Note: Descriptions are shown in the official language in which they were submitted.
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DISULFIDE CORE POLYPEPTIDES
BACKGROUND OF THE INVENTION
Protein inhibitors are classified into a series
of families based on extensive sequence homologies among
the family members and the conservation of intrachain
disulfide bridges, see Laskowski and Kato, Ann. Rev.
Biochem. 49: 593-626 (1980). An example of a serine
proteinase inhibitor is the serine proteinase inhibitor
aprotinin which is used therapeutically in the treatment/
of acute pancreatitis, various states of shock syndrome,
hyperfibrinolytic hemorrhage and myocardial infarction.
Administration of aprotinin in high doses significantly
reduces blood loss in connection with cardiac surgery,
including cardiopulmonary bypass operations.
However, when administered in vivo, aprotinin
has been found to have a nephrotoxic effect in rats,
rabbits and dogs after repeated injections of relativel~,~
high doses. The nephrotoxicity (appearing, i.e., in thEs
form of lesions) observed for aprotinin might be ascribE~d
to the accumulation of aprotinin in the proximal tubulus
cells of the kidneys as a result of the high positive nest
charge of aprotinin, which causes it to be bound to the
negatively charged surfaces of 'the tubuli. This
nephrotoxicity makes aprotinin :less suitable for clinical
purposes, particularly in those uses requiring
administration of large doses o.f the inhibitor (such as
cardiopulmonary bypass operations). Furthermore,
aprotinin is a bovine protein, which may induce an immune
response upon administration to humans.
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Thus there is a need for serine proteinase
inhibitors which are not toxic for the treatment of acute
pancreatitis, various states of shock syndrome,
hyperfibrinolytic hemorrhage and myocardial infarction.
SUMMARY OF THE INVENTION
The present invention fills this need by
providing for a new class of proteinase inhibitors called
disulfide core proteinase inhibitors (hereinafter referred
to as a Zdscl polypeptide). Murine Zdscl, SEQ ID NOs: 1
and 2 has a signal sequence extending from the methioni:ne
at position 1 thraugh and including the alanine at
position 24 of SEQ ID N0:2. The mature murine Zdscl
polypeptide is also depicted by SEQ ID N0:3. SEQ ID N0:4
and 5 are examples of a mature human Zdscl polypeptide ;end
polynucleotide which encodes it. A generic Zdscl
polypeptide is exemplified by S:EQ ID N0:6.
Within one aspect of the invention there is
provided an isolated polypeptide. The polypeptide being
comprised of a sequence of amino acids containing the
sequence of SEQ ID N0:2, SEQ ID N0:3 or SEQ ID N0:5.
Within another aspect of the invention there is
provided an isolated polynucleotide which encodes a
polypeptide comprised of a sequence of amino acids
containing the sequence of SEQ :LD N0:2, SEQ ID N0:3 or SEQ
ID N0:5.
Within an additional aspect of the invention
there is provided a polynucleotide sequence which
hybridizes under stringent cand:itions to either SEQ ID
NO:1 or SEQ ID N0:4 or to a complementary sequence of SF;Q
ID NO:l or to a complementary sequence of SEQ ID N0:4.
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Within an additional aspect of the invention
there is provided a polynucleot_ide sequence which is at
least 90%, 950, or 99% homologous to a polynucleotide
sequence which encodes the polypeptide of SEQ ID N0:3 or
SEQ ID N0:4.
Within another aspects of the invention there is
provided an expression vector comprising (a) a
transcription promoter; (b) a DNA segment encoding a
Zdscl polypeptide, containing an amino acid sequence as
described above.
Within another aspect of the invention there is
provided a cultured eukaryotic, bacterial, fungal or other
cell into which has been introduced an expression vector
as disclosed above, wherein said cell expresses a
mammalian Zdscl polypeptide encoded by the DNA segment.
Within another aspect of the invention there is
provided a chimeric polypeptide consisting essentially of
a first portion and a second portion joined by a peptide'
bond. The first portion of the chimeric polypeptide
consists essentially of a Zdscl polypeptide as described
above. The invention also provides expression vectors
encoding the chimeric polypeptides and host cells
transfected to produce the chimeric polypeptides.
Within an additional aspect of the invention
there is provided an antibody that specifically binds to a
polypeptide as disclosed above and an anti-idiotypic
antibody of an antibody which specifically binds to a
Zdscl antibody.
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These and other aspects of the invention will
become evident upon reference to the following detailed
description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
All references cited herein are
incorporated in their entirety herein by reference.
Prior to setting forth the invention in detail,
it may be helpful to the understanding thereof to define
the following terms.
The term "affinity tag" is used herein to denote
a polypeptide segment that can be attached to a second
polypeptide to provide for purification or detection of
the second polypeptide or provide sites for attachment of
the second polypeptide to a substrate. In principal, a:ny
peptide or protein for which an antibody or other specific
binding agent is available can be used as an affinity t,ag.
Affinity tags include a poly-histidine tract, protein A,
Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,
Methods Enzymol. 198:3 (1991), glutathione S transferase,
Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity
tag, Grussenmeyer et al., Proc. Natl. Acad. Sci. USA
82:7952-4 (1985) , substance P, FlagT"' peptide {Hopp et all. ,
Biotechnology 6:1204-10 (1988), streptavidin binding
peptide, or other antigenic epitope or binding domain.
