Note: Descriptions are shown in the official language in which they were submitted.
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COMPoaXPICNS 4WD XETROD5 FOR EFFECTING TIE
LEVWLB OF BLOW DENSITY LIZPOPROTBXN (EDL) CUOLESTWROL
AND APOLIPOPROTEIN Al, VERY LOW DENSITY LXPOFROTEIN (VLDL)
OEDLU5T.EROL AND LOW DIESITY LIPOPROTEIN (LDL) CEOLEOTEROL
FIELD 07 T11E INVENTION
This invention relates to methods and compositions for
increasing the level of high density lipoprotein (NDL)
cholesterol and apolipoprotsin Al in a patient and to methods
and compositions for lowering the levels of very low density
lipoprotein (VLDL) cholesterol, and low density lipoprotein
(LDL) cholesterol in a patient. The invention includes
within its scope methods and compositions which lower the
expression of, or inhibit the activity of, a gene, LIPG,
which encodes a lipase enzyme that lowers the levels of {DL
cholesterol and apolipoprotein hl. The invention
additionally includes within its scope methods and
compositions to increase the expression of, or enhance the
activity of, the lipase enzyme, resulting in lower levels of
VDT, and LDL cholesterol.
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Lipids
Lipids are water-insoluble organic biomolecules, which
are essential components of diverse biological functions,
including the storage, transport, and metabolism of energy,
and membrane structure and fluidity. Lipids are derived from
two sources in man and other animals: some lipids are
ingested as dietary fats and oils and other lipids are
biosynthesized by the human or animal. In mammals, at least
10% of the body weight is lipid, the bulk of which is in the
form of triacylglycerols.
Triacylglycerols, also known as triglycerides and
triacylglycerides, are made up of three fatty acids
esterified to glycerol. Dietary triacylglycerols are stored
in adipose tissues as a source of energy, or hydrolyzed in
the digestive tract by triacyiglycerol lipases, the most
important of which is pancreatic lipase. Triacylglycerols
are transported between tissues in the form of lipoproteins.
Lipoproteins are micelle-like assemblies found in plasma
which contain varying proportions of different types of
lipids and proteins (called apoproteins). There are five
main classes of plasma lipoproteins, the major function of
which is lipid transport. These classes are, in order of
increasing density: chylomicrons; very low density
lipoproteins (VLDL); intermediate-density lipoproteins (IDL);
low density lipoproteins (LDL); and high density lipoproteins
(HDL). Although many types of lipid are found associated
with each lipoprotein class, each class transports
predominantly one type of lipid: triacylglycerols described
above are transported in chylomicrons, VLDL, and IDL; whereas
phospholipids and cholesterol esters are transported in HDL
and LDL respectively.
Phospholipids are di-fatty acid esters of glycerol
phosphate which contain a polar group coupled to the
phosphate. Phospholipids are important structural components
of cellular membranes. Phospholipids are hydrolyzed by
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enzymes called phospholipases. Phosphatidylcholine, an
exemplary phospholipid, is a major component of most
eukaryotic cell membranes.
Cholesterol is the metabolic precursor of steroid
hormones and bile acids as well as an essential constituent
of cell membranes. In man and other animals, cholesterol is
ingested in the diet and is synthesized also by the liver and
other tissues. Dietary cholesterol is transported from the
intestine to the liver by large lipoprotein molecules in the
blood. The liver secretes Very Low Density Lipoprotein
(VLDL) which transports cholesterol and cholesterol ester and
various other compounds into the bloodstream. VLDL is
partially converted in adipose tissue to Low Density
Lipoprotein (LDL). LDL transports both free and esterified
cholesterol to body tissues. High Density Lipoprotein (HDL)
transports cholesterol to the liver to be broken down and
excreted.
Membranes surround every living cell and serve as a
barrier between the intracellular and extracellular
compartments. Membranes also enclose the eukaryotic nucleus,
make up the endoplasmic reticulum, and serve specialized
functions such as in the myelin sheath that surrounds axons.
A typical membrane contains about 40% lipid and 60% protein,
but there is considerable variation. The major lipid
components are phospholipids, specifically
phosphatidylcholine and phosphatidylethanolamine, and
cholesterol. The physicochemical properties of membranes,
such as fluidity, can be changed by modification of either
the fatty acid profiles of the phospholipids or the
cholesterol content. Modulating the composition and
organization of membrane lipids also modulates membrane-
dependent cellular functions, such as receptor activity,
endocytosis, and cholesterol flux.
Enzymes
The triacylglycerol lipases are a family of enzymes
which play several pivotal roles in the metabolism of lipids
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in the body. Three members of the human triacyiglycerol
lipase family have been described: pancreatic lipase,
lipoprotein lipase, and hepatic lipase (Goldberg, I.J., Le,
N.-A., Ginsberg, H.N., Krauss, R.M., and Lindgren, F.T.
(1988) J. C1in. Invest. 81,561-568; Goldberg, I.J., Le, N.,
Paterniti J.R., Ginsberg, H.N., Lindgren, F.T., and Brown,
W.V. (1982) J. Clin. Invest. 70,1184-1192; Hide, W.A., Chan,
L., and Li, W.-H. (1992) J. Lipid. Res. 33,167-178).
Pancreatic lipase is primarily responsible for the hydrolysis
of dietary lipids. Variants of pancreatic lipase have been
described, but their physiological role has not been
determined (Giller, T., Buchwald, P., Blum-Kaelin, D., and
Hunziker, W. (1992) J. Biol. Chem. 267,16509-16516).
Lipoprotein lipase is the major enzyme responsible for the
distribution and utilization of triglycerides in the body.
Lipoprotein lipase hydrolyzes triglycerides in both
chylomicrons and VLDL. Hepatic lipase hydrolyzes
triglycerides in IDL and HDL and is responsible for
lipoprotein remodeling. Hepatic lipase also functions as a
phospholipase and hydrolyzes phospholipids in HDL.
Phospholipases play important roles in the catabolism
and remodeling of the phospholipid component of lipoproteins
and the phospholipids of membranes. Phospholipases also play
a role in the release of arachidonic acid and the subsequent
formation of prostaglandins, leukotrienes, and other lipids
which are involved in a variety of inflammatory processes.
The aforementioned lipases are approximately 450 amino
acids in length and have leader signal peptides to facilitate
secretion. The lipases are comprised of two principal
domains (Winkler, K., D'Arcy, A., and Hunziker, W. (1990)
Nature 343, 771-774). The amino terminal domain contains the
catalytic site while the carboxyl domain is believed to be
responsible for substrate binding, cofactor association, and
interaction with cell receptors (Wong, H., Davis, R.C.,
Nikazy, J., Seebart, K.E., and Schotz, M.C. (1991) Proc.
Natl. Acad. Sci. USA 88,11290-11294; van Tilbeurgh, H.,
Roussel, A., Lalouel, J.-M., and Cambillau, C. (1994) J.
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-
Biol. Chem. 269,4626-4633; Wong, H., Davis, R.C., Thuren,
T., Goers, J.W., Nikazy, J., Waite, M., and Schotz, M.C.
(1994) J. Biol. Chem. 269,10319-10323; Chappell, D.A.,
Inoue, I., Fry, G.L., Pladet, M.W., Bowen, S.L., Iverius, P.-
5 H., Lalouel, J.-M., and Strickland, D.K. (1994) J. Biol.
Chem. 269, 18001-18006). The overall level of amino acid
homology between members of the family is 22-65%, with local
regions of high homology corresponding to structural
homologies which are linked to enzymatic function.
The naturally occurring lipoprotein lipase is
glycosylated. Glycosylation is necessary for LPL enzymatic
activity (Semenkovich, C.F., Luo, C.-C., Nakanishi, M.K.,
Chen, S.-H., Smith, L C., and Chan L. (1990) J. Biol. Chem.
265, 5429-5433). There are two sites for N-linked
glycosylation in hepatic and lipoprotein lipase and one in
pancreatic lipase. Additionally, four sets of cysteines form
disulfide bridges which are essential in maintaining
structural integrity for enzymatic activity (Lo, J.-Y.,
Smith, L.C., and Chan, L. (1995) Biochem. Biophys. Res.
Commun. 206, 266-271; Brady, L., Brzozowski, A.M.,
Derewenda, Z.S., Dodson, E., Dodson G., Tolley, S.,
Turkenburg, J.P., Christiansen, L., Huge-Jensen B., Norskov,
L., Thim, L., and Menge, U. (1990) Nature 343, 767-770).
Members of the triacylglycerol lipase family share a
number of conserved structural features. One such feature is
the "GXSXG" motif, in which the central serine residue is one
of the three residues comprising the "catalytic triad"
(Winkler, K., D'Arcy, A., and Hunziker, W. (1990) Nature 343,
771-774; Faustinella, F., Smith, L.C., and Chan, L. (1992)
Biochemistry 31,7219-7223). Conserved aspartate and
histidine residues make up the balance of the catalytic
triad. A short span of 19-23 amino acids (the "lid region")
forms an amphipathic helix structure and covers the catalytic
pocket of the enzyme (Winkler, K., D'Arcy, A., and Hunziker,
W. (1990) Nature 343, 771-774). This region diverges
significantly between members of the family. It has been
determined recently that the span confers substrate
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specificity to the enzymes (Dugi, K.A., Dichek H.L., and
Santamarina-Fojo, S. (1995) J. Biol. Chem. 270, 25396-25401).
Comparisons between hepatic and lipoprotein lipase have
demonstrated that differences in triacylglycerol lipase and
phospholipase activities of the enzymes are in part mediated
by this lid region (Dugi, K.A., Dichek H.L., and Santamarina-
Fojo, S. (1995) J. Biol. Chem. 270, 25396-25401).
The triacylglycerol lipases possess varying degrees of
heparin binding activity. Lipoprotein lipase has the highest
affinity for heparin. This binding activity has been mapped
to stretches of positively charged residues in the amino
terminal domain (Ma, Y., Henderson, H.E., Liu, M.-S., Zhang,
H., Forsythe, I.J., Clarke-Lewis, I., Hayden, M.R., and
Brunzell, J.D. J. Lipid Res. 35, 2049-2059).' The
localization of lipoprotein lipase to the endothelial surface
(Cheng, C.F., Oosta, G.M., Bensadoun, A., and Rosenberg, R.D.
(1981) J. Biol. Chem. 256, 12893-12896) is mediated primarily
through binding to surface proteoglycans (Shimada K., Gill,
P.J., Silbert, J.E., Douglas, W.H.J., and Fanburg, B.L.
(1981) J. Clin. Invest. 68, 995-1002; Saxena, U., Klein,
M.G., and Goldberg, I.J. (1991) J. Biol. Chem. 266, 17516-
17521; Eisenberg, S., Sehayek, E., Olivecrona, T., and
Vlodavsky, I. (1992) J. Clin Invest. 90,2013-2021). It is
this binding activity which allows the enzyme to accelerate
LDL uptake by acting as a bridge between LDL and the cell
surface (Mulder, M., Lombardi, P., Jansen, H., vanBerkel
T.J., Frants R.R., and Havekes, L.M. (1992) Biochem.
Biophys. Res. Comm. 185, 582-587; Rutledge, J.C., and
Goldberg, I.J., (1994) J. Lipid Res. 35. 1152-1160; Tsuchiya,
S., Yamabe, M., Yamaguchi, T., Kobayashi, Y., Konno, T., and
Tada, K. (1980) Int. J. Cancer 26,171-176).
Lipoprotein lipase and pancreatic lipase are both known
to function in conjunction with co-activator proteins:
apolipoprotein CII for lipoprotein lipase; and colipase for
pancreatic lipase.
The genetic sequences encoding human pancreatic lipase,
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hepatic lipase and lipoprotein lipase have been reported
(Genbank accession #M93285, #J03540, and #M15856
respectively). The messenger RNAs of human hepatic lipase
and pancreatic lipase are approximately 1.7 and 1.8 kilobases
in length respectively. Two mRNA transcripts of 3.6 and 3.2
kilobases are produced from the human lipoprotein lipase
gene. These two transcripts utilize alternate
polyadenylation signals and differ in their translational
efficiency (Ranganathan, G., Ong, J.M., Yukht, A.,
Saghizadeh, M., Simsolo, R.B., Pauer, A., and Kern, P.A.
(1995) J. Biol. Chem. 270, 7149-7155).
Physiological processes
The metabolism of lipids involves the interaction of
lipids, apoproteins, lipoproteins, and enzymes.
Hepatic lipase and lipoprotein lipase are
multifunctional proteins which mediate the binding, uptake,
catabolism, and remodeling of lipoproteins and phospholipids.
Lipoprotein lipase and hepatic lipase function while bound to
the luminal surface of endothelial cells in peripheral
tissues and the liver respectively. Both enzymes participate
in reverse cholesterol transport, which is the movement of
cholesterol from peripheral tissues to the liver either for
excretion from the body or for recycling. Genetic defects in
both hepatic lipase and lipoprotein lipase are known to be
the cause of familial disorders of lipoprotein metabolism.
Defects in the metabolism of lipoproteins result in serious
metabolic disorders, including hypercholesterolemia,
hyperlipidemia, and atherosclerosis.
REPORTED DEVELOPMENTS
Atherosclerosis is a complex, polygenic disease which is
defined in histological terms by deposits (lipid or
fibrolipid plaques) of lipids and of other blood derivatives
in blood vessel walls, especially the large arteries (aorta,
coronary arteries, carotid). These plaques, which are more
or less calcified according to the degree of progression of
the atherosclerotic process, may be coupled with lesions and
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are associated with the accumulation in the vessels of fatty
deposits consisting essentially of cholesterol esters. These
plaques are accompanied by a thickening of the vessel wall,
hypertrophy of the smooth muscle, appearance of foam cells
(lipid-laden cells resulting from uncontrolled uptake of
cholesterol by recruited macrophages) and accumulation of
fibrous tissue. The atheromatous plaque protrudes markedly
from the wall, endowing it with a stenosing character
responsible for vascular occlusions by atheroma, thrombosis
or embolism, which occur in those patients who are most
affected. These lesions can lead to serious cardiovascular
pathologies such as infarction, sudden death, cardiac
insufficiency, and stroke.
High density lipoprotein (HDL)
cholesterol levels and atherosclerotic diseases
High density lipoprotein (HDL) cholesterol levels are
inversely associated with risk of atherosclerotic
cardiovascular disease (Gordon et al., N. Engl. J. Med., 321,
1311-1316 (1989)). At least 50% of the variation in HDL
cholesterol levels is.genetically determined (Breslow, J.L.,
The Metabolic Basis of Inhereited Disease, 2031-2052, McGraw-
Hill, New York (1995); Heller et al., N. Engl. J. Med., 328,
1150-1156 (1993)), but the genes responsible for variation in
HDL levels have not been fully elucidated. Lipoprotein
lipase (LPL) and hepatic lipase (HL), two members of the
triacylglycerol (TG) lipase family, both influence HDL
metabolism (Breslow, supra; Murthy et al., Pharmacol. Ther.,
70, 101-135 (1996); Goldberg, J.I., J. Lipid Res., 37, 693-
707 (1996); Bensadoun et al., Curr. Opin. Lipidol., 7, 77-81
(1996)) and the HL (LIPC) locus has been associated with
variation in HDL cholesterol levels in humans (Cohen et al.,
J. C1in. Invest., 94, 2377-2384 (1994); Guerra et al., Proc.
Natl. Acad. Sci. USA, 94, 4532-4537 (1997)). The normal
range for HDL cholesterol is about 35 to 65 mg/dL, and the
HDL level should account for more than 25% of the total
cholesterol.
Very low density lipoprotein (VLDL)
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and low density lipoprotein (LDL)
cholesterol levels and atherosclerotic diseases
High levels of circulating LDL and VLDL cholesterol are
associated with increased risk of atherosclerosis.
VLDL are the precursors of LDL. Therapeutic agents that
lower plasma VLDL and LDL cholesterol levels are highly
desirable because of the known strong association between
these lipid parameters and coronary heart disease.
Epidemiologic studies have demonstrated a strong
relationship between elevated LDL cholesterol and coronary
heart disease (CHD) and other atherosclerotic vascular
diseases (Kannell, W.B., An. J. Cardiol., 76, 69C-77C
(1995)). Three major secondary prevention trials performed
with statins have demonstrated that reduction of LDL
cholesterol levels result in significant reduction in CHD
events and total mortality (Scandinavian Simvastatin Survival
Study Group, Lancet, 344, 1383-1389 (1994); Sacks et al., N.
Engl. J. Med., 335, 1001-1009 (1996); Tonkin et al., N. Engl.
J. Med., 339, 1349-1357 (1998); Grundy, S.M., Circulation,
1436-1439 (1998)). Two large primary prevention trials with
statins have also demonstrated significant benefit of LDL
cholesterol reduction with statins in reducing cardiovascular
events (Grundy, supra; Shepherd et al., N. Engl. J. Med.,
333, 1301-1307 (1995); Downs et al., JAMA, 279, 1615-1622
(1998)). However, current therapies do not adequately reduce
LDL cholesterol levels in all persons. VLDL cholesterol
levels have also been recognized to be associated with
increased risk of CHD (Kannel, supra). Current therapies do
not have as much effect in reducing VLDL cholesterol as LDL
cholesterol. Therefore, new approaches to reducing both LDL
cholesterol and VLDL cholesterol are still needed.
Ideally, the range for VLDL cholesterol is about 1 to 30
mg/dL and the range for LDL cholesterol is about 60 to 160
mg/dL. The LDL to HDL ratio is ideally less than 3.5.
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The Role of Triacylglycerol
Lipases in Atherosclerotic Diseases
The role of triacylglycerol lipases in vascular
pathologies such as atherosclerosis has been an area of
intense study (reviewed in Olivecrona, G., and Olivecrona, T.
(1995) Curr. Opin. Lipid. 6,291-305). Generally, the
action of the lipoprotein lipase is believed to be
antiatherogenic because this enzyme lowers serum
triacylglycerol levels and promote HDL formation. Transgenic
animals expressing human lipoprotein lipase have decreased
levels of plasma triglycerides and an increased level of high
density lipoprotein (HDL) (Shimada, M., Shimano, H., Gotoda,
T., Yamamoto, K., Kawamura, M., Inaba, T., Yazaki, t., and
Yamada, N. (1993) J. Biol. Chem. 268, 17924-17929; Liu, M.-
S., Jirik, F. R., LeBoeuf, R. C., Henderson, H.,
Castellani, L.W., Lusis, A. J., ma, Y., Forsythe, I.J.,
Zhang, H., Kirk, E., Brunzell, J.D., and Hayden, M.R. (1994)
J. Biol. Chem. 269, 11417-11424). Humans with genetic
defects resulting in decreased levels of lipoprotein lipase
activity have been found to have hypertriglyceridemia, but no
increased risk of coronary heart disease. This is reported
to be due to the lack of production of intermediate-sized,
atherogenic lipoproteins which could accumulate within the
subendothelial space (Zilversmit, D.B. (1973) Circ. Res.
33,633-638).
In contrast to lipoprotein lipase (LPL), the physiologic
function of HL appears to be related to the metabolism of
lipoprotein remnants and HDL (Bensadoun et al., Curr. Opin.
Lipidol., 7, 77-81 (1996)). Genetic deficiency of HL is
associated with modestly increased levels of remnants and HDL
cholesterol in humans (Hegele et al., Arterioscler. Thromb.,
13, 720-728 (1993)) and mutant mice (Homanics et al., J.
Biol. Chem., 270, 2974-2980 (1.995)). Despite increased
plasma cholesterol levels, HL deficiency is associated with
reduced atherosclerosis in apoE mutant mice (Mezdour et al.,
J. Biol. Chem., 272, 13570-13575 (1997)). Transgenic animals
overexpressing HL have decreased HDL (Busch et al., J. Biol.
Chem., 269, 16376-16382 (1994); Fan et al., Proc. Natl. Acad.
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Sci. USA, 91, 8724-8728 (1994)). Increased HL activity in
humans is associated with low HDL cholesterol. The HL locus
on chromosome 15g21 has been associated with variation in
plasma HDL cholesterol levels in humans (Cohen et al., J.
Clin. Invest., 94, 2377-2384 (1994); Guerra et al., Proc.
Natl. Acad. Sci. USA, 94 4532-4537 (1997)), but accounts for
only a portion of the genetic contribution to variation in
HDL cholesterol levels. There is at least one major locus
influencing HDL cholesterol levels in humans that is distinct
from the HL locus (Mahaney et al., Arterioscler. Thromb.
Vasc. Biol., 15, 1730-1739 (1995)).
In the localized area of an atherosclerotic lesion, the
increased level of lipase activity is hypothesized to
accelerate the atherogenic process (Zilversmit, D.B. (1995)
Clin. Chem. 41,153-158; Zambon, A., Torres, A., Bijvoet, S.,
Gagne, C., Moojani, S., Lupien, P.J., Hayden M.R., and
Brunzell, J.D. (1993) Lancet 341, 1119-1121). This may be
due to an increase in the binding and uptake of lipoproteins
by vascular tissue mediated by lipases (Eisenberg, S.,
Sehayek, E., Olivecrona, T. Vlodavsky, I. (1992) J. C1in.
Invest. 90,2013-2021; Tabas, I., Li, I., Brocia R.W., Xu,
S.W., Swenson T.L. Williams, K.J. (1993) J. Biol. Chem.
268,20419-20432; Nordestgaard, B.G., and Nielsen, A.G. (1994)
Curr. Opin. Lipid. 5,252-257; Williams, K.J., and Tabas, I.
(1995) Art. Thromb. and Vasc. Biol. 15,551-561).
Additionally, a high local level of lipase activity may
result in cytotoxic levels of fatty acids and
lysophosphatidylcholine being produced in precursors of
atherosclerotic lesions.
Despite the understanding that has evolved regarding the
role of lipase enzyme activity in regulating the levels of
lipids and the various plasma lipoproteins, there is a need
to identify and develop therapies which can increase the
levels of HDL cholesterol, as well as lower the levels of
VLDL and LDL cholesterol to reduce the risk of developing
atherosclerotic cardiovascular diseases.
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SUMMARY OF THE INVENTION
In accordance with the present invention, there is
provided a composition for lowering the expression of the
LIPG gene in a patient comprising an antisense nucleic acid,
including for example, an expression vector which includes
said antisense nucleic acid. Examples of preferred
expression vectors are retroviral vectors, adenoviral
vectors, adeno-associated viral vectors, herpesviral vectors,
and naked DNA vectors. The antisense nucleic acid can be,
for example, an oligonucleotide which contains chemically
modified bases.
Another aspect of the present invention is the provision
of a composition for lowering the enzymatic activity of the
LIPG polypeptide in a patient comprising a neutralizing
antibody capable of binding to the LIPG polypeptide and
lowering its enzymatic activity, including, for example, an
expression vector which includes a DNA sequence encoding said
antibody. Examples of preferred expression vectors are
retroviral vectors, adenoviral vectors, adeno-associated
viral vectors, herpesviral vectors, and naked DNA vectors.
Still another aspect of the present invention is the
provision of a composition for lowering the enzymatic
activity of the LIPG polypeptide in a patient comprising an
intracellular binding protein, including, for example, an
expression vector which includes a DNA sequence encoding said
intracellular binding protein. Examples of preferred
expression vectors are retroviral vectors, adenoviral
vectors, adeno-associated viral vectors, herpesviral vectors,
and naked DNA vectors.
Yet other aspects of the present invention are the
provision of: (A) a composition which comprises an inhibitor
that is capable of inhibiting the enzymatic activity of the
LIPG polypeptide in a patient; (B) a composition which
comprises an inhibitor that is capable of lowering the
expression of the LIPG gene in a patient; and (C) a
composition which is capable of lowering the expression of
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LIPG in a patient and which comprises a ribozyme, including,
for example, an expression vector which includes a DNA
sequence encoding said ribozyme. Examples of preferred
expression vectors are retroviral vectors, adenoviral
vectors, adeno-associated viral vectors, herpesviral vectors,
and naked DNA vectors. A preferred ribozyme is a hammerhead
ribozyme.
The present invention provides also: (D) a composition
which increases the level of LIPG polypeptide in a patient
and which comprises an expression vector that includes a DNA
sequence encoding the LIPG polypeptide or an enhancer that is
capable of increasing the expression of the LIPG gene; and
(E) a composition which increases the enzymatic activity of
LIPG polypeptide in a patient which comprises an enhancer
that binds to and enhances the enzymatic activity of the LIPG
polypeptide.
In addition, the present invention provides a method for
raising the level of high density lipoprotein (HDL)
cholesterol and apolipoprotein AI in a patient by
administering to the patient a composition which lowers the
enzymatic activity of LIPG in said patient, for example, by
lowering the level of LIPG polypeptide in the patient. In
preferred form, the method involves the use of a composition
which comprises an antisense nucleic acid, particularly one
that is modified to increase the chemical stability of the
nucleic acid. The aforementioned method can be practiced
also by use of a composition which comprises a neutralizing
antibody capable of binding to the LIPG polypeptide and
lowering its enzymatic activity or a composition which
comprises an inhibitor which inhibits the enzymatic activity
of LIPG polypeptide, for example, a compound which lowers the
expression of the LIPG gene or a composition which comprises
a ribozyme that cleaves mRNA encoding LIPG, or a composition
which comprises a DNA molecule and a liposome, for example, a
cationic liposome.
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In preferred form, the aforementioned method comprises
also the administration of a composition which is capable of
expressing apolipoprotein AI in said patient.
Another aspect of the present invention is the provision
of a method for lowering the level of very low density
lipoprotein (VLDL) cholesterol in a patient by administering
to the patient a composition which is capable of increasing
the enzymatic activity of LIPG in said patient, for example,
by use of a composition which comprises an LIPG polypeptide
and a pharmaceutically acceptable carrier and which includes
preferably an expression vector that is capable of expressing
an LIPG polypeptide, preferably a retroviral vector, an
adenoviral vector, or an adeno-associated viral vector. The
aforementioned method can be practiced by use of a
composition which comprises an enhancer that enhances the
enzymatic activity of LIPG polypeptide or an enhancer that
increases expression of the LIPG gene.
Still another aspect of the present invention is the
provision of a method for lowering the level of low density
lipoprotein (LDL) cholesterol in a patient by administering
to the patient a composition which is capable of increasing
the enzymatic activity of LIPG in the patient, preferably by
use of an LIPG polypeptide, for example, by use of an
expression vector that is capable of expressing the LIPG
polypeptide, preferably by use of a retroviral vector, an
adenoviral vector, or an adeno-associated viral vector. The
aforementioned method includes preferably the use of a
composition which comprises an enhancer that enhances the
enzymatic activity of LIPG polypeptide or an enhancer which
increases the expression of the LIPG gene.
The present invention provides also a method for
lowering the level of LDL cholesterol in a patient by
administering to the patient an enhancer which preferentially
enhances the enzymatic reactions between LIPG polypeptide and
LDL cholesterol relative to the enzymatic reactions between
LIPG polypeptide and HDL cholesterol and apolipoprotein AI.
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In addition, the present invention provides a method for
lowering the level of VLDL cholesterol in a patient by
administering to the patient an enhancer which preferentially
enhances the enzymatic reactions between LIPG polypeptide and
VLDL cholesterol relative to the enzymatic reactions between
LIPG polypeptide and HDL cholesterol and apolipoprotein AI.
Still another aspect of the present invention is the
provision of a method for diagnosing a predisposition to low
HDL cholesterol and apolipoprotein AI levels by obtaining a
tissue sample from a patient and measuring the level of LIPG
polypeptide in the sample, for example, by use of blood
tissue and the use of an immunoassay for measurement. In
another aspect of the present invention, the levels of LIPG
polypeptide are measured by measuring the levels of LIPG
mRNA.
An additional aspect of the present invention is the
provision of a method for determining whether a test compound
can inhibit the enzymatic reaction between the LIPG
polypeptide and HDL cholesterol and apolipoprotein AI
comprising: (A) comparing the level of HDL cholesterol and
apolipoprotein AI in a first sample comprising: (1) HDL
cholesterol and apolipoprotein AI, (2) LIPG polypeptide, and
(3) the test compound with the level of HDL cholesterol and
apolipoprotein AI in another sample comprising: (4) HDL
cholesterol and apolipoprotein AI, and (5) LIPG polypeptide;
and (B) identifying whether or not the test compound is
effective in inhibiting the enzymatic reaction between the
LIPG polypeptide and HDL cholesterol and apolipoprotein AI by
observing whether or not the first sample has a higher level
of HDL cholesterol and apolipoprotein AI than that of said
other sample.
The present invention provides also a method for
determining whether a test compound can enhance the enzymatic
reaction between the LIPG polypeptide and VLDL cholesterol
comprising: (A) comparing the level of VLDL cholesterol in a
first sample comprising: (1) VLDL cholesterol, (2) LIPG
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polypeptide, and (3) the test compound with the level of VLDL
cholesterol in another sample comprising: (4) VLDL
cholesterol, and (5) LIPG polypeptide; and (B) identifying
whether or not the test compound is effective in enhancing
the enzymatic reaction between the LIPG polypeptide and VLDL
cholesterol by observing whether or not the first sample has
a lower level of VLDL cholesterol than that of said other
sample.
Still another aspect of the present invention is the
provision of a method for determining whether a test compound
can enhance the enzymatic reaction between the LIPG
polypeptide and LDL cholesterol comprising: (A) comparing the
level of LDL cholesterol in a. first sample comprising: (1)
LDL cholesterol, (2) LIPG polypeptide, and (3) the test
compound with the level of LDL cholesterol in another sample
comprising: (4) LDL cholesterol, and (5) LIPG polypeptide;
and (B) identifying whether or not the test compound is
effective in enhancing the enzymatic reaction between the
LIPG polypeptide and LDL cholesterol by observing whether or
not the first sample has a lower level of LDL cholesterol
than that of said other sample.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the sequences (SEQ ID Nos: 17-31) of the
primers used in the exemplified PCR amplifications.
