat ggtggtggcc agtgacaaaa 1980
gcgggcagcc tgtgacagca gatgatttgg gggtgacagg tgctttgaca gttttgatga 2040
aagatgcaat aaaaccaaac ctgatgcaga ccctggaagg gacacctgtg ttcgtgcatg 2100
cgggcccttt tgctaacatt gctcacggca actcttcagt gttggctgat aaaattgccc 2160
tgaaactggt tggtgaagaa ggatttgtag tgaccgaagc tggctttggt gctgacatcg 2220
gaatggagaa attcttcaac atcaagtgcc gagcttccgg cttggtgccc aacgtggttg 2280
tgttagtggc aacggtgcga gctctgaaga tgcatggagg cgggccaagt gtaacggctg 2340
gtgttcctct taagaaagaa tatacagagg agaacatcca gctggtggca gacggctgct 2400
gtaacctcca gaagcaaatt cagatcactc agctctttgg ggttcccgtt gtggtggctc 2460
tgaatgtctt caagaccgac acccgcgctg agattgactt ggtgtgtgag cttgcaaagc 2520
gggctggtgc ctttgatgca gtcccctgct atcactggtc ggttggtgga aaaggatcgg 2580
tggacttggc tcgggctgtg agagaggctg cgagtaaaag aagccgattc cagttcctgt 2640
atgatgttca ggttccaatt gtggacaaga taaggaccat tgctcaggct gtctatggag 2700
ccaaagatat tgaactctct cctgaggcac aagccaaaat agatcgttac actcaacagg 2760
gttttggaaa tttgcccatc tgcatggcaa agacccacct ttctctatct caccaacctg 2820
acaaaaaagg tgtgccaagg gacttcatct tacctatcag tgacgtccgg gccagcatag 2880
gcgctgggtt catttaccct ttggtcggaa cgatgagcac catgccagga ctgcccaccc 2940
ggccctgctt ttatgacata gatcttgata ccgaaacaga acaagttaaa ggcttgttct 3000
aagtggacaa ggctctcaca ggacccgatg cagactcctg aaacagacta ctctttgcct 3060
ttttgctgca gttggagaag aaactgaatt tgaaaaatgt ctgttatgca atgctggaga 3120
catggtgaaa taggccaaag atttcttctt cgttcaagat gaattctgtt cacagtggag 3180
tatggtgttc ggcaaaagga cctccaccaa gactgaaaga aactaattta tttctgtttc 3240
tgtggagttt ccattatttc tactgcttac actttagaat gtttatttta tggggactaa 3300
gggattaaga gtgtgaacta aaaggtaaca ttttccactc tcaagttttc tactttgtct 3360
ttgaactgaa aataaacatg gatctagaaa accaaaaaaa 3400
<210> 17
<211> 3395
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1436543CB1
<400> 17
caggtctggg ctacaaaagt atggccgctt ctgaggcggc ggtggtgtct tcgccgtctt 60
tgaaaacaga cacatcccct gtccttgaaa ctgcaggaac ggtcgcagca atggctgcga 120
ccccgtcagc aagggctgca gccgcggtgg ttgcggccgc ggccaggacc ggatccgaag 180
ccagggtctc caaggccgct ttggctacca agctgctgtc cttgagcggc gtgttcgccg 240
tgcacaagcc caaagggccc acttcagccg agctgctgaa tcggttgaag gagaagctgc 300
tggcagaagc tggaatgcct tctccagaat ggaccaagag gaaaaagcag actttgaaaa 360
20/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
ttgggcatgg agggactcta gacagcgcag cccgaggagt tctggttgtt ggaattggaa 420
gcggaacaaa aatgttgacc agtatgttgt cagggtccaa gagatatact gccattggag 480
aactggggaa agctactgat acactagatt ctacggggag ggtaacagaa gaaaaacctt 540
acgataaaat aacacaagaa gatattgaag gcattctaca gaaatttact ggaaatataa 600
tgcaagtgcc ccccctctat tctgcattaa agaaagatgg acaaagactt tcgactttga 660
tgaagagagg tgaagtcgta gaagcaaaac ctgccaggcc agtgactgta tacagtatct 720
cccttcaaaa attccagcca ccatttttca cattagatgt tgaatgtgga ggaggttttt 780
atatcagaag cttggtcagt gacattggaa aagaactatc ttcctgtgcc aatgtgctag 840
agctgacccg aaccaaacag ggaccattta cgctagaaga acatgccctt cctgaagaca 900
aatggacaat tgatgacatt gcacagtctc ttgagcattg ctcatctctt ttcccagcag 960
agttggcact taaaaaatca aaacctgagt ctaatgaaca ggttttgagc tgtgaatata 1020
taactctaaa tgagccaaag agagaagatg atgtaattaa gacgtgttga gattggcctg 1080
ggaatatcat cattttctag ttgacatttg aatcctgtgt gcagatgcag aatgacaagc 1140
tgcattcaaa agacaaacaa tatgtctttt ttttttttgc atgaagaaaa atgtctatca 1200