See, in general, Ford et aI, Protein Expression and
Purification 2: 95-107 (1991). DNAs encoding affinity
tags are available from commercial suppliers, (e. g.,
Pharmacia Biotech, Piscataway, NJ).
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The term "allelic variant" is used herein to
denote any of two or more alternative forms of a gene
occupying the same chromosomal locus. Allelic variation
arises naturally through mutation, and may result in
5 phenotypic polymorphism within :populations. Gene
mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered
amino acid sequence. The term allelic variant is also
used herein to denote a protein encoded by an allelic
variant of a gene.
The terms "amino-terminal" and "carboxyl-
terminal" are used herein to denote positions within
polypeptides. Where the context allows, these terms are
used with reference to a particular sequence or portion of
a polypeptide to denote proximity or relative position.
For example, a certain sequence positioned carboxyl-
terminal to a reference sequence within a polypeptide i:~
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus
of the complete polypeptide.
The term "complement/anti-complement pair"
denotes non-identical moieties that form a non-covalentl_y
associated, stable pair under appropriate conditions. F'or
instance, biotin and avidin (or streptavidin) are
prototypical members of a complement/anti-complement pair.
Other exemplary complement/anti--complement pairs include:
receptor/ligand pairs, antibody/antigen (or hapten or
epitope) pairs, sense/antisense polynucleotide pairs, and
the like. Where subsequent dis:~ociation of the
complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding
affinity of <109 M-1.
The term "complements of a polynucleotide
molecule" is a polynucleotide molecule having a
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complementary base sequence and reverse orientation as
compared to a reference sequenc:e. For example, the
sequence 5' ATGCACGGG 3~ is complementary to 5' CCCGTGC'AT
3'.
The term "contig" denotes a polynucleotide that
has a contiguous stretch of identical or complementary
sequence to another polynucleotide. Contiguous sequences
are said to "overlap" a given stretch of polynucleotide
sequence either in their entirety or along a partial
stretch of the polynucleotide.
The term "degenerate nucleotide sequence"
denotes a sequence of nucleotides that includes one or
more degenerate codons (as compared to a reference
polynucleotide molecule that encodes a polypeptide).
Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.~e.,
GAU and GAC triplets each encode Asp).
The term "expression vector" is used to denote a
DNA molecule, linear or circular, that comprises a segm<~nt
encoding a polypeptide of interest operably linked to
additional segments that provida_ for its transcription.
Such additional segments include promoter and terminator
sequences, and may also include one or more origins of
replication, one or more selectable markers, an enhancer_,
a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may
contain elements of both.
The term "isolated", when applied to a
polynucleotide, denotes that the polynucleotide has been
removed from its natural genetir_ milieu and is thus free
of other extraneous or unwanted coding sequences, and i~~
in a form suitable for use within genetically engineered
protein production systems. Such isolated molecules are
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those that are separated from their natural environment
and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes
with which they are ordinarily associated, but may include
naturally occurring 5' and 3' u.ntranslated regions such as
promoters and terminators. The identification of
associated regions will be evident to one of ordinary
skill in the art (see for example, Dynan and Tijan, Nature
316:774-7$ (1985).
An 'isolated" polypeptide or protein is a
polypeptide or protein that is found in a condition other
than its native environment, such as apart from blood a:nd
animal tissue. In a preferred form, the isolated
polypeptide is substantially free of other polypeptides,
particularly other polypeptides of animal origin. It i;s
preferred to provide the polypeptides in a highly purified
form, i.e. greater than 95o pure, more preferably greater
than 99o pure. When used in this context, the term
"isolated" does not exclude the presence of the same
polypeptide in alternative physical forms, such as dime_rs
or alternatively glycosylated or derivatized forms.
The term "operably linked", when referring to
DNA segments, indicates that the=_ segments are arranged ;~o
that they function in concert for their intended purposes,
e.g., transcription initiates in the promoter and proceeds
through the coding segment to the terminator.
The term "ortholog" denotes a polypeptide or
protein obtained from one species that is the functiona7_
counterpart of a polypeptide or protein from a different.
species. Sequence differences among orthologs are the
result of speciation.
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"Paralogs" are distinct but structurally related
proteins made by an organism. Paralogs are believed to
arise through gene duplication. For example, a-globin, (3-
globin, and myoglobin are paral.ogs of each other.
A "polynucleotide" is a single- or double-
stranded polymer of deoxyribonucleotide or ribonucleoti.de
bases read from the 5' to the ?.' end. Polynucleot.ides
include RNA and DNA, and may be isolated from natural
sources, synthesized in vitro, or prepared from a
combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviate:d
"bp"), nucleotides ("nt"), or k:ilobases ("kb"). Where the
context allows, the latter two terms may describe
polynucleotides that are single-stranded or double-
stranded. When the term is applied to double-stranded
molecules it is used to denote overall length and will be
understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the
two strands of a double-stranded polynucleotide may differ
slightly in length and that the ends thereof may be
staggered as a result of enzymatic cleavage; thus all
nucleotides within a double-stranded polynucleotide
molecule may not be paired. Such unpaired ends will in
general not exceed 20 nt in length.
A "polypeptide" is a polymer of amino acid
residues joined by peptide bonds, whether produced
naturally or synthetically. Polypeptides of less than
about 10 amino acid residues are commonly referred to as
"peptides".