Figure 2 shows the nucleic acid sequence (SEQ ID NO: 1)
and the deduced amino acid sequence (SEQ ID NO: 2) of the
differential display RT-PCR product containing the LIPG gene
cDNA. The sequences corresponding to the two primers used in
the amplification are underlined. The termination codon and
polyadenylation signal are boxed. The GAATTC motifs and
flanking sequence are from the pCRII vector into which the
product was cloned.
Figure 3 shows the nucleic acid sequence (SEQ ID NO: 3)
and the deduced amino acid sequence (SEQ ID NO: 4) of the
5'RACE extension of the LIPG cDNA. The sequences
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corresponding to the two primers used in the amplification
are underlined. The GAATTC motifs and flanking sequence are
from the pCRII vector into which the product was cloned.
Figure 4 shows the sequence (SEQ ID NO: 7) of the cDNA
containing the complete open reading frame of the LIPG gene,
LLGXL. The start codon (ATG) and termination codon (TGA) are
boxed. The Dral site (TTTAAA) and SrfI site (GCCCGGGC) used
in the construction of the expression vectors are underlined.
Figure 5 shows the deduced amino acid sequence (SEQ ID
NO: 8) of the LLGXL protein. The predicted signal sequence
is underlined.
Figure 6 shows a protein sequence alignment of the
members of the triacylglycerol lipase gene family (SEQ ID
Nos: 13-15). Shaded residues are identical to the LLGXL
protein (SEQ ID NO: 8). The deduced amino acid sequence of
human LIPG(EL) is provided on the top line and is compared
with the other major members of the TG lipase family, LPL, HL
and PL. EL residues identical to those in at least one other
member of the family are shaded as well as the corresponding
residue in the other family member. Amino acids are numbered
according to convention beginning with the initial residue of
the secreted protein. The predicted sites of signal peptide
cleavage are marked with a solid line between amino acid
residues. The GXSXG lipase motif containing the active
serine is boxed. The amino acids of the catalytic triad are
marked with an asterisk. The conserved cysteines are marked
with filled circles. Potential N-linked glycosylation sites
are marked with arrowheads. The lid region is indicated by a
bold line. Gaps were introduced into the sequences to
maximize the alignment values using the CLUSTAL program.
Figure 7 shows a northern analysis of LIPG mRNA in THP-1
cells. Cells were stimulated with either PMA or PMA and
oxidized LDL (PMA + oxLDL). Numbers at the left indicate the
positions of RNA standards (in kilobases).
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Figure 8 shows a northern-blot analysis of expression of LIPG mRNA
compared with LPL in human tissues. A blot containing mRNA from the indicated
human tissues was incubated with radiolabelled LPL and 6-actin (ACTB) probes
as
described.
b
Figure 9 shows a Northern-blot analysis of cultured cell lines. The panel on
the left (lanes 1-6) was hybridized with thc. LIPG(EL) probe and that on the
right
(lanes 7-12) with the LPL probe. Lanes 1, 7, unstimulated HUVEC; lanes 2, 8,
HUVEC stimulated with PMA; lanes 3, 9, EIU VEC stimulated with thrombin; lanes
4, 10, unstimulated HCAEC; lanes 5, 11, HCAEC stimulated with PMA; lanes 6,
12,
TB?-1 stimulated with PMA.
Figure 10 shows the sequence of the immunizing peptide (SEQ ID NO: 16)
and its relation to the LLGXL protein sequence. The peptide is shown in the
shaded
box. The terminal cysteine was introduced to aid coupling of the
peptide to the carrier protein.
Figure 11 shows the results obtained when conditioned media from HUVI+ C
and HCAEC were subjected to immunoblot analysis with rabbit anti-EL peptide
antiserum. Lane 1, unconditioned media; lane 2, unatimulated HUVEC; lane 3,
HUVEC stimulated with PHA; lane 4, unstimulated HCAEC; lane 5, HCAEC
stimulated with PMA.
Figure 12 shows a western analysis of heparin-Sepharoee''m bound proteins in
conditioned medium from COS-7 cells transiently transfeeted with an expression
vector containing a cDNA for LLGN or LLGXL or no DNA (Mock). Proteins from
PMA-stimulated endothelial cells (HCAEC' + PMA) were included for size
reference.
Numbers to the left indicate the apparent molecular weight of the major
immunoreactive proteins as determined by a comparison to protein standards.
Figure 13 shows the sequence of the rabbit LIPG PCR product (RLLG.SEQ,
SEQ ID NO: 12) and the sequence alignmE-nt between the rabbit LIPG PC1 product
and the corresponding
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sequence in the human cDNA (LLG7742A). Identical nucleotides
are shaded.
Figure 14 shows the phospholipase A activity of human
EL-AS, EL and LPL using a phosphatidylcholine substrate. To
perform the assay 700gl of conditioned medium harvested from
COS-7 cells transiently transfected with either
pcDNA3.0/LIPG-AS, LIPG, or LPL expression constructs were
assayed in triplicate for phospholipase activities as
described below. Following a two hour incubation at 37 C,
reactions were terminated, and 14C labeled free fatty-acid
was extracted, and counted to determine the amount of free
fatty-acid produced.
Figure 15 shows the triacylglyceride lipase activity of
human EL-AS, EL and LPL using a triolein substrate. To
perform the assay 700 l of conditioned medium harvested from
COS-7 cells transiently transfected with either
pcDNA3.0/LIPG-AS, LIPG, or LPL expression constructs was
assayed in triplicate for triglyceride activities described
below. Following a two hour incubation at 37 C, reactions
were terminated, and 14C labeled free fatty-acid was
extracted, and counted to determine the amount of free fatty-
acid produced.
Figure 16 shows the hybridization of LIPG and LPL probes
to genomic DNAs from different species.
Figure 17 shows expression of LIPG in the liver of a
wild-type mouse 5 days after AdhEL injection. Lane 1, liver
from mouse injected with Adnul.i; lane 2, liver from mouse
injected with AdhEL.
Figure 18 shows plasma levels of HDL cholesterol in
AdhEL- and Adnull-injected wild-type mice.
Figure 19 shows lipoprotein profiles in wild-type mice
injected with AdhEL and Adnull at baseline before injection
(left) and 14 days after injection (right).
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Figure 20 shows HDL cholesterol levels in human apoA-I
transgenic mice after injection with Adnull or AdhEL.
Figure 21 shows ApoA-I levels in human apoA-1 transgenic
mice after injection with Adnull or AdhEL.
Figure 22 shows the effect of injection of AdhEL in LDL
receptor-deficient mice on VLDL/LDL cholesterol levels.
Figure 23 shows the effect of AdhEL on HDL receptor-
deficient mice on HDL cholesterol levels.
Figure 24 is a western blot analysis-of LIPG(EL)
demonstrating the effect of glycosidase and tunicamycin on
EL. Moving from left to right, the lanes are: untreated EL
control; EndoF-treated EL; EndoH-treated EL; neuraminidase-
treated EL; marker, plasma sample; EL untreated lysate; EL
tunicamycin-treated lysate; EL untreated pellet; EL
tunicamycin-treated pellet; EL untreated medium; EL
tunicamycin-treated medium, marker; plasma.
Figure 25 is a western blot illustrating the effect on
EL of heparin administration. "Pre" lanes are blood samples
taken from mice before heparin injection; "post" lanes are
blood samples taken after heparin injection. "Control virus"
did not include EL; "EL virus" was an adenoviral vector
expressing EL.
Figures 26A and 26B are bar graphs illustrating the
triglyceridase activity (Fig. 26A) and the phospholipase
activity (Fig. 26B) of LPL (lipoprotein lipase), HL (hepatic
lipase) and EL (endothelial lipase).
Figures 27A and 27B are graphs illustrating the effect
of human serum on lipoprotein lipase triglyceridase activity
(Fig. 27A) and endothelial lipase triglyceridase activity
(Fig. 27B).
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Figures 28A and 28B illustrate expression of EL at
different virus doses. Figure 28A is a western blot
illustrating the EL present at the different viral doses.
Figure 28B illustrates phospholipase activity at the
different viral dosages.
DETAILED DESCRIPTION OF THE INVENTION
The detailed description which follows sets forth the
basis for the present invention, followed by a definitions
section. Following the definitions section, the various
compositions useful in the practice of the invention are
discussed, followed by a discussion of the methods used to
lower or raise the levels of LIPG activity.
The Enzymatic Activity of the LIPG Gene Product
The present invention relates to methods for regulating
the levels of HDL cholesterol and apolipoprotein AI, VLDL
cholesterol and LDL cholesterol utilizing methods and
compositions which lower or raise the activity of the LIPG
lipase enzyme. In particular, the present invention is based
in part on the discovery of the enzymatic activity of the
polypeptide products of the LIPG gene on HDL cholesterol and
apolipoprotein AI, VLDL cholesterol and LDL cholesterol. The
polypeptide products of LIPG are members of the
triacylglycerol lipase family and comprise an approximately
39 kD catalytic domain of the triacylglycerol lipase family,
e,g., having the sequence SEQ ID NO: 10. Because this newly
discovered lipase was found to be synthesized by endothelial
cells and this is a unique feature compared with other
members of the triacylglycerol lipase family, this lipase has
been named "endothelial lipase" (EL). Because the LIPG gene
will be discussed extensively in the sections which follow,
EL will he hereinafter referred to as LIPG polypeptide, for
the purposes of clarity. In general, the LIPG polypeptide is
found in two major forms, referred to hereinafter as "the
LLGN polypeptide" and "the LLGXL polypeptide." The LLGN
polypeptide, has 354 amino acids. The LLGXL polypeptide has
500 amino acids and exhibits 43% similarity to human
lipoprotein lipase and 37% similarity to human hepatic
lipase. As used herein, the term "LIPG polypeptide" or "LIPG
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protein" encompasses both LLGN and LLGXL.
The sequence of the LIPG polypeptide contains the
characteristic GXSXG lipase motif, a conserved catalytic
triad, a 19-residue lid region, conserved heparin and
lipoprotein binding sites and 5 potential N-linked
glycosylation sites. The region with the greatest sequence
divergence in the triacylglycerol lipase family is the lid
domain, which forms an amphipathic helix covering the
catalytic pocket of the enzyme (Winkler et al., Nature, 343,
771-774 (1990); van Tilbeurgh et al., J. Biol. Chem., 269,
4626-4633 (1994)) and confers substrate specificity to the
enzymes of this family (Dugi et al., J. Biol. Chem., 270,
25396-25401 (1995)). The 19-residue lid region of LIPG is
three residues shorter and less amphipathic than those found
in lipoprotein lipase and hepatic lipase, consistent with a
different enzymatic profile. The predicted molecule weight
of the mature protein is approximately 55 kD; a 68 kD form is
likely to be a glycosylated form, whereas a 40 kD form may be
the product of a specific proteolytic cleavage.
The LIPG polypeptide has the ability to lower the levels
of HDL cholesterol and apolipoprotein AI as well as the
levels of VLDL cholesterol and LDL cholesterol. It is well
established that lowered HDL cholesterol levels result in
increased susceptibility to atherosclerosis and increased
levels of HDL cholesterol can dramatically reduce
susceptibility to atherosclerosis.
One physiologic role of LIPG may be to hydrolyse HDL
phospholipid in peripheral tissues and in liver to facilitate
selective uptake of HDL cholesteryl ester via the HDL
receptor SR-BI (Kozarsky et al., Nature, 387, 414-417
(1997)). Another possible role is the facilitation of apoB-
containing remnant lipoprotein uptake, similar to the role of
hepatic lipase (Mahley et al., J. Lipid Res., 40, 1-16
(1999)). In addition, LIPG is abundantly expressed in the
placenta, and a role for this enzyme in development is
possible, given the importance of lipid transport in fetal
development (Farese et al., Trends Genet., 14, 115-120
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(1998)).
Based on HDL cholesterol's beneficial properties, it is
desirable to raise HDL cholesterol levels by lowering the
enzymatic activity of LIPG. Accordingly, the present
invention is directed to methods and compositions which lower
the activity of LIPG in the body by lowering the expression
of the LIPG gene or lowering the enzymatic activity of the
LIPG polypeptide.
Given the ability of the LIPG polypeptide to reduce the
levels of VLDL cholesterol and LDL cholesterol and the
studies demonstrating the correlation between high levels of
these compounds and atherosclerotic diseases, it is desirable
to lower the level of these compounds in a patient.
Accordingly, the present invention additionally provides
methods and compositions for increasing the expression of the
LIPG gene and increasing the enzymatic activity of the LIPG
polypeptides.
There are set forth hereafter definitions of terms used
herein and descriptions of preferred embodiments of the
present invention.
Definitions
The following defined terms are used throughout the
present specification and should be helpful in understanding
the scope and practice of the present invention.
A "polypeptide" is a polymeric compound comprised of
covalently linked amino acid residues. Amino acids are
classified into seven groups on the basis of the side chain:
(1) aliphatic side chains, (2) side chains containing a
hydroxylic (OH) group, (3) side chains containing sulfur
atoms, (4) side chains containing an acidic or amide group,
(5) side chains containing a basic group, (6) side chains
containing an aromatic ring, and (7) proline, an imino acid
in which the side chain is fused to the amino group.
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A "protein" is a polypeptide which plays a structural or
functional role in a living cell.
The polypeptides and proteins of the invention may be
glycosylated or unglycosylated.
"Homology" means similarity of sequence reflecting a
common evolutionary origin. Polypeptides or proteins are
said to have homology, or similarity, if a substantial number
of their amino acids are either (1) identical, or (2) have a
chemically similar side chain. Nucleic acids are said to
have homology if a substantial number of their nucleotides
are identical.
"Isolated polypeptide" or "isolated protein" is a
polypeptide or protein which is substantially free of those
compounds that are normally associated therewith in its
natural state (e.g., other proteins or polypeptides, nucleic
acids, carbohydrates, lipids). "Isolated" is not meant to
exclude artificial or synthetic mixtures with other
compounds, or the presence of impurities which do not
interfere with biological activity, and which may be present,
for example, due to incomplete purification, addition of
stabilizers, or compounding into a pharmaceutically
acceptable preparation.
A molecule is "antigenic" when it is capable of
specifically interacting with an antigen recognition molecule
of the immune system, such as an immunoglobulin (antibody) or
T cell antigen receptor. An antigenic polypeptide contains
at least about 5, and preferably at least about 10, amino
acids. An antigenic portion of a molecule can be that
portion that is immunodominant for antibody or T cell
receptor recognition, or it can be a portion used to generate
an antibody to the molecule by conjugating the antigenic
portion to a carrier molecule for immunization. A molecule
that is antigenic need not be itself immunogenic, i.e.,
capable of eliciting an immune response without a carrier.
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"LLGN polypeptide" and "LLGN protein" mean a
polypeptide including the sequence SEQ ID NO: 6, said
polypeptide being glycosylated or non-glycosylated.
"LLGXL polypeptide" and "LLGXL protein" mean a
polypeptide including the sequence SEQ ID NO: 8, said
polypeptide being glycosylated or non-glycosylated.
"LIPG polypeptide" and "LIPG protein" describe the
lipase enzyme encoded by the LIPG gene and generically
describes both the LLGN polypeptide and the LLGXL
polypeptide.
"Endothelial lipase," or "EL", refer to the lipase
enzyme encoded by the LIPG gene and is equivalent to the term
LIPG polypeptide.
The LIPG polypeptide or protein of the invention
includes any analogue, fragment, derivative, or mutant which
is derived from an LIPG polypeptide and which retains at
least one biological property of the LIPG polypeptide.
Different variants of the LIPG polypeptide exist in nature.
These variants may be allelic variations characterized by
differences in the nucleotide sequences of the structural
gene coding for the protein, or may involve differential
splicing or post-translational modification. The skilled
artisan can produce variants having single or multiple amino
acid substitutions, deletions, additions, or replacements.
These variants may include, inter alia: (a) variants in which
one or more amino acid residues are substituted with
conservative or non-conservative amino acids, (b) variants in
which one or more amino acids are added to the LIPG
polypeptide, (c) variants in which one or more of the amino
acids includes a substituent group, and (d) variants in which
the LIPG polypeptide is fused with another polypeptide such
as serum albumin. Other LIPG polypeptides of the invention
include variants in which amino acid residues from one
species are substituted for the corresponding residue in
another species, either at conserved or non-conserved
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positions. In another embodiment, amino acid residues at
non-conserved positions are substituted with conservative or
non-conservative residues. The techniques for obtaining
these variants, including genetic (suppressions, deletions,
mutations, etc.), chemical, and enzymatic techniques, are
known to persons having ordinary skill in the art.
If such allelic variations, analogues, fragments,
derivatives, mutants, and modifications, including
alternative mRNA splicing forms and alternative post-
translational modification forms result in derivatives of the
LIPG polypeptide which retain any of the biological
properties of the LIPG polypeptide, they are included within
the scope of this invention.
A "nucleic acid" is a polymeric compound comprised of
covalently linked subunits called nucleotides. Nucleic acid
includes polyribonucleic acid (RNA) and polydeoxyribonucleic
acid (DNA), both of which may be single-stranded or double-
stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and
semi-synthetic DNA. The sequence of nucleotides that encodes
a protein is called the sense sequence.
An "antisense nucleic acid" is a sequence of nucleotides
that is complementary to the sense sequence. Antisense
nucleic acids can be used to down regulate or block the
expression of the polypeptide encoded by the sense strand.
"Isolated nucleic acid" means a nucleic acid which is
substantially free of those compounds that are normally
associated therewith in its natural state. "Isolated" is not
meant to exclude artificial or synthetic mixtures with other
compounds, or the presence of impurities which do not
interfere with biological activity, and which may be present,
for example, due to incomplete purification, addition of
stabilizers, or compounding into a pharmaceutically
acceptable preparation.
The phrase "a nucleic acid which hybridizes at high
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stringency" means that the hybridized nucleic acids are able
to withstand a washing under high stringency conditions. An
example of high stringency washing conditions for DNA-DNA
hybrids is 0.1X SSC, 0.5% SDS at 68 C. Other conditions of
high stringency washing are known to persons having ordinary
skill in the art.
"Regulatory region" means a nucleic acid sequence which
regulates the expression of a nucleic acid. A regulatory
region may include sequences which are naturally responsible
for expressing a particular nucleic acid (a homologous
region) or may include sequences of a different origin
(responsible for expressing different proteins or even
synthetic proteins). In particular, the sequences can be
sequences of eukaryotic or viral genes or derived sequences
which stimulate or repress transcription of a gene in a
specific or non-specific manner and in an inducible or non-
inducible manner. Regulatory regions include origins of
replication, RNA splice sites, enhancers, transcriptional
termination sequences, signal sequences which direct the
polypeptide into the secretory pathways of the target cell,
and promoters.
A regulatory region from a "heterologous source" is a
regulatory region which is not naturally associated with the
expressed nucleic acid. Included among the heterologous
regulatory regions are regulatory regions from a different
species, regulatory regions from a different gene, hybrid
regulatory sequences, and regulatory sequences which do not
occur in nature, but which are designed by one having
ordinary skill in the art.
A "vector" is any means for the transfer of a nucleic
acid according to the invention into a host cell. The term
"vector" includes both viral and nonviral means for
introducing the nucleic acid into a prokaryotic or eukaryotic
cell in vitro, ex vivo or in vivo. Non-viral vectors include
plasmids, liposomes, electrically charged lipids
(cytofectins), DNA-protein complexes, and biopolymers. Viral
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vectors include retrovirus, adeno-associated virus, pox,
baculovirus, vaccinia, herpes simplex, Epstein-Barr and
adenovirus vectors. In addition to nucleic acid according to
the invention, a vector may also contain one or more
regulatory regions, and/or selectable markers useful in
selecting, measuring, and monitoring nucleic acid transfer
results (transfer to which tissues, duration of expression,
etc.).
A "recombinant cell" is a cell which contains a nucleic
acid which is not naturally present in the cell.
"Recombinant cell" includes higher eukaryotic cells such as
mammalian cells, lower eukaryotic cells such as yeast cells,
prokaryotic cells, and archaebacterial cells.
"Pharmaceutically acceptable carrier" includes diluents
and fillers which are pharmaceutically acceptable for methods
of administration, are sterile, and may be aqueous or
oleaginous suspensions formulated using suitable dispersing
or wetting agents and suspending agents. The particular
pharmaceutically acceptable carrier and the ratio of active
compound to carrier are determined by the solubility and
chemical properties of the composition, the particular mode
of administration, and standard pharmaceutical practice.
A "lipase" is a protein which can enzymatically cleave a
lipid substrate.
A "phospholipase" is a protein which can enzymatically
cleave a phospholipid substrate.
A "triacylglycerol lipase" is a protein which can
enzymatically cleave a triacylglyceride substrate.
"Phosphatidylcholine" is a glycerol phospholipid.
Phosphatidylcholine is also known as lecithin.
"Lipid profile" means the set of concentrations of
cholesterol, triglyceride, lipoprotein cholesterol and other
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lipids in the body of a human or other animal.
An "undesirable lipid profile" is the condition in which
the concentrations of cholesterol, triglyceride, or
lipoprotein cholesterol are outside of the age- and gender-
adjusted reference ranges. Generally, a concentration of
total cholesterol > 200 mg/dl, of plasma triglycerides > 200
mg/dl, of LDL cholesterol > 130 mg/dl, of HDL cholesterol <
39 mg/dl, or a ratio of total cholesterol to HDL cholesterol
> 4.0 is considered to, be an undesirable lipid profile. An
undesirable lipid profile is associated with a variety of
pathological conditions, including hyperlipidaemias, diabetes
hypercholesterolaemia, atherosclerosis, and other forms of
coronary artery disease.
A "ribozyme" is an RNA molecule which can function as an
enzyme.
A "neutralizing antibody" is an antibody which can bind
to an LIPG polypeptide and lower or eliminate the enzymatic
activity of the LIPG polypeptide. These antibodies may be
monoclonal antibodies or polyclonal antibodies. The present
invention includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments and the products of an
Fab expression library, and Fv fragments and the products of
an Fv expression library.
An "inhibitory molecule" or "inhibitor" is a molecule
which lowers or eliminates the expression of the LIPG
polypeptide or which lowers or eliminates the enzymatic
activity of the LIPG polypeptide.
An "enhancer molecule" or "enhancer" is a molecule which
increases the expression of the LIPG polypeptide or which
increases the enzymatic activity of the LIPG polypeptide.
A "liposome" is is an artificial or naturally-occurring
phospholipid vesicle.
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A "cationic liposome" is a liposome having a net
positive electrical charge.
The sections which follow discuss the elements used in
the claimed methods and compositions and the preferred
embodiments of these elements.
Polypeptides
The present invention utilizes polypeptides encoded by
LIPG which are members of the triacylglycerol lipase family,
and which comprise a 39 kD catalytic domain of the
triacylglycerol lipase family, e.g., having the sequence SEQ
ID NO: 10. In certain embodiments of the present invention,
an isolated LIPG polypeptide comprising the sequence SEQ ID
NO: 6 and having an apparent molecular weight of about 40 kD
on a 10% SDS-PAGE gel is utilized. In another embodiment of
the present invention, an isolated LIPG polypeptide
comprising the sequence SEQ ID NO: 8 and having an apparent
molecular weight of about 55 kD or 68 kD on a 10% SDS-PAGE
gel is utilized. In yet another embodiment, the polypeptides
utilized in the present invention are subfragments of these
polypeptides. In still yet another embodiment, the
polypeptides used in the present invention are antibodies
capable of binding to an LIPG polypeptide.
The polypeptides and proteins utilized in the present
invention may be recombinant polypeptides, natural
polypeptides, or synthetic polypeptides, and may be of human,
rabbit, or other animal origin. The polypeptides are
characterized by a reproducible single molecular weight
and/or multiple set of molecular weights, chromatographic
response and elution profiles, amino acid composition and
sequence, and biological activity.
The polypeptides utilized in the present invention may
be isolated from natural sources, such as placental extracts,
human.plasma, or conditioned media from cultured cells such
as macrophages or endothelial cells, by using the
purification procedures known to one of skill in the art.
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Alternatively, the polypeptides utilized in the present invention may be
prepared utilizing recombinant DNA technology, which comprises combining a
nucleic acid encoding the polypeptide thereof in a suitable vector, inserting
the
resulting vector into a suitable host cell, recovering the polypeptide
produced by
S the resulting host cell, and purifying the polypeptide recovered.
Nucleic Acids
The present invention utilizes isolated nucleic acids which encode LIPG
polypeptides.
The present invention also utilize:i antisense nucleic acids which can be
used to down regulate or block the expression of LIPG polypeptides in vitrq ex
vivo or in vivo.
The techniques of recombinant DNA technology are known to those of
ordinary skill in the art. General methods for the cloning and expression of
recombinant molecules are described in Maniatis (Molecular Cloning, Cold
Spring Harbor Laboratories, 1982), and in Ausubel (Current Protocols in
Molecular Biology, Wiley and Sons, 1987).
The nucleic acids of the present invention may be linked to one or more
regulatory regions. Selection of the appropriate regulatory region or regions
is a
routine matter, within the level of ordinary skill in the art. Regulatory
regions include promoters, and may include enhancers, suppressors, etc..
Promoters that may be used in the, present invention include both
constituitive promoters and regulated (inducible) promoters. The promoters may
be prokaryotic or eukary otic depending on the host. Among the prokaryotic
(including bacteriophage) promoters useitul for practice of this invention are
lacl,
80 lacZ, T3. T7, lambda Pr, Pr, and trp promoters. Among the eukaryotic
(including
viral) promoters useful for practice of this invention are ubiquitous
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promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate
filament promoters (e.g. desmin, neurofilaments, keratin,
GFAP), therapeutic gene promoters (e.g. MDR type, CFTR,
factor VIII), tissue-specific promoters (e.g. actin promoter
in smooth muscle cells, or Flt and Flk promoters active in
endothelial cells), including animal transcriptional control
regions, which exhibit tissue specificity and have been
utilized in transgenic animals: elastase I gene control
region which is active in pancreatic acinar cells (Swift et
al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring
Harbor Symp. Quant. Biol., 50:399-409; MacDonald, 1987,
Hepatology 7:425-515); insulin gene control region which is
active in pancreatic beta cells (Hanahan, 1985, Nature
315:115-122), immunoglobulin gene control region which is
active in lymphoid cells (Grosschedl et al., 1984, Cell
38:647-658; Adames et al., 1985, Nature 318:533-538;
Alexander et al., 1987, Mot. Cell. Biol., 7:1436-1444), mouse
mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al.,
1986, Cell 45:485-495), albumin gene control region which is
active in liver (Pinkert et al., 1987, Genes and Devel.
1:268-276), alpha-fetoprotein gene control region which is
active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.,
5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha
1-antitrypsin gene control region which is active in the
liver (Kelsey et al., 1987, Genes and Devel., 1:161-171),
beta-globin gene control region which is active in myeloid
cells (Mogram et al., 1985, Nature 315:338-340; Kollias et
al., 1986, Cell 46:89-94), myelin basic protein gene control
region which is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-712), myosin light chain-
2 gene control region which is active in skeletal muscle
(Sani, 1985, Nature 314:283-286), and gonadotropic releasing
hormone gene control region which is active in the
hypothalamus (Mason et al., 1986, Science 234:1372-1378).
Other promoters which may be used in the practice of the
invention include promoters which are preferentially
activated in dividing cells, promoters which respond to a
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stimulus (e.g. steroid hormone receptor, retinoic acid receptor), tetracycline-
regulated transcriptional modulators, cytomegalovirus immediate-early,
retroviral LTR, metallothionein, SV-40, 9la, and MLP promoters. Tetracycline-
regulated transcriptional modulators and CMV promoters are described in WO
96/01313, US 5,168,062 and 5,385,839.
Viral Vector evatems
Preferably, the viral vectors used in the gene therapy methods of the
present invention are replication defective, that is, they are unable to
replicate
autonomously in the target cell. In general, the genome of the replication
defective viral vectors which are used within the scope of the present
invention
lack at least one region which is necessary for the replication of the virus.
in the
infected cell. These regions can either be eliminated (in whole or in part),
or be
rendered non-functional by any technique known to a person skilled in the art.
These techniques include the total removal, substitution (by other sequences,
in
particular by the inserted nucleic acid), partial deletion or addition of one
or
more bases to an essential (for replication) region. such techniques may be
performed in vitro (on the isolated DNA) or in situ, using the techniques of
genetic manipulation or by treatment with mutagenic agents.
Preferably, the replication defective virus retains the sequences of its
genome which are necessary for encapsictating the viral particles.
The retroviruses are integrating viruses which infect dividing cells. The
retrovirus genome includes two LTRs, an encapaidation sequence and three
coding regions (gag, pol and env). The construction of recombinant retroviral
vectors has been described= see, in particular, EP 453242, P178220, Bernstein
et
al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, etc. In
recombinant retroviral
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vectors, the gag, pol and env genes are generally deleted, in
whole or in part, and replaced with a heterologous nucleic
acid sequence of interest. These vectors can be constructed
from different types of retrovirus, such as, MoMuLV ("murine
Moloney leukaemia virus" MSV ("murine Moloney sarcoma
virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis
virus"); RSV ("Rous sarcoma virus") and Friend virus.
Lentivirus vector systems may also be used in the practice of
the present invention. The lentiviral genome is a positive-
strand polyadenylated RNA of 9,000 to 10,000 base pairs
containing three structural genes organized 5' to 3' (gag,
pol, env), typical of all retroviruses. For an extensive
review of lentiviral systems, see Fields Virology, Second
Edition, Volume 2, Chapter 55, "Lentiviruses," pp. 1571-1589,
Raven Press, New York, 1990.