tttacagttt caatagcaca taatttattt tctatgcatt ataaatggcc ttgcagttgg 1260
ctcagttgtt tgctgtgttg tgaaatgttt taggattttt tgtattgtga aaatatgaat 1320
atgattggat tcagaaaaat taactttctg aatttgatct gtcttcagtc ttgtgaaaaa 1380
gttgaacaaa tttcctaatc aaagaaaaaa gtatgagctc catgtttctt tagtttcaca 1440
aaaatgacca taatttagtg ttatttttac tttatttagg cttcctggtg gcttcatttt 1500
attgaaattc tttaaattgt ttaaagtggc cattattgat ctctttcttc tgttttggag 1560
agtttattat taaaaacatt tctttgataa aatggccatc atctagtaat acctgtgttt 1620
gtttagatct tggaaatgaa taagctttga taatatttgt aaatgaacca aattattact 1680
gctaccacta acaggttgta aatagaagac taatacttaa ttaaagtcac cttcctacca 1740
ttagagcaga agacagctcc tatagttttg tattttggca gctatgagat attttcatgg 1800
taatgtcaac atggtcaagc actttgtacc aagttattaa gtaacataat ttttaaaatt 1860
taaagaatgt gtcttcaact aaaaacttta ttctttagca tttatttata tttctctgta 1920
gggtgttccc tgtgacattg tctctttagt ttgctctttc aagagatact tacagatgtt 1980
gagatggctg ccctgcattt ccagctaatc tcttctgctc taaatattta aaaacagttc 2040
ttctcaaaca ttttcattca gatagctttc tgaaagttcc ctatccctct ttaccataat 2100
tttttaaatg tagccacatt gtaatagtaa acttcatata taatgagtgc ttcatatttt 2160
tgttatggga aagcaatata ttatgcagcc agtctgtaga aacattcaga tccctcttcc 2220
tttactcaaa tacagtttca aaaggaagac tcatgagaaa tttcataaaa tacaagtttt 2280
tagatgttta tgctttgcct ttctttttaa aggtgttttc ctgctttgta gtctctaact 2340
ctgaaattta aaatatgtaa actaaagtgg ttttatttgt gcttaaccca atttaaactc 2400
aatgtaaaat gttatatatg catcagtaca gcattttcaa catattggca acatatttta 2460
aatgaaaaca ctaaaacaat tcttagtatg agacaaaact gtaaggaaaa agagtgttaa 2520
taccatgatg cattaacata aaatatcaaa cacacaaagt cataaaatga aaatttacag 2580
ttttacctgt tcatatctag tgccccacag tgtgtgtcaa ccaaaggtgg cagtggctac 2640
atctgcctgt tggactggta caggttacaa tatgtcctct tccattgcaa attaaagtcc 2700
aaatagagaa atacttaggt tttagaacac atcagaggta tttctgctgt atttttcacc 2760
ttaaaaattg acacagagtt tactaataga ggagtagaga ttgttgacca tttttaaaaa 2820
acgatagcca ctctttttct tttatgttta aaactgaagt tttgccaaat gggaaaatta 2880
ctgttacctc taccatctta atgtagtaac tttagaattt aaatttttat attactattt 2940
tcctttttgt tgttcacata gtcttaaggc acctatactt ttaaattgac tttttcattt 3000
gatattatct atatgtatgt agttgtgata atgattattt taattatatt actttatact 3060
cttaatttat ttagagtatt tctctattgc tgaatactta agtagtttta aattttatta 3120
tgataaattc ctgggagggg gattatttag tgaaataata tgaagaactt tatgacttat 3180
gtttgcctta ttgcattccc aaagagttgt aacattttac agtgttacca tttgagtagg 3240
ggttttatat gttgttgcta atttagtaaa cataggagag aaatcaaagt ttttctgatt 3300
tgcttttatg tgatttatct gtatactttg ttcatttata taaataaatg tcttaatggt 3360
ttctatacat aaaaaaaaaa aaaaaaaaaa aaaaa 3395
<210> 18
<211> 2283
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7491063CB1
<400> 18
gtactatttt gccgcttcct gtgagtctat aattatttca aaataaaaag attatttaaa 60
21/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
aaagaatgtg ggggccagga gcattggctc atgcctataa tccaagcact ttgggaggct 120
gaggtgggcg gattgcttga gctcaggagt ttgagaccag cctgggcaac atggcaaaac 180
cctgtctcta aaaaaagaaa aaagaaaaag aatgtgggga aaaatcattt ggagacttaa 240
aatgtagaaa caaagaacat aaatgtggta taagaagttt taggtgacaa tatagttttg 300
aggctaaacc aaaaaggaaa aggagatata tctgactgta tatctaagaa ctcaccaata 360
agaaagacgt gggccctgct ctgttgcctg cctttgctcc ccaggccccg agtggttcac 420