The term "promoter" is used herein for its art-
recognized meaning to denote a portion of a gene
containing DNA sequences that provide for the binding of
RNA polymerase and initiation of transcription. Promoter
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sequences are commonly, but not: always, found in the 5'
non-coding regions of genes.
A "protein" is a macromolecule comprising one or
more polypeptide chains. A protein may also comprise non-
peptidic components, such as carbohydrate groups.
Carbohydrates and other non-peptidic substituents may be
added to a protein by the cell in which the protein is
produced, and will vary with the type of cell. Proteins
la are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are
generally not specified, but may be present nonetheless.
The term "receptor" denotes a cell-associated
protein that binds to a bioactive molecule (i.e.. a
ligand) and mediates the effect of the ligand on the cell.
Membrane-bound receptors are characterized by a multi-
domain (Frank Grant suggests "mufti-peptide" in that
subunit binding and signal transduction can be separate
subunits) structure comprising an extracellular ligand-
binding domain and an intracellular effector domain that
is typically involved in signal transduction. Binding of
ligand to receptor results in a conformational change in
the receptor that causes an interaction between the
effector domain and other molecules) in the cell. Thi;
interaction in turn leads to an alteration in the
metabolism of the cell. Metabolic events that are link<~d
to receptor-ligand interactions include gene
transcription, phosphorylation, dephosphorylation,
increases in cyclic AMP production, mobilization of
cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inosito:l lipids and hydrolysis of
phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e. g., thyroid
stimulating hormone receptor, beta-adrenergic receptor) or
multimeric (e. g., PDGF receptor, growth hormone receptor,
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IL-3 receptor, GM-CSF receptor, G-CSF receptor,
erythropoietin receptor and IL-6 receptor).
The term "secretory signal sequence" denotes a
5 DNA sequence that encodes a polypeptide (a "secretory
peptide") that, as a component of a larger polypeptide,
directs the larger polypeptide through a secretory pathway
of a cell in which it is synthesized. The larger
polypeptide is commonly cleaved. to remove the secretory
10 peptide during transit through the secretory pathway.
The term "splice variant" i_s used herein to
denote alternative forms of RNA transcribed from a gene.
Splice variation arises naturally through use of
alternative splicing sites within a transcribed RNA
molecule, or less commonly between separately transcribed
RNA molecules, and may result in several mRNAs transcribed
from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term
splice variant is also used herein to denote a protein
encoded by a splice variant of an mRNA transcribed from a
gene.
Molecular weights and lengths of polymers
determined by imprecise analytical methods (e.g., gel
electrophoresis) will be understood to be approximate
values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be
understood to be accurate to ~100.
Serine proteinase inhibitors regulate the
proteolytic activity of target proteinases by occupying
the active site and thereby preventing occupation by
normal substrates. Although serine proteinase inhibitors
fall into several unrelated structural classes, they al:L
possess an exposed loop (variously termed an "inhibitor
loop", a "reactive core", a "reactive site", a "binding
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loop") which is stabilized by intermolecular interactions
between residues flanking the binding loop and the protein
core. See Bode, W. and Huber, R. "Natural protein
proteinase inhibitors and their interactions with
proteinases", Eur. J. Biochem., 204: 433-451 (1992).
Interaction between inhibitor and enzyme produces a stable
complex which disassociates very slowly, producing either
virgin or a modified inhibitor which is cleaved at the
scissile bond of the binding loop.
l0
Serine proteinase inhibitors fall into various str~actu:ral
families, for example, the Kunitz family, the Kazal family, and.
the Hirudin family. The protein Zdscl is a member of a new
subfamily, which appears to be closely related the Chelonianin
family. The Chelonianin family is characterized by a common
structural motif which comprises two adjacent beta-hairpin
motifs, each consisting of two antiparallel beta strand:
connected by a loop region. The secondary structure of this
motif is depicted by beta-sheet topology K (Branden, C. and
Tooze, J. Introduction to Protein Structure. p. 28
(GarlandPublishing, Inc., 1991). The beta strands are linked by
intra-chain hydrogen bonding and by a network of four disul:Eide
bonds. These disulfide bonds stabilize the structure of the=_
proteinase inhibitor and render it less susceptible to
degradation. This structural feature has caused the Chelonianin
family to be referred to as the "four-disulfide core" family of
proteinase inhibitors. This family includes human
antileukoproteinase, human elafin, guinea pig caltrin-like
protein, human kallman syndrome protein, sea turtle che7_onianin,
and human epididymal secretory protein E4, and trout TOFU-2, and
C. Elegans C08G9. Several of these family members contain
several copies of this structural motif.
Imbalances between native proteinases and a
proteinase inhibitor is seen in patients where levels ofd
human antileukoproteinase inhibitor are compromised by
genetic background or by air contamination. In these
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patients, severe lung damage can result due to unmitigated
activity of proteinases. The elastase inhibitory domain
of antileukoproteinase inhibitor falls into the four-
disulfide core family, which is related to the three-
s disulfide core family of zdscl. As another example, human
elafin (also in the four-disulfide core family) is a
specific inhibitor of leukocyte elastase and pancreatic
elastase. These proteinases have the ability to cleave
the connective tissue protein elastin and therefore ela:Ein
may prevent excessive elastase-mediated tissue proteoly:~is
and damage.