In general, in order to construct recombinant
retroviruses containing a sequence encoding LIPG, a plasmid
is constructed which contains the LTRs, the encapsidation
sequence and the coding sequence. This construct is used to
transfect a packaging cell line, which cell line is able to
supply in trans the retroviral functions which are deficient
in the plasmid. In general, the packaging cell lines are
thus able to express the gag,.pol and env genes. Such
packaging cell lines have been described in the prior art, in
particular the cell line PA317 (US4,861,719); the PsiCRIP
cell line (W090/02806) and the GP+envAm-12 cell line
(W089/071.50). In addition, the recombinant retroviral
vectors can contain modifications within the LTRs for
suppressing transcriptional activity as well as extensive
encapsidation sequences which may include a part of the gag
gene (Bender et al., J. Virol. 61 (1987) 1639). Recombinant
retroviral vectors are purified by standard techniques known
to those having ordinary skill in the art.
The adeno-associated viruses (AAV) are DNA viruses of
relatively small size which can integrate, in a stable and
site-specific manner, into the genome of the cells which they
infect. They are able to infect a wide spectrum of cells
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without inducing any effects on cellular growth, morphology
or differentiation, and they do not appear to be involved in
human pathologies. The AAV genome has been cloned, sequenced
and characterized. It encompasses approximately 4700 bases
and contains an inverted terminal repeat (ITR) region of
approximately 145 bases at each end, which serves as an
origin of replication for the virus. The remainder of the
genome is divided into two essential regions which carry the
encapsidation functions: the left-hand part of the genome,
which contains the rep gene involved in viral replication and
expression of the viral genes; and the right-hand part of the
genome, which contains the cap gene encoding the capsid
proteins of the virus.
The use of vectors derived from the AAVs for
transferring genes in vitro and in vivo has been described
(see WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP
488 528). These publications describe various AAV-derived
constructs in which the rep and/or cap genes are deleted and
replaced by a gene of interest, and the use of these
constructs for transferring the said gene of interest in
vitro (into cultured cells) or in vivo, (directly into an
organism). The replication defective recombinant AAVs
utilized in the present invention can be prepared by
cotransfecting a plasmid containing the nucleic acid sequence
of interest flanked by two AAV inverted terminal repeat (ITR)
regions, and a plasmid carrying the AAV encapsidation genes
(rep and cap genes), into a cell line which is infected with
a human helper virus (for example an adenovirus). The AAV
recombinants which are produced are then purified by standard
techniques. The invention also relates, therefore, to an AAV-
derived recombinant virus whose genome encompasses a sequence
encoding an LIPG polypeptide flanked by the AAV ITRs. The
invention also relates to a plasmid encompassing a sequence
encoding an LIPG polypeptide flanked by two ITRs from an AAV.
Such a plasmid can be used as it is for transferring the LIPG
sequence, with the plasmid, where appropriate, being
incorporated into a liposomal vector (pseudo-virus).
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In a preferred embodiment, the vector utilized in the
present invention is an adenovirus vector.
Adenoviruses are eukaryotic DNA viruses that can be
modified to efficiently deliver a nucleic acid to a variety
of cell types.
Various serotypes of adenovirus exist. Of these
serotypes, preference is given, within the scope of the
present invention, to using type 2 or type 5 human
adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin
(see W094/26914). Those adenoviruses of animal origin which
can be used within the scope of the present invention include
adenoviruses of canine, bovine, murine (example: Mavl, Beard
et al., Virology 75 (1990) 81), ovine, porcine, avian, and
simian (example: SAV) origin. Preferably, the adenovirus of
animal origin is a canine adenovirus, more preferably a CAV2
adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800),
for example).
Preferably, the replication defective adenoviral vectors
comprise the ITRs, an encapsidation sequence and the nucleic
acid of interest. Still more preferably, at least the El
region of the adenoviral vector is non-functional. The
deletion in the El region preferably extends from nucleotides
455 to 3329 in the sequence of the Ads adenovirus. Other
regions may also be modified, in particular the E3 region
(W095/02697), the E2 region (W094/28938), the E4 region
(W094/28152, W094/3.2649 and W095/02697), or in any of the
late genes Ll-L5. Defective retroviral vectors are disclosed
in W095/02697.
In a preferred embodiment, the adenoviral vector has a
deletion in the El and E4 regions. In another preferred
embodiment, the adenoviral vector has a deletion in the El
region into which the E4 region and the sequence encoding LLG
are inserted (see FR94 13355).
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The replication defective recombinant adenoviruses can
be prepared by any technique known to the person skilled in
the art (Levrero et al., Gene 101 (1991) 195, EP 185 573;
Graham, EMBO J. 3 (1984) 2917). In particular, they can be
prepared by homologous recombination between an adenovirus
and a plasmid which carries, inter alia, the DNA sequence of
interest. The homologous recombination is effected following
cotransfection of the said adenovirus and plasmid into an
appropriate cell line. The cell line which is employed
should preferably (i) be transformable by the said elements,
and (ii) contain the sequences which are able to complement
the part of the genome of the replication defective
adenovirus, preferably in integrated form in order to avoid
the risks of recombination. Examples of cell lines which may
be used are the human embryonic kidney cell line 293 (Graham
et al., J. Gen. Virol. 36 (1977) 59) which contains the left-
hand portion of the genome of an Ad5 adenovirus (12%)
integrated into its genome, and cell lines which are able to
complement the El and E4 functions, as described in
applications W094/26914 and W095/02697. Recombinant
adenoviruses are recovered and purified using standard
molecular biological techniques, which are well known to one
of ordinary skill in the art.
Antisense Nucleic Acids
The down regulation of gene expression using antisense
nucleic acids can be achieved at the translational or
transcriptional level. Antisense nucleic acids of the
invention are preferably nucleic acid fragments capable of
specifically hybridizing with all or part of a nucleic acid
encoding LIPG or the corresponding messenger RNA. In
addition, antisense nucleic acids may be designed or
identified which decrease expression of the LIPG gene by
inhibiting splicing of its primary transcript. With
knowledge of the structure and partial sequence of the LIPG
gene, such antisense nucleic acids can be designed and tested
for efficacy.
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The antisense nucleic acids are preferably
oligonucleotides and may consist entirely of deoxyribo-
nucleotides, modified deoxyribonucleotides, or some
combination of both. The antisense nucleic acids can be-
synthetic oligonucleotides. The oligonucleotides may be
chemically modified, if desired, to improve stability and/or
selectivity. Since oligonucleotides are susceptible to
degradation by intracellular nucleases, the modifications can
include, for example, the use of a sulfur group to replace
the free oxygen of the phosphodiester bond. This
modification is called a phosphorothioate linkage.
Phosphorothioate antisense oligonucleotides are water
soluble, polyanionic, and resistant to endogenous nucleases.
In addition, when a phosphorothioate antisense
oligonucleotide hybridizes to its target site, the RNA-DNA
duplex activates the endogenous enzyme ribonuclease (Rnase)
H, which cleaves the mRNA component of the hybrid molecule.
In addition, antisense oligonucleotides with
phosphoramidite and polyamide (peptide) linkages can be
synthesized. These molecules should be very resistant to
nuclease degradation. Furthermore, chemical groups can be
added to the 2' carbon of the sugar moiety and the 5 carbon
(C-5) of pyrimidines to enhance stability and facilitate the
binding of the antisense oligonucleotide to its target site.
Modifications may include 2' deoxy, O-pentoxy, O-propoxy, 0-
methoxy, fluoro, methoxyethoxy phosphoro-thioates, modified
bases, as well as other modifications known to those of skill
in the art.
The antisense nucleic acids can also be DNA sequences
whose expression in the cell produces RNA complementary to
all or part of the LIPG mRNA. Antisense nucleic acids can be
prepared by expression of all or part of a sequence selected
from the group consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ
ID No. 7, or SEQ ID No. 11., in the opposite orientation, as
described in EP 140308. Any length of antisense sequence is
suitable for practice of the invention so long as it is
capable of down-regulating or blocking expression of LIPG.
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-$s
Preferably, the antisense sequence is at least 20 nucleotides in length. The
preparation and use of antisense nucleic acids, DNA encoding antisense RNAs
and the use of oligo and genetic antisense is disclosed in W092/15680.
6 One approach to determining the optimum fragment of LIPG to use in an
antisense nucleic acid treatment method involves preparing random fragments
of LIPG eDNA by mechanical shearing, enzymatic treatment, and cloning the
fragment into any of the vector systems described herein. Individual clones or
pools of clones are used to infect LIPG=expressing cells, and effective
antisense
LIPG cDNA fragments are identified by monitoring LIPG expression at the RNA
or protein level.
The retroviral, adeno-associated viral, and adenoviral vector systems
discussed hereinabove may all be used to introduce and express antisense
nucleic acids in cells. Antisense synthetic oligonucleotides may be introduced
in
a variety of ways, including the meth) ids discussed hereinbelow.
Ribozvmes
Reductions in the levels of LIPG polypeptide may be accomplished using
ribozyrnes_ Ribozymes are catalytic RNA molecules (RNA enzymes) that have
separate catalytic and substrate binding domains. The substrate binding
sequence combines by nucleotide complementarity and, possibly, nonhydrogen
bond interactions with its target sequence. The catalytic portion cleaves the
target RNA at a specific site. The substrate domain of a ribozyme can be
engineered to direct it to a specified nRNA sequence. The ribozyme
recognizes and then binds a target mitNA through complementary base-pairing.
Once it is bound to the correct target -{ite, the ribozyme acts enzymatically
to cut
the target mRNA. Cleavage of the LII'G mRNA by a. ribozyme destroys its
ability to direct synthesis of LIFG polypeptide. Once the ribozyme has cleaved
its
target sequence, it is released and cart repeatedly bind and cleave at other
LIPG
mRNAs.
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In preferred embodiments of this invention, the ribozyme
is formed in a hammerhead motif. Other forms include a
hairpin motif, a hepatitis delta virus, group I intron or
RnaseP RNA (in association with an RNA guide sequence) motif
or Neurospora VS RNA motif. Hammerhead motifs are described
by Rossi et al., 1992, Aids Research and Human Retroviruses,
8, 183. Hairpin motifs are described in Hampel and Tritz,
1989, Biochemistry, 28, 4929, and Hampel et al., 1990,
Nucleic Acids Res., 18, 299. The hepatitis delta virus motif
is described by Perrotta and Been, 1992, Biochemistry, 31,
16, the RnaseP motif is described by Guerrier-Takada et al.,
1983, Cell, 35, 849, the Neurospora VS RNA ribozyme motif is
described by Collins (Saville and Collins, 1990, Cell, 61,
685-696; Saville and Collins, 1991, Proc. Natl. Acad. Sci.
USA, 88, 8826-8830; Collins and olive, 1993, Biochemistry,
32, 2795-2799) the Group I intron motif is described by Cech
et al., U.S. Patent No. 4,987,071.
One approach in preparing a ribozyme is to chemically
synthesize an oligodeoxyribonucleotide with a ribozyme
catalytic domain (-20 nucleotides) flanked by sequences that
hybridize to the target LIPG mRNA after transcription. The
oligodeoxyribonucleotide is amplified by using the substrate
binding sequences as primers. The amplification product is
cloned into a eukaryotic expression vector.
Ribozymes possessing a hammerhead or hairpin structure
are readily prepared since these catalytic RNA molecules can
be expressed within cells from eukaryotic promoters (e.g.,
Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-
5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15;
Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et
al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc.
Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic
Acids Res., 20, 4581-9; Sarver et al., 1990, Science, 247,
1222-1225)). A ribozyme of the present invention can be
expressed in eukaryotic cells from the appropriate DNA
vector. If desired, the activity of the ribozyme may be
augmented by its release from the primary transcript by a
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second ribozyme (Ohkawa et al., 1992, Nucleic Acids Symp.
Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19,
5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-
55).
In one approach to preparing ribozymes, ribozymes are
expressed from transcription units inserted into DNA, RNA, or
viral vectors. Transcription of the ribozyme sequences are
driven from a promoter for eukaryotic RNA polymerase I (pol
(I), RNA polymerase II (pol II), or RNA polymerase III (pol
III). Transcripts from pol II or pol III promoters will be
expressed at high levels in all cells; the levels of a given
pol II promoter in a given cell type will depend on nearby
gene regulatory sequences. Prokaryotic RNA polymerase
promoters are also used, providing that the prokaryotic RNA
polymerase enzyme is expressed in the appropriate cells
(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87,
6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72;
Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et
al., 1990, Mot. Cell. Biol., 10, 4529-37). It has been
demonstrated that ribozymes expressed from these promoters
can function in mammalian cells (Kashani-Sabet et al., 1992,
Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc.
Natil. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992 Nucleic
Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.
Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11,
4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. USA,
90, 8000-4).
In one embodiment of the present invention, a
transcription unit expressing a ribozyme that cleaves LIPG
RNA is inserted into a plasmid DNA vector, a retrovirus
vector, an adenovirus DNA viral vector or an adeno-associated
virus vector. The recombinant vectors are preferably DNA
plasmids or adenovirus vectors. However, other mammalian
cell vectors that direct the expression of RNA may be used
for this purpose. The vectors are delivered as recombinant
viral particles. DNA may be delivered alone or complexed
with various vehicles. The DNA, DNA/vehicle complexes, or
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the recombinant virus particles are locally administered to the site of
treatment,
as discussed below. Preferably, recombinant vectors capable of expressing the
ribozymes are locally delivered as described below, and persist in target
cells.
Once expressed, the ribozymes cleave the target LIPG mRNA.
Ribozymes may be administered I o a patient by a variety of methods.
They may be added directly to target tis.;ues, complexed with cationic lipids,
packaged within liposomes, or elivered to target cells by other methods known
in
the art. Localized administration to the desired tissues may be done by
catheter,
infusion pump or stent, with or without incorporation of the ribozyme in
biopolymers as discussed hereinbelow. Alternative routes of delivery include,
but
are not limited to, intravenous injection, intramuscular injection,
subcutaneous
injection, aerosol inhalation, oral (tablet or pill form), topical, systemic,
ocular,
intraperitoneal and/or intrathecal delivery. More detailed descriptions of
ribozyrne delivery and administration are provided in Sullivan et al., PCT
W094102595 and Draper et al., PCT W0,93/23569.
Non-Viral De);v~rv Systems
Certain non viral systems have been used in the art and can facilitate
introduction of DNA encoding the LIPG polypeptides or antisense nucleic acids
into a patient.
A DNA vector encoding a desired LIPG polypeptide or antisense sequence
can be introduced in vivo by lipofection. For the past decade, there has been
increasing use of liposomee for eneapsulittion and transfection of nucleic
acids in
vitro. Synthetic cationic lipids designed to limit the difficulties and
dangers
encountered with liposome mediated transfection can be used to prepare
liposomes for in vivo transfection of a gene encoding a marker (FeIgner, et.
al.,
Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey, et al., Proc.
NarJ.
Acad. Sci. U.S.A. 85:8027-8031
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(1988); Ulmer et al., Science 259:1745-1748 (1993)]. The use
of cationic lipids may promote encapsulation of negatively
charged nucleic acids, and also promote fusion with
negatively charged cell membranes [Feigner and Ringold,
Nature 337:387-388 (1989)]. Particularly useful lipid
compounds and compositions for transfer of nucleic acids are
described in International Patent Publications W095/18863 and
W096/17823, and in U.S. Patent No. 5,459,127. The use of
lipofection to introduce exogenous genes into the specific
organs in vivo has certain practical advantages. Molecular
targeting of liposomes to specific cells represents one area
of benefit. It is clear that directing transfection to
particular cell types would be particularly advantageous in a
tissue with cellular heterogeneity, for example, pancreas,
liver, kidney, and the brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting [see
Mackey, et. al., supra]. Targeted peptides, e.g., hormones
or neurotransmitters, and proteins for example, antibodies,
or non-peptide molecules could be coupled to liposomes
chemically.
Other molecules are also useful for facilitating
transfection of a nucleic acid in vivo, for example, a
cationic oligopeptide (e.g., International Patent Publication
W095/21931), peptides derived from DNA binding proteins
(e.g., International Patent Publication W096/25508), or a
cationic polymer (e.g., International Patent Publication
W095/21931).
It is also possible to introduce A DNA vector encoding a
LIPG polypeptide or antisense sequence in vivo as a naked DNA
plasmid (see U.S. Patents 5,693,622, 5,589,466 and
5,580,859). Naked DNA vectors for gene therapy can be
introduced into the desired host cells by methods known in
the art, e.g., transfection, electroporation, microinjection,
transduction, cell fusion, DEAE dextran, calcium phosphate
precipitation, use of a gene gun, or use of a DNA vector
transporter [see, e.g., Wilson et al., J. Biol. Chem.
267:963-967 (1992); Wu and Wu, J. Biol. Chem. 263:14621-14624
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(1988); Hartmut et al., Canadian Patent Application No.
2,012,311, filed March 15, 1990; Williams et al., Proc. Natl.
Acad. Sci. USA 88:2726-2730 (1991)]. Receptor-mediated DNA
delivery approaches can also be used [Curiel et al., Hum.
Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem.
262:4429-4432 (1987)].
Antibodies
The present invention provides antibodies against the
LIPG polypeptide. These antibodies may be monoclonal
antibodies or polyclonal antibodies. The present invention
includes chimeric, single chain, and humanized antibodies, as
well as Fab fragments and the products of an Fab expression
library, and Fv fragments and the products of an Fv
expression library.
Polyclonal antibodies may be prepared against an
antigenic fragment of an LIPG polypeptide, as described in
the Examples section hereinbelow. Antibodies may also be
generated against the intact LIPG protein or polypeptide, or
against a fragment, derivative, or epitope of the protein or
polypeptide. Antibodies may be obtained following the
administration of the protein, polypeptide, fragment,
derivative, or epitope to an animal, using the techniques and
procedures known in the art.
Monoclonal antibodies may be prepared using the method
of Mishell, B. B., et al., Selected Methods In Cellular
Immunology, (W.H. Freeman, ed.) San Francisco (1980).
Briefly, a polypeptide of the present invention is used to
immunize spleen cells of Balb/C mice. The immunized spleen
cells are fused with myeloma cells. Fused cells containing
spleen and myeloma cell characteristics are isolated by
growth in HAT medium, a medium which kills both parental
cells, but allows the fused products to survive and grow.
The monoclonal antibodies of the present invention may
be "humanized" to prevent the host from mounting an immune
response to the antibodies. A "humanized antibody" is one in
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which the complementarity determining regions (CDRs) and/or
other portions of the light and/or heavy variable domain
framework are derived from a non-human immunoglobulin, but
the remaining portions of the molecule are derived from one
or more human immunoglobulins. Humanized antibodies also
include antibodies characterized by a humanized heavy chain
associated with a donor or acceptor unmodified light chain or
a chimeric light chain, or vice versa. The humanization of
antibodies may be accomplished by methods known in the art
(see, e.g. G.E. Mark and E.A. Padlan, "Chapter 4.
Humanization of Monoclonal Antibodies", The Handbook of
Experimental Pharmacology Vol. 113, Springer-Verlag, New
York, 1994). Transgenic animals may be used to express
humanized antibodies.
Techniques known in the art for the production of single
chain antibodies can be adapted to produce single chain
antibodies to the immunogenic polypeptides and proteins of
the present invention.
In a preferred embodiment, an anti-LIPG antibody is used
to bind to and inhibit the enzymatic activity of LIPG in a
patient.
The anti-LIPG antibodies are also useful in assays for
detecting or quantitating levels of LIPG. In one embodiment,
these assays provide a clinical diagnosis and assessment of
LIPG in various disease states and a method for monitoring
treatment efficacy. These anti-LIPG antibodies may
additionally be used to quantitate LIPG in a tissue sample in
order to predict further susceptibility to lowered levels of
HDL cholesterol and apolipoprotein AI.
Methods of Identifying and Utilizing
Inhibitory Molecules and Enhancer Molecules
The present invention provides methods of screening
small molecule libraries or natural product sources for
enhancers (agonists) or co-activators including proteinaceous
co-activators or inhibitors (antagonists) of LIPG activity.
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A potential enhancer or inhibitor is contacted with LIPG
protein and a substrate of LIPG, and the ability of the
potential enhancer or inhibitor to enhance or inhibit LIPG
activity is measured.
These screening methods may also be used to determine if
a compound can function as a substrate specific enhancer or
inhibitor, that is, whether a compound can enhance the
enzymatic activity of LIPG toward one substrate while
lowering or maintaining a given level of enzymatic activity
for a different substrate, for example, the LIPG polypeptide
of the present invention utilizes HDL cholesterol as a
substrate and also utilizes LDL cholesterol and VLDL
cholesterol as substrates. In certain embodiments, it is
desirable to isolate and identify substrate specific
enhancers or inhibitors which enhance the enzymatic activity
of the LIPG polypeptide towards LDL cholesterol or VLDL
cholesterol while lowering or maintaining the normal level of
enzymatic activity for HDL cholesterol.
The LIPG protein used in these methods can be produced
recombinantly in a variety of host cells, including mammalian
cells, baculovirus-infected insect cells, yeast, and
bacteria. LIPG expression in stably transfected CHO cells
can be optimized by methotrexate amplification of the cells.
LIPG protein can also be purified from natural sources such
as human plasma, placental extracts, or conditioned media
from cultured endothelial cells, THP-1 cells, or macrophages.
The optimization of assay parameters including pH, ion
concentrations, temperature, concentration of substrate, and
emulsification conditions are determined empirically by one
having ordinary skill in the art.
The fatty acid substituents of the substrates may vary
in chain length as well as in degree and position of
unsaturation. The substrates may be radiolabelled in any of
several positions. Phospholipid substrates such as
phosphatidylcholine can be radiolabelled, for example, in the
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Sn-1 or Sn-2 fatty acid position, or in the glycerol,
phosphate, or polar head group (choline in the case of
phosphatidylcholine).
As an alternative to radiolabeled substrates, other
classes of labeled substrates, such as fluorescent substrates
or thio-containing substrates, can also be used in the
screening methods.
Fluorescent substrates are particularly useful in
screening assays because enzymatic catalysis can be measured
continuously by measuring fluorescence intensity, without the
physical separation (extraction) of the products from the
substrates. An example of a fluorescent phosphatidylcholine
substrate is C6NBD-PC{l-acyl-2-[6-(nitro-2,1,3-benzoxadiazol-
4-yl)amino] caproylphosphatidylcholine.
The thio-containing substrates include 1,2-
bis(hexanoylthio)-1, 2-dideoxy-sn-glycero-3-phosphorylcholine
(L.J. Reynolds, W.N. Washburn, R.A. Deems, and E.A. Dennis,
1991. Methods in Enzymology 197: 3-23; L. Yu and E.A.
Dennis, 1991. Methods in Enzymology 197: 65-75; L.A.
Wittenauer, K. Shirai, R.L. Jackson, and J.D. Johnson, 1984.
Biochem. Biophys. Res. Commun. 118: 894-901).
In addition to inhibitory and enhancer molecules which
operate at the level of enzymatic activity, there are
inhibitory and enhancer molecules which operate at the level
of expression of the LIPG gene. One method for identifying
compounds which are able to enhance or inhibit the expression
of LIPG is to use a reporter gene system. These systems
utilize reporter gene expression vectors which include a
cloning site into which a given promoter may be cloned
upstream of a "reporter gene" which can be easily detected
and quantified. One of skill in the art could readily
identify and subclone the promoter for the LIPG gene as well
as other control sequences into a commercially available
reporter gene expression vector. The expression vector is
transferred into host cells and the cells are exposed to a
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test compound (a putative inhibitor or enhancer molecule) to
determine the effect of the test compound on expression of
the reporter gene product. In particular, the cells are
assayed for the presence of the reporter gene product by
directly measuring the amount of reporter mRNA, the reporter
protein itself or the enzymatic activity of the reporter
protein. Ideally, the reporter gene is not endogenously
expressed in the cell type of interest and lends itself to
sensitive, quantitative and rapid assays. A variety of
reporter assay constructs are commercially available and
several reporter genes and assays have been developed and can
be readily prepared by those of skill in the art. The most
popular systems for monitoring genetic activity in eukaryotic
cells include the chloramphenicol acetyltransferase (CAT), 3-
galactosidase, firefly luciferase, growth hormone (GH), (3-
glucurorudase (GUS), alkaline phosphatase (AP), green
fluorescent protein (GFP) and Renilla luciferase. Reporter
assay constructs can be purchased from a variety of sources
including Promega and Invitrogen.
As mentioned above, reporter gene activity can be
detected by assaying for the reporter mRNA or the reporter
protein. The reporter mRNA can be detected by northern blot
analysis, ribonuclease protection assays or RT-PCR. While
these assays are more direct than measuring protein
expression, many assays have been developed to measure the
presence of the reporter protein rather than the mRNA present
in a cell. Reporter proteins can be assayed by
spectrophotometry or by detecting enzymatic activity.
Reporter protein levels may also be measured with antibody-
based assays. In general, the enzymatic assays are very
sensitive and are a preferred method of monitoring reporter
gene expression.
Compositions
The present invention provides compositions in a
biologically compatible (biocompatible) solution, comprising
the polypeptides, nucleic acids, vectors, and antibodies of
the invention. A biologically compatible solution is a
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solution in which the polypeptide, nucleic acid, vector, or
antibody of the invention is maintained in an active form,
e.g., in a form able to effect a biological activity. For
example, a polypeptide of the invention would have
phospholipase activity; a nucleic acid would be able to
replicate, translate a message, or hybridize to a
complementary nucleic acid; a vector would be able to
transfect a target cell; an antibody would bind a polypeptide
of the invention. Generally, such a biologically compatible
solution will be an aqueous buffer, e.g., Tris, phosphate, or
HEPES buffer, containing salt ions. Usually the
concentration of salt ions will be similar to physiological
levels. In a specific embodiment, the biocompatible solution
is a pharmaceutically acceptable composition. Biologically
compatible solutions may include stabilizing agents and
preservatives.
Such compositions can be formulated for administration
by topical, oral, parenteral, intranasal, subcutaneous, and
intraocular, routes. Parenteral administration is meant to
include intravenous injection, intramuscular injection,
intraarterial injection or infusion techniques. The
composition may be administered parenterally in dosage unit
formulations containing standard, well known nontoxic
physiologically acceptable carriers, adjuvants and vehicles
as desired.
The preferred sterile injectable preparations can be a
solution or suspension in a nontoxic parenterally acceptable
solvent or diluent. Examples of pharmaceutically acceptable
carriers are saline, buffered saline, isotonic saline (e.g.
monosodium or disodium phosphate, sodium, potassium, calcium
or magnesium chloride, or mixtures of such salts), Ringer's
solution, dextrose, water, sterile water, glycerol, ethanol,
and combinations thereof. 1,3-butanediol and sterile fixed
oils are conveniently employed as solvents or suspending
media. Any bland fixed oil can be employed including
synthetic mono- or di-glycerides. Fatty acids such as oleic
acid also find use in the preparation of injectables.
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The composition medium can also be a hydrogel which is prepared from
any biocompatible or non=cytotoxic (home or hetero) polymer, such as a
hydrophilic polyaciylic acid polymer that can act as a drug absorbing sponge.
Such polymers have been described, for example, in application
W093/08845. Certain of them, such as, in particular, those obtained from
ethylene and/or propylene oxide are commercially available. A hydrogel can be
deposited directly onto the surface of the tissue to be treated, for example
during
surgical intervention.
Another preferred embodiment of the present invention relates to a
pharmaceutical composition comprising a replication defective recombinant
virus
and poloxamer. More specifically, the invention relates to a composition
comprising a replication defective recombinant virus comprising a nucleic acid
encoding an LIPG polypeptide and poloxamer. A preferred poloxamer is
Poloxamer 407, which is commercially available (BASF, Parsippany, NJ) and is a
non-toxic, biocompatible polyol, and is most preferred. A poloxamer
impregnated
with recombinant viruses may be deposit ed directly on the surface of the
tissue
to be treated, for example during a surgical intervention.
Poloxamer possesses essentially the same advantages as hydrogel while having a
lower viscosity.
Methods of Treatment
The present invention provides methods of treatment which comprise the
administration to a human or other animal of an effective amount of a
composition of the invention.
Effective amounts may vary, depending on the age, type and severity of
the condition to be treated, body weight, desired duration of treatment,
method
of administration, and other parameters. Effective amounts are determined by a
physician or other qualified medical professional. In most cases, the dosage
levels may be adjusted so that the desired levels of HDL cholesterol and
apolipoprotein Al are achieved
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and maintained. Similarly, the dosage levels may be adjusted
to lower the VLDL cholesterol and LDL cholesterol levels to
acceptable levels and bring the ratio HDL cholesterol to LDL
cholesterol and VLDL cholesterol to within desirable levels.
Polypeptides according to the invention are generally
administered in doses of about 0.01 mg/kg to about 100 mg/kg,
preferably about 0.1 mg/kg to about 50 mg/kg, and most
preferably about 1 mg/kg to about 10 mg/kg of body weight per
day.
Neutralizing antibodies according to the invention may
be delivered as a bolus only, infused over time or both
administered as a bolus and infused over time. Although the
dosage amount will vary based on the parameters above, and on
the binding ability of the antibody, a dose 0.2 to 0.6 mg/kg
may be given as a bolus followed by a 2 to 12 hour infusion
period. Alternatively, multiple bolus injections are
administered every other day or every third or fourth day as
needed. Dosage levels may be adjusted as determined by HDL
cholesterol levels and/or VLDL and LDL cholesterol levels.