tgagccatga agacagattc caggcaccag gaattgcacc tccaatccca gatgctatgt 480
tcagcaaagg ctccatggtt ctggcctaca gtgccggcct ggacacctcc tgcatcctcg 540
tgtggctgaa ggaacaaggc tatgacatta ttgcctacct ggccaatgtt ggccagaagg 600
aagacttcaa ggaagccagg aagaaggcac tgaaccttgg gtccaaaaag gtgttcattg 660
aggacgtcag caaggagttc gtggaggagt tcatctggcc ttatgaggac tgctacctcc 720
tgggcccctc tcttcccagg ccttacatca cccgcaaaca agtggaaatt gctcagtggg 780
agggggcaaa gtatgtgtcc cacagtgcca tgggaaaggg gaacgatcag gtctggtttg 840
agctcgcctg ctactcgctg gccccccaga ttaaggtcat tgctcccggg aggatccctg 900
agttctacaa ccagtccaag ggccgcagtg atctgatgga atatgcagag aaacatggga 960
ttcccatccc agtcactctg aagcacccat ggaacatgga cgagaacctc atgcacatca 1020
gccacgaggg ctggaatctt ggagaacccc aagaaccaga ggcaccttca ggtctctaca 1080
tgaagattca ggacctggcc aaagccccca acacccccaa cattttcaag actgaggggg 1140
tccctgtgaa ggtgaccagc atcaaggatg gcaccaccca ccagacctcc ttggtgctct 1200
tacatgtaac ctggaatgaa gtcgcaggca agcacagcgt gggccatatt gacatcgtgg 1260
agaaccgctt cattgaaatg aatatctgca agaccccagc aggcaccatc ctttaccacc 1320
ctcatttaga cattgagggc tttgccatgg aacaggaagt gcgcaaaatc aaacaaggcc 1380
tgggcttgaa atttgctgag ttggtgtaca ccggtttctg gcacaaccct cagtgtgact 1440
ttgcccacca ctgcattgcc aagtcccagg accgagtgga agggaaagtg caggtatcca 1500
tcttcaaggg ccaggtgtac atcctctgcc aggagccccc actgtctctc tacagtggag 1560
agcaggtgag catgaacgtg gagggcaatg atgagccagc cagtcgtctc atcaacatca 1620
attccctcag gatgaaggaa tatcatcatc tccagagcaa ggtcactgcc aaatagaccc 1680
ctgtacaatg gggagctagg gccacctcac tttgcagatt ccccaagtac aggcactaat 1740
tgttgtgata atttgtaatt gtgacttgtt ctccctggct aagagtgtag tggggctgcc 1800
gggccccagc tttgttccct ggtccccctg aaatggtcat caaagggaag ggtgaggggc 1860
agctacagtg gggagctata aaatgacaat taaaaaaaaa aaaaaaaaag actaaggcca 1920
gccacggggg ctcacgtcgg taatcccggc acttggggag accgaggtgg gcaagatcac 1980
cggaggtcag gcagtctggc gcaccagccg ggccacaacg tggggaaacc ccatctctac 2040
gtaaagcaat cccacaaatt agcggggcgc ggtgttgtgt gcctggtaaa cccagcaaat 2100
ggcgagggct tgaggccaga agaatagctg ggagcctggg cagccaacgg gtcgacggag 2160
gcagaaattc ggcacatgga cagcccagcg ggggtcaaga catcgactcc gcgctagaaa 2220
cacagaacaa gcgacagaga gcggctcaac aacctgtcga aacggcgcaa ggatgcgcga 2280
caa 2283
<210> 19
<211> 1193
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7625645CB1
<400> 19
gaagaacttc atggtcggcg caggcagcat ccaggcggcg gtcgagaact tcgtgcgtga 60
ggtgaaggcg cgcaccttcc caagtccgga atactgctac tgacgcggcc tcaaggcgcg 120
tactcgtccg tcatggagat catccggacc atagccgagc tgcgggttcg tctcgcacgc 180
gaatcgaaca tcgcgctggt gcccaccatg ggcaacatcc acgacggcca cctgagcctc 240
gtgcgcattg gtctcaagcg cgccccgttc acggtgacca gcatcttcgt gaaccgcctc 300
cagttcatcc agggcgagga cttcgacaag tacccgcgca ccttcgagga ggattgcgag 360
aagctcgagg cggccggcaa cagcgtggtg ttcgcgccgg acgaaaccca gatgtatcct 420
gagcggcagg tgttcatcgt cgagccgccg cccatcgcga acaagctgga gggccgcttt 480
cgtccaggac atttccgcgg cgtgtccacg gtcgtcctca agctcttcaa catggtgcaa 540
ccgcaggtcg cggtgttcgg caagaaggac taccagcagc tttccatcgt gcgccatatg 600
gtggaccagc tcgcgttgcc cctgtccatc attcccgccg agacggtgcg cgccgaagat 660
ggcctggcac tttcgtcgcg caaccgctac ctgagccccg aggaacgcag cgaggccccg 720
cgcctcaacg aagcgctgcg ccaggtgaag caggccgtgg aatcgggtga tcgcaacttc 780