Serine proteinase inhibitor activity can be
measured using the method essentially described by Norris
et al . , Biol. Chem. Hoppe-Seyle.r 371: 37-42 (1990) .
Briefly, various fixed concentrations of the Kunitz-type
inhibitor are incubated in the presence of serine
proteinases at the concentrations listed in Table 1 in x_00
mM NaCl, 50 mM Tris HC1, 0.01% TWEEN80
(Polyoxyethylenesorbitan monoleate) (pH 7.4) at 25°C.
After a 30 minute incubation, the residual enzymatic
activity is measured by the degradation of a solution of:
the appropriate substrate as li:~ted in Table 1 in assay
buffer. The samples are incubated for 30 minutes after
which the absorbance of each sample is measured at 405 nm.
An inhibition of enzyme activity is measured as a decrease
in absorbance at 405 nm or fluorescence Em at 460 nm.
From the results, the apparent inhibition constant Ki i~;
calculated.
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Table 1
Protease (concentration) Substrate (concentration)
Source Source
Trypsin (8 nM) H-D-Val-Leu-Lys-pNA (0.6 mM)
Novo Nordisk A/S, Kabi
Bagsvaerd, Denmark
Chymotrypsin (2.5 nM) Me0-Suc-Arg-Pro-Tyr-pNA (0.6
mM)
Novo Nordisk A/S Kabi
GL Kallikrein (1 U/ml) H-D-Val-Leu-Arg-pNA (0.6 mM)
Sigma, St Louis, MO Kabi
Plasmin (10 nM) H-D~-Val-Leu-Lys-pNA (0.6 mM)
Kabi Kabi
Urokinase (5 nM) <Glu-Gly-Arg-pNA (0.6 mM)
Serono Kabi
Frei urg, Germany
rec. Protein Ca (5 nM) <Glu-Pro-Arg-pNA (0.6 mM)
Novo Nordisk A/S Kabi
PL Kallikrein (3 nM) H-D-Pro-Phe-Arg-pNA (0.6 mM)
Kabi Kabi
human Factor Xlla (30 nM) H-D-Pro-Phe-Arg-pNA (0.6 mM)
Dr. Walt Kisiel Kabi
University of New Mexico,
Albuquerque, NM
human Factor Xla (1 nM) Boc-Glu(OBzI)-Ala-Arg-MCA (0.12
Dr. Kazuo Fujikawa mM)
University of Washington, Peptide Institute
Seattle, WA Osaka, Japan
human Factor Xa (3 nM) Me0-CO-CHA-Gly-Arg-pNA (0.3
mM)
Dr. I. Schousboe NycoMed
Copenhagen, Denmark Oslo, Norway
rec. human Factor Vlla (300 H-D-lle-Pro-Arg-pNA (0.6 mM)
nM)
Novo Nordisk A/S Kabi
Leukocyte Elastase Me0-Suc-Ala-Ala-Pro-Val-pNA
(0.6 mM)
purified at Novo Nordisk (SEQ JD N0:14)
A/S
using the method of Sigma Chemical Co.
Baugh and Travis St. Louis, MO
(Biochemistry _15: 836-843,
1976)
Cathepsin G Suc-Ala-Ala-Pro-Phe-pNA (0.6
mM)
purified at Novo Nordisk (SEQ ID N0:15
A/S
using the method of Sigma Chemical Co.
Baugh and Travis
{Biochemistry 15: 836-843,
1976)
Abbreviations in Table 1: rec. refers to recombinant, GL kallikrein refers to
glandular kallikrein,
and PL kallikrein refers to plasma kallikrein.
Inhibition assays were performed in microtiter
wells in a total volume of 300 ~tl in 10 mM NaCl, 50 mM
Tris-HC1 (pH 7.4), 0.01% TWEEN80 (Polyoxyethylenesorbitan
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monoleate). Each reaction contained 1 ~M of the sample
inhibitor and one of the proteases at the concentration
listed in Table 1. The reactions were incubated at 25°C
for ten minutes after which the appropriate chromogenic
substrate was added to the final concentration listed in
Table 1 and the final reaction was incubated for thirty
minutes at 25°C. Amidolytic activity was measured at 405
nm or by fluorescence Em at 460 nm. Percent inhibition
was determined relative to reactions carried out in the
absence of inhibitor representing 1000 activity or 0%
inhibition.
The serine proteinase inhibitors of the present
invention may be used in the disclosed methods for the
treatment of, inter alia, acute pancreatitis, various
states of shock syndrome, hyperfibrinolytic hemorrhage ;end
myocardial infarction. The amyloid protein precursor
homologues of the present invention may be used, inter
alia, to generate antibodies fo:r use in demonstrating
tissue distribution of the precursor or for use in
purifying such proteins.
Cysteines 3-8 in members of the four disulfide core family
occur according to the motif:
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys ('ys
Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys (SEQ ID N0:17)
The residue Xaa can by any amino acid residue except for
cysteine.