As discussed hereinabove, recombinant viruses may be
used to introduce both DNA encoding LIPG and subfragments of
LIPG as well as antisense nucleic acids. Recombinant viruses
according to the invention are generally formulated and
administered in the form of doses of between about 104 and
about 1014 pfu. In the case of AAVs and adenoviruses, doses
of from about 106 to about 1011 pfu are preferably used. The
term pfu ("plaque-forming unit") corresponds to the infective
power of a suspension of virions and is determined by
infecting an appropriate cell culture and measuring the
number of plaques formed. The techniques for determining the
pfu titre of a viral solution are well documented in the
prior art.
Ribozymes according to the present invention may be
administered in amounts ranging from about 5 to about 50
mg/kg/day in a pharmaceutically acceptable carrier. Dosage
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levels may be adjusted based on the measured therapeutic
efficacy.
Appropriate levels of inhibitor or enhancer molecules
may be determined by qualified medical personnel using the
parameters discussed above.
The present invention provides compositions and methods
for increasing the level of HDL cholesterol and
apolipoprotein AI and lowering the levels of VLDL and LDL
cholesterol in a patient. The present invention further
provides methods of treating a human or other animal having
an undesirable lipid profile, wherein said undesirable lipid
profile is the result of abnormally high expression of LIPG
polypeptide activity.
Methods and Compositions for
Lowering Levels of LIPG Polypeptide Activity
The methods for decreasing the expression of LIPG
polypeptide in order to increase the levels of HDL
cholesterol and apolipoprotein AI and correct those
conditions in which LIPG polypeptide activity contributes to
a disease or disorder associated with an undesirable lipid
profile include but are not limited to administration of a
composition comprising an antisense nucleic acid,
administration of a composition comprising an intracellular
binding protein such as an antibody, administration of an
inhibitory molecule which inhibits the enzymatic activity of
LIPG, for example, a composition comprising an expression
vector encoding a subfragment of LIPG, for example, LLGN
polypeptide or a small molecular weight molecule, including
administration of a small molecular weight compound which
down regulates LIPG expression at the level of transcription,
translation or post-translation, and administration of a
ribozyme which cleaves mRNA encoding LIPG.
Methods Utilizing Antisense Nucleic Acids
In one embodiment, a composition comprising an antisense
nucleic acid is used to down-regulate or block the expression
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of LIPG. In one preferred embodiment, the nucleic acid encodes antisense RNA
molecules. In this embodiment, the nuch-ic acid is operably linked to signals
enabling expression of the nucleic acid sequence and is introduced
into a cell utilizing, preferably, recombinant vector constructs, which will
express
the antisense nucleic acid once the vector is introduced into the cell.
Examples of
suitable vectors includes plasmids, adew viruses, adenoassociated viruses,
retroviruses, and herpes viruses. Preferably, the vector is an adenovirus.
Most
preferably, the vector is a replication defective adenovirus comprising a
deletion
in the E 1 and/or E3 regions of the virus.
In another embodiment, the antisense nucleic acid is synthesized and may
be chemically modified to resist degradation by intracellular nucleases, as
discussed above. Synthetic antisense oligonucleotides can be introduced to a
cell
using liposomes. Cellular uptake occurs when an antisense oligonucleotide is
encapsulated within a liposome. with an effective delivery system, low, non-
toxic
concentrations of the antisense molecule can be used to
inhibit translation of the target mRNA. Moreover, liposomes that are
conjugated
with cell-specific binding sites direct an antisense oligonucleotide to a
particular
tissue.
Methods Utilizing Neutralizing
Antibodies and Ot1v;r Binding Proteins
In another embodiment, the expression of LIPG is downregulated or
blocked by the expression of a nucleic acid sequence encoding an intracellular
binding protein which is capable of selectively interacting with LIPG. WO
94/29446 and WO 94/02610, disclose eelli der transfection with genes encoding
an
intracellular binding protein. An intracellular binding protein includes any
protein capable of selectively interacting, or binding, with LIPG in the cell
in
which it is expressed and of neutralizing the function of bound LLG.
Preferably,
the intracellular binding protein is a neutralizing antibody or a fragment of
a
neutralizing
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antibody. More preferably, the intracellular binding protein
is a single chain antibody.
WO 94/02610 discloses preparation of antibodies and
identification of the nucleic acid encoding a particular
antibody. Using LIPG or a fragment thereof, a specific
monoclonal antibody is prepared by techniques known to those
skilled in the art. A vector comprising the nucleic acid
encoding an intracellular binding protein, or a portion
thereof, and capable of expression in a host cell is
subsequently prepared for use in the method of this
invention.
Alternatively, LIPG activity can be blocked by
administration of a neutralizing antibody into the
circulation. Such a neutralizing antibody can be
administered directly as a protein, or it can be expressed
from a vector (with a secretory signal).
Methods Utilizing an Inhibitory Molecule
which Inhibits the Enzymatic Activity of LIPG
In another embodiment, LIPG activity is inhibited by the
administration of a composition comprising a subfragment of
LIPG polypeptide, for example, LLGN. This composition may be
administered in a convenient manner, such as by the oral,
topical, intravenous, intraperitoneal, intramuscular,
subcutaneous, intranasal, or intradermal routes. The
composition may be administered directly or it may be
encapsulated (e.g. in a lipid system, in amino acid
microspheres, or in globular dendrimers). The polypeptide
may, in some cases, be attached to another polymer such as
serum albumin or polyvinyl pyrrolidone.
In another embodiment, LIPG activity is inhibited
through the use of gene therapy, that is, through the
administration of a composition comprising a nucleic acid
which encodes and directs the expression of a subfragment of
LIPG, for example, LLGN.
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In another embodiment, LIPG activity is inhibited
through the use of inhibitory molecules. These low molecular
weight compounds interfere with LIPG's enzymatic properties
or prevent its appropriate recognition by cellular binding
sites.
In a specific embodiment, the LIPG polypeptide of the
present invention also has an affinity for heparin. LIPG
polypepti.de binding to extracellular heparin in the lumen of
blood vessels would permit LIPG to bind to and accelerate LDL
uptake by acting as a bridge between LDL and the
extracellular heparin. In the localized area of an
atherosclerotic lesion, an increased level of lipase activity
is hypothesized to accelerate the atherogenic process
(Zilversmit, D.B. (1995) Clin. Chem. 41,153-158; Zambon,
A., Torres, A., Bijvoet, S., Gagne, C., Moojani, S., Lupien,
P.J., Hayden M.R., and Brunzell, J.D. (1993) Lancet 341,
1119-1121). This may be due to an increase in the binding
and uptake of lipoproteins by vascular tissue mediated by
lipases (Eisenberg, S., Sehayek, E., Olivecrona, T.
Vlodavsky, I. (1992) J. Clin. Invest. 90,2013-2021; Tabas,
I., Li, I., Brocia R.W., Xu, S.W., Swenson T.L. Williams,
K.J. (1993) J.Biol. Chem. 268,20419-20432; Nordestgaard,
B.G., and Nielsen, A.G. (1994) Curr. Opin. Lipid. 5,252-257;
Williams, K.J., and Tabas, I. (1995) Art. Thromb. and Vasc.
Biol. 15,551-561). Additionally, a high local level of
lipase activity may result in cytotoxic levels of fatty acids
and lysophosphatidylcholine being produced in precursors of
atherosclerotic lesions. This particular activity of LLG may
contribute to the development or progression of
atherosclerosis, particularly in the context of excessive
lipid levels in a subject due to dietary or genetic factors.
Thus, the present invention permits inhibition of lipoprotein
accumulation by inhibiting LIPG polypeptide expression or
binding to lipoprotein (e.g., LDL).
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Methods Utilizing an Inhibitory
Molecule which Prevents LIPG Gene Expression
In another embodiment, inhibitory molecules, including
small molecular weight compounds, are able to down regulate
LIPG expression at the level of transcription, translation or
post-translation. In order to identify such inhibitory
molecules, the reporter gene systems described above may be
used. These inhibitory molecules may be combined with a
pharmaceutically acceptable carrier and administered using
conventional methods known in the art.
Methods Utilizing Ribozymes
Ribozymes may be administered to cells by encapsulation
in liposomes, by iontophoresis, by incorporation into
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres or by any of a variety of other
methods dicussed above. The ribozyme may be delivered to a
target tissue by direct injection or by use of a catheter,
infusion pump or stent. Alternative routes of delivery
include intravenous injection, intramuscular injection,
subcutaneous injection, aerosol inhalation, oral (tablet or
pill form), topical, systemic, ocular, intraperitoneal and/or
intrathecal delivery.
In preferred embodiments, a ribozyme-encoding sequence
is cloned into a DNA expression vector. Transcription of the
ribozyme sequence is driven from an eukaryotic RNA polymerase
II (pol II), or RNA polymerase III (pol III) promoter. The
expression vector can be incorporated into a variety of
vectors including the viral DNA vectors such as adenovirus or
adeno-associated virus vectors discussed above.
In a preferred embodiment of the invention, a
transcription unit expressing a ribozyme that cleaves LIPG
RNA is inserted into an adenovirus DNA viral vector. The
vector is delivered as recombinant viral particles and is
locally administered to the site of treatment, through the
use of a catheter, stent or infusion pump.
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Administration, of-Apolinonrotein AI
In another embodiment, any of the methods discussed above for lowering
the levels of LWPG polypeptide activity are utilized in combination with
administration of apolipoprotein Al or an expression system capable of
expressing apolipoprotein AI in a patient (see, for example, U.S. Patent No.
5,866,551).
Methods and Compositions for
Increasing Levels of LI.PG po ypeptide Activity
The methods for increasing the expression or activity of LIPG polypeptide
to lower the levels of'VLDL and LDL cholesterol include, but are not limited
to,
administration of a composition comprising the LIPG polypeptide,
administration of a composition comprising an expression vector which encodes
the LIPG polypeptide, administration of a composition comprising an enhancer
molecule which enhances the enzymatic activity of the LIPO polypeptide and
administration of an enhancer molecule which increases expression of the L1PG
gene.
Methods utilizingLUPG DO entides
In one embodiment, the level of LIPG activity is increased through the
administration of a composition comprising the LIPG polypeptide. This
composition may be administered in a convenient manner, such as by the oral,
topical, intravenous, intraperitoneal, intramuscular, subcutaneous,
intranasal,
or intradermal routes. The composition may be administered directly or it may
be encapsulated (e.g. in a lipid system, in amino acid microspherco, or in - -
- -
globular dendrimers). The polypeptide may, in some cases, be attached to
another polymer such as serum albumin or polyvinyl pyrrolidone.
Methods it 1'z' g Vectors that Express LIPG
In another embodiment, the level of LIPG is increased through the use of gene
therapy, that is, through the administration of composition comprising a
nucleic
acid which
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encodes and directs the expression of the LIPG polypeptide.
In this embodiment, the LIPG polypeptide is cloned into an
appropriate expression vector. Possible vector systems and
promoters are extensively discussed above. The expression
vector is transferred into the target tissue using one of the
vector delivery systems discussed above. This transfer is
carried out either ex vivo in a procedure in which the
nucleic acid is transferred to cells in the laboratory and
the modified cells are then administered to the human or
other animal, or in vivo in a procedure in which the nucleic
acid is transferred directly to cells within the human or
other animal. In preferred embodiments, an adenoviral vector
system is used to deliver the expression vector. If desired,
a tissue specific promoter is utilized in the expression
vector as described above.
Non-viral vectors may be transferred into cells using
any of the methods known in the art, including calcium
phosphate coprecipitation, lipofection (synthetic anionic and
cationic liposomes), receptor-mediated gene delivery, naked
DNA injection, electroporation and bioballistic or particle
acceleration.
Methods Utilizing an Enhancer Molecule
which Enhances the Enzymatic Activity of LIPG
In another embodiment, the activity of LIPG is enhanced
by enhancer molecules that increase the enzymatic activity of
LIPG or increase its appropriate recognition by cellular
binding sites. These enhancer molecules may be introduced by
the same methods discussed above for the administration of
polypeptides.
Methods Utilizing an Enhancer
Molecule which Increases LIPG Gene Expression
In another embodiment, the level of LIPG is increased
through the use of small molecular weight compounds, which
can upregulate LIPG expression at the level of transcription,
translation, or post-translation. These compounds may be
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administered by the same methods discussed above for the
administration of polypeptides.
Treatment Methods Relating
to Impaired Biliary Excretion
Intrahepatic cholestasis can be characterized by
increased serum cholesterol and phospholipid levels. A
recently described, phalloidin drug-induced intrahepatic
cholestasis model in rats demonstrated significant increases
in the serum levels of cholesterol and phospholipid
(Ishizaki, K., Kinbara, S., Miyazawa, N., Takeuchi, Y.,
Hirabayashi, N., Kasai, H., and Araki, T. (1997) Toxicol.
Letters 90, 29-34). The products of this invention may be
used to treat intrahepatic cholestasis in patients that have
increased serum cholesterol and/or phospholipid. In
addition, this rat model also exhibited a severe decrease in
biliary cholesterol excretion rates. The LIPG polypeptide
and nucleic acid products of this invention may be used to
treat patients with an impaired,biliary excretion system.
.Intrahepatic cholestasis is also characterized by
impaired bile flow from the liver. Recently, the loci for
progressive familial intrahepatic cholestasis (PFIC or Byler
disease) and benign recurrent intrahepatic cholestasis (BRIC)
were mapped to 18q2l-q22 (Carlton, V.E.H., Knisely, A.S., and
Freimer, N.B. (1995) Hum. Mol.. Genet. 4, 1049-1053 and
Houwen, R.H., Baharloo, S., Blankenship, K., Raeymaekers, P.,
Juyn, J., Sandkuijl, L.A., and Freimer, N.B. (1994) Nature
Genet. 8, 380-386, respectively). As LLG gene maps within
this chromosomal region at 18g21, the LLG gene or products of
this invention may be used to treat patients with
intrahepatic cholestasis that is caused by a mutation or
defective expression of the PFIC/BRIC disease gene(s).
In another embodiment, the LLG gene or polypeptide
products of this invention may be used to treat patients with
intrahepatic cholestasis that is not due to a defect in the
PFIC/BRIC disease gene(s) at 18g21-q22. A recent study
suggested that another locus, located outside of the 18g21-
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q22 region may also produce the PFIC phenotype (Strautnieks,
S.S., Kagalwalla, A.F., Tanner, M.S., Gardiner, R.M., and
Thompson, R.J. (1996) J. Med. Genet. 33, 833-836).
Nevertheless, administration of LLG polypeptide, either
directly or via gene therapy, may alleviate this form of the
condition.
Methods and Compositions for
Diagnosing a Predisposition to Low HDL Levels
Given the ability of LIPG polypeptide to lower the
levels of HDL cholesterol and apolipoprotein AI, the level of
LIPG polypeptide in the body may be used to determine whether
an individual is predisposed to low levels of HDL cholesterol
and apolipoprotein AI. In this method, a tissue sample is
taken from the patient. The tissue may be blood or one of
the tissues which has been demonstrated to express LIPG as
discussed in the Examples section. Measurement of the level
of LIPG may be performed by a variety of methods known to
those of skill in the art. In preferred embodiments, an
antibody directed against LIPG polypeptide may be used to
measure the level of LIPG in a tissue sample.
EXAMPLES
The following examples illustrate the invention. These
examples are illustrative only, and do not limit the scope of
the invention.
EXAMPLE 1 - Identification of a Differentially Expressed cDNA
RNA Preparation
Human monocytic THP-1 cells (Smith, P.K., Krohn, R.I.,
Hermanson, G.T., Mallia, A.K., Gartner, F.H. Provenzano,
M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk,
D.C. (1985) Anal. Biochem. 150,76-85) were cultured in RPMI-
1640 medium (GIBCO) with 25 mM HEPES, 10% fetal bovine serum,
100 units/ml penicillin G sodium and 100 units/ml
streptomycin sulfate. Cells were plated onto 15 cm tissue
culture dishes at 1.5x107 cells/plate, and treated with 40
ng/ml phorbol 12-myristate 13-acetate (Sigma) for 48 hours to
induce differentiation of the cells. Human low density
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lipoproteins (LDL) were purchased from Calbiochem, and were
dialyzed exhaustively versus PBS at 4 C. The LDL was then
diluted to 500 g/ml and dialyzed versus 5 M CuSO4 in PBS at
37 C for 16 hours. To stop oxidation, the LDL was dialyzed
exhaustively versus 150 mM NaCl, 0.3 mM EDTA, then filter
sterilized. Protein concentration was determined by the BCA
method (Schuh, J. Fairclough, G.F., and Haschemeyer, R.H.
(1978) Proc. Natl. Acad. Sci. USA 75, 3173-3177) (Pierce).
The degree of oxidation was determined by TBARS (Chomczynski,
P. (1993) Biotechniques 15,532-537), and was between 25-30
nmol MDA equivalents/mg protein. The differentiated THP-1
cells were exposed for 24 hours to.either 50 Ag/ml oxidized
LDL or NaCl-EDTA buffer in RPMI medium with 10% lipoprotein-
deficient fetal bovine serum (Sigma). To harvest the RNA,
the plates were rinsed with 10 ml of PBS, then 14 ml of
TRIZOL (Liang, P. and Pardee, A.B. (1992) Science 257,967-
971) (GIBCO) were added to each plate. The solution was
pipetted several times to mix, then like samples were pooled
into centrifuge tubes and 3 ml chloroform per plate were
added and mixed. The tubes were centrifuged for 15 minutes
at 12000 x g. After centrifugation the upper layer was
transferred to a new tube and 7.5 ml isopropanol per plate
was added and mixed. The tubes were centrifuged at 12000 x g
for 20 minutes. The pellet was rinsed with ice-cold 70%
ethanol and dried at room temperature. The pellets were
suspended in 500 Al TE (Tris-EDTA) and treated with 200 units
RNase-free DNAse I and 200 units RNasin placental RNase
inhibitor (Promega) for 30 minutes at 37 C. The RNA was
purified by sequential extractions with phenol,
phenol/chloroform/isoamyl alcohol (25:24:1), and
chloroform/isoamyl alcohol (24:1) followed by ethanol
precipitation.
cDNA Synthesis
cDNA synthesis and PCR amplification were accomplished
using protocols from the Differential Display Kit, version
1.0 (Display Systems Biotechnology, Inc.) This system is
based on the technique originally described by Liang and
Pardee (Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A.,
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and Smith, L.M., (1991) BiolTechnology 9,657-663). The primer pairs which
yielded the cDNA fragment containing the first information of the lipase like
gene were downstream primer 7 and upstream primer 15. The cDNA for the
amplification was synthesized as follows, using RNA derived from FMA treated
THP-1 cells exposed to either buffer or oxidized LDL: 3 l of 25 pM downstream
primer 7 and 7.5 gl of diethylpyrocarbonate (DEPC)-treated water were added to
300 ng (3.0pl) RNA from either sample of THP-1 RNA. This was heated to 70 C
for 10 minutes then chilled on ice. To this tube were added 8 pl of 5x PCR
buffer
(250 mM Tris-HC1 pH 8.3, 375 mM KC1) (CIBCO), 3 l25 mM MgC12, 3 l OA M
DTT, 1.2 pl 500 pM dNTPs, 0.7 pl RNasia, and 5.6 p1 DEPC-treated water. The
tubes were incubated for 2 minutes at room temperature, after which 1. 5 pl
(300
units) Superscript II RNase H- reverse transcriptase (GIBCO) were added. The
tubes were incubated sequentially at room temperature for 2 minutes, 60
minutes at 37 C, and 5 minutes at 95 C, followed by chilling on ice. PCR
amplification was performed using a master mix containing 117 p1 10x FCR
buffer (500 mM KC1, 100 mM Tris-HC1 pH 8.3, 15 mM MgClg, and 0.01% (w/v)
gelatin), 70.2 p125 mM MgC12, 5.9 pl alpha-33P dATP (10m Ci/ml, DuPont
NEN), 4.7 15O0 pM dNTP mix, 111l AmpliTagTm DNA polymerase (5 units/pl,
Perkin-Elmer), and 493.3 p1 DEPC-treated water. For each reaction, 12 p1 of
the
master mix was added to 2 pl downstream pricier #7, 1 pl of cDNA, and 5 p1 of
upstream primer #15. The reaction mixes were heated to 94 C for 1 minute, then
thermoeyeled 40 times with a denaturing step of 94 C for 15 seconds, annealing
step of 40 C for 1 minute, and an extension step of 72 C for 30 seconds.
Following the 40 cycles, the reactions were incubated at 72 C for 5 minutes
and
stored at 10 C. The PCR reactions were performed in a Perldn=Elmer GeneAmp
System 9600 thermocycler.
Four microliters of the amplification reaction were mixed with an equal
volume of loading buffer (0-2%
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bromphenol blue, 0.2% Xylene cyanol, 10 mM EDTA pH 8.0, and 20% glycerol).
Four microliters of this mix was run on :t 6% nondenaturing acrylamide
sequencing format gel for 3 hours at 1200 volts (constant voltage). The gel
was
dried at 80 C for 1.5 hours and exposed to KodakTM XAR film. An amplification
product found only in the reaction containing cDNA from THF-1 cells exposed to
oxidized LDL was identified and excised from the gel. 1O0 1 of DEPC-treated
water was added to a microcentrifuge tube containing the excised gel fragment
and was incubated for 30 minutes at room temperature followed by 15 minutes
at 96 C.
To reaniplify the PCR product, 26.5 microliters of the eluted DNA were
used in a amplification reaction that alai) included 5 gl lox PCR buffer, 3 l
25mM MgCI2, 5 160O M dNTPs, 5 p1 2 M downstream primer 7, 7.5 pl
upstream primer 15, and 0.5 l AmpiiTagTM polymerase. The PCR cycling
parameters and instrument were as described above- Following amplification, 20
l of the reamplification was analyzed on an agarose gel and 4 l was used to
subelone the PCR products into the vector pCWI using the TA cloning
system (Frohman, M.A., Dush; M.K., and Martin, G.R. (1988) Proc. Natl. Acad.
Sci. USA 85,8998-9002) Unvitrogen). Following an overnight ligation at 14 C,
the
ligation products were used to transform L ski. Resulting transformants were
picked and 3 ml overnight cultures were used in plaamid minipreparations.
Insert sizes were determined using EcoRI digestions of the plasmids and clones
containing inserts of the approximate size of the original, PCR product were
sequenced using fluorecent dye-terminator reagents (Prism, Applied Biosystems)
and an Applied Biosystems 373 DNA sequencer. The sequence of the PCR
product is shown in Figure 2. The sequence of the amplification primers is
underlined-
6' RACE Reaction
Extension of the eDNA identified through RT-PCR was accomplished
using the 5'RACE system (Loh, E.Y., Eliot,
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J.F., Cwirla, S., Lanier, L.L., and Davis, M.M. (1989)
Science 243, 217-219; Simms, D., Guan, N., and Sitaraman,
K., (1991) Focus 13, 99-100) (GIBCO). One microgram of the
THP-1 RNA (oxidized LDL treated) used initially in the
differential display reactions was utilized in the 5'RACE
procedure:
l l (1 g) of RNA was combined with 3 gl (3 pmol) primer
2a and 11 gl DEPC-treated water and heated to 70 C for 10
minutes followed by 1 minute on ice. 2.5 gl lOx reaction
buffer (200 mM Tris-HCl pH 8.4, 500 mM KC1), 3 gl 25 mM
MgC12r 1 gl 10 mM dNTP mix, and 2.5 gl 0.1 M DTT were added.
The mix was incubated at 42 C for 2 minutes, then 1 gl
Superscript II reverse transcriptase was added. The
reaction was incubated for an additional 30 minutes at 42 C,
15 minutes at 70 C, and on ice for 1 minute. One microliter
of RNase H (2 units) was added and the mixture was incubated
at 55 C for 10 minutes. The cDNA was purified using the
GlassMax columns (Sambrook, J. Fritsch, E.F., and Maniatis,
T. (1989) Molecular Cloning: A Laboratory Manual, second
edition, Cold Spring Harbor Laboratory Press, Plainview, NY)
included in the kit. The cDNA was eluted from the column in
50 pl dH2O, lyophilized, and resuspended in 21 pl dH2O.
Tailing of the cDNA was. accomplished in the following
reaction: 7.5 tl dH,O, 2.5 j.tl reaction buffer (200 mM Tris-
HC1 pH 8.4, 500 mM KC1), 1.5 l 25 mM MgCl2, 2.5 gl 2 mM
dCTP, and 10 l of the cDNA were incubated at 94 C for 3
minutes, then 1 minute on ice. 1 gl (10 units) of terminal
deoxynucleotidyl transferase was added and the mixture was
incubated for 10 minutes at 37 C. The enzyme was heat
inactivated by incubation at 70 C for 10 minutes and the
mixture was placed on ice. PCR amplification of the cDNA
was performed in the following steps: 5 tl of the tailed
cDNA was included in a reaction which also contained 5 l
lOx PCR buffer (500 mM KC1, 100 mM Tris-HCl pH 8.3, 15 MM
MgC12, and 0.01% (w/v) gelatin), 1 tl 10 mM dNTP mix, 2 pl
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(10 pmol) anchor primer, 1 l (20 pmol) primer 3a, and 35 l
dH2O. The reaction was heated to 95 C for 1 minute, then 0.9
gl (4.5 units) Amplitaq polymerase was added. The reaction
was cycled 40 times under the following conditions: 94 C for
5 seconds, 50 C for 20 seconds, and 72 C for 30 seconds. One
microliter of this reaction was used in a nested
reamplification to increase levels of specific product for
subsequent isolation. The reamplification included: 1 gl
primary amplification, 5 l lOx PCR buffer, 1 l 10 mM dNTP
mix, 2 Al (20 pmol) universal amplification primer, 2 l (20
pmol) primer 4a, and 38 41 dH2O. The reaction was heated to
95 C for 1 minute, then 0.7 l (3.5 units) Amplitaq
polymerase was added. The reaction was cycled 40 times
under these conditions; 94 C for 5 seconds, 50 C for 20
seconds, and 72 C for 30 seconds. The amplification products
were analyzed via 0.8% agarose gel electrophoresis. A
predominant product of approximately 1.2 kilobase pairs was
detected. Two microliters of the reaction products were
cloned into the pCRII vector from the TA cloning kit
(Invitrogen) and incubated at 14 C overnight. The ligation
products were used to transform E. coli. The insert sizes
of the resulting transformants were determined following
EcoRI digestion. Clones containing inserts of the
approximate size of the PCR product were sequenced using
fluorescent dye-terminator reagents (Prism, Applied
Biosystems) and an Applied Biosystems 373 DNA sequencer.
The sequence of the RACE product including the EcoRI sites
from the TA vector are shown in Figure 3. The sequences of
the amplimers (universal amplification primer and the
complement to 5'RACE primer 4a) are underlined.
EXAMPLE 2 - Cloning and Chromosomal
Localization of the LIPG Gene
cDNA library screening
A human placental cDNA library (Oligo dT and random
primed, Cat #5014b, Lot #52033) was obtained from Clontech
(Palo Alto, CA). A radiolabeled probe was created by
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excising the insert of a plasmid containing the 5'RACE
reaction PCR product described above. The probe was
radiolabeled using the random priming technique: the DNA
fragment (50-100 ng) was incubated with 1 pg of random
hexamers (Gibco) at 95 C for 10 minutes followed by 1 minute
on ice. At room temperature the following were added: 3 l
10x Klenow buffer (100 mM Tris-HC1 pH 7.5, 50 mM MgCL2, 57 mM
dithiothreitol; New England Biolabs), 3 gl 0.5 mM dATP,
dGTP, dTTP), 100 pCi a -32PdCTP (3000 Ci/mmol, New England
Nuclear), and 1 pl Klenow fragment of DNA polymerase I (5
units, Gibco). The reaction was incubated for 2-3 hours at
room temperature and the reaction was then stopped by
increasing the volume to 100 pl with TE pH 8.0 and adding
EDTA to a final concentration of 1 mM. The unincorporated
nucleotides were removed by raising the reaction volume to
100 pl and passing over a G-50 spin column (Boehringer
Mannheim). The resulting probes had a specific activity
greater than 5x108 cpm/ g DNA.
The library was probed using established methods
(Walter, P., Gilmore, R., and Blobel, G. (1984) Cell 38,5-
8). Briefly, the filters were hybridized for 24 hours at
65 C in 4.8X SSPE (20X SSPE = 3.6 M NaCl, 0.2 M NaH2PO4, 0.02
M EDTA, pH 7.7), 20 mM Tris-HC1 pH 7.6, 1X Denhardt's
solution (100X= 2% Ficoll 400, 2% polyvinylpyrrolidone, 2%
BSA), 10% dextran sulfate, 0.1% SDS, 100 pg/ml salmon sperm
DNA, and 1x106 cpm/ml radiolabelled probe. Filters were then
washed three times for 15 minutes at room temperature in 2X
SSC (1X SSC = 150 mM NaCl, 15 mM sodium citrate pH 7.0),
0.1% sodium dodecyl sulfate (SDS) followed by three washes
for 15 minutes each at 65 C in 0.5X SSC, 0.1% SDS. Phage
which hybridized to the probe were isolated and amplified.
DNA was purified from the amplified phage using LambdaSorb
reagent (Promega) according to the manufacturer's
instructions. The inserts were excised from the phage DNA
by digestion with EcoRI. The inserts were subcloned into
the EcoRI site of a plasmid vector (Bluescript II SK,
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Stratagene). The sequence of the open reading frame
contained within the 2.6 kb EcoRI fragment of the cDNA was
determined by automated sequencing as described above. The
sequence is shown in Figure 4. The amino acid sequence of
the predicted protein encoded by the open reading frame is
shown in Figure 5 and has been termed LLGXL. The first
methionine is predicted to be encoded by nucleotide pairs
252-254. The predicted protein is 500 amino acids in
length. The first 18 amino acids form a sequence
characteristic of a secretory signal peptide (Higgins, D.G.,
and Sharp, P.M. (1988) Gene 73, 237-244). The propeptide is
predicted to have a molecular weight of 56,800 Daltons.