gaggcgctgg agttcgcagc cgatgcgctg cttgcgcgcc atcgttggca cgtggactat 840
22/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
gtcgtggtgc gcagccgccg cacgctgatg cagccggacc cggacgagcg cgaactggtg 900
gtgctgggtg cggcgcgtct tggacagacg cggctcatcg acaacctgga gatcctggcg 960
cccgcctgaa cgtcggcagg gcgctcatga aggcaagccc atgagccgtc ctgcaccgtt 1020
tcccttttcg gccatcgtcg gtcaggacga gatgaagctc gccctgctga tcgcggccgt 1080
ggacccgacc gtcggtgggg tgctcgtgtt cggcgatcgc ggtaccggca agtccacagc 1140
cgtgcgggca ctcgcggctc tgctgccacc catgcgggcg gtcgcgggtt gcc 1193
<210> 20
<211> 2092
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5730123CB1
<400> 20
cggacgcgtg ggcggacgcg tggggggcag gtctggcggg actgcgcggc ggcaacagca 60
gacatgtcgg gggtccgggg cctgtcgcgg ctgctgagcg ctcggcgcct ggcgctggcc 120
aaggcgtggc caacagtgtt gcaaacagga.acccgaggtt ttcacttcac tgttgatggg 180
aacaagaggg catctgctaa agtttcagat tccatttctg ctcagtatcc agtagtggat 240
catgaatttg atgcagtggt ggtaggcgct ggaggggcag gcttgcgagc tgcatttggc 300
ctttctgagg cagggtttaa tacagcatgt gttaccaagc tgtttcctac caggtcacac 360
actgttgcag cacagggagg aatcaatgct gctctgggga acatggagga ggacaactgg 420
aggtggcatt tctacgacac cgtgaagggc tccgactggc tgggggacca ggatgccatc 480
cactacatga cggagcaggc ccccgccgcc gtggtcgagc tagaaaatta tggcatgccg 540
tttagcagaa ctgaagatgg gaagatttat cagcgtgcat ttggtggaca gagcctcaag 600
tttggaaagg gcgggcaggc ccatcggtgc tgctgtgtgg ctgatcggac tggccactcg 660
ctattgcaca ccttatatgg aaggtctctg cgatatgata ccagctattt tgtggagtat 720
tttgccttgg atctcctgat ggagaacggg gagtgccgtg gtgtcatcgc actgtgcata 780
gaggacgggt ccatccatcg cataagagca aagaacactg ttgttgccac aggaggctac 840
gggcgcacct acttcagctg cacgtctgcc cacaccagca ctggcgacgg cacggccatg 900
atcaccaggg caggccttcc ttgccaggac ctagagtttg ttcagttcca ccccacaggc 960
atatatggtg ctggttgtct cattacggaa ggatgtcgtg gagagggagg cattctcatt 1020
aacagtcaag gcgaaaggtt tatggagcga tacgcccctg tcgcgaagga cctggcgtct 1080
agagatgtgg tgtctcggtc gatgactctg gagatccgag aaggaagagg ctgtggccct 1140
gagaaagatc acgtctacct gcagctgcac cacctacctc cagagcagct ggccacgcgc 1200
ctgcctggca tttcagagac agccatgatc ttcgctggcg tggacgtcac gaaggagccg 1260
atccctgtcc tccccaccgt gcattataac atgggcggca ttcccaccaa ctacaagggg 1320
caggtcctga ggcacgtgaa tggccaggat cagattgtgc ccggcctgta cgcctgtggg 1380
gaggccgcct gtgcctcggt acatggtgcc aaccgcctcg gggcaaactc gctcttggac 1440
ctggttgtct ttggtcgggc atgtgccctg agcatcgaag agtcatgcag gcctggagat 1500
aaagtccctc caattaaacc aaacgctggg gaagaatctg tcatgaatct tgacaaattg 1560
agatttgctg atggaagcat aagaacatcg gaactgcgac tcagcatgca gaaggtgcgg 1620
attgatgagt acgattactc caagcccatc caggggcaac agaagaagcc ctttgaggag 1680
cactggagga agcacaccct gtcctttgtg gacgttggca ctgggaaggt cactctggaa 1740
tatagacccg taatcgacaa aactttgaac gaggctgact gtgccaccat cccgccagcc 1800
attcgctcct actgatgaga caagatgtgg tgatgacaga atcagctttt gtaattatgt 1860
ataatagctc atgcatgtgt ccatgtcata actgtcttca tacgcttctg cactctgggg 1920
aagaaggagt acattgaagg gagattggca cctagtggct gggagcttgc caggaaccca 1980
gtggccaggg agcgtggcac ttacctttgt cccttgcttc attcttgtga gatgataaaa 2040
ctgggcacag ctcttaaata aaatataaat gaacaaaaaa aaaaaaacag gg 2092
<210> 21
<211> 4213
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7481031CB1
<400> 21
23/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