The spacing between cysteines 1--2 and between cysteines 2-
3 in this family is variable. Cysteines 1-3 have been
observed to occur according to one of the following
motifs:
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Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Cys (SEQ ID N0:18)
Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys~ Xaa Xaa Xaa Xaa Xaa Xaa
5 Xaa Xaa Cys (SEQ ID N0:19)
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Cys (SEQ ID N0:20)
10 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys
(SEQ ID N0:21)
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Cys (SEQ ID N0:22)
The 8 cysteines in the four-disulfide core are bonded
according to the pattern:
1-6, 2-7, 3-5, 4-8
Zdscl
The protein Zdscl is a member of a new related
subfamily, which will be referred to as the "three-
disulfide core" family. This family is distinct from the
four-disulfide core family due to the absence of cysteines
1 and 6. The remaining 6 cysteines occur according to t:he
pattern:
Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys {SEQ ID
N0:23) .
Zdscl is related by sequence homology to most of the four-
disulfide core proteins, having the highest similarity t.o
trout TOP-2 and mouse WDMN1 protein. See Garczynski, M.
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and Goetz, F. Molecular characterization of a RNA
transcript that is highly up-regulated at the time of
ovulation in the brook trout ovary, Biology of
Reproduction, 57: 856-864 (1997).
To further characterize the three-dimensional
structure of Zdscl, including the disulfide bonding
pattern and binding loop, we have constructed a homology
model based on the NMR structure for porcine elafin, FLE,
IO Francart, C. et al "Solution structure of R-elafin, a
specific inhibitor of elastase", J. Mol. Biol. 26~: 666-
677 (1997). The multiple alignment between the three
proteins is given below. By analogy with the known and
predicted structure/function relationships in elafin and
the crystal structure of antileukoproteinase complexed
with chymotrypsin certain features of Zdscl/2 can be
predicted. See Grutter, M. et a.l., "The 2.5A X-ray
crystal structure of the acid-stable proteinase inhibitor
from human mucous secretions analyzed in its complex wii=h
bovine alpha-chymotrypsin", EMBO J., 7: 345-351 (1988).
The 6 cysteines in Zdscl are bonded according to the
pattern:
2-7, 3-5, 4-8
The reactive binding loop of Zdscl includes the
sequence LQLLGT (SEQ ID NO: 9). Their active binding loop
of human Zdsc includes the sequence DRLLGT (SEQ ID NO:
20). In Zdscl flanking residues around this binding loop
are expected to interact with the target proteinase. Tree
scissile bond is in the reactive binding loop between the
two Leucines. Substitution at t:he P1 position (the second
Leucine) is not tolerated as this residue is predicted t:o
influence specificity towards the target proteinase, Bocle,
W. and Huber, R. "Natural protein proteinase inhibitor;
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and their interactions with proteinases", Eur. J.
Biochem., 204: 433-451 (1992). Substitution of any
cysteine residue is not tolerated as this is predicted to
significantly destabilize the structure.
To predict the variation acceptable from
positions G1n30 through Cys60 in Zdscl we have created a
generalized motif which enumerates the permissible
substitutions at each position.
MKLGAFLLLVSLITLSLEVQELQA (SEQ ID NO: 8)
(The predicted signal sequence for Zdscl)
3 4 56 7 8
FLE . IILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ
ID NO: 7)
ZDSCl(m): AVRPLQLLGTCAELCRGDWDCGPEEQCVSIGCSHICTTN (SEQ
ID N0:3)
ZDSC1(h): AGDRLLGTCVELCTGDWDCNPGDHCVSNGCGHECVAG (SEQ
ID N0:5)
2 3 4 5 7 8
Multiple alignment between porcine elafin, and
the predicted mature peptide for Zdscl. Cysteines 3-8 of
FLE are labeled on the top of the alignment. Cysteines 1-
6 of Zdscl are labeled on the bottom of the alignment,
using the standard numbering for four-disulfide core
proteins. Based upon the analysis of Zdscl and Zdse2 the
following generic protein has been deduced as shown below
in SEQ ID N0: 6.
SEQ ID N0:6
Xaa Xaa Xaa Xaa Xaa Xaa Leu Leu Gly Thr Cys Xaa Glu Leu
5 10
Cys Xaa Gly Asp Trp Asp Cys Xaa Pro Xaa Xaa Xaa Cys Val
15 20 25
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Ser Xaa Gly Cys Xaa His Xaa Cys Xaa Xaa Xaa
30 3S
wherein
Xaa at amino acid position 1 is Ala or is absent;
Xaa at amino acid position 2 is Val or is absent;
Xaa at amino acid position 3 is Arg or Ala;
Xaa at amino acid position 4 is Pro or Gly;
Xaa at amino acid position 5 is Leu or Asp;
Xaa at amino acid position 6 is Gln, Arg, Lys or Glu;
Xaa at amino acid position 12 is Val, Ala, Ile, Leu, Met:
or Ser;
Xaa at amino acid position 16 is Thr, Arg, Ala, Asn, Ser,
Val, Gln, Glu, His or Lys;
Xaa at amino acid position 22 is Asn, Gly, Asp, His or
Ser;
Xaa at amino acid position 24 i~~ Ala, Arg, Asn, Asp, Glu.,
Gln, Gly, His, Lys, Pro, Ser, or- Thr;
Xaa at amino acid position 25 i~~ Asp or Glu
Xaa at amino acid position 26 is His, Gln Tyr or Glu;
Xaa at amino acid position 30 is Ala, Arg, Asn, Asp, Gln,
Glu, Gly His, Ile, Leu, Lys, Met., Phe, Ser, Thr, Tyr, or
Val;
Xaa at amino acid position 33 is Gly, Ser, Ala, Asn, Thr;
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19
Xaa at amino acid position 35 i:~ Ala, Arg, Asn, Asp, Glu,
Gln, Gly, His, Ile, Leu, Lys, Met Phe, Pro, Ser, Thr, Trp,
Tyr or Val;
Xaa at amino acid position 37 i:~ Val or Thr;
Xaa at amino acid position 38 is Ala or Thr; and
Xaa at amino acid position 39 i:~ Asn or Gly.