Assuming cleavage of the signal peptide at position 18, the
unmodified mature protein has a molecular weight of 54,724
Daltons.
The overall similarities between this protein and the
other known members of the triacylglycerol lipase family is
illustrated in Figure 6 and Table 1. In the alignment shown
in Figure 6, LIPG is the polypeptide (SEQ ID NO: 6) encoded
by the cDNA (SEQ ID NO: 5) described in Example 1, and
hereafter referred to as LLGN. This protein is identical
with the LLGXL protein in the amino terminal 345 residues.
Nine unique residues are followed by a termination codon,
producing a propolypeptide of. 39.3 kD and a mature protein
of 37.3 kD. The sequences which are common to LLGN and
LLGXL are nucleic acid sequence SEQ ID NO: 9 and amino acid
sequence SEQ ID NO: 10.
Interestingly, the position at which the LLGN and LLGXL
proteins diverge is at a region known from the structure of
the other lipase to be between the amino and carboxy domains
of the proteins. Therefore, the LLGN protein appears to
consist of only one of the two domains of triaclyglycerol
lipases. This sequence contains the characteristic "GXSXG"
lipase motif at positions 167--171 as well as conservation of
the catalytic triad residues at Ser 169, Asp 193, and His
274. Conservation of cysteine residues (positions 64, 77,
252, 272, 297, 308, 311, 316, 463, and 483) which have been
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implicated in disulfide linkage in the other lipases
suggests that the LLGXL protein has structural similarities
to the other enzymes. There are five predicted sites for N-
linked glycosylation; at amino acid positions 80, 136, 393,
469, and 491. The protein sequences used in the comparisons
are human lipoprotein lipase (LPL; Genbank accession
#M15856, SEQ ID NO: 13), Human hepatic lipase (HL; Genbank
accession #J03540, SEQ ID NO: 14), human pancreatic lipase
(PL; Genbank accession # M93285, SEQ ID NO: 15), human
pancreatic lipase related protein-i (PLRP-1; Genbank
accession # M93283), and human pancreatic lipase related
protein-2 (PLRP-2; Genbank accession # M93284).
TABLE 1. Similarity of triacylglycerol lipase gene family
LLGXL LPL HL PL PLRP1 PLRP2
LLGXL - 42.7 36.5 24.5 22.5 22.6
LPL 42.7 - 40.0 22.8 22.7 20.9
HL 36.5 40.0 - 22.8 24.0 22.0
PL 24.5 22.8 22.8 - 65.2 62.2
PLRP1 22.5 22.7 24.0 65.2 - 61.7
PRLP2 22.6 20.9 22.0 62.2 61.7 -
Percent similarity was based on pairwise alignment using the
Clustal algorithm (Camps, L., Reina, M., Llobera, M.,
Vilaro, S., and Olivecrona, T. (1990) Am. J. Physiol.
258,C673-C681) in the Megalign program of the Lasergene
Biocomputing Software Suite (Dnastar).
Chromosomal Localization
DNA from a P1 clone (Sternberg, N., Ruether, J. and
DeRiel, K. The New Biologist 2:151-62, 1990) containing
genomic LLG DNA was labelled with digoxigenin UTP by nick
translation. Labelled probe was combined with sheared human
DNA and hybridized to PHA stimulated peripheral blood
lymphocytes from a male donor in a solution containing 50%
formamide, 10% dextran sulfate, and 2X SSC. Specific
hybridization signals were detected by incubating the
hybridized cells in fluoresceinated antidigoxigenin
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antibodies' followed by counterstaining with DAPI. This
initial experiment resulted in specific labeling of a group
E chromosome, which was believed to be chromosome 18 on the
basis of DAPI staining.
A second experiment was conducted in which a biotin
labelled probe specific for the centromere of chromosome 18
was cohybridized with the LLG probe. This experiment
resulted in the specific labeling of the chromosome 18
centromere in red and the long arm of chromosome 18 in
green. Measurements of 11 specifically labelled hybridized
chromosomes 18 demonstrated that LLG has a Flter of 0.67
(Franke measurement of 0.38), which corresponds to band
18q2l. Several genetic diseases, including intrahepatic
cholestasis, cone rod dystrophy, and familial expansile
osteolysis, are believed to involve defects in this
chromosomal region.
EXAMPLE 3 - LIPG RNA Analysis
Expression of LIPG RNA in THP-1 cells
Analysis of the mRNA from which the cDNA was derived was
performed by northern analysis of THP-1 RNA. RNA from these
cells was prepared as described above. The mRNA was
purified from the total RNA through the use of a poly-dT-
magnetic bead system (Polyattract system, Promega). Three
micrograms of poly (A)-containing mRNA was electrophoresed
on a 1% agarose-formaldehyde gel. The gel was washed for 30
minutes in dH2O. RNAs were vacuum transferred to a nylon
membrane using alkaline transfer buffer (3M NaCl, 8 mM NaOH,
2 mM sarkosyl). After transfer, the blot was neutralized by
incubation for 5 minutes in 200 mM phosphate buffer pH 6.8.
The RNA was crosslinked to the membrane using an ultraviolet
crosslinker apparatus (Stratagene).
A probe was made by excising the insert of a plasmid
containing the 51RACE reaction PCR product described above.
The probe was radiolabeled using the random priming
technique described in Example 2.
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The filters were prehybridized in QuikHyb rapid
hybridization solution (Stratagene) for 30 minutes at 65 C.
The radiolabeled probe (1-2 x 106 cpm/ml) and sonicated
salmon sperm DNA (final concentration 100 g/ml) were
denatured by heating to 95 C for 10 minutes and quick-chilled
on ice before adding to the filter in QuikHyb.
Hybridization was for 3 hours at 65 C. The unhybridized
probe was removed by washing the filters two times for 15
minutes with 2X SSC, 0.1 % sodium dodecyl sulfate at room
temperature followed by two times for 15 minutes in 0.1X
SSC, 0.1% SDS at 62 C. Following the washes, the filters
were allowed to dry briefly and then exposed to Kodak XAR-2
film with intensifying screens at -80 C. The results are
shown in Figure 7, which shows a major mRNA species of
approximately 4.5 kilobases. Minor species of 4.3 and 1.6
kilobases are also present. The expected size of the LLGN
cDNA is 1.6 kb. The LLGXL sequence is likely to be encoded
by the major species of mRNA detected.
Expression of LIPG RNA in various human tissues
A commercially prepared filter containing 3 g each of
mRNAs from human tissues (heart, brain, placenta, lung,
liver, skeletal muscle, kidney, and pancreas) was obtained
from Clontech (Catalog #7760-1). This filter was probed and
processed as described above. After probing with the
radiolabeled LLG fragment and autoradiography, the probe was
stripped by washing in boiling O.1X SSC, 0.1% SDS for 2 x15
min. in a 65 C incubator. The membranes were then probed
with a 1.4 kilobase pair DNA fragment encoding human
lipoprotein lipase. This fragment was obtained by RT-PCR of
the THP-1 RNA (PMA and oxLDL treated) using the 5'LPL and
3'LPL primers described in Figure 1. and the RT-PCR
conditions described above. After autoradiography, the
membranes were stripped again and reprobed with a
radiolabeled fragment of the human beta actin cDNA to
normalize for RNA content. The results of these analyses
are shown in Figure 8. The highest levels of LIPG message
were detected in placental RNA, with lower levels found in
RNAs derived from lung, liver, and kidney tissue. In
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agreement with previous studies by others (Verhoeven,
A.J.M., Jansen, H. (1994) Biochem. Biophys. Acta 1211,121-
124), lipoprotein lipase message was found in many tissues,
with highest levels found in heart and skeletal muscle
tissue. Results of this analysis indicates that the tissue
distribution of LIPG expression is very different from that
of LPL. The pattern of LIPG expression is also different
from that of either hepatic lipase or pancreatic lipase, as
reported by others (Wang, C.-S., and Hartsuck, J.A. (1993)
Biochem. Biophys. Acta 1166,1-19; Semenkovich, C.F., Chen,
S.-W., Wims, M., Luo C.-C., Li, W.-H., and Chan, L. (1989)
J. Lipid Res. 30,423-431; Adams., M.D., Kerlavage, A.R.,
Fields, C., and Venter, C. (1993) Nature Genet. 4,256-
265).
To determine the expression pattern in additional human
tissues, another commercially prepared membrane was probed
with LLGXL cDNA. This dot blot (Human RNA Master Blot,
Clontech Cat. # 7770-1) contains 100-500 ng mRNA from 50
different tissues and is normalized for equivalent
housekeeping gene expression (Chen, L., and Morin, R. (1971)
Biochim. Biophys. Acta 231,194-197). A 1.6 kb DraI-SrfI
fragment of the LLGXL cDNA was labeled with 32PdCTP using a
random oligonucleotide priming system (Prime It II,
Stratagene) according to the manufacturer's instructions.
After 30 minutes prehybridization at 65 C, the probe was
added to QuikHyb hybridization solution at 1.3x106cpm/ml.
Hybridization was for 2 hours at 65 C . The unhybridized
probe was removed by washing the filters two times for 15
minutes with 2X SSC, 0.1 % sodium dodecyl sulfate at room
temperature followed by two times for 15 minutes in 0.1X
SSC, 0.1% SDS at 62 C. Following the washes, the filters
were allowed to dry briefly and then exposed to Kodak XAR-2
film with intensifying screens at -80 C. for varying amounts
of time. The resulting images were quantitated by
densitometry. The results are shown in Table 2. The
relative expression levels of tissues represented in both
the multiple tissue northern and the multiple tissue dot
blot are similar, with highest levels in placenta, and lower
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levels in lung, liver and kidney. Fetal liver, kidney, and
lung also express roughly the same levels as the adult
tissues. Surprisingly, thyroid tissue expression levels
were the highest of all tissues represented, with expression
of 122% of that in placental tissue. While there is
precedence for lipase expression by the placenta (Rothwell,
J.-E., Elphick, M.C. (1982) J. Dev. Physiol. 4,153-159;
Verhoeven, A.J.M., Carling D., and Jansen H. (1994) J. Lipid
Res. 35, 966-975; Burton, B.K., Mueller, H.W. (1980)
Biochim. Biophys. Acta 618,449-460), the thyroid was not
previously known to express any lipase. These results
suggest that LIPG expression may be involved in maintenance
of the placenta, where LIPG may serve to liberate free fatty
acids from substrates such as phospholipids as a source of
energy. The LIPG expressed in the thyroid may provide
precursors for the synthesis of bioactive molecules by that
gland.
Table 2. Expression of LIPG mRNA in various human tissues
whole brain N.D. substantial N.D. uterus N.D. rnanunary N.D. lung 29
nigm gland
amygdala N.D. temporal N.D. prostate 5 kidney 44 trachea 12
lobe
caudate N.D. thalamus N.D. stomach N.D. liver 61 placenta 100
nucleus
cerebellum 4 sub-thalamic N.U. testes 9 small 6 fetal brain 5
nucleus intestine
cerebral N. D. spinal cord N.D ovary N.D. spleen N. D. fetal heart N.D.
cortex
frontal lobe N.D. heart N.D. pancreas N.D. thymus N.D. fetal kidney 56
hippocampus N.D.
1 aorta N.D. pituitary N.D. peripheral N.D. fetal liver 14
land leukocyte
medulla N.D. skeletal N.D. adrenal gland N.D. lymph node N. D. fetal spleen
N.D.
oblongata muscle
'tal lobe N.D. colon 8 thyroid and 122 bone marrow ND. fetal thymus N.D.
putamen N.D. bladdet N.D. salivary N.U. appendix 7 fetal lung 8
gland
Values given are percentage of expression with levels in
placental tissue arbitrarily set at 100%. Values are
average of densitometric measurements from two
autoradiographic exposures. N.D. = not detectable.
Expression of LIPG RNA in cultured endothelial cells
Human umbilical vein endothelial cells (HUVEC) and human
coronary arterial endothelial cells (HCAEC) were obtained
from Clonetics. HUVECs were propagated in a commercially
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prepared endothelial cell growth medium (EGM, Clonetics)
supplemented with 3 mg/ml bovine brain extract (Maciag, T.,
Cerundolo, J., Ilsley, S., Kelley, P.R., and Forand, R.
(1979) Proc. Natl. Acad. Sci. USA 76, 5674-5678),
Clonetics), while HCAECs were propagated in EGM supplemented
with 3 mg/ml bovine brain extract and 3% fetal bovine serum
(5% final concentration). Cells were grown to confluence,
then the medium was changed to EGM without bovine brain
extract. Cultures were stimulated by adding 100 ng/ml of
phorbol myristate (Sigma). After 24 hours incubation, the
RNAs were extracted from the cells via the Trizol method
described above. Twenty micrograms of total RNA was
electrophoresed and transferred to the membrane for
analysis. The membranes were probed with LIPG and LPL
probes as described above. The results are shown in Figure
9. Twenty micrograms of total RNA from THP-1 cells
stimulated with PMA was run on the blot for comparison. RNA
hybridizing to the LIPG probe was detected in unstimulated
and PMA stimulated HUVEC cells. In contrast, detectable
levels of LIPG mRNA were only found in HCAEC cultures after
stimulation with PMA. In agreement with previous studies
of others, no detectable lipoprotein lipase mRNA was
detected in any of the endothelial RNAs (Verhoeven, A.J.M.,
Jansen, H. (1994) Biochem. Biophys. Acta 1211,121-124).
EXAMPLE 4 - LIPG Protein Analysis
Antibody preparation
Antisera were generated to peptides with sequences
corresponding to a region of the predicted protein encoded
by the LIPG cDNA open reading frame. This peptide was
chosen because of its high predicted antigenicity index
(Jameson B.A., and Wolf, H. (1988) Comput. Applic. in the
Biosciences 4,181-186). The sequence of the immunizing
peptide was not found in any protein or translated DNA
sequence in the Genbank database. Its corresponding
position in the LIPG protein is shown in Figure 10. The
carboxy terminal cysteine of the peptide does not correspond
to the residue in the LIPG putative protein, but was
introduced to facilitate coupling to the carrier protein.
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The peptide was synthesized on a Applied Biosystems Model
433A peptide synthesizer. Two milligrams of peptide was
coupled to two milligrams of maleimide-activated keyhole
limpet hemocyanin following the protocols included in the
Imject Activated Immunogen Conjugation Kit (Pierce
Chemical). After desalting, one-half of the conjugate was
emulsified with an equal volume of Freund's complete
adjuvant (Pierce). This emulsification was injected into a
New Zealand White rabbit. Four weeks after the initial
inoculation, a booster inoculation was made with an
emulsification made exactly as described above except
Freund's incomplete adjuvant (Pierce) was used. Two weeks
after the boost, a test bleed was made and titers of
specific antibodies were determined via ELISA using
immobilized peptide. A subsequent boost was made one month
after the first boost.
Western analysis of medium
from endothelial cell cultures
HUVEC and HCEAC cells were cultured and stimulated with
PMA as described in Example 3C, except that the cells were
stimulated with PMA for 48 hours. Samples of conditioned
medium (9 ml) were incubated with 500 l of a 50% slurry of
heparin-Sepharose CL-6B in phosphate buffered saline (PBS,
150 mM sodium chloride, 100 mM sodium phosphate, pH 7.2).
Heparin-Sepharose was chosen to partially purify and
concentrate-the LIPG proteins because of the conservation of
residues in the LLGXL sequence which have been identified as
critical for the heparin-binding activity of LPL (Ma, Y.,
Henderson, H.E., Liu, M.-S., Zhang, H., Forsythe, I.J.,
Clarke-Lewis, I., Hayden, M.R., and Brunzell, J.D. J. Lipid
Res. 35, 2049-2059; and Fig. 6.). After rotation at 4 C for
1 hour, the samples were centrifuged for 5 minutes at 150 x
g. The medium was aspirated and the Sepharose was washed
with 14 ml PBS. After centrifugation and aspiration, the
pelleted heparin-Sepharose was suspended in 200 l 2x SDS
loading buffer (4% SDS, 20% glycerol, 2% P-mercaptoethanol,
0.002% bromphenol blue, and 120 mM Tris pH 6.8). The
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samples were heated to 95 C for 5 minutes and 40 l was loaded onto a 10%
Tris-Glycine SDS gel. After electrophoresis at 140 V for approximately 90
minutes, the proteins were transferred to nitrocellulose membranes via a
Novex electroblotting apparatus (210 V, 1 hour). The membranes were blocked
for 30 minutes in blocking buffer (5% nonfat dried milk, 0.1% TweenTM 20, 150
mM sodium chloride, 25 roM Tris pH 7.2). Antipeptide antisera and normal
rabbit serum was diluted 1:5000 in blocking buffer and was incubated with the
membranes overnight at 4 C with gentle agitation. The membranes were then
washed dx 15 minutes with TBST (0.1% TweenTM 20, 150 mM sodium chloride,
25 mM Tris pH 7.2). Goat antirabbit peroxidase conjugated antisera (Boehringer
Mannheim) was diluted 1:5000 in blocking buffer and incubated with the
membrane for 1 hour with agitation. The membranes were washed as above,
reacted with Renaissance chemilumines+ent reagent (DuPont NEN), and
exposed to KodakTM XAR-2 film. The results are shown in Figure 11. Two species
of immunoreactive proteins are present in the samples from unatimulated
HTJVEC and HCAEC cells. Levels of imniunoreactive protein in the
unatimulated HCAEC samples are much lower than the corresponding HUVEC
sample. Upon stimulation with PMA, three immunoreactive proteins are
secreted by the endothelial cell cultures. PMA exposure greatly increased the
level of LIPG proteins produced by the H CAEC cultures. PMA induction of LLG
proteins was not as dramatic in the HUV'EC cultures.
EXAMPLE 5 - Rowmbinant LIPG Protein Production
LIPG expression constructs
The cDNAs encoding the LLGN and LLGXL proteins were cloned into the
mammalian expression vector pCDNA3 (Invitrogen). This vector allows
expression of foreign genes in many mammalian cells through the use of the
cytomegalovirus major late promoter. The LLGN 5'RACE product was cloned
into the EcoRI site of pCDNA3. The LLGXL cDNA was digested with Dral and
Sri! to yield a 1.55 kb cDNA (see Figure 1.). The vector was digested with the
restriction enzyme EcoRV and the vector and insert were ligated using T4
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DNA ligase and reagents from the Rapid Ligation Kit
(Boehringer Mannheim) according to the manufacturers.
instructions. The ligation products were used to transform
competent E. coli . Resultant colonies were screened by
restriction analysis and sequencing for the presence and
orientation of the insert in the expression vector.
Transient transfection of LIPG in COS-7 cells
The LIPG expression vectors were introduced into COS-7
cells through the use of Lipofectamine cationic lipid
reagent (GIBCO). Twenty-four hours before the transfection,
COS-7 cells were plated onto 60 mm tissue culture dishes at
a density of 2x105 cells/plate. The cells were propagated in
Dulbecco's modified Eagle's medium (DMEM; GIBCO)
supplemented with 10% fetal calf serum, 100 U/ml penicillin,
100 gg/ml streptomycin. One microgram of plasmid DNA was
added to 300 pl of Optimem I serum-free medium (Gibco). Ten
microliters of Lipofectamine reagent were diluted into 300
gl of Optimem I medium and this was combined with the DNA
solution and allowed to sit at room temperature for 30
minutes. The medium was removed from the plates and the
cells were rinsed with 2 ml of Optimem medium. The DNA-
Lipofectamine solution was added to the plates along with
2.7 ml Optimem medium and the plates were incubated for 5
hours at 37 C. After the incubation, the serum free medium
was removed and replaced with DMEM supplemented with 2% FBS
and antibiotics. Twelve hours post-transfection, some of
the cultures were treated with either 0.25 mM Pefabloc SC
(Boehringer Mannheim), a protease inhibitor, or 10 U/ml
heparin. Thirty minutes before harvest, the heparin treated
samples were treated with an additional 40 U/ml heparin.
The medium was removed from the cells 60 hours after
transfection. Heparin-Sepharose CL-4B (200 l of a 50%
slurry in PBS pH 7.2) was added to 1 ml of medium and was
mixed at 4 C for 1 hour. The Sepharose was pelleted by low
speed centrifugation and was washed three times with 1 ml
cold PBS. The Sepharose was pelleted and suspended in 100
l 2x loading buffer. The samples were heated to 95 C for 5
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minutes. 40 l of each sample was loaded onto a 10% S-DSPAGE gel.
Electrophoresis and western analysis wfts performed using the anti-LIPG
antiserum as described above. The results are shown in Figure 12. Proteins
from
HCAEC conditioned medium were included for size references. LLGN migrates
at approximately 40 kD, corresponding lo the lowest band in HCAEC. The
medium from COS cells transfected with LLGXL cDNA contains both 68 kD and
40 kD species. When these cells were treated with heparin, the amount of both
68 kD and 40 kD proteins recovered from the medium increased dramatically,
indicating either the release of proteoglycanbound protein from the call
surface
or stabilization of the proteins by heparin. When the cells were treated with
the
protease inhibitor PefablocTM, the amount of 68 kD protein increased relative
to
that of the 40 kD species. This suggests that the lower molecular weight
protein
produced by these cells is a proteolysis product of the larger 68 kD form. The
role
of the mENA identified through differential display which encodes a shorter,
40 kD species is not known. There has, however, been a report of an
alternately-
spliced form of hepatic lipase which app,uently is expressed in a tissue-
specific
manner and would create a truncated protein.
EXAMPLE 6 - LIPG in Animal species
Cloning the Rabbit Homolog of L1PG
A commercially available lambda cDNA library derived from rabbit lung
tissue (Clontech, Cat. #TL1010b) was used to isolate a fragment of the rabbit
homolog of the LIPG gene. Five microliters of the stock library were added to
45
l water and heated to 95 C for 10 minutes. The following were added in a final
volume of 100 1. 200 pM dNTPs, 20 mM Tri,s-HCI pH 8.4, 50 mM KC1, 1.5 mM
MgC1s, 100 #M each primer DL1P774 and LLGgen2a, and 2.5 U Taq polyinerase
(GIBCO). The reaction was thermocyelecl 35 times with the parameters of 15
seconds at 940C, 20 seconds at 50 C and 30 seconds at 72 C. Ten microliters of
the reaction was analyzed via agerose g(:1 electrophoresis. A product of
approximately 300 basepairs was detected. A portion (4 .tI) of the reaction
mix
was used
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to clone the product via the TA cloning system. The insert
of a resulting clone was sequenced (SEQ ID NO: 11). An
alignment between the deduced rabbit amino acid sequence (SEQ
ID NO: 12) and the corresponding sequence of the human cDNA
is also shown in Figure 14. Of the nucleotides not part of
either amplification primer, there is an 85.8% identity
between the rabbit and human LLG sequences. The predicted
protein encoded by this rabbit cDNA shares 94.6 % identity
with that of the human protein, with most of the nucleotide
substitutions in the third or "wobble" positions of the
codons. Notably, this region spans the "lid" sequence of the
predicted LLG proteins and is a variable domain in the lipase
gene family. This is evidence that there is a high degree of
conservation of this gene between species.
LIPG in Other Species
To demonstrate the presence of LLG genes in other
species, genomic DNAs from various species were restriction
digested with EcoRI, separated by electrophoresis in agarose
gels, and blotted onto nitrocellulose membranes.
The membranes were hybridized overnight at 65 C with 2.5
x 106 cpm/ml of random primed 32 P-LLG or 32 P-LPL (lipoprotein
lipase) probe in a hybridization solution of 6X SSC, 10%
dextran sulfate, 5 X Dendardt's solution, 1% SDS, and 5 g/ml
salmon sperm DNA. The membranes were washed with O.1X SSC,
0.5% SDS for ten minutes at room temperature, then
sequentially for ten minutes at 40 C, 50 C, and 55 C.
Autoradiograms of the blots are shown in Figure 16.
Figure 16 shows the presence of LLG and LPL genes in all
species examined, with the exception that no hybridization
was observed with the LLG probe against rat DNA. The
exceptional data from rat may represent an artifact caused by
generation of abnormally sized restriction fragments
containing LLG sequences. Such fragments may be outside of
the fractionation range of the agarose gel or may blot
inefficiently. The different bands detected by the two
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probes indicate that LPL and LIPG are separate,
evolutionarily conserved genes.
EXAMPLE 7 - Enzymatic Activity of LLGXL
Phospholipase Activity
Conditioned media from COS-7 cells transiently
expressing human lipoprotein lipase (LPL), LLGN, or LLGXL
were assayed for phospholipase activity. MEM containing 10%
FBS (MEM) was used as the blank, and conditioned media from
COS-7 cells transfected with an antisense LLGXL plasmid (AS)
was used as a negative control.
A phosphatidylcholine (PC) emulsion was made up using
10 gl phosphatidylcholine (10 mM), 40 pl 14C-
phosphatidylcholine, dipalmitoyl (2 LCi), labeled at the sn 1
and 2 positions, and 100 gl Tris-TCNB [100 mM Tris, 1%
Triton, 5 mM CaC121 200 mM NaCl, 0.1% BSA). The emulsion was
evaporated for 10 minutes, then brought to a final volume of
1 ml in Tris-TCNB.
Reactions were performed in duplicate and contained 50
gl PC emulsion and 950 gl medium. Samples were incubated in a
shaking water bath for 2-4 hours at 37 C. The reactions were
terminated by adding 1 ml iN HC1, then extracted with 4 ml of
2-propanol:hexane (1:1). The upper 1.8 ml hexane layer was
passed through a silica gel column, and the liberated 14C-free
fatty acids contained in the flow-thru fraction were
quantitated in a scintillation counter. The results of these
assays are shown in Figure 14.
Triacylglycerol Lipase Activity
Conditioned media from COS-7 cells transiently
expressing human lipoprotein lipase (LPL), LLGN, or LLGXL
were assayed for triglycerol lipase activity. MEM containing
10% FBS was used as the blank, and conditioned media from
COS-7 cells transfected with an antisense LLGXL plasmid (AS)
was used as a negative control.
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A concentrated substrate was prepared as an anhydrous
emulsion of labeled triolein, [9,10- 3H(N)] and unlabeled
triolein (final total triolein = 150 mg with 6.25 x 108cpm),
which was stabilized by adding 9 mg of lecithin in 100%
glycerol. 0.56 ml of 3H-triolein, (0.28 mCi) was mixed with
0.17 ml of unlabeled triolein and 90 pl of lecithin (9 mg).
The mixture was evaporated under a stream of nitrogen. The
dried lipid mixture was emulsified in 2.5 ml 100% glycerol by
sonication (30 second pulse level 2 followed by 2 second
chill cycles over 5 minutes].
The assay substrate was prepared by dilution of 1 volume
of concentrated substrate with 4 volumes of 0.2M Tris-HC1
buffer (pH 8.0) containing 3% w/v fatty acid free bovine
serum albumin. The diluted substrate was vortexed vigorously
for 5 seconds.
Reactions were performed in duplicate in a total volume
of 0.2 ml containing 0.1 ml of assay substrate and 0.1 ml of
the indicated conditioned media. The reactions were
incubated for 90 minutes at 37 C. The reactions were
terminated by adding 3.25 ml of methanol-chloroform-heptane
1.41:1.25:1 (v/v/v) followed by 1.05 ml of 0.1M potassium
carbonate-borate buffer (pH 10.5). After vigorous mixing for
15 seconds, the samples were centrifuged for 5 minutes at
1000 rpm. A 1.0 ml aliquot of the upper aqueous phase was
counted in a scintillation counter. The results of these
assays are shown in Figure 15.
EXAMPLE 8 - Use of LIPG Polypeptide
to Screen for Enhancers or Inhibitors
Recombinant LIPG is produced in baculovirus-infected
insect cells or stably transfected CHO cells or other
acceptable mammalian host cells. Recombinant LIPG is
purified from the serum-containing or serum-free conditioned
medium by chromatography on heparin-Sepharose, followed by
chromatography on a cation exchange resin. A third
chromatographic or further chromatographic steps, such as
molecular sieving, is used in the purification of LIPG if
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needed. During purification, anti-peptide antibodies are
used to monitor LIPG protein and the phospholipase assay is
used to follow LIPG activity.
In the fluorescent assay, the final assay conditions are
approximately 10 mM Tris-HC1 (pH 7.4), 100 mM KC1, 2 mM CaC12,
5 M C6NBD-PC{1-acyl-2-[6-(nitro-2,1,3-benzoxadiazol-4-
yl)amino] caproylphosphatidylcholine, and LIPG protein
(approx. 1-100 ng). The reaction is subjected to
fluorescence excitation at 470 nm, and enzyme activity, as
measured by the fluorescence emission at 540 nm is
continuously monitored. Compounds and/or substances to be
tested for stimulation and/or inhibition of LIPG activity are
added as 10-200 mM solutions in dimethylsulfoxide. Compounds
which stimulate or inhibit LIPG activity are identified as
causing an increased or decreased fluorescence emission at
540 nm.
In the thio assay, the final assay conditions are
approximately 25 mM Tris-HC1 (pH 8.5), 100 mM KC1, 10 mM
CaCl2, 4.24 mM Triton X-100, 0.5 mM 1,2-bis(hexanoylthio)-
1,2-dideoxy-sn-glycero-3-phosphorylcholine, 5 mM 4,4'-
dithiobispyridine (from a 50 mM stock solution in ethanol),
and 1-100 ng recombinant LIPG. Phospholipase activity is
determined by measuring the increase in absorption at 342 nm.
Compounds and/or substances to be tested for stimulation
and/or inhibition of LIPG activity are added as 10-200 mM
solutions in dimethylsulfoxide. Compounds which stimulate or
inhibit LIPG activity are identified as causing an increased
or decreased absorption at 342 nm.
EXAMPLE 9 - Transgenic Mice Expressing Human LIPG
To further study the physiological role of LIPG,
transgenic mice expressing human LIPG are generated.