ggttgggtgg ggctttgggt cccatccatt cccgagcact gcggccctgg gcgaagggta 60
gggttggagg gatggggcag gattgactct gggctgcccg gcagaatcat gcggaagcag 120
ccgaccgtcg tcctgggtgt ggcgcacact gtggtgaaga ggatgtcgac cttcgtgcgg 180
caaatcgact ttgccctgga ctgggtggag gtggaggccg ggcgagcaat atacaggcag 240
ggggacaagt ccgactgcac gtacatcatg ctcagcggcc ggctgcgctc tgtgatccgg 300
aaggatgatg ggaagaagcg cctggccggg gagtacggcc gaggagacct cgtcggcgtg 360
cctggtggtt ccagctccat ccaccggatg aaggctcaca ctccaggccg gatgccggca 420
gggaggcctc agtgccagag ggcagggctc ccgcaggctg agggacacat cttaaaaaga 480
ggagttctcc aggccgggcg cggcggttca cgcctgtaat cccagcactt tgggaggccg 540
aggcggcgga ccatgaggtc aggagatcga gaccatcctg gtatgtggga ggcgggattg 600
ggcggggagg ggccagggct agtcagaggc cggcactcgc ttgcactcac ctgctggccc 660
ggcagatgca gaggcctggt ggccggtggg ctggtccaga gcgcgttcag gtgcgcaggg 720
cactgtgggg accctgctcc ctgcgtggcc ccgaatgggc tgcaccagac accagggagt 780
gcgtgcaaac aaggttgctt acggctgcct ccccagcaga tcacgggggc ggccgcccgg 840
cagctgctgg gcccctgctg aagaggagcc actccgtccc cgcgccttcc attcgcaaac 900
agatcttgga ggagctggag aagcccgggg caggtgaccc tgacccttcg gccccacaag 960
ggggcccagg cagtgccact tctgatctgg ggatggcatg tgaccgtgcc agggtcttcc 1020
tgcactcgga cgagcacccc gggagctccg tggccagcaa gtccaggaaa agcgtgatgg 1080
ttgcagagat accctccacg gtctcccagc actcagagag tcacacggat gagaccctgg 1140
ccagcaggaa gtcggatgcc atcttcagag ctgccaagaa ggacctgctc accctgatga 1200
agctggaaga ctcatctctg ttggatggcc gggtggcgct tctgcacgtt cctgcaggca 1260
cggtggtgtc aaggcaggga gaccaggacg ccagcatcct gttcgtggtc tcggggctgc 1320
tgcacgtgta ccagcggaag atcggcagcc aggaggacac ctgcttgttc ctcacgcgcc 1380
ccggggagat ggtgggccag ctggccgtgc tcaccgggga gcctctcatc ttcaccgtca 1440
aggccaacag ggactgcagc ttcctgtcca tctccaaggc ccacttctat gaaatcatgc 1500
ggaagcagcc gaccgtcgtc ctgggtgtgg cgcacactgt ggtgaagagg atgtcgtcct 1560
tcgtgcggca aatcgacttt gccctggact gggtggaggt ggaggccggg cgagcaatat 1620
acaggcaggg ggacaagtcc gactgcacgt acatcatgct cagcggccgg ctgcgctctg 1680
tgatccggaa ggatgatggg aagaagcgcc tggccgggga gtacggccga ggagacctcg 1740
tcggcgtggt ggagacactg acccaccagg cccgggcgac cacggtgcat gccgttcggg 1800
actcagaatt ggccaagctg ccggcaggag ccctcacgtc catcaagcgc aggtacccac 1860
aggtggtgac tcggctgatt catctcttgg gtgagaagat cctgggcagc ctccagcagg 1920
gacctgtgac aggccaccag cttgggctcc ccacggaggg cagcaagtgg gacttgggga 1980
acccggctgt caacctgtcc acggtggcag tgatgcccgt gtcagaggaa gtgcccctca 2040
ccgccttcgc cctggagctg gagcatgccc tcagcgccat cggcccgacc ctgctgctga 2100
ctagtgacaa cataaaacgg cgccttggct ccgctgccct ggacagtgtt cacgagtacc 2160
ggctgtccag ctggctgggg cagcaggagg acacccacag gatcgtgctc taccaggcag 2220
atggcacgct cacaccctgg acccagcgct gcgtgcgcca ggccgactgc atcctcatcg 2280
tgggcctggg tgaccaggag cccacagtgg gcgagctgga gcggatgctg gagagcacag 2340
ctgtgcgtgc ccagaagcag ctgatcctgc tgcacaggga ggagggcccg gcgccagcgc 2400
gcaccgtgga gtggctcaac atgcggagct ggtgctccgg ccacctgcac ctctgctgcc 2460
cgcgccgcgt cttctccagg aggagcctgc ccaagctggt ggagatgtac aagcatgtct 2520
tccagcggcc cccggaccga cactcagact tctcccgcct ggcgagggtg ctgacgggca 2580
acgccattgc cctggtgctt gggggagggg gagcaagagg ctgtgcccag gtgggcgttc 2640
tcaaggcctt ggcggagtgc ggcatccctg tggacatggt gggaggcacg tccatcgggg 2700
ccttcgtggg tgccctgtac tctgaggagc ggaactacag ccagatgcgg atccgggcca 2760