Any resultant polypeptide based upon SEQ ID NO: 8
must be at least 80%, preferably 90 or 95o and most
preferably 99o identical to SEQ ID NO: 3, SEQ ID NO: 5 or
to SEQ ID N0:7.
POLYNUCLEOTIDES
The present invention also provides
polynucleotide molecules, including DNA and RNA molecules,
that encode the Zdsc polypeptide~s disclosed herein. Those
skilled in the art will readily recognize that, in view of
the degeneracy of the genetic cade, considerable sequence
variation is possible among these polynucleotide
molecules. Polynucleotides, generally a cDNA sequence, of
the present invention encode the described polypeptides
herein. A cDNA sequence which encodes a polypeptide of
the present invention is comprised of a series of codons,
each amino acid residue of the polypeptide being encoded.
by a codon and each codon being comprised of three
nucleotides. The amino acid residues are encoded by their
respective codons as follows.
Alanine (Ala) is encoded by GCA, GCC, GCG or
GCT;
Cysteine (Cys) is encoded by TGC or TGT;
Aspartic acid (Asp) is encoded by GAC or GAT;
Glutamic acid (Glu) is encoded by GAA or GAG;
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Phenylalanine (Phe) is encoded by TTC or TTT;
Glycine (Gly) is encoded by GGA, GGC, GGG or
GGT;
Histidine (His) is encoded by CAC or CAT;
5 Isoleucine (Ile) is encoded by ATA, ATC or AT'.C;
Lysine (Lys) is encoded by AAA, or AAG;
Leucine (Leu) is encoded by TTA, TTG, CTA, CTC:,
CTG or CTT;
Methionine (Met) is encoded by ATG;
10 Asparagine (Asn) is encoded by AAC or AAT;
Proline (Pro) is encoded by CCA, CCC, CCG or
CCT;
Glutamine (Gln) is encoded by CAA or CAG;
Arginine (Arg) is encoded by AGA, AGG, CGA, CC~C,
15 CGG or CGT;
Serine (Ser) is encoded by AGC, AGT, TCA, TCC,
TCG or TCT;
Threonine (Thr) is encoded by ACA, ACC, ACG or
ACT;
20 Valine (Val) is encoded by GTA, GTC, GTG or GTT;
Tryptophan (Trp) is encoded by TGG; and
Tyrosine (Tyr) is encoded by TAC or TAT.
It is to be recognized that according to the
present invention, when a polynucleotide is claimed as
described herein, it is understood that what is claimed
are both the sense strand, the anti-sense strand, and the
DNA as double-stranded having both the sense and anti-
sense strand annealed together by their respective
hydrogen bonds. Also claimed is the messenger RNA (mRNA)
which encodes the polypeptides of the president invention,
and which mRNA is encoded by the' cDNA described herein.
Messenger RNA (mRNA) will encodes a polypeptide using the
same codons as those defined herein, with the exception
that each thymine nucleotide (T) is replaced by a uracil
nucleotide (U).
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21
One of ordinary skill in the art will also
appreciate that different species can exhibit
"preferential codon usage." In general, see, Grantham, et
al., Nuc. Acids Res. 8:1893-912 (1980); Haas, et al. Curr.
Biol. 6:315-24 (1996); Wain-Hobson, et al., Gene 1.3:355-64
(1981); Grosjean and Fiers, Gene 18:199-209 (1982); Holm,
Nuc. Acids Res. 14:3075-87 (1986); Ikemura, J. Mol. Bio.l.
158:573-97 (1982). As used herein, the term "preferent:ial
codon usage" or "preferential codons" is a term of art
referring to protein translation codons that are most
frequently used in cells of a certain species, thus
favoring one or a few representatives of the possible
codons encoding each amino acid. For example, the amino
acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or
ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yea:~t,
viruses or bacteria, different 'L'hr codons may be
preferential. Preferential codons for a particular
species can be introduced into the polynucleotides of the
present invention by a variety of methods known in the
art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of
the protein by making protein translation more efficient.
within a particular cell type or species. Sequences
containing preferential codons c:an be tested and optimized
for expression in various species, and tested for
functionality as disclosed herein.
Within preferred embodiments of the invention
the isolated polynucleotides wil.1 hybridize to similar
sized regions of SEQ ID NO:1, SE:Q ID N0:4, or a sequence
complementary thereto, under stringent conditions. In
general, stringent conditions are selected to be about 5°C
lower than the thermal melting point (Tm) for the specific
sequence at a defined ionic strength and pH. The Tm is
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22
the temperature (under defined .ionic strength and pH) at.
which 500 of the target sequence hybridizes to a perfectly
matched probe. Typical stringent conditions are those ~_n
which the salt concentration is up to about 0.03 M at pF3 7
and the temperature is at least about 60°C.