The 1.53 kb DraI/SrfI restriction fragment encoding
LLGXL (see Figure 4) was cloned into a plasmid vector (pHMG)
downstream of the promoter for the ubiquitously expressed 3-
hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene.
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Transgenic mice expressing different levels of human LLGXL
are generated using standard methods (see, e.g., G.L. Tremp
et al. Gene 156:199-205, 1995). The transgenic mice are used
to determine the impact of LLGXL overexpression on lipid
profile, vascular pathology, rate of development and severity
of atherosclerosis, and other physiological parameters.
EXAMPLE 10 - Expression of LIPG in Atherosclerotic Tissues
LLGXL expression in atherosclerosis was examined by
performing a reverse transcription-polymerase chain reaction
(RT-PCR) using mRNA isolated from vascular biopsies from four
patients with atherosclerosis. The tissue samples were from
the aortic wall (one sample), the iliac artery (two samples),
and the carotid artery (one sample).
Atherosclerosis biopsies were received from
Gloucestershire Royal Hospital, England, and polyA+ mRNA was
prepared and resuspended in diethylpyrocarbonate (DEPC)
treated water at a concentration of 0.5 g/ l mRNA. Reverse
transcriptase reactions were performed according to the
GibcoBRL protocol for Superscript Preamplification system for
First Strand cDNA Synthesis. Briefly, the cDNA was
synthesized as follows: 2 l of each mRNA was added to 1 l
oligo (dT)12-18 primer and 9 l of DEPC water. The tubes were
incubated at 70 C for 10 minutes and put on ice for 1 minute.
To each tube, the following components were added: 2 l lOX
PCR buffer, 2 l 25 MM MgC12r 1 l 10 mM dNTP mix and 2 l
O.1M DTT. After 5 minutes at 42 C, 1 l (200 units) of Super
Script II reverse transcriptase was added. The reactions
were mixed gently, then incubated at 42 C for 50 minutes.
The reactions were terminated by incubation at 70 C for 15
minutes then put on ice. The remaining mRNA was destroyed by
the addition of 1 l of RNase H to each tube and incubated
for 20 minutes at 37 C.
PCR amplifications were performed using 2 41 of the cDNA
reactions. To each tube the following were added: 5 l lOX
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PCR buffer, 5 l 2 mM dNTPs, 1 gl hllg-gspl primer (20
pmol/ml, see Figure 1), 1 l hllg-gsp2a primer (20 pmol/ml,
see Figure 1), 1.5 l 50 mM MgCl2, 0.5 gl Taq polymerase
(5U/ml) and 34 Al water. After holding the reactions at 95 C
for 2 minutes, thirty cycles of PCR were performed as
follows: 15 seconds at 94 C, 20 seconds at 52 C, and 30
seconds at 72 C. The finished reactions were held for 10
minutes at 72 C before analysis by agarose gel
electrophoresis. The hllg-gsp primers are specific for LIPG
and yield an expected product of 300 bp. In a parallel PCR
to show that the cDNA synthesis reactions had been
successful, primers specific for the housekeeping gene, G3PDH
(human glyceraldehyde 3-phosphate dehydrogenase) were used (1
l each at 20 pmol/ml).
The G3PDH primers (see Figure 1) yielded the expected
product of 983 bp in all four vascular biopsy samples. LIPG
expression was detected in three of the four samples, with no
expression being detected in the carotid artery sample.
Example 11 - Differential display,
RT-PCR and cDNA library screening
To perform the experiments discussed in Examples 12 to
16, the following procedure (based on the procedure outlined
in Example 1) was used to obtain the cDNA for LIPG. THP-1
cells were plated in the presence of phorbol 12-myristate 13-
acetate (PMA, 40 ng/ml; Sigma) for 48 hours. The
differentiated THP-1 cells were exposed for 24 hours to
either oxLDL (50 gg/ml) or control medium. Total RNAs were
collected and purified using standard procedures. Poly(A)+
RNA was purified from total RNA using a poly-dT magnetic bead
system (Promega). cDNA synthesis and PCR amplification were
accomplished using protocols from the Differential Display
kit, version 1.0 (Display Systems Biotechnology). The primer
pairs that yielded the initial cDNA fragment of EL were
downstream primer 7 (5'-TTTTTTTTTTTGA-3') and upstream primer
15 (5'-GATCCAATCGC-3'). The amplification reaction was
fractionated on a 6% nondenaturing acrylamide sequencing
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format gel and an amplification product found only in the
reaction containing cDNA from THP-1 cells exposed to oxLDL
was identified and excised from the gel. A reamplification
using the same primers was performed and the product was
excised and subcloned into the pCRII vector using the TA
cloning system (Invitrogen). Insert sizes were determined
using EcoRI digestions of the plasmids, and clones containing
inserts of the approximate size of the original PCR product
were sequenced using fluorescent dye-terminator reagents
(Prism, Applied Biosystems) and an Applied Biosystems 373 DNA
sequencer. We extended the cDNA sequence of the original,
gel-excised cDNA using the 51-RACE system (GIBCO). RNA (1
pg)from the THP-1 cells used initially in the differential
display reactions was used in the 5'-RACE procedure using a
gene-specific primer (5'-TAGGACATGCACAGTGTAATCTG-3') for
first strand cDNA synthesis. We performed PCR amplification
of the cDNA using an anchor primer and gene-specific primer 2
(5'-GATTGTGCTGGCCACTTCTC-3'). This reaction (1 gl)was used
in a nested re-amplification using the universal
amplification primer (5'-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3')
and the gene-specific primer 3 (5'-GACACTCCAGGGACTGAAG-3') to
increase levels of specific product for subsequent isolation.
The reaction products were cloned into the pCRII vector from
the TA cloning kit and determined the sequence. A human
placental cDNA library (oligo dT and random primed) was
obtained from Clontech and probed with the 5'-RACE reaction
PCR product. The DNA from hybridizing clones was purified
using LambdaSorb reagent (Promega). Inserts were excised
from the phage DNA by digestion with EcoRI, subcloned into
the EcoRI site of the Bluescript II SK plasmid vector
(Stratagene) and sequenced.
Example 12 - Antibody Preparation
A 17-residue peptide (GPEGRLEDKLHKPKATC) was synthesized
corresponding to residues 8-23 of the secreted LIPG gene
product on a Model 433A peptide synthesizer (Applied
Biosystems). Peptide (2 mg) was coupled to maleimide-
activated keyhole limpet haemocyanin (2 mg) following the
protocols included in the Imject Activated Immunogen
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Conjugation kit (Pierce Chemical). After desalting, one-half
of the conjugate was emulsified with an equal volume of
Freund's complete adjuvant (Pierce) and injected into a New
Zealand White rabbit. Four weeks after the initial
inoculation, a booster inoculation was administered with an
emulsification made exactly as described above except for the
use of Freund's incomplete adjuvant (Pierce). Two weeks
after the boost, the titres of specific antibodies were
determined in a test bleed via ELISA using immobilized
peptide.
Example 13 - Gene expression studies
HUVECs were propagated in a commercially prepared
endothelial cell growth medium (EGM, Clonetics) supplemented
with bovine brain extract (3 mg/ml; Clonetics), whereas
HCAECs were propagated in EGM with bovine grain extract (3
mg/ml) and 5% fetal bovine serum. Cultures were stimulated
by addition of PMA (100 ng/ml). After 24 hours incubation,
RNA was extracted from the cells via the Trizol method,
electrophoresed on a 1% agarose-formaldehyde gel, transferred
to Nytran membrane on a Turboblotter apparatus (Schleicher
and Schuell) and crosslinked to the membrane using a
Stratalinker ultraviolet crosslinker (Stratagene). The 5'-
RACE reaction PCR product was radiolabelled using the random
priming technique. The radiolabelled probe (1-2x106 cpm/ml)
was denatured by heating to 95 C for 10 minutes and quick-
chilled on ice before adding to the filter in QuikHyb.
Hybridization was allowed to proceed for 3 hours at 65 C.
Filters were exposed to Kodak XAR-2 film with intensifying
screens at -80 C. We incubated HUVEC- and HCEAC-conditioned
medium with heparin-Sepharose CL-6B at 4 C for 1 hour.
After centrifugation, the pelleted heparin-Sepharose was
suspended in SDS loading buffer, heated to 95 C for 5
minutes and loaded onto a 10% Tris-Glycine SDS gel (NOVEX).
After electrophoresis at 140 V for 90 minutes, the proteins
were transferred to nitrocellulose membranes and detected
with rabbit anti-LIPG peptide antisera (1:5,000), with goat
anti-rabbit peroxidase conjugated antisera (1:5,000;
Boehringer) as the secondary antibody. The membranes were
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reacted with Renaissance chemiluminescent reagent (DuPont
NEN) and exposed to Kodak XAR-2 film. A commercially
prepared filter containing poly (A)` RNAs (3 g each) from
human heart, brain, placenta, lung, liver, skeletal muscle,
kidney and pancreas (Clontech)was hybridized with a
radiolabelled fragment and processed as described above.
Following autoradiography, the blot was stripped by washing
in boiling 0.1xSSC, 0.1% SDS for 2x15 minutes at 65 C and
then probed as described above with a 1.4-kb cDNA fragment
encoding human LPL. This fragment was obtained by RT-PCR of
the THP-1 RNA (PMA and oxLDL treated) using the 5' LPL and 3'
LPL primers 5'-ACCACCATGGAGAGCAAAGCCCTG-3' and 5'-
CCAGTTTCAGCCTGACTTCTTATTC-3', respectively. After exposure
to film, the membranes were stripped again and reprobed with
a radiolabelled fragment of human R actin cDNA to normalize
to RNA content.
Human umbilical vein endothelial cells (HUVEC) were
negative for LPL mRNA expression as expected, but were found
to constitutively express a high level of mRNA for the LIPG
gene (Figure 9).
Human coronary artery endothelial cells (HCAEC) were
also found to express the mRNA which was further upregulated
on treatment of these cells with phorbol ester (Figure 9).
Conditional medium from stimulated HUVEC and HCAEC
contained immunoreactive proteins of approximately 68 kD and
40 kD, as well as a less prominent band of 55 kD (Figure 11).
To determine the tissue sites of LIPG production in
vivo, a multiple human tissue northern blot analysis with
probes for both LIPG and LPL was performed. Abundant levels
of LIPG mRNA were found in lung, liver and kidney (Figure 8)
tissues, which showed low levels of LPL expression. LIPG was
also expressed at high levels in the placenta (Figure 8),
suggesting the potential for a role in development.
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In tissues such as heart and skeletal muscle, which
expressed the highest amount of LPL (confirming previous
reports, Goldberg, J.I., J. Lipid Res., 37, 693-707 (1996)),
we did not detect LIPG expression. This analysis indicated
that the tissue distribution of LIPG expression is very
different from that of LPL, as well as that reported for HL
and PL. We found no LIPG mRNA in adrenals or ovaries, but
did find a very low level of LIPG mRNA in the testes (data
not shown). We also found that HepG2 cells express LIPG mRNA
and protein in vitro (data not shown), but at levels less
than 10% of that expressed by HUVECs.
Example 14 - Lipase Assays
The cDNA and the 1.4-kb LPL cDNA were cloned into the
EcoRV site of the mammalian expression vector pCDNA3
(Invitrogen). An antisense pCDNA3 vector was used as
negative control. The recombinant expression vectors (3 g)
were mixed with lipofectamine (Life Technologies) and
transfected in quadruplicate into semiconfluent COS7 cells in
60-mm dishes. Established methods were used to assay samples
of conditioned media from transfected COST cells for TG
lipase and phospholipase activities (Goldberg, J.I., J. Lipid
Res., 37, 693-707 (1996)). for the TG lipase assay, 9,10-
3H(N)-triolein (250 DCi; NEN) was mixed with unlabeled
triolein (150 mg) and type IV-S-a lecithin (9 mg; Sigma) in
glycerol. The mixture was evaporated under nitrogen and
emulsified in glycerol (2.5 ml) by sonication with a Branson
Sonifier 450. The assay substrate was prepared by combining
one volume of the emulsified substrate, four volumes of Tris-
HC1 (0.2 M, pH 8.0) containing 3% (w/v) fatty acid-free
bovine serum albumin (BSA) and one volume of heat-inactivated
bovine serum. Reactions were performed in triplicate in a
total volume (0.2 ml) containing assay substrate (0.1 ml) and
conditioned media (0.1 ml). The reactions were incubated for
2 hours at 37 C and terminated by adding methanol-chloroform-
heptane (1.41:1.25:1; 3.25 ml) followed by potassium
carbonate-borate buffer (1.05 ml; 0.1 M, pH 10.5). After
vigorous mixing for 15 seconds, the samples were centrifuged
for 5 minutes at 1,000 rpm and the upper aqueous phase (1.0
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ml) was counted in a scintillation counter. For the
phospholipase assay, a phosphatidylcholine (PC) emulsion was
made by combining 14C-dipalmitoyl PC (2 DCi; NEN) and lecithin
(10 gl)with Tris-TCNB (100 l; 100 mM Tris-HC1 pH 7.4, 1%
Triton X-100, 5 mM CaC12, 200 mM NaCl, 0.1% BSA). The
mixture was vortexed for 2 minutes and then evaporated under
nitrogen. The dried lipid was reconstituted with TCNB (1 ml)
and vortexed for 10 seconds. Reactions were performed in
triplicate and contained PC emulsion (50 l), conditioned
media (600 Al) and MEM (350 Al). Samples were incubated at
37 C for 2 hours, terminated by addition of HC1 (1 ml) and
extracted with 2-propanol:hexane (1:1; 4m1). A sample (1.8
ml) of the upper hexane layer was passed through a silica gel
column, and the liberated 14 C-free fatty acids contained in
the flow-through fraction were quantitated in a scintillation
counter. For both assays, MEM containing 10% FBS was used as
a blank and conditioned media from COS7 cells transfected
with an antisense plasmid (AS) was used as a negative
control.
Example 15 - Recombinant Adenovirus
Construction and Animal Studies
A recombinant adenovirus encoding human LIPG was
constructed as described (Tsukamoto et al., J. Clin. Invest.,
100, 107-114 (1997); Tsukamoto et al., J. Lipid Res., 38,
1869-1876 (1997)). In brief, the full-length human cDNA was
subcloned into the shuttle plasmid vector pAdCMVLinkl. After
screening for the appropriate orientation by restriction
analysis, the plasmid was linearized with NheI and
cotransfected into 293 cells along with adenoviral DNA
digested with ClaI. Cells were overlaid with agar and
incubated at 37 C for 15 days. Six plaques were picked and
screened by PCR; two plaques positive for cDNA were subjected
to a second round of plaque purification. After confirmation
of the presence of cDNA, the recombinant adenovirus was
expanded in 293 cells at 37 C. Cell lysates were used to
infect HeLa cells for confirmation of the expression of human
LIPG by western blot of conditioned media. The recombinant
adenovirus (AdhEL) was further expanded in 293 cells and
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purified by cesium chloride ultracentrifugation. Control
adenovirus containing no cDNA insert (Adnull) was also
subjected to plaque purification and purified as described
above. The purified viruses were stored in 10% glycerol/PBX
at -80 C. Wild-type C57BL/6, human apoA-I transgenic and
LDL receptor mutant mice were obtained from Jackson
Laboratory. All mice were fed chow diets. Wild-type and
human apoA-I transgenic mice were injected intravenously via
the tail vein with AdhEL or Adnull 1x1011 particles
(approximately 2x109 pfu) and LDLR-deficient mice were
injected with 1x1010 particles. In all experiments, blood was
obtained from the retro-orbital plexus 1 day before injection
and at multiple time points following injection.
Intravenous injection of AdhEL into wild-type C57BL/6
mice resulted in expression in the liver (Figure 17) and
reduction of plasma levels of HDL cholesterol that remained
significantly lower than control virus-injected mice through
at least 41 days post-injection (Figure 18). Lipoproteins
were separated by FPLC gel filtration, demonstrating that HDL
was undetectable 14 days after adenovirus injection (Figure
19). Injection of recombinant LIPG adenovirus into human
apoA-I transgenic mice (which have much higher levels of HDL
cholesterol and apoA-I) reduced both HDL cholesterol (Figure
20) and apoA-I (Figure 21) levels. To determine the relative
effects of LIPG expression on HDL compared with the apoB-
containing lipoproteins VLDL and LDL, we injected a lower
dose of the LIPG adenovirus into chow-fed LDL receptor-
deficient mice, which have approximately 70% of cholesterol
in VLDL/LDL and approximately 30% in HDL. As before,
expression of LIPG reduced HDL cholesterol levels (Figure
23). Although LIPG expression reduced VLDL/LDL cholesterol
levels in the same mice (Figure 24), the effect was
proportionately less. Overexpression of LIPG reduced
VLDL/LDL cholesterol, therefore a role of LIPG in the
modulation of apoB-containing lipoproteins cannot be
excluded.
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Example 16 - Lipid/Lipoprotein Analyses
The plasma total cholesterol and HDL cholesterol levels
were measured enzymatically on a Cobas Fara (Roche Diagnostic
Systems) using Sigma reagents. ApoA-I was quantitated using
a turbidometric assay (Sigma) on a Cobas Fara. Pooled plasma
samples were subjected to fast protein liquid chromatography
(FPLC) gel filtration (Pharmacia LKB Biotechnology) using two
Superose 6 columns in series as described (Tsukamoto et al.,
J. Cl.in. Invest., supra). Fractions (0.5 ml) were collected,
and cholesterol concentrations were determined using an
enzymatic assay (Wako Pure Chemical Industries).
Example 17 - Identification of Inhibitors of LIPG
Modulators of EL activity may be found using the
following method:
Recombinant LIPG would be purified from the conditioned
medium of stably transfected Chinese hamster ovary cells,
from baculovirus infected insect cells, yeast (Pichia
pastoris, Kluveromyces Lactis) or other sources. Non-
recombinant sources of LIPG (such as human plasma,
endothelial cell conditioned media, etc.) could also be
employed. An example of a primary screen to look for
modulators of LIPG activity would utilize the soluble
fluorescent substrate 4-methylumberiferyl hepatanoate. This
assay is continuous and homogeneous. Hydrolysis of this
substrate by LIPG results in the production of highly
fluorescent 4-methylumbelliferone that can be measured in a
microplate fluorimeter. Other primary screening assay
formats that could be used are a scintillation proximity
assay (Amersham) that measures phospholipase activity, the
lower-throughput radiometric phospholipase assay described in
Example 7 (and proposed below as a secondary assay), or the
alternative phospholipase assays described in Example 8.
The catalytic center of LIPG, like other TG lipases,
consists of the same catalytic triad (ser, his, asp) found in
serine proteases. Indeed, other TG lipases, such as
lipoprotein lipase, are inhibited by serine protease
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inhibitors such as PMSF and DFP. Either one of these
compounds may serve as a positive control for inhibitors of
LIPG activity.
Secondary assay: Compounds active in the esterase assay or
alternative screening assays described above will be assayed
in a standard, radiometric phospholipase A assay. This assay
measures the release of radiolabelled palmitic acid from
mixed micelles containing [14C]-dipalmitoyl-
phosphatidy1choline. Other assay formats could be envisioned
which utilize fluorescent substrates and which would be
amenable to a greater throughput.
Selectivity assays: Compounds would be assayed for
inhibition of the related enzymes lipoprotein lipase (LPL)
and pancreatic lipase (PL). Human PL and bovine LPL are
commercially available and assays could be readily
implemented. The phospholipase activity of PL is measured in
exactly the same way as described above for the secondary
assay of LIPG. Since LPL is primarily a TG lipase, the
secondary assay would measure radiolabelled fatty acid (oleic
acid) release from a radiolabelled TG (triolein) substrate
(described in Example 7). This assay has a similar capacity
and may be adapted to other assay formats which utilize
fluorescent substrates and which would be amenable to a
greater throughput.
Phospholipase activity of LIPG would be tested on its in
vivo substrate, HDL, in an in vitro assay. Radiolabelled HDL
could be generated by exchange with a radiolabelled
phospholipid, and then used to measure LIPG phosphospholipase
activity and the activity of compounds emerging from the
screens.
An additional assay could measure the impact of
preincubation of LIPG, HDL, +/- compounds on radiolabelled
cholesterol efflux from cultured cells such as the rat Fu5AH
hepatoma line.
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In vivo assays for assessment of compounds can be run in
wild-type, LIPG-overexpressing, and as control, LIPG null
mice. If, as in the case of adenoEL expression, the
transgenic mice exhibit decreased HDL relative to control
mice, then treatment of transgenic mice with LIPG inhibitory
compounds would be expected to raise HDL to the levels of
control mice. It is also possible that compounds could be
tested for their LIPG inhibitory activity (elevation of HDL)
in other animals such as the LDLR-/- mouse, apoAl transgenic
mice hamsters, or rabbits. Compounds which elevated LIPG or
LIPG activity would be expected to raise HDL in these or
other animal models.
Example 18 - Inhibitory Small Molecule Treatment Method
A small molecule (hereafter an "inhibitory small
molecule") identified in the screening outlined in Example 17
as able to inhibit the LIPG polypeptide in vitro is tested
for its ability to inhibit the LIPG polypeptide in vivo.
Wild-type and LIPG transgenic mice will be studied by
administering the small molecule orally (if orally
bioavailable) or by intravenous injection. Activity of the
LIPG polypeptide will be measured in plasma before and after
heparin injection (to release the enzyme from bound sites).
In addition, cholesterol, VLDL, LDL and HDL cholesterol and
apoA-I levels will be monitored in animals receiving the
inhibitory small molecule. Finally, LDL receptor deficient
mice will be fed an atherogenic diet and administered the
inhibitory small molecule or placebo for a period of 8 weeks.
Atherosclerosis will be quantitated in the aortas of the mice
in order to determine whether administration of the
inhibitory small molecule recudes the progression or induces
regression of atherosclerosis. Based on these preclinical
data, additional animal models such as hamsters, rabbits, or
pigs will be studied for the ability of the inhibitory small
molecule to raise HDL cholesterol levels, reduce VLDL and LDL
cholesterol levels, and/or inhibit the progression of
atherosclerosis.
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Those inhibitory small molecules found to have the
desired properties will be administered to patients in
combination with pharmaceutically acceptable carriers. The
inhibitory small molecules may be administered in a variety
of ways, including oral administration and intravenous
injection. The patients' HDL, VLDL and LDL cholesterol
levels will be monitored to determine efficacy of the
inhibitory small molecule and to optimize dosage and
administration protocols.
Example 19 - Inhibitory Peptide Treatment Method
Therapeutic peptides are identified by testing fragments
of the LIPG polypeptide to determine which of these fragments
inhibit LIPG polypeptide activity in vitro. Once identified,
an "inhibitory peptide" is then tested for its ability to
inhibit the LIPG polypeptide in vivo. Inhibitory peptides
will be produced recombinantly in E.coli and purified by
methods known in the art. The effect of the inhibitory
peptides will be studied in wild-type and LIPG transgenic
mice by administering the inhibitory peptide by intravenous
injection. Activity of the LIPG polypeptide will be measured
in plasma before and after heparin injection (to release the
enzyme from bound sites). In addition, cholesterol, VLDL,
LDL and HDL cholesterol and apoA-I levels will be monitored
in animals receiving the inhibitory peptide. Finally, LDL
receptor deficient mice will be fed an atherogenic diet and
administered the inhibitory peptide or placebo for a period
of 8 weeks. Atherosclerosis will be quantitated in the
aortas of the mice in order to determine whether
administration of the inhibitory peptide reduces the
progression or induces regression of atherosclerosis. Based
on these preclinical data, additional animal models such as
hamsters, rabbits or pigs will be studied for the ability of
the inhibitory small molecule to raise HDL cholesterol
levels, reduce VLDL and LDL cholesterol levels, and/or
inhibit the progression of atherosclerosis.
Those inhibitory peptides found to have the desired
properties will be administered to patients in combination
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with pharmaceutically acceptable carriers. The inhibitory
peptides may be administered in a variety of ways, including
oral administration and intravenous injection. The patients'
HDL, VLDL and LDL cholesterol levels will be monitored to
determine efficacy of the inhibitory peptides and to optimize
dosage and administration protocols.
Example 20 - Antisense Treatment Method
A series of antisense oligonucleotides, each
complementary to about 20 bases of the LIPG cDNA sequence are
chemically synthesized by standard techniques. To determine
the most efficient oligonucleotide to use therapeutically,
each oligonucleotide is individually transfected into cells
expressing the LIPG gene, using standard transfection
protocols.
At about 24-48 hours following transfection of the
oligonucleotides, the LIPG mRNA level in cells is determined
by quantitative PCR, northern blot, RNAse protection, or
other appropriate methods. Alternatively, LIPG expression
may be monitored with specific antibodies, which can be used
to screen for effective antisense oligonucleotides.
Oligonucleotides which effectively reduce LIPG mRNA levels
are then formulated for in vivo delivery as therapeutics.
Antisense LIPG sequences may be delivered in a gene
therapy vector, such as adenovirus, adeno-associated virus,
retrovirus, naked DNA, or other systems discussed in the
detailed description. Such fragments can be used
therapeutically when delivered in gene therapy vectors.
Hepatic expression of such recombinant vectors is a preferred
approach. Alternatively, synthetic antisense
oligonucleotides may be formulated for in vivo delivery as
therapeutics as described above.
Antisense oligonucleotides may be administered by the
following routes: intravenous, subcutaneous, introdermal,
pulmonary, oral, intraventricular, intrathecal, and topical.
The route of administration may include direct administration
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to vessel walls (i.e., endothelium and/or vascular smooth
muscle). As an example, patients with low HDL-C could
receive a dose of 0.5-2 mg/kg of an effective antisense
oligonucleotide, infused intravenously, every other day for
up to 2-3 weeks. As LIPG is expressed in the liver, it may
be desireable to deliver antisense reagents to the portal
circulation. This may be accomplished by conjugating or
complexing the oligonucleotide with a liver-targeting moiety,
such as asialoglycoprotein. Dose and timing of therapy would
depend on efficiency of antisense delivery, as well as
parameters such as half life, specificity and toxicology of
the antisense oligonucleotide.
Increase in HDL-C can be monitored using standard
clinical laboratory procedures. The original dosing schedule
(such as that described above) is repeated as often as
required to maintain HDL-C above 35 mg/dL.
Example 21 - Ribozyme Treatment Method
Based on the LIPG cDNA sequence, hammerhead ribozymes
which effectively reduce LIPG mRNA levels are prepared.
These consist of two "arms" of 6-7 bases each of nucleotide
sequence complementary to LIPG mRNA, separated by the
catalytic moiety of the ribozyme. Examples of such
hammerhead motifs are described by Rossi et al., 1992, Aids
Research and Human Retroviruses, 8, 183. The ribozymes are
expressed in eukaryotic cells from an appropriate DNA vector.
The ribozymes may be administered encapsulated in
liposomes, as discussed above.
The ribozyme/liposome composition is delivered to the
liver by direct injection or by use of a catheter, infusion
pump or stent. The route of administration may include
direct administration to vessel walls (i.e., endothelium
and/or vascular smooth muscle). Patients are treated for up
to 2 weeks with 5-50 mg/kg/day ribozyme in a pharmaceutically
effective carrier. Increase in HDL-C and dosing regimen are
monitored and determined as for antisense oligonucleotides.
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Example 22 - Neutralizin Antibody Treatment Method
Anti-LIPG antibodies, antibody fragments, or chimeric
antibodies consisting of at least one LIPG-binding moiety,
prepared as described in Example 12, are used to inhibit LIPG
activity in vivo. The antibodies may be delivered as a
bolus only, infused over time, or both. Typically a dose of
0.2-0.6 mg/kg is given as bolus, followed by a 2 to 12-hour
infusion. Alternatively, multiple bolus injections are
administered every other day, or every third of fourth day,
as required to reduce LIPG and raise HDL-C. Repeat dosing is
performed as determined by measurement of HDL-C levels.
Antibodies to LIPG may also be delivered in a gene therapy
vehicle to facilitate expression in vivo. The level of
expression of the antibody is determined indirectly by
measuring HDL-C levels and additional vectors may be
introduced as needed.
Example 23 - Use of Inhibitory
Molecules or Enhancer Molecules
Fragments of LIPG protein, which can inhibit LIPG
activity by competing for binding to intact LIPG, required
coactivator molecules, cell surface receptors or binding
proteins, may be delivered as therapeutic recombinant
proteins or from gene therapy vectors.
As an example, the LLGN polypeptide based on LIPG is
cloned into a recombinant adenovirus as described (Tsukamoto
et al., J. Clin. Invest., 100, 107-114 (1997); Tsukamoto et
al., J. Lipid Res., 38, 1869-1876 (1997)). The LLGN cDNA is
cloned into the shuttle plasmid vector pAdCMVLinkl. After
screening for the appropriate orientation by restriction
analysis, the plasmid is linearized with NheI and
cotransfected into 293 cells along with adenoviral DNA
digested with ClaI. Cells are then overlaid with agar and
incubated at 37 C for 15 days. Plaques are picked and
screened by PCR; plaques positive for cDNA are subjected to a
second round of plaque purification. After confirmation of
the presence of cDNA, the recombinant adenovirus is expanded
in 293 cells at 37 C. Cell lysates are used to infect HeLa
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cells for confirmation of the expression of human EL by
western blot of conditioned media. The recombinant
adenovirus is further expanded in 293 cells and purified by
cesium chloride ultracentrifugation. The purified viruses are
stored in 10% glycerol/PBX at -80 C. The patient is injected
intravenously with the recombinant adenovirus 1x1011 particles
(approximately 2x109 pfu).