agcagtgggc cgagggcatg acgtccttga tgaaggccgc gctggacctc acctacccca 2820
tcacgtccat gttctccgga gccggcttca acagcagcat cttcagcgtc ttcaaggacc 2880
agcagatcga ggacctgtgg attccttatt tcgccatcac caccgacatc acagcctcgg 2940
ccatgcgggt ccacaccgac ggctccctgt ggcggtacgt gcgtgccagc atgtccctgt 3000
ccggttacat gccccctctc tgtgacccga aggacggaca cctgctgatg gacgggggct 3060
acatcaacaa cctcccagcg gatgtggccc ggtccatggg ggcaaaagtg gtgatcgcca 3120
ttgacgtggg cagccgagat gagacggacc tcaccaacta tggggatgcg ctgtctgggt 3180
ggtggctgct gtggaaacgc tggaacccct tggccacgaa agtcaaggtg ttgaacatgg 3240
cagagattca gacgcgcctg gcctacgtgt gttgcgtgcg gcagctggag gtggtgaaga 3300
gcagtgacta ctgcgagtac ctgcgccccc ccatcgacag ctacagcacc ctggacttcg 3360
gcaagttcaa cgagatctgc gaagtgggct accagcacgg gcgcacggtg tttgacatct 3420
ggggccgcag cggcgtgctg gagaagatgc tgcgcgacca gcaggggccg agcaagaagc 3480
ccgcgagtgc ggtcctcacc tgtcccaacg cctccttcac ggaccttgcc gaaattgtgt 3540
ctcgcattga gcccgccaag cccgccatgg tggatgacga atctgactac cagacggagt 3600
acgaggagga gctgctggac gtccccaggg atgcatacgc agacttccag agcacctcag 3660
cccagcaggg ctcagacttg gaggacgagt cctcactgcg gcatcgacac cccagtctgg 3720
ctttcccaaa actgtctgag ggctcctctg accaggacgg gtagaggcct ctgctaaaga 3780
24/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
gcccggatgc agcgtcttcc gtgggactgt ccccaaggct gaggctcctg ccaagtccta 3840
ggggcctctg tacctgccct gctggaagcc ctgacttccc cggggcccca ggctgtgtta 3900
gggttctctg ggcctcttct ttgtaccagc agccctgcat acagggccct gtgagccccc 3960
ctgcagtcct gtgaggcccc tgaagctctg tgaggcccct gaagctctgt gaaccccctg 4020
cagccctgtg aggccccccg aagccctgtg aggccccccg aagccctgtg aaccacctgc 4080
tgccctgtga ggcccccaaa gccctgtgaa ctgcctgctg tcctgtgaac tgcctgctgc 4140
cctgtgaggt gtgggagccc tgatgctgcc gtgtgatgtt tcaataaagg tggatctcac 4200
tgttaaaaca aaa 4213
<210> 22
<211> 1784
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7491216CB1
<400> 22
gcttcctctc agcccgctct gagctggaag cagcatgtgg gacctggccc tgatcttcct 60
cgcagcagcc tgcgtgttct cactaggggt cactctgtgg gtcatttgca gccatttttt 120
cactgtgcac atccctgcag cggttggcca ccctgtgaaa ctgagagtcc tccattgcat 180
cttccagctg ctgttgactt gggggatgat atttgagaag ctcagaatct gttctatgcc 240
ccaatttttc tgtttcatgc aagatctgcc tccgctaaag tatgaccccg atgttgtggt 300
cacggatttc cgctttggga caatccctgt gaagctgtac caacccaagg catccacctg 360
caccctgaag cctggcatcg tgtactacca cggtggcggg ggcgtcatgg ggagtttgaa 420
aacccaccat ggcatatgct ctcgtttgtg caaggagagt gactccgtgg ttctggcagt 480
tggttaccgc aagttaccta agcataagtt tccagtgcca gtaagagact gcttggtggc 540
caccatccac ttcctgaagt ccctggatgc atatggagtg gatccagccc gggttgtggt 600
ctgcggtgac agtttcggag gggcaatagc cgcagtggtt tgtcaacaac ttgtggacag 660
gccagatctg ccccggatcc gggctcagat cctgatctat gccattctcc aagccctgga 720
tttacaaacc ccttcgtttc aacagaggaa aaacatccca ctgctcacct ggagtttcat 780
ctgctactgt ttttttcaaa acctggattt cagctcctcc tggcaagagg tcatcatgaa 840
aggtgcccat ttgcctgctg aagtctggga aaagtacaga aagtggttgg gcccagaaaa 900
catccctgag aggtttaagg agaggggtta ccaactgaag ccccatgagc ccatgaatga 960
agctgcttac ttggaagtaa gtgttgtcct ggatgtgatg tgctcgcccc tgattgcaga 1020
agatgacata gtgtctcagc tcccggaaac ctgcatcgtg agctgtgagt atgatgctct 1080
ccgggacaat tcactgttgt acaagaaaag gctggaagac ctgggagtgc ccgtgacctg 1140
gcaccatatg gaggatggtt tccatggagt gctcaggacc attgacatga gcttcttgca 1200