As previously noted, t=he isolated
polynucleotides of the present invention include DNA and
RNA. Methods for preparing DNA and RNA are well known in
the art. In general, RNA is isolated from a tissue or
cell that produces large amount: of Zdsc1 RNA. Such
tissues and cells are identified by Northern blotting,
Thomas, Proc. Natl. Acad. Sci. LISA 77:5201 (1980), and
include high expression of human Zdscl in the liver.
Total RNA can be prepared using guanidine HCl extractior.~.
followed by isolation by centrifugation in a CsCl
gradient, Chirgwin et al., Biochemistry 18:52-94, 1979).
Poly (A)+ RNA is prepared from total RNA using the method
of Aviv and Leder, Proc . Na t1 . ficad . Sci . USA 69 : 14 0 8 -1412
(1972). Complementary DNA (cDNA) is prepared from
poly(A)+ RNA using known methods. In the alternative,
genomic DNA can be isolated. Polynucleotides encoding
Zdsc polypeptides are then identified and isolated by, for
example, hybridization or PCR.
A full-length clone encoding Zdscl polypeptide
can be obtained by conventianal cloning procedures.
Complementary DNA (cDNA) clones are preferred, although
for some applications (e. g., expression in transgenic
animals) it may be preferable to use a genomic clone, or
to modify a cDNA clone to include at least one genomic
intron. Methods for preparing cDNA and genomic clones are
well known and within the level of ordinary skill in the
art, and include the use of the sequence disclosed herein,
or parts thereof, for probing or priming a library.
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23
Expression libraries can be probed with antibodies to
Zdsc, receptor fragments, or other specific binding
partners.
The polynucleotides of the present invention can
also be synthesized using gene machines. Currently the
method of choice is the phosphoramidite method. If
chemically synthesized double st=randed DNA is required f_or
an application such as the synthesis of a gene or a gene
fragment, then each complementary strand is made
separately. The production of short genes (60 to 80 bp) is
technically straightforward and can be accomplished by
synthesizing the complementary :strands and then annealing
them. For the production of longer genes (>300 bp),
however, special strategies must. be invoked, because the
coupling efficiency of each cycle during chemical DNA
synthesis is seldom 1000. To overcome this problem,
synthetic genes (double-stranded) are assembled in modular
form from single-stranded fragments that are from 20 to
100 nucleotides in length. The double-stranded constructs
are sequentially linked to one another to form the entire
gene sequence. Because it is absolutely essential that a.
chemically synthesized gene have the correct sequence of
nucleotides, each double-stranded fragment and then the
complete sequence is characterized by DNA sequence
analysis. See Glick and Pasternak, Molecular
Biotechnology, Principles & App.l.ications of Recombinant
DNA, (ASM Press, Washington, D.C. 1994); Itakura et al.,
Annu. Rev. Biochem. 53: 323-56 (1984) and Climie et al.,
Proc. Natl. Acad. Sci. USA 87:633-637 (1990).
The present invention further provides
counterpart polypeptides and polynucleotides from other
species (orthologs). These species include, but are not
limited to mammalian, avian, amphibian, reptile, fish,
insect and other vertebrate and invertebrate species. Of
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24
particular interest are Zdsc polypeptides from other
mammalian species, including murine, porcine, ovine,
bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human Zdsc can be cloned using
information and compositions provided by the present
invention in combination with conventional cloning
techniques. For example, a cDNA can be cloned using mRN'A
obtained from a tissue or cell type that expresses Zdsc as
disclosed herein. Suitable sources of mRNA can be
identified by probing Northern f>lots with probes designed
from the sequences disclosed herein. A library is then
prepared from mRNA of a positive tissue or cell line. A
Zdsc-encoding cDNA can then be isolated by a variety of
methods, such as by probing with. a complete or partial
human cDNA or with one or more sets of degenerate pi°obes
based on the disclosed sequences. A cDNA can also be
cloned using the polymerase chain reaction, or PCR
(Mullis, U.S. Patent No. 4,683,202), using primers
designed from the representative human Zdsc sequence
disclosed herein. Within an additional method, the cDNA
library can be used to transform or transfect host cells,
and expression of the cDNA of interest can be detected
with an antibody to Zdscl polypeptide. Similar techniques
can also be applied to the isolation of genomic clones.
Those skilled in the art will recognize that t:he
sequences disclosed in SEQ ID NO:1 and SEQ ID N0:4
represent a single alleles of murine Zdscl and human Zdscl
respectively, and that allelic variation and alternative
splicing are expected to occur. Allelic variants of thia
sequence can be cloned by probing cDNA ar genomic
libraries from different individuals according to standard
procedures. Allelic variants of the DNA sequence shown
in SEQ ID NO:1, including those ~~ontaining silent
mutations and those in which mutations result in amino
acid sequence changes, are within the scope of the present
invention, as are proteins which are allelic variants of
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SEQ ID N0:2. cDNAs generated from alternatively spliced
mRNAs, which retain the properties of the Zdscl
polypeptide are included within the scope of the present:
invention, as are polypeptides Encoded by such cDNAs and
5 mRNAs. Allelic variants and sp7_ice variants of these
sequences can be cloned by probing cDNA or genomic
libraries from different individuals or tissues according
to standard procedures known in the art.