Example 24 - Methods of Increasing the
Level of LIPG in a Patient by
Expression of LIPG from an Expression Vector
The full length LIPG cDNA is cloned into a recombinant
adenovirus (Tsukamoto et al., J. Clin. Invest., 100, 107-114
(1997); Tsukamoto et al., J. Lipid Res., 38, 1869-1876
(1997)) encoding human LIPG. The full-length human LIPG cDNA
is cloned into the shuttle plasmid vector pAdCMVLinki. After
screening for the appropriate orientation by restriction
analysis, the plasmid is linearized with NheI and
cotransfected into 293 cells along with adenoviral DNA
digested with ClaI. Cells are overlaid with agar and
incubated at 37 C for 15 days. Plaques are picked and
screened by PCR; plaques positive for cDNA are subjected to a
second round of plaque purification. After confirmation of
the presence of cDNA, the recombinant adenovirus is expanded
in 293 cells at 37 C. Cell lysates are used to infect HeLa
cells for confirmation of the expression of human LIPG
polypeptide by western blot of conditioned media. The
recombinant adenovirus (AdhEL) is further expanded in 293
cells and purified by cesium chloride ultracentrifugation.
the purified viruses are stored in 10% glycerol/PBX at -80 C.
Patients are injected intravenously with AdhEL or Adnull
1x1011 particles (approximately 2x109 pfu).
Example 25 - Methods of Increasing the Level
of LIPG Activity by Administration
of a Full-Length Wild-Type or
Engineered Recombinant LIPG Protein
The wild-type LIPG protein reduces VLDL and LDL
cholesterol levels and LIPG could be engineered to act
specifically on VLDL and LDL cholesterol without having
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effects on HDL cholesterol. Administration of wild-type or engineered
recombinant
LIPG could in certain circumstances be used as a therapy for reducing VLDL
and/or
LDL cholesterol levels. Wild-type and/or engineered LIPG protein ("recombinant
LIPG protein") will be produced recombinantly in E. coli and purified using
methods
known in the art. Wild-type mice will be studied by administering the
recombinant
LIPG protein by intravenous injection. Activity of LIPG will be measured in
plasma.
In addition, cholesterol, VLDL, LDL and HDL cholesterol and apoA-I levels will
be
monitored in animals receiving the recombinant LIPG protein. Finally, LDL
receptor
deficient mice will be fed an atherogenic diet and administered the
recombinant
LIPG protein or placebo for a period of 8 weeks. Atherosclerosis will be
quantitated
in the aortas of the mice in order to determine whether administration of the
recombinant LIPG protein reduces the progression or induces regression of
atherosclerosis. Based on these preclinical data, additional animal models
such as
hamsters, rabbits, or pigs will be studied for the ability of the recombinant
LIPG
protein to reduce VLDL and LDL cholesterol levels and/or inhibit the
progression of
atherosclerosis. Those recombinant LIPG proteins found to have the desired
ability
to reduce VLDL and LDL cholesterol levels and/or inhibit the progression of
atherosclerosis will be combined with a pharmaceutically acceptable carrier
and
administered to patients. The recombinant LIPG polypeptides may be
administered
in a variety of ways, including oral administration and intravenous injection.
One skilled in the art will readily appreciate the present invention is well
adapted to carry out the objects and obtain the ends and advantages mentioned,
as
well as those inherent therein. The scope of the claims should not be limited
by the
preferred embodiments set forth in the examples, but should be given the
broadest
interpretation consistent with the description as a whole.
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Example 26 - Demonstration of Glycosylation
and Biological Activity of LIPG
A number of experiments were conducted to further characterize LIPG. The
experiments which follow further define the biological activity of LIPG
(referred to
in these experiments and in the figures as "endothelial lipase," or "EL").
Figure 24 demonstrates that endothelial lipase is a glycoprotein. EL was
treated with the glycosidases EndoF, EndoH, and neuraminidase. This resulted
in
reductions in the size of the EL band on western blotting, consistent with the
removal of carbohydrate from the polypeptide chain. Glycosylation was also
inhibited with tunicamycin in EL-expressing cells. This resulted in a
substantial
reduction in the size of the EL band. The glycosidase and tunicamycin
experiments
indicate that EL is a glycoprotein.
Figure 25 demonstrates that endothelial lipase is a heparin binding protein.
An adenoviral vector expressing EL AdhEL (see Example 15) was injected into
mice.
Blood was drawn before (pre) and after (post) heparin injection. The intensity
of the
full-length 68 kD EL band greatly increased after heparin injection consistent
with
EL bound to heparin sulfate proteoglycans and released by the administration
of
heparin.
Figures 26A and 26B and 27A and 27B illustrate the lipolytic activity of
endothelial lipase relative to lipoprotein lipase (LPL) and hepatic lipase
(HL). This
experiment was conducted in order to determine whether endothelial lipase has
triglyceride (TG) lipase activity (Fig, 27A). In multiple studies on different
conditioned media, unequivocal evidence of TG lipase activity was
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repeatedly found. The phospholipase activity of endothelial
lipase was assayed in the same conditioned media samples
(Fig. 27B). The ratio of phospholipase to TG lipase activity
was highly consistent as assayed in different batches of
conditioned media. In absolute terms, the moles of fatty
acid generated per volume conditioned media was significantly
greater for phospholipase than for TG lipase activity. The
ability of serum to effect endothelial lipase activity was
then tested (Figures 27A and 27B). As expected, the addition
of serum significantly increased lipoprotein lipase TG lipase
activity in the absence of any other source of apoC-II.
Interestingly, serum reproducibly and substantially reduced
the activity of endothelial lipase. This confirms that
neither apoC-II nor any other serum factor is a required co-
factor for endothelial lipase. It also indicates that there
is an endogenous inhibitor of endothelial lipase in serum.
Figures 28A and 28B illustrate that expression of EL
increased post-heparin plasma phospholipase activity in a
dose dependent fashion. Injection of three different doses
(1x1011, 3x101 or 1x10'" particles) of AdhEL into mice
resulted in the presence of EL protein in the post-heparin
plasma. Major bands of 68 kD and 40 kD and a minor band of
55 kD were observed (Fig. 28A). The 68 kD band is believed
to be the full-length glycosylated form of EL and the 40 kD
band a proteolytic fragment. The amount of EL protein in
plasma assessed by western blot is proportional to the dose
of vector injected (Fig. 28B). The expression of different
levels of human EL resulted in a significant and dose-
dependent increase in post-heparin plasma phospholipase
activity, compared to Adnull injected mice (9779 +/- 733,
6501 +/- 1299, 3963 +/- 796 ninol.ml-l.hr-1 vs 952 +/- 258
nmol.mo-l.hr-1, respectively). The post-heparin phospholipase
activity correlated with levels of human EL protein for the
68 kD (r=0.98, P<0.01), 55 kD (r=0.83, P<0.05), and 40 kD
(r=0.94, P<0.05) bands.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jaye, Michael C.
Lynch, Kevin J.
Amin, Dilip V.
Doan, Kim-Anh T.
Marchadier, Dawn
Maugeais, Cyrille
Rader, Daniel J.
Krawiec, John A.
South, Victoria J.
(ii) TITLE OF INVENTION: COMPOSITIONS'AND METHODS FOR EFFECTING THE
LEVELS
OF HIGH DENSITY LIPOPROTEIN (HDL) CHOLESTEROL AND APOLIPOPROTEIN
AI,
VERY LOW DENSITY LIPOPROTEIN (VLDL)CHOLESTEROL AND LOW DENSITY
LIPOPROTEIN (LDL) CHOLESTEROL
(iii) NUMBER OF SEQUENCES: 31
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Deeth Williams Wall LLP
(B) STREET: Suite 400 National Bank Building, 150 York Street
(C) CITY: Toronto
(D) STATE: ON
(E) COUNTRY: CANADA
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(C) REFERENCE/DOCKET NUMBER: 3207 0007
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 416-941-9440
(B) TELEFAX: 416-941-9443
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 367 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 22..180
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GAATTCGGCT TGATCAATCG C TTC AAA AAG GGG ATC TGT CTG AGC TGC CGC 51
Phe Lys Lys Gly Ile Cys Leu Ser Cys Arg
1 5 10
AAG AAC CGT TGT AAT AGC ATT GGC TAC AAT GCC AAG AAA ATG AGG AAC 99
Lys Asn Arg Cys Asn Ser Ile Gly Tyr Asn Ala Lys Lys Met Arg Asn
15 20 25
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AAG AGG AAC AGC AAA ATG TAC CTA AAA ACC CGG GCA GGC ATG CCT TTC 147
Lys Arg Asn Ser Lys Met Tyr Leu Lys Thr Arg Ala Gly Met Pro Phe
30 35 40
AGA GGT AAC CTT CAG TCC CTG GAG TGT CCC TGA GGAAGGCCCT TAATACCTCC 200
Arg Gly Asn Leu Gln Ser Leu Glu Cys Pro
45 50
TTCTTAATAC CATGCTGCAG AGCAGGGCAC ATCCTAGCCC AGGAGAAGTG GCCAGCACAA 260
TCCAATCAAA TCGTTGCAAA TCAGATTACA CTGTGCATGT CCTAGGAAAG GGAATCTTTA 320
CAAAATAAAC AGTGTGGACC CCTCAAAAAA AAAAAAAAGC CGAATTC 367
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Phe Lys Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Ser
1 5 10 15
Ile Gly Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys Met
20 25 30
Tyr Leu Lys Thr Arg Ala Gly Met Pro Phe Arg Gly Asn Leu Gln Ser
35 40 45
Leu Glu Cys Pro
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1382 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 312..1370
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
gaattcggct tctactacta ctaggccacg cgtcgcctag tacggggggg gggggggggg 60
tcagcgagtc cttgcctccc ggcggctcag gacgagggca gatctcgttc tggggcaagc 120
cgttgacact cgctccctgc caccgcccgg gctccgtgcc gccaagtttt cattttccac 180
cttctctgcc tccagtcccc cagcccctgg ccgagagaag ggtcttaccg gccgggattg 240
ctggaaacac caagaggtgg tttttgtttt ttaaaacttc tgtttcttgg gagggggtgt 300
ggcggggcag g atg agc aac tcc gtt cct ctg ctc tgt ttc tgg agc ctc 350
Met Ser Asn Ser Val Pro Leu Leu Cys Phe Trp Ser Leu
1 5 10
tgc tat tgc ttt get gcg ggg agc ccc gta cct ttt ggt cca gag gga 398
Cys Tyr Cys Phe Ala Ala Gly Ser Pro Val Pro Phe Gly Pro Glu Gly
15 20 25
cgg ctg gaa gat aag ctc cac aaa ccc aaa get aca cag act gag gtc 446
Arg Leu Glu Asp Lys Leu His Lys Pro Lys Ala Thr Gln Thr Glu Val
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30 35 40 45
aaa cca tct gtg agg ttt aac ctc cgc acc tcc aag gac cca gag cat 494
Lys Pro Ser Val Arg Phe Asn Leu Arg Thr Ser Lys Asp Pro Glu His
50 55 60
gaa gga tgc tac ctc tcc gtc ggc cac agc cag ccc tta gaa gac tgc 542
Glu Gly Cys Tyr Leu Ser Val Gly His Ser Gln Pro Leu Glu Asp Cys
65 70 75
agt ttc aac atg aca get aaa acc ttt ttc atc att cac gga tgg acg 590
Ser Phe Asn Met Thr Ala Lys Thr Phe Phe Ile Ile His Gly Trp Thr
80 85 90
atg agc ggt atc ttt gaa aac tgg ctg cac aaa ctc gtg tca gcc ctg 638
Met Ser Gly Ile Phe Glu Asn Trp Leu His Lys Leu Val Ser Ala Leu
95 100 105
cac aca aga gag aaa gac gcc aat gta gtt gtg gtt gac tgg ctc ccc 686
His Thr Arg Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro
110 115 120 125
ctg gcc cac cag ctt tac acg gat gcg gtc aat aat acc agg gtg gtg 734
Leu Ala His Gln Leu Tyr Thr Asp Ala Val Asn Asn Thr Arg Val Val
130 135 140
gga cac agc att gcc agg atg ctc gac tgg ctg cag gag aag gac gat 782
Gly His Ser Ile Ala Arg Met Leu Asp Trp Leu Gln Glu Lys Asp Asp
145 150 155
ttt tct ctc ggg aat gtc cac ttg atc ggc tac agc ctc gga gcg cac 830
Phe Ser Leu Gly Asn Val His Leu Ile Gly Tyr Ser Leu Gly Ala His
160 165 170
gtg gcc ggg tat gca ggc aac ttc gtg aaa gga acg gtg ggc cga atc 878
Val Ala Gly Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg Ile
175 180 185
aca ggt ttg gat cct gcc ggg ccc atg ttt gaa ggg gcc gac atc cac 926
Thr Gly Leu Asp Pro Ala Gly Pro Met Phe Glu Gly Ala Asp Ile His
190 195 200 205
aag agg ctc tct ccg gac gat gca gat ttt gtg gat gtc ctc cac acc 974
Lys Arg Leu Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu His Thr
210 215 220
tac acg cgt tcc ttc.ggc ttg agc att ggt att cag atg cct gtg ggc 1022
Tyr Thr Arg Ser Phe Gly Leu Ser Ile Gly Ile Gln Met Pro Val Gly
225 230 235
cac att gac atc tac ccc aat ggg ggt gac ttc cag cca ggc tgt gga 1070
His Ile Asp Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro Gly Cys Gly
240 245 250
ctc aac gat gtc ttg gga tca att gca tat gga aca atc aca gag gtg 1118
Leu Asn Asp Val Leu Gly Ser Ile Ala Tyr Gly Thr Ile Thr Glu Val
255 260 265
gta aaa tgt gag cat gag cga gcc gtc cac ctc ttt gtt gac tct ctg 1166
Val Lys Cys Glu His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu
270 275 280 285
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gtg aat cag gac aag ccg agt ttt gcc ttc cag tgc act gac tcc aat 1214
Val Asn Gln Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn
290 295 300
cgc ttc aaa aag ggg atc tgt ctg agc tgc cgc aag aac cgt tgt aat 1262
Arg Phe Lys Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn
305 310 315
agc att ggc tac aat gcc aag aaa atg agg aac aag agg aac agc aaa 1310
Ser Ile Gly Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys
320 325 330
atg tac cta aaa acc cgg gca ggc atg cct ttc aga ggt aac ctt cag 1358
Met Tyr Leu Lys Thr Arg Ala Gly Met Pro Phe Arg Gly Asn Leu Gln
335 340 345
tcc ctg gag tgt caagccgaat tc 1382
Ser Leu Glu Cys
350
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 353 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ser Asn Ser Val Pro Leu Leu Cys Phe Trp Ser Leu Cys Tyr Cys
1 5 10 15
Phe Ala Ala Gly Ser Pro Val Pro Phe Gly Pro Glu Gly Arg Leu Glu
20 25 30
Asp Lys Leu His Lys Pro Lys Ala Thr Gln Thr Glu Val Lys Pro Ser
35 40 45
Val Arg Phe Asn Leu Arg Thr Ser Lys Asp Pro Glu His Glu Gly Cys
50 55 60
Tyr Leu Ser Val Gly His Ser Gln Pro Leu Glu Asp Cys Ser Phe Asn
65 70 75 80
Met Thr Ala Lys Thr Phe Phe Ile Ile His Gly Trp Thr Met Ser Gly
85 90 95
Ile Phe Glu Asn Trp Leu His Lys Leu Val Ser Ala Leu His Thr Arg
100 105 110
Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro Leu Ala His
115 120 125
Gln Leu Tyr Thr Asp Ala Val Asn Asn Thr Arg Val Val Gly His Ser
130 135 140
Ile Ala Arg Met Leu Asp Trp Leu Gln Glu Lys Asp Asp Phe Ser Leu
145 150 155 160
Gly Asn Val His Leu Ile Gly Tyr Ser Leu Gly Ala His Val Ala Gly
165 170 175
Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg Ile Thr Gly Leu
180 185 190
Asp Pro Ala Gly Pro Met Phe Glu Gly Ala Asp Ile His Lys Arg Leu
195 200 205
Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu His Thr Tyr Thr Arg
210 215 220
Ser Phe Gly Leu Ser Ile Gly Ile Gln Met Pro Val Gly His Ile Asp
225 230 235 240
Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro Gly Cys Gly Leu Asn Asp
245 250 255
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Val Leu Gly Ser Ile Ala Tyr Gly Thr Ile Thr Glu Val Val Lys Cys
260 265 270
Glu His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu Val Asn Gln
275 280 285
Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn Arg Phe Lys
290 295 300
Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Ser Ile Gly
305 310 315 320
Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys Met Tyr Leu
325 330 335
Lys Thr Arg Ala Gly Met Pro Phe Arg Gly Asn Leu Gln Ser Leu Glu
340 345 350
Cys
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1065 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1065
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATG AGC AAC TCC GTT CCT CTG CTC TGT TTC TGG AGC CTC TGC TAT TGC 48
Met Ser Asn Ser Val Pro Leu Leu Cys Phe Trp Ser Leu Cys Tyr Cys
1 5 10 15
TTT GCT GCG GGG AGC CCC GTA CCT TTT GGT CCA GAG GGA CGG CTG GAA 96
Phe Ala Ala Gly Ser Pro Val Pro Phe Gly Pro Glu Gly Arg Leu Glu
20 25 30
GAT AAG CTC CAC AAA CCC AAA GCT ACA CAG ACT GAG GTC AAA CCA TCT 144
Asp Lys Leu His Lys Pro Lys Ala Thr Gln Thr Glu Val Lys Pro Ser
35 40 45
GTG AGG TTT AAC CTC CGC ACC TCC AAG GAC CCA GAG CAT GAA GGA TGC 192
Val Arg Phe Asn Leu Arg Thr Ser Lys Asp Pro Glu His Glu Gly Cys
50 55 60
TAC CTC TCC GTC GGC CAC AGC CAG CCC TTA GAA GAC TGC AGT TTC AAC 240
Tyr Leu Ser Val Gly His Ser Gln Pro Leu Giu Asp Cys Ser Phe Asn
65 70 75 80
ATG ACA GCT AAA ACC TTT TTC ATC ATT CAC GGA TGG ACG ATG AGC GGT 288
Met Thr Ala Lys Thr Phe Phe Ile Ile His Gly Trp Thr Met Ser Gly
85 90 95
ATC TTT GAA AAC TGG CTG CAC AAA CTC GTG TCA GCC CTG CAC ACA AGA 336
Ile Phe Glu Asn Trp Leu His Lys Leu Val Ser Ala Leu His Thr Arg
100 105 110
GAG AAA GAC GCC AAT GTA GTT GTG GTT GAC TGG CTC CCC CTG GCC CAC 384
Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro Leu Ala His
115 120 125
CAG CTT TAC ACG GAT GCG GTC AAT AAT ACC AGG GTG GTG GGA CAC AGC 432
Gln Leu Tyr Thr Asp Ala Val Asn Asn Thr Arg Val Val Gly His Ser
130 135 140
ATT GCC AGG ATG CTC GAC TGG CTG CAG GAG AAG GAC GAT TTT TCT CTC 480
Ile Ala Arg Met Leu Asp Trp Leu Gln Glu Lys Asp Asp Phe Ser Leu
145 150 155 160
GGG AAT GTC CAC TTG ATC GGC TAC AGC CTC GGA GCG CAC GTG GCC GGG 528
Gly Asn Val His Leu Ile Gly Tyr Ser Leu Gly Ala His Val Ala Gly
165 170 175
TAT GCA GGC AAC TTC GTG AAA GGA ACG GTG GGC CGA ATC ACA GGT TTG 576
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Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg Ile Thr Gly Leu
180 185 190
GAT CCT GCC GGG CCC ATG TTT GAA GGG GCC GAC ATC CAC AAG AGG CTC 624
Asp Pro Ala Gly Pro Met Phe Glu Gly Ala Asp Ile His Lys Arg Leu
195 200 205
TCT CCG GAC GAT GCA GAT TTT GTG GAT GTC CTC CAC ACC TAC ACG CGT 672
Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu His Thr Tyr Thr Arg
210 215 220
TCC TTC GGC TTG AGC ATT GGT ATT CAG ATG CCT GTG GGC CAC ATT GAC 720
Ser Phe Gly Leu Ser Ile Gly Ile Gln Met Pro Val Gly His Ile Asp
225 230 235 240
ATC TAC CCC AAT GGG GGT GAC TTC CAG CCA GGC TGT GGA CTC AAC GAT 768
Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro Gly Cys Gly Leu Asn Asp
245 250 255
GTC TTG GGA TCA ATT GCA TAT GGA ACA ATC ACA GAG GTG GTA AAA TGT 816
Val Leu Gly Ser Ile Ala Tyr Gly Thr Ile Thr Glu Val Val Lys Cys
260 265 270
GAG CAT GAG CGA GCC GTC CAC CTC TTT GTT GAC TCT CTG GTG AAT CAG 864
Glu His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu Val Asn Gln
275 280 285
GAC AAG CCG AGT TTT GCC TTC CAG TGC ACT GAC TCC AAT CGC TTC AAA 912
Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn Arg Phe Lys
290 295 300
AAG GGG ATC TGT CTG AGC TGC CGC AAG AAC CGT TGT AAT AGC ATT GGC 960
Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Ser Ile Gly
305 310 315 320
TAC AAT GCC AAG AAA ATG AGG AAC AAG AGG AAC AGC AAA ATG TAC CTA 1008
Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys Met Tyr Leu
325 330 335
AAA ACC CGG GCA GGC ATG CCT TTC AGA GGT AAC CTT CAG TCC CTG GAG 1056
Lys Thr Arg Ala Gly Met Pro Phe Arg Gly Asn Leu Gln Ser Leu Glu
340 345 350
TGT CCC TGA 1065
Cys Pro
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ser Asn Ser Val Pro Leu Leu Cys Phe Trp Ser Leu Cys Tyr Cys
1 5 10 15
Phe Ala Ala Giy Ser Pro Val Pro Phe Gly Pro Glu Gly Arg Leu Glu
20 25 30
Asp Lys Leu His Lys Pro Lys Ala Thr Gln Thr Glu Val Lys Pro Ser
35 40 45
Val Arg Phe Asn Leu Arg Thr Ser Lys Asp Pro Glu His Glu Gly Cys
50 55 60
Tyr Leu Ser Val Gly His Ser Gln Pro Leu Glu Asp Cys Ser Phe Asn
65 70 75 80
Met Thr Ala Lys Thr Phe Phe Ile Ile His Gly Trp Thr Met Ser Gly
85 90 95
Ile Phe Glu Asn Trp Leu His Lys Leu Val Ser Ala Leu His Thr Arg
100 105 110
Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro Leu Ala His
115 120 125
Gln Leu Tyr Thr Asp Ala Val Asn Asn Thr Arg Val Val Gly His Ser
130 135 140
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Ile Ala Arg Met Leu Asp Trp Leu Gln Glu Lys Asp Asp Phe Ser Leu
145 150 155 160
Gly Asn Val His Leu Ile Gly Tyr Ser Leu Gly Ala His Val Ala Gly
165 170 175
Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg Ile Thr Gly Leu
180 185 190
Asp Pro Ala Gly Pro Met Phe Glu Gly Ala Asp Ile His Lys Arg Leu
195 200 205
Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu His Thr Tyr Thr Arg
210 215 220
Ser Phe Gly Leu Ser Ile Gly Ile Gln Met Pro Val Gly His Ile Asp
225 230 235 240
Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro Gly Cys Gly Leu Asn Asp
245 250 255
Val Leu Gly Ser Ile Ala Tyr Gly Thr Ile Thr Glu Val Val Lys Cys
260 265 270
Glu His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu Val Asn Gln
275 280 285
Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn Arg Phe Lys
290 295 300
Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Ser Ile Gly
305 310 315 320
Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys Met Tyr Leu
325 330 335
Lys Thr Arg Ala Gly Met Pro Phe Arg Gly Asn Leu Gln Ser Leu Glu
340 345 350
Cys Pro
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2565 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 252..1754
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAATTCGCGG CCGCGTCGAC GGCGGCTCAG GACGAGGGCA GATCTCGTTC TGGGGCAAGC 60
CGTTGACACT CGCTCCCTGC CACCGCCCGG GCTCCGTGCC GCCAAGTTTT CATTTTCCAC 120
CTTCTCTGCC TCCAGTCCCC CAGCCCCTGG CCGAGAGAAG GGTCTTACCG GCCGGGATTG 180
CTGGAAACAC CAAGAGGTGG TTTTTGTTTT TTAAAACTTC TGTTTCTTGG GAGGGGGTGT 240
GGCGGGGCAG G ATG AGC AAC TCC GTT CCT CTG CTC TGT TTC TGG AGC CTC 290
Met Ser Asn Ser Val Pro Leu Leu Cys Phe Trp Ser Leu
1 5 10
TGC TAT TGC TTT GCT GCG GGG AGC CCC GTA CCT TTT GGT CCA GAG GGA 338
Cys Tyr Cys Phe Ala Ala Gly Ser Pro Val Pro Phe Gly Pro Glu Gly
15 20 25
CGG CTG GAA GAT AAG CTC CAC AAA CCC AAA GCT ACA CAG ACT GAG GTC 386
Arg Leu Glu Asp Lys Leu His Lys Pro Lys Ala Thr Gln Thr Glu Val
30 35 40 45
AAA CCA TCT GTG AGO TTT AAC CTC CGC ACC TCC AAG GAC CCA GAG CAT 434
Lys Pro Ser Val Arg Phe Asn Leu Arg Thr Ser Lys Asp Pro Glu His
50 55 60
GAA GGA TGC TAC CTC TCC GTC GGC CAC AGC CAG CCC TTA GAA GAC TGC 482
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Glu Gly Cys Tyr Leu Ser Val Gly His Ser Gln Pro Leu Glu Asp Cys
65 70 75
AGT TTC AAC ATG ACA GCT AAA ACC TTT TTC ATC ATT CAC GGA TGG ACG 530
Ser Phe Asn Met Thr Ala Lys Thr Phe Phe Ile Ile His Gly Trp Thr
80 85 90
ATG AGC GGT ATC TTT GAA AAC TGG CTG CAC AAA CTC GTG TCA GCC CTG 578
Met Ser Gly Ile Phe Glu Asn Trp Leu His Lys Leu Val Ser Ala Leu
95 100 105
CAC ACA AGA GAG AAA GAC GCC AAT GTA GTT GTG GTT GAC TGG CTC CCC 626
His Thr Arg Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro
110 115 120 125
CTG GCC CAC CAG CTT TAC ACG GAT GCG GTC AAT AAT ACC AGG GTG GTG 674
Leu Ala His Gln Leu Tyr Thr Asp Ala Val Asn Asn Thr Arg Val Val
130 135 140
GGA CAC AGC ATT GCC AGG ATG CTC GAC TGG CTG CAG GAG AAG GAC GAT 722
Gly His Ser Ile Ala Arg Met Leu Asp Trp Leu Gln Glu Lys Asp Asp
145 150 155
TTT TCT CTC GGG AAT GTC CAC TTG ATC GGC TAC AGC CTC GGA GCG CAC 770