ctttccctgc tccatgagaa ttctgagtgc attagttcaa tttgtaaagg gactgtgacc 1260
atctttcttc tctgctggta ctgcggtgtg gattccactg gcatccagcc tcccacaggg 1320
ctctctgttg ctgatttagg tggtgcatag tggggctagg gaggaggtag aggttgctgt 1380
cacctttctg gtccaggttc tagaaccaca caatgcatgc tcctgatgtc cagaggacgt 1440
ggtagaaaag acaggtttgg aggtgggagt gtggctgtct ctattctctg ttgggaaaac 1500
ctgggctgac aatattcagt ggccatttgt gggagtgaat cagccggtaa gagctgttct 1560
cagcctctct aaggggcagt tcaggctccc agattgatcc agactgtgtg tgactttcgt 1620
ccatttgact tgactttgga atagcacaag ggcatcacgt acttcacgag gctttcccaa 1680
tgtggctcag aggcaggagc tctgatgctc taggctgctg tgaggtggtg gtggtggtgg 1740
agaaactggc ttcacccacc tactcttctg tgaacagtag tgac 1784
<210> 23
<211> 2141
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71624817CB1
<400> 23
ctcttttgtg ctgattcctg aggactagga aggtgccccg aaaagaattc agagtgagta 60
cagtgaaaca gaaacctgct cagcttctaa gtgcaggaag gacttcacag ggaggcatga 120
tcagaacttg aaaaaaggac ctgacaatgt ccctgtattg tggaatagct tgcaggagaa 180
25/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
aatttttttg gtgctatagg ctgctgtcaa cctatgttac taagacacgg tatttatttg 240
aactgaagga agatgatgat gcatgtaaaa aagcccagca aacaggagcg ttttacctct 300
ttcatagtct ggctcctctg cttcagactt cagcacatca atacctggcc ccccggcaca 360
gcctgttaga gttggaaagg ctcctgggta aatttggaca ggatgcacaa agaatagaag 420
attctgtgct gattggatgc tctgagcagc aggaagcatg gtttgctctg gatctaggtc 480
tggatagctc cttttccata agtgcctcct tacacaaacc tgaaatggag acagagctca 540
aggggtcttt cattgagctg agaaaggcac tctttcaact caatgcaagg gatgcctcct 600
tgctgtccac ggctcaagct cttctccgct ggcatgatgc tcatcagttc tgcagcagaa 660
gtgggcagcc caccaagaag aacgtggctg gcagcaagcg tgtgtgccct tccaataata 720
taatctatta tccacagatg gctcctgtgg cgatcacgct ggtgtcagat gggacccgat 780
gcctgcttgc ccgccaaagc tcctttccca agggaatgta ttctgccttg gcaggttttt 840
gtgatatagg tgaaagtgtg gaagagacca tccgccgaga agttgcagaa gaggtgggat 900
tggaggtgga aagcctgcag tactatgcat cccagcattg gcccttccct agtggctcac 960
tcatgattgc ttgccatgca actgtgaaac cagggcagac agaaatccag gtgaacttga 1020
gagaattaga gacagctgcc tggttcagtc atgatgaggt agccacagcc ctgaagagaa 1080
agggccccta tactcagcaa cagaatggga ctttcccatt ctggctgccc cctaagttag 1140
ccatctccca ccaactgatt aaggagtggg tggaaaaaca gacctgttct tccctgcctg 1200
cttagcccgg atcaagtcac ttagatcgct ccttggtatt cctgagggac aaactagaga 1260
tcagttgaca aaggagaagt gacaaaagat aagctgcaga aggacctcag aagggcagag 1320
caaagggtga gcctacagta agacacttct atcagcagtg ttaatggaag aaattcctac 1380
caatgggcaa tcaaaaaagc cagtgtgaga agaaaactga tgagctgtca actgtcaaaa 1440
atcaggggga agggggaagc attagtttgg atgtaggccc ttgttcagtc attttctcta 1500
aggccttgga agcaaacatc ttctggcata tggcttgtta atgttgtgtt tacactaact 1560
gccaaaatgt gcctgttgta agtttggtta aaacttttat cttagtattg aaaatatata 1620
gcaagtttta agtcattact gagttacttg ttacccttac agaattaaga aaaatagcac 1680
agtatgacat tttaaaattc atgtacaggt gcattctagt tacatgaaca tgctagttaa 1740
ataaaagtca caattaagtc attatgactc agagtacttt ataataaatc agatgccctg 1800
aggctctgtt gaggaagtag aatcccaatg ggactccaga cagtaaatct ttttcgctgt 1860
tttcagctct gtcacatgct cagtttgtga cccagggaaa agactattgc tttgccatgc 1920
ctgttttcct aaataaaaca gttgctggca tttgcacatc cacaacgtgt tgattaacaa 1980
agggattggt ggagataaac agatgccaaa cctgacctat ttcttaaact ttatggaata 2040
aactaaattt aggatttctc atcattcata tatgatgcca taagggagaa gagtttattt 2100
ggggaaaata aaagaaattt ccacaaaaaa aaaaaaaaaa a 2141
<210> 24
<211> 2518
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6945964CB1
<400> 24
tgaagacagc ttccatcacg ttactaattc gattcgctcg agccactgag gcttttacaa 60
gaggaaagga gttgctctta gcagatgaca gacacttctc aaaagacagc ttttcttcct 120
ggagaacaga ctttttcagc aggattttcc tttcagtgaa acataatttg acttgaaagg 180
aacccaggga aaagtgtcca ggtgtgagca tgagcgggta gaggtgtgcc cttgtttgct 240
tcaggctgtc tgcttttcgc ccctgactgt tttttctgtt tctggccatg gaggaagaga 300
aagatgacag cccacaggct gacttctgcc tgggcaccgc cctgcactct tggggactgt 360
ggttcacgga ggaaggttca ccgtccacca tgctgacggg gattgcagtt ggagccctcc 420
tggccctggc cttggttggt gtcctcatcc ttttcatgtt cagaaggctt agacaatttc 480
gacaagcaca gcccactcct cagtaccggt tccggaagag agacaaagtg atgttttacg 540
gccggaagat catgaggaag gtgaccacac tccccaacac ccttgtggag aacactgccc 600
tgccccggca gcgggccagg aagaggacca aggtgctgtc tttggccaag aggattctgc 660
gtttcaagaa ggaatacccg gccctgcagc ccaaggagcc cccgccctcc ctgctggagg 720
ccgacctcac ggagtttgac gtgaagaatt ctcacctgcc atcggaagtt ctgtacatgc 780
tgaaaaacgt tcgggtcctg ggccactttg agaagccgct gttcctggag ctttgcaaac 840
acatcgtctt tgtgcagctg caggaagggg agcacgtctt ccagcccagg gagccggacc 900
ccagcatctg tgtggtgcag gacgggcggc tggaggtctg catccaggac actgacggca 960
ccgaggtggt ggtgaaagag gttctggcgg gagacagcgt ccacagcctg ctcagcatcc 1020
tggacatcat caccggccat gctgcacctt acaaaacggt ctccgtccgc gcggccatcc 1080
cgtccaccat cctccggctt ccagctgcgg cttttcatgg agtttttgag aaatatccgg 1140
26/27
CA 02443244 2003-10-07
WO 02/083873 PCT/US02/15253
aaactctggt gagggtggtg cagatcatca tggtgcggct gcagagggtg acctttctgg 1200
ctctgcacaa ctacctcggc ctgaccacag agctcttcaa cgctgagagc caggccatcc 1260
ctctcgtgtc tgtagccagt gtggctgccg ggaaggccaa gaagcaggtg ttctatggcg 1320
aagaagagcg gcttaaaaag ccaccgcggc tccaggagtc ctgtgactca ggtactgtcc 1380
tgcaccaagg agggcaatgt ccagccccag agtgggggat ggagtggaca gagtggagcg 1440
agtgagtggg ggatggagtg gacagagtgg agtgagtggg ggatggagtg agcagagttc 1500
gagaccagcc cgggcaacat agtgagaccc tgtctctatg aataaaattt gaaaaatgat 1560
ccaggcatgt tagcacacac ctgtcgtccc agctatgcag agggttgagg tgggaggatc 1620
gtttgggcct cggagttcga ggttacagtg agctatgatc gcgccaccgc agtccagcct 1680
ggtcaacaga gcgagaccct gcctcaaaac acacacacaa aaaggtgttt ttctctgtct 1740
ttagttttca gaaatgtgat ttggaagtgt ctgagcatga atactttgga tctgtcctgt 1800
ttggggtttc ttcgtcttgg acccgtaggt ttatagcttt taacacattt tggaagtgtt 1860
cagtctatat gtcttcaaac gattcttcca ctcgtgtgcc cctccacctc ttctggaaat 1920
aaagtggcac aagtgttaga cctcgcgtta tgtccccaag gtctctgggg cactgctcac 1980
tttccttaaa cctttttgtt ctctctctga tgttaagatg gaattgtttc ggtttgatct 2040
atcttcacgg atctttggct tgtttcgtca tctcgattct agctttgagg cctatcagag 2100
agttttaaaa aaatgttctg tcatcgtgtt ttggttcttc tgtgtgcctc ctgtttcttt 2160
gctaggacgt tctctctttg tctttgtctc aggagtgttc aggattgctg gttctagctg 2220
gtaatgataa cagctttatc gtcctggaga aataattccg tctgtgacat gcggtcgttt 2280
ttatctgtgg attgttttta ctcctctggt tcagatcctc cttgctcttg gcatactggg 2340
tcaccatgta gtcgtcgtac tgttcactgg aattatccgg atactgtcgt catatctggt 2400
gttcgaatat tgtgacgacg gataaccact gtgcactcat ggacacaagt ctaagtaccc 2460
actgagttac ccaagccagt gtgctcgagt ccagaacaca cctgacgacc gcccagct 2518
27/27