Phe Ser Leu Gly Asn Val His Leu Ile Gly Tyr Ser Leu Gly Ala His
160 165 170
GTG GCC GGG TAT GCA GGC AAC TTC GTG AAA GGA ACG GTG GGC CGA ATC 818
Val Ala Gly Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg Ile
175 180 185
ACA GGT TTG GAT CCT GCC GGG CCC ATG TTT GAA GGG GCC GAC ATC CAC 866
Thr Gly Leu Asp Pro Ala Gly Pro Met Phe Glu Gly Ala Asp Ile His
190 195 200 205
AAG AGG CTC TCT CCG GAC GAT GCA GAT TTT GTG GAT GTC CTC CAC ACC 914
Lys Arg Leu Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu His Thr
210 215 220
TAC ACG CGT TCC TTC GGC TTG AGC ATT GGT ATT CAG ATG CCT GTG GGC 962
Tyr Thr Arg Ser Phe Gly Leu Ser Ile Gly Ile Gln Met Pro Val Gly
225 230 235
CAC ATT GAC ATC TAC CCC AAT GGG GGT GAC TTC CAG CCA GGC TGT GGA 1010
His Ile Asp Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro Gly Cys Gly
240 245 250
CTC AAC GAT GTC TTG GGA TCA ATT GCA TAT GGA ACA ATC ACA GAG GTG 1058
Leu Asn Asp Val Leu Gly Ser Ile Ala Tyr Gly Thr Ile Thr Glu Val
255 260 265
GTA AAA TGT GAG CAT GAG CGA GCC GTC CAC CTC TTT GTT GAC TCT CTG 1106
Val Lys Cys Glu His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu
270 275 280 285
GTG AAT CAG GAC AAG CCG AGT TTT GCC TTC CAG TGC ACT GAC TCC AAT 1154
Val Asn Gln Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn
290 295 300
CGC TTC AAA AAG GGG ATC TGT CTG AGC TGC CGC AAG AAC CGT TGT AAT 1202
Arg Phe Lys Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn
305 310 315
AGC ATT GGC TAC AAT GCC AAG AAA ATG AGG AAC AAG AGG AAC AGC AAA 1250
Ser Ile Gly Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys
320 325 330
ATG TAC CTA AAA ACC CGG GCA GGC ATG CCT TTC AGA GTT TAC CAT TAT 1298
Met Tyr Leu Lys Thr Arg Ala Gly Met Pro Phe Arg Val Tyr His Tyr
335 340 345
CAG ATG AAA ATC CAT GTC TTC AGT TAC AAG AAC ATG GGA GAA ATT GAG 1346
Gln Met Lys Ile His Val Phe Ser Tyr Lys Asn Met Gly Glu Ile Glu
350 355 360 365
CCC ACC TTT TAC GTC ACC CTT TAT GGC ACT AAT GCA GAT TCC CAG ACT 1394
Pro Thr Phe Tyr Val Thr Leu Tyr Gly Thr Asn Ala Asp Ser Gln Thr
370 375 380
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CTG CCA CTG GAA ATA GTG GAG CGG ATC GAG CAG AAT GCC ACC AAC ACC 1442
Leu Pro Leu Glu Ile Val Glu Arg Ile Glu Gln Asn Ala Thr Asn Thr
385 390 395
TTC CTG GTC TAC ACC GAG GAG GAC TTG GGA GAC CTC TTG AAG ATC CAG 1490
Phe Leu Val Tyr Thr Glu Glu Asp Leu Gly Asp Leu Leu Lys Ile Gln
400 405 410
CTC ACC TGG GAG GGG GCC TCT CAG TCT TGG TAC AAC CTG TGG AAG GAG 1538
Leu Thr Trp Glu Gly Ala Ser Gln Ser Trp Tyr Asn Leu Trp Lys Glu
415 420 425
TTT CGC AGC TAC CTG TCT CAA CCC CGC AAC CCC GGA CGG GAG CTG AAT 1586
Phe Arg Ser Tyr Leu Ser Gln Pro Arg Asn Pro Gly Arg Glu Leu Asn
430 435 440 445
ATC AGG CGC ATC CGG GTG AAG TCT GGG GAA ACC CAG CGG AAA CTG ACA 1634
Ile Arg Arg Ile Arg Val Lys Ser Gly Glu Thr Gln Arg Lys Leu Thr
450 455 460
TTT TGT ACA GAA GAC CCT GAG AAC ACC AGC ATA TCC CCA GGC CGG GAG 1682
Phe Cys Thr Glu Asp Pro Glu Asn Thr Ser Ile Ser Pro Gly Arg Glu
465 470 475
CTC TGG TTT CGC AAG TGT CGG GAT GGC TGG AGG ATG AAA AAC GAA ACC 1730
Leu Trp Phe Arg Lys Cys Arg Asp Gly Trp Arg Met Lys Asn Glu Thr
480 485 490
AGT CCC ACT GTG GAG CTT CCC TGA GGGTGCCCGG GCAAGTCTTG CCAGCAAGGC 1784
Ser Pro Thr Val Glu Leu Pro
495 500
AGCAAGACTT CCTGCTATCC AAGCCCATGG AGGAAAGTTA CTGCTGAGGA CCCACCCAAT 1844
GGAAGGATTC TTCTCAGCCT TGACCCTGGA GCACTGGGAA CAACTGGTCT CCTGTGATGG 1904
CTGGGACTCC TCGCGGGAGG GGACTGCGCT GCTATAGCTC TTGCTGCCTC TCTTGAATAG 1964
CTCTAACTCC AAACCTCTGT CCACACCTCC AGAGCACCAA GTCCAGATTT GTGTGTAAGC 2024
AGCTGGGTGC CTGGGGCCTC TCGTGCACAC TGGATTGGTT TCTCAGTTGC TGGGCGAGCC 2084
TGTACTCTGC CTGACGAGGA ACGCTGGCTC CGAAGAGGCC CTGTGTAGAA GGCTGTCAGC 2144
TGCTCAGCCT GCTTTGAGCC TCAGTGAGAA GTCCTTCCGA CAGGAGCTGA CTCATGTCAG 2204
GATGGCAGGC CTGGTATCTT GCTCGGGCCC TGGCTGTTGG GGTTCTCATG GGTTGCACTG 2264
ACCATACTGC TTACGTCTTA GCCATTCCGT CCTGCTCCCC AGCTCACTCT CTGAAGCACA 2324
CATCATTGGC TTTCCTATTT TTCTGTTCAT TTTTTAATTG AGCAAATGTC TATTGAACAC 2384
TTAAAATTAA TTAGAATGTG GTAATGGACA TATTACTGAG CCTCTCCATT TGGAACCCAG 2444
TGGAGTTGGG ATTTCTAGAC CCTCTTTCTG TTTGGATGGT GTATGTGTAT ATGCATGGGG 2504
AAAGGCACCT GGGGCCTGGG GGAGGCTATA GGATATAAGC AGTCGACGCG GCCGCGAATT 2564
C 2565
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 500 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:B:
Met Ser Asn Ser Val Pro Leu Leu Cys Phe Trp Ser Leu Cys Tyr Cys
1 5 10 15
Phe Ala Ala Gly Ser Pro Val Pro Phe Gly Pro Glu Gly Arg Leu Glu
20 25 30
Asp Lys Leu His Lys Pro Lys Ala Thr Gln Thr Glu Val Lys Pro Ser
35 40 45
Val Arg Phe Asn Leu Arg Thr Ser Lys Asp Pro Glu His Glu Gly Cys
50 55 60
Tyr Leu Ser Val Gly His Ser Gln Pro Leu Glu Asp Cys Ser Phe Asn
65 70 75 80
Met Thr Ala Lys Thr Phe Phe Ile Ile His Gly Trp Thr Met Ser Gly
85 90 95
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Ile Phe Glu Asn Trp Leu His Lys Leu Val Ser Ala Leu His Thr Arg
100 105 110
Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro Leu Ala His
115 120 125
Gln Leu Tyr Thr Asp Ala Val Asn Asn Thr Arg Val Val Gly His Ser
130 135 140
Ile Ala Arg Met Leu Asp Trp Leu Gln Glu Lys Asp Asp Phe Ser Leu
145 150 155 160
Gly Asn Val His Leu Ile Gly Tyr Ser Leu Gly Ala His Val Ala Gly
165 170 175
Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg Ile Thr Gly Leu
180 185 190
Asp Pro Ala Gly Pro Met Phe Glu Gly Ala Asp Ile His Lys Arg Leu
195 200 205
Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu His Thr Tyr Thr Arg
210 215 220
Ser Phe Gly Leu Ser Ile Gly Ile Gln Met Pro Val Gly His Ile Asp
225 230 235 240
Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro Gly Cys Gly Leu Asn Asp
245 250 255
Val Leu Gly Ser Ile Ala Tyr Gly Thr Ile Thr Glu Val Val Lys Cys
260 265 270
Glu His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu Val Asn Gln
275 280 285
Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn Arg Phe Lys
290 295 300
Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Ser Ile Gly
305 310 315 320
Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys Met Tyr Leu
325 330 335
Lys Thr Arg Ala Gly Met Pro Phe Arg Val Tyr His Tyr Gln Met Lys
340 345 350
Ile His Val Phe Ser Tyr Lys Asn Met Gly Glu Ile Glu Pro Thr Phe
355 360 365
Tyr Val Thr Leu Tyr Gly Thr Asn Ala Asp Ser Gln Thr Leu Pro Leu
370 375 380
Glu Ile Val Glu Arg Ile Glu Gln Asn Ala Thr Asn Thr Phe Leu Val
385 390 395 400
Tyr Thr Glu Glu Asp Leu Gly Asp Leu Leu Lys Ile Gln Leu Thr Trp
405 410 415
Glu Gly Ala Ser Gln Ser Trp Tyr Asn Leu Trp Lys Glu Phe Arg Ser
420 425 430
Tyr Leu Ser Gln Pro Arg Asn Pro Gly Arg Glu Leu Asn Ile Arg Arg
435 440 445
Ile Arg Val Lys Ser Gly Glu Thr Gln Arg Lys Leu Thr Phe Cys Thr
450 455 460
Glu Asp Pro Glu Asn Thr Ser Ile Ser Pro Gly Arg Glu Leu Trp Phe
465 470 475 480
Arg Lys Cys Arg Asp Gly Trp Arg Met Lys Asn Glu Thr Ser Pro Thr
485 490 495
Val Glu Leu Pro
500
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1035 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1035
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATG AGC AAC TCC GTT CCT CTG CTC TGT TTC TGG AGC CTC TGC TAT TGC 48
Met Ser Asn Ser Val Pro Leu Leu Cys Phe Trp Ser Leu Cys Tyr Cys
1 5 10 15
TTT GCT GCG GGG AGC CCC GTA CCT TTT GGT CCA GAG GGA CGG CTG GAA 96
Phe Ala Ala Gly Ser Pro Val Pro Phe Gly Pro Glu Gly Arg Leu Glu
20 25 30
GAT AAG CTC CAC AAA CCC AAA GCT ACA CAG ACT GAG GTC AAA CCA TCT 144
Asp Lys Leu His Lys Pro Lys Ala Thr Gln Thr Glu Val Lys Pro Ser
35 40 45
GTG AGG TTT AAC CTC CGC ACC TCC AAG GAC CCA GAG CAT GAA GGA TGC 192
Val Arg Phe Asn Leu Arg Thr Ser Lys Asp Pro Glu His Glu Gly Cys
50 55 60
TAC CTC TCC GTC GGC CAC AGC CAG CCC TTA GAA GAC TGC AGT TTC AAC 240
Tyr Leu Ser Val Gly His Ser Gln Pro Leu Glu Asp Cys Ser Phe Asn
65 70 75 80
ATG ACA GCT AAA ACC TTT TTC ATC ATT CAC GGA TGG ACG ATG AGC GGT 288
Met Thr Ala Lys Thr Phe Phe Ile Ile His Gly Trp Thr Met Ser Gly
85 90 95
ATC TTT GAA AAC TGG CTG CAC AAA CTC GTG TCA GCC CTG CAC ACA AGA 336
Ile Phe Glu Asn Trp Leu His Lys Leu Val Ser Ala Leu His Thr Arg
100 105 110
GAG AAA GAC GCC AAT GTA GTT GTG GTT GAC TGG CTC CCC CTG GCC CAC 384
Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro Leu Ala His
115 120 125
CAG CTT TAC ACG GAT GCG GTC AAT AAT ACC AGG GTG GTG GGA CAC AGC 432
Gln Leu Tyr Thr Asp Ala Val Asn Asn Thr Arg Val Val Gly His Ser
130 135 140
ATT GCC AGG ATG CTC GAC TGG CTG CAG GAG AAG GAC GAT TTT TCT CTC 480
Ile Ala Arg Met Leu Asp Trp Leu Gln Glu Lys Asp Asp Phe Ser Leu
145 150 155 160
GGG AAT GTC CAC TTG ATC GGC TAC AGC CTC GGA GCG CAC GTG GCC GGG 528
Gly Asn Val His Leu Ile Gly Tyr Ser Leu Gly Ala His Val Ala Gly
165 170 175
TAT GCA GGC AAC TTC GTG AAA GGA ACG GTG GGC CGA ATC ACA GGT TTG 576
Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg Ile Thr Gly Leu
180 185 190
GAT CCT GCC GGG CCC ATG TTT GAA GGG GCC GAC ATC CAC AAG AGG CTC 624
Asp Pro Ala Gly Pro Met Phe Glu Gly Ala Asp Ile His Lys Arg Leu
195 200 205
TCT CCG GAC GAT GCA GAT TTT GTG GAT GTC CTC CAC ACC TAC ACG CGT 672
Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu His Thr Tyr Thr Arg
210 215 220
TCC TTC GGC TTG AGC ATT GGT ATT CAG ATG CCT GTG GGC CAC ATT GAC 720
Ser Phe Gly Leu Ser Ile Gly Ile Gln Met Pro Val Gly His Ile Asp
225 230 235 240
ATC TAC CCC AAT GGG GGT GAC TTC CAG CCA GGC TGT GGA CTC AAC GAT 768
Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro Gly Cys Gly Leu Asn Asp
245 250 255
GTC TTG GGA TCA ATT GCA TAT GGA ACA ATC ACA GAG GTG GTA AAA TGT 816
Val Leu Gly Ser Ile Ala Tyr Gly Thr Ile Thr Glu Val Val Lys Cys
260 265 270
GAG CAT GAG CGA GCC GTC CAC CTC TTT GTT GAC TCT CTG GTG AAT CAG 864
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Glu His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu Val Asn Gln
275 280 285
GAC AAG CCG AGT TTT GCC TTC CAG TGC ACT GAC TCC AAT CGC TTC AAA 912
Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn Arg Phe Lys
290 295 300
AAG GGG ATC TGT CTG AGC TGC CGC AAG AAC CGT TGT AAT AGC ATT GGC 960
Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Ser Ile Gly
305 310 315 320
TAC AAT GCC AAG AAA ATG AGG AAC AAG AGG AAC AGC AAA ATG TAC CTA 1008
Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys Met Tyr Leu
325 330 335
AAA ACC CGG GCA GGC ATG CCT TTC AGA 1035
Lys Thr Arg Ala Gly Met Pro Phe Arg
340 345
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 345 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Ser Asn Ser Val Pro Leu Leu Cys Phe Trp Ser Leu Cys Tyr Cys
1 5 10 15
Phe Ala Ala Gly Ser Pro Val Pro Phe Gly Pro Glu Gly Arg Leu Glu
20 25 30
Asp Lys Leu His Lys Pro Lys Ala Thr Gln Thr Glu Val Lys Pro Ser
35 40 45
Val Arg Phe Asn Leu Arg Thr Ser Lys Asp Pro Glu His Glu Gly Cys
50 55 60
Tyr Leu Ser Val Gly His Ser Gln Pro Leu Glu Asp Cys Ser Phe Asn
65 70 75 80
Met Thr Ala Lys Thr Phe Phe Ile Ile His Gly Trp Thr Met Ser Gly
85 90 95
Ile Phe Glu Asn Trp Leu His Lys Leu Val Ser Ala Leu His Thr Arg
100 105 110
Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro Leu Ala His
115 120 125
Gin Leu Tyr Thr Asp Ala Val Asn Asn Thr Arg Val Val Gly His Ser
130 135 140
Ile Ala Arg Met Leu Asp Trp Leu Gln Glu Lys Asp Asp Phe Ser Leu
145 150 155 160
Gly Asn Val His Leu Ile Gly Tyr Ser Leu Gly Ala His Val Ala Gly
165 170 175
Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg Ile Thr Gly Leu
180 185 190
Asp Pro Ala Gly Pro Met Phe Glu Gly Ala Asp Ile His Lys Arg Leu
195 200 205
Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu His Thr Tyr Thr Arg
210 215 220
Ser Phe Gly Leu Ser Ile Gly Ile Gln Met Pro Val Gly His Ile Asp
225 230 235 240
Ile Tyr Pro Asn Gly Gly Asp Phe Gln Pro Gly Cys Gly Leu Asn Asp
245 250 255
Val Leu Gly Ser Ile Ala Tyr Gly Thr Ile Thr Glu Val Val Lys Cys
260 265 270
Glu His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu Val Asn Gln
275 280 285
Asp Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn Arg Phe Lys
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290 295 300
Lys Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Ser Ile Gly
305 310 315 320
Tyr Asn Ala Lys Lys Met Arg Asn Lys Arg Asn Ser Lys Met Tyr Leu
325 330 335
Lys Thr Arg Ala Gly Met Pro Phe Arg
340 345
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..225
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CTG GGA TCC ATC GCC TAT GGC ACG ATC GCG GAG GTG GTG AAG TGC GAG 48
Leu Gly Ser Ile Ala Tyr Gly Thr Ile Ala Glu Val Val Lys Cys Glu
1 5 10 15
CAT GAG CGG GCC GTG CAT CTC TTT GTG GAC TCC CTG GTG AAC CAG GAC 96
His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu Val Asn Gln Asp
20 25 30
AAG CCG AGC TTT GCC TTC CAG TGC ACA GAC TCC AAC CGC TTC AAA AAA 144
Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn Arg Phe Lys Lys
35 40 45
GGG ATC TGT CTC AGC TGC CGG AAG AAC CGC TGT AAC GGC ATC GGC TAC 192
Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Gly Ile Gly Tyr
50 55 60
AAT GCT AAG AAG ACG AGG AAT AAG AGG AAC ACC 225
Asn Ala Lys Lys Thr Arg Asn Lys Arg Asn Thr
65 70 75
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Leu Gly Ser Ile Ala Tyr Gly Thr Ile Ala Glu Val Val Lys Cys Glu
1 5 10 15
His Glu Arg Ala Val His Leu Phe Val Asp Ser Leu Val Asn Gln Asp
20 25 30
Lys Pro Ser Phe Ala Phe Gln Cys Thr Asp Ser Asn Arg Phe Lys Lys
35 40 45
Gly Ile Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Gly Ile Gly Tyr
50 55 60
Asn Ala Lys Lys Thr Arg Asn Lys Arg Asn Thr
65 70 75
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 475 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Glu Ser Lys Ala Leu Leu Val Leu Thr Leu Ala Val Trp Leu Gln
1 5 10 15
Ser Leu Thr Ala Ser Arg Gly Gly Val Ala Ala Ala Asp Gln Arg Arg
20 25 30
Asp Phe Ile Asp Ile Glu Ser Lys Phe Ala Leu Arg Thr Pro Glu Asp
35 40 45
Thr Ala Glu Asp Thr Cys His Leu Ile Pro Gly Val Ala Glu Ser Val
50 55 60
Ala Thr Cys His Phe Asn His Ser Ser Lys Thr Phe Met Val Ile His
65 70 75 80
Gly Trp Thr Val Thr Gly Met Tyr Glu Ser Trp Val Pro Lys Leu Val
85 90 95
Ala Ala Leu Tyr Lys Arg Glu Pro Asp Ser Asn Val Ile Val Val Asp
100 105 110
Trp Leu Ser Arg Ala Gln Glu His Tyr Pro Val Ser Ala Gly Tyr Thr
115 120 125
Lys Leu Val Gly Gln Asp Val Ala Arg Phe Ile Asn Trp Met Glu Glu
130 135 140
Glu Phe Asn Tyr Pro Leu Asp Asn Val His Leu Leu Gly Tyr Ser Leu
145 150 155 160
Gly Ala His Ala Ala Gly Ile Ala Gly Ser Leu Thr Asn Lys Lys Val
165 170 175
Asn Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro Asn Phe Glu Tyr Ala
180 185 190
Glu Ala Pro Ser Arg Leu Ser Pro Asp Asp Ala Asp Phe Val Asp Val
195 200 205
Leu His Thr Phe Thr Arg Gly Ser Pro Gly Arg Ser Ile Gly Ile Gln
210 215 220
Lys Pro Val Gly His Val Asp Ile Tyr Pro Asn Gly Gly Thr Phe Gln
225 230 235 240
Pro Gly Cys Asn Ile Gly Glu Ala Ile Arg Val Ile Ala Glu Arg Gly
245 250 255
Leu Gly Asp Val Asp Gln Leu Val Lys Cys Ser His Glu Arg Ser Ile
260 265 270
His Leu Phe Ile Asp Ser Leu Leu Asn Glu Glu Asn Pro Ser Lys Ala
275 280 285
Tyr Arg Cys Ser Ser Lys Glu Ala Phe Glu Lys Gly Leu Cys Leu Ser
290 295 300
Cys Arg Lys Asn Arg Cys Asn Asn Leu Gly Tyr Glu Ile Asn Lys Val
305 310 315 320
Arg Ala Lys Arg Ser Ser Lys Met Tyr Leu Lys Thr Arg Ser Gln Met
325 330 335
Pro Tyr Lys Val Phe His Tyr Gln Val Lys Ile His Phe Ser Gly Thr
340 345 350
Glu Ser Glu Thr His Thr Asn Gln Ala Phe Glu Ile Ser Leu Tyr Gly
355 360 365
Thr Val Ala Glu Ser Glu Asn Ile Pro Phe Thr Leu Pro Glu Val Ser
370 375 380
Thr Asn Lys Thr Tyr Ser Phe Leu Ile Tyr Thr Glu Val Asp Ile Gly
385 390 395 400
Glu Leu Leu Met Leu Lys Leu Lys Trp Lys Ser Asp Ser Tyr Phe Ser
405 410 415
Trp Ser Asp Trp Trp Ser Ser Pro Gly Phe Ala Ile Gln Lys Ile Arg
420 425 430
Val Lys Ala Gly Glu Thr Gln Lys Lys Val Ile Phe Cys Ser Arg Glu
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435 440 445
Lys Val Ser His Leu Gln Lys Gly Lys Ala Pro Ala Val Phe Val Lys
450 455 460
Cys His Asp Lys Ser Leu Asn Lys Lys Ser Gly
465 470 475
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 499 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Asp Thr Ser Pro Leu Cys Phe Ser Ile Leu Leu Val Leu Cys Ile
1 5 10 15
Phe Ile Gln Ser Ser Ala Leu Gly Gln Ser Leu Lys Pro Glu Pro Phe
20 25 30
Gly Arg Arg Ala Gln Ala Val Glu Thr Asn Lys Thr Leu His Glu Met
35 40 45
Lys Thr Arg Phe Leu Leu Phe Gly Glu Thr Asn Gln Gly Cys Gln Ile
50 55 60
Arg Ile Asn His Pro Asp Thr Leu Gln Glu Cys Giy Phe Asn Ser Ser
65 70 75 80
Leu Pro Leu Val Met Ile Ile His Gly Trp Ser Val Asp Gly Val Leu
85 90 95
Glu Asn Trp Ile Trp Gln Met Val Ala Ala Leu Lys Ser Gln Pro Ala
100 105 110
Gln Pro Val Asn Val Gly Leu Val Asp Trp Ile Thr Leu Ala His Asp
115 120 125
His Tyr Thr Ile Ala Val Arg Asn Thr Arg Leu Val Gly Lys Glu Val
130 135 140
Ala Ala Leu Leu Arg Trp Leu Glu Glu Ser Val Gln Leu Ser Arg Ser
145 150 155 160
His Val His Leu Ile Gly Tyr Ser Leu Gly Ala His Val Ser Gly Phe
165 170 175
Ala Gly Ser Ser Ile Gly Gly Thr His Lys Ile Gly Arg Ile Thr Gly
180 185 190
Leu Asp Ala Ala Gly Pro Leu Phe Glu Gly Ser Ala Pro Ser Asn Arg
195 200 205
Leu Ser Pro Asp Asp Ala Asn Phe Val Asp Ala Ile His Thr Phe Thr
210 215 220
Arg Glu His Met Gly Leu Ser Val Gly Ile Lys Gln Pro Ile Gly His
225 230 235 240
Tyr Asp Phe Tyr Pro Asn Gly Gly Ser Phe Gln Pro Gly Cys His Phe
245 250 255
Leu Glu Leu Tyr Arg His Ile Ala Gln His Gly Phe Asn Ala Ile Thr
260 265 270
Gln Thr Ile Lys Cys Ser His Glu Arg Ser Val His Leu Phe Ile Asp
275 280 285
Ser Leu Leu His Ala Gly Thr Gln Ser Met Ala Tyr Pro Cys Gly Asp
290 295 300
Met Asn Ser Phe Ser Gln Gly Leu Cys Leu Ser Cys Lys Lys Gly Arg
305 310 315 320
Cys Asn Thr Leu Gly Tyr His Val Arg Gln Glu Pro Arg Ser Lys Ser
325 330 335
Lys Arg Leu Phe Leu Val Thr Arg Ala Gln Ser Pro Phe Lys Val Tyr
340 345 350
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His Tyr Gin Leu Lys Ile Gln Phe Ile Asn Gln Thr Glu Thr Pro Ile
355 360 365
Gln Thr Thr Phe Thr Met Ser Leu Leu Gly Thr Lys Glu Lys Met Gln
370 375 380
Lys Ile Pro Ile Thr Leu Gly Lys Gly Ile Ala Ser Asn Lys Thr Tyr
385 390 395 400
Ser Phe Leu Ile Thr Leu Asp Val Asp Ile Gly Glu Leu Ile Met Ile
405 410 415
Lys Phe Lys Trp Glu Asn Ser Ala Val Trp Ala Asn Val Trp Asp Thr
420 425 430
Val Gln Thr Ile Ile Pro Trp Ser Thr Gly Pro Arg His Ser Gly Leu
435 440 445
Val Leu Lys Thr Ile Arg Val Lys Ala Gly Giu Thr Gln Gin Arg Met
450 455 460
Thr Phe Cys Ser Glu Asn Thr Asp Asp Leu Leu Leu Arg Pro Thr Gln
465 470 475 480
Giu Lys Ile Phe Val Lys Cys Glu Ile Lys Ser Lys Thr Ser Lys Arg
485 490 495
Lys Ile Arg
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Leu Pro Leu Trp Thr Leu Ser Leu Leu Leu Gly Ala Val Ala Gly
1 5 10 15
Lys Glu Val Cys Tyr Glu Arg Leu Gly Cys Phe Ser Asp Asp Ser Pro
20 25 30
Trp Ser Gly Ile Thr Glu Arg Pro Leu His Ile Leu Pro Trp Ser Pro
35 40 45
Lys Asp Val Asn Thr Arg Phe Leu Leu Tyr Thr Asn Glu Asn Pro Asn
50 55 60
Asn Phe Gln Glu Val Ala Ala Asp Ser Ser Ser Ile Ser Giy Ser Asn
65 70 75 80
Phe Lys Thr Asn Arg Lys Thr Arg Phe Ile Ile His Gly Phe Ile Asp
85 90 95
Lys Gly Glu Glu Asn Trp Leu Ala Asn Val Cys Lys Asn Leu Phe Lys
100 105 110
Val Glu Ser Val Asn Cys Ile Cys Val Asp Trp Lys Gly Gly Ser Arg
115 120 125
Thr Gly Tyr Thr Gln Ala Ser Gln Asn Ile Arg Ile Val Gly Ala Glu
130 135 140
Val Ala Tyr Phe Val Glu Phe Leu Gln Ser Ala Phe Gly Tyr Ser Pro
145 150 155 160
Ser Asn Val His Val Ile Gly His Ser Leu Gly Ala His Ala Ala Gly
165 170 175
Glu Ala Gly Arg Arg Thr Asn Gly Thr Ile Gly Arg Ile Thr Gly Leu
180 185 190
Asp Pro Ala Glu Pro Cys Phe Gln Gly Thr Pro Glu Leu Val Arg Leu
195 200 205
Asp Pro Ser Asp Ala Lys Phe Val Asp Val Ile His Thr Asp Gly Ala
210 215 220
Pro Ile Val Pro Asn Leu Gly Phe Gly Met Ser Gln Val Val Gly His
225 230 235 240
Leu Asp Phe Phe Pro Asn Gly Gly Val Glu Met Pro Gly Cys Lys Lys
245 250 255
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Asn Ile Leu Ser Gln Ile Val Asp Ile Asp Gly Ile Trp Glu Gly Thr
260 265 270
Arg Asp Phe Ala Ala Cys Asn His Leu Arg Ser Tyr Lys Tyr Tyr Thr
275 280 285
Asp Ser Ile Val Asn Pro Asp Gly Phe Ala Gly Phe Pro Cys Ala Ser
290 295 300
Tyr Asn Val Phe Thr Ala Asn Lys Cys Phe Pro Cys Pro Ser Gly Gly
305 310 315 320
Cys Pro Gln Met Gly His Tyr Ala Asp Arg Tyr Pro Gly Lys Thr Asn
325 330 335
Asp Val Gly Gln Lys Phe Tyr Leu Asp Thr Gly Asp Ala Ser Asn Phe
340 345 350
Ala Arg Trp Arg Tyr Lys Val Ser Val Thr Leu Ser Gly Lys Lys Val
355 360 365
Thr Gly His Ile Leu Val Ser Leu Phe Gly Asn Lys Gly Asn Ser Lys
370 375 380
Gln Tyr Glu Ile.Phe Lys Gly Thr Leu Lys Pro Asp Ser Thr His Ser
385 390 395 400
Asn Glu Phe Asp Ser Asp Val Asp Val Gly Asp Leu Gln Met Val Lys
405 410 415
Phe Ile Trp Tyr Asn Asn Val Ile Asn Pro Thr Leu Pro Arg Val Gly
420 425 430
Ala Ser Lys Ile Ile Val Glu Thr Asn Val Giy Lys Gln Phe Asn Phe
435 440 445
Cys Ser Pro Glu Thr Val Arg Glu Glu Val Leu Leu Thr Leu Thr Pro
450 455 460
Cys
465
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Gly Pro Glu Gly Arg Leu Glu Asp Lys Leu His Lys Pro Lys Ala Thr
1 5 10 15
Cys
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TTTTTTTTTT TGA 13
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
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(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GATCAATCGC 10
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
TAGGACATGC ACAGTGTAAT CTG 23
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GATTGTGCTG GCCACTTCTC 20
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GACACTCCAG GGACTGAAG 19
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(ix) FEATURE:
(A) NAME/KEY: modified-base
(B) LOCATION: 36
(D) OTHER INFORMATION: /mod base= i
(ix) FEATURE:
(A) NAME/KEY: modified-base
(B) LOCATION: 37
(D) OTHER INFORMATION: /mod base= i
(ix) FEATURE:
(A) NAME/KEY: modified-base
(B) LOCATION: 41
(D) OTHER INFORMATION: /mod base= i
(ix) FEATURE:
(A) NAME/KEY: modified-base
(B) LOCATION: 42
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(D) OTHER INFORMATION: /mod-base= i
(ix) FEATURE:
(A) NAME/KEY: modified-base
(B) LOCATION: 46
(D) OTHER INFORMATION: /mod base= i
(ix) FEATURE:
(A). NAME/KEY: modified-base
(B) LOCATION: 47
(D) OTHER INFORMATION: /mod base= i
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CUACUACUAC UAGGCCACGC GTCGACTAGT ACGGGNNGGG NNGGGNNG 48
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CACACACAGG CCACGCGTCG ACTAGTAC 28
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ACCACCATGG AGAGCAAAGC CCTG 24
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CCAGTTTCAG CCTGACTTCT TATTC 25
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GGCTGTGGAC TCAACGATGT C 21
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
CCGGGTGGGT AGGTACATTT TG 22
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
GGGGGTGACT TCCAGCCAGG CTGTG 25
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
AACTCTGAAA GGCATGCCTG CCCGG 25
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
TGAAGGTCGG AGTCAACGGA TTTGGT 26
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
CATGTGGGCC ATGAGGTCCA CCAC 24