10 The present invention also provides isolated
Zdscl polypeptides that are substantially identical to the
polypeptides of SEQ ID N0:2, SEQ ID N0:3 and SEQ ID N0:5
and their orthologs. The term ''substantially identical"
is used herein to denote polypeptides having 50%,
15 preferably 600, more preferably at least 800, sequence
identity to the sequences shown in SEQ ID N0:2 or their
orthologs. Such polypeptides will more preferably be at
least 90% identical, and most preferably 95% or more
identical to SEQ ID N0:2 or its orthologs.) Percent
20 sequence identity is determined by conventional methods.
See, for example, Altschul et al., Bull. Math. Bio. 48:
603-616 (1986) and Henikoff and Henikoff, Proc. Natl.
Acad. Sci. USA 89:10915-10929 (1992). Briefly, two amino
acid sequences are aligned to optimize the alignment
25 scores using a gap opening penalty of 10, a gap extension
penalty of 1, and the "blosum 62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 2 (amino
acids are indicated by the standard one-letter codes).
The percent identity is then calculated as:
Total number of identical matches
x 100
[length of the longer sequence plus the
number of gaps introduced into the longer
sequence in order to align the two sequences]
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WO 99/63091 PCT/US99/12545
~r
~ N M
r-~ I
L(7 N N O
I I
~ t-1 M N N
I I
I~ '-1 ~ V' M N
I i I I
lD d' N N n-I M ri
I I I
tll O N ri rl rl '-1 ri
I I I I I
tll ,-1 M r-I O rl M N N
I I I f I i I
a d'NNOMNr-iNri~
I I I I I I
VI N M r~ O M N '-I M rl M
N I I f 1 I I
x. CO M M r-1 N rl N rl N N N M
I I I r I I I I I I
C7 lf~ N ~ d' N M M N O N N M M
I J I I I I I I I i I
w f~ N O M M rl N M ri O ri frl N N
I I I i 1 f I I I I
l!1 N N O M N ~-1 O M rl O r-i N '-1 N
I I I 1 I i I I I
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I I i I I I I 1 I I I I I i I
Ga l0 M O N e-~I ri M dl rl M M r-I O r-1 ~ M M
I I I I i I I I I r I I I
z lD '-1 M O O O rl M M O N M N ~-1 O d~ N M
i I I I I I t I I
A-i Lll O N M r-~ O N O M N N r-I M N rl '-i M N M
I I I I I I I I I I I I I
~. d' I-I N N O r-W -1 O N r-I ri r1 rl N ,-r ri O M N O
I i I 1 I I 1 1 I 1 I I I
I~ rx z c~ v a w c~ x H a x ~ ~, ~, ~n ~I 3 ~, ~
N
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'"i f-i N
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27
Those skilled in the art appreciate that there
are many established algorithms to align two amino acid
sequences. The "FASTA" similarity search algorithm of
Pearson and Lipman is a suitable protein alignment method
for examining the level of ident=ity shared by an amino
acid sequence and the amino acid sequence of a putative
variant. The FASTA algorithm is described by Pearson anc~
Lipman, Proc. Nat'1 Acad. Sci. C~SA 85:2444 (1988), and by
Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA
first characterizes sequence sirnilarity by identifying
regions shared by the query sequence (e. g., SEQ ID N0:2)
and a test sequence that have either the highest densit~~
of identities (if the ktup variable is 1) or pairs of
identities (if ktup=2), without considering conservative
amino acid substitutions, insertions or deletions. 'The t:en
regions with the highest density of identities are then
rescored by comparing the similarity of all paired amino
acids using an amino acid substitution matrix, and the
ends of the regions are "trimmed" to include only those
residues that contribute to the highest score. If there
are several regions with scores greater than the "cvstoff"
value (calculated by a predetermined formula based upon
the length of the sequence and t:he ktup value), then the
trimmed initial regions are examined to determine whether
the regions can be joined to form an approximate alignment
with gaps. Finally, the highest scoring regions of she two
amino acid sequences are aligned using a modification of
the Needleman-Wunsch-Sellers algorithm (Needleman and
Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM ~7.
Appl. Math. 26:787 (1974), which allows for amino acid
insertions and deletions. Illustrative parameters for
FASTA analysis are: ktup=l, gap opening penalty=10, gap
extension penalty=1, and substitution matrix=BLOSUM62.
These parameters can be introduced into a FASTA program by
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28
modifying the scoring matrix file (~~SMATRIX"), as
explained in Appendix 2 of Pearson, Meth. Enzymol. 183:Ei3
(1990) .
FASTA can also be used to determine the sequence
identity of nucleic acid molecules using a ratio as
disclosed above. For nucleotide sequence comparisons, the
letup value can range between one to six, preferably from
four to six.
The present invention includes nucleic acid
molecules that encode a polypept:ide having one or more
conservative amino acid changes, compared with the amino
acid sequence of SEQ ID N0:3 or with the amino acid
sequence of SEQ ID N0:5. The BLOSUM62 table is an amino
acid substitution matrix derived from about 2,000 1_ocal
multiple alignments of protein ~~equence segments,
representing highly conserved regions of more than 500
groups of related proteins [Heni.koff and Henikoff, Proc.
Nat'1 Acad. Sci. USA 89:10915 (1.992)]. Accordingly, the
BLOSUM62 substitution frequencies can be used to define
conservative amino acid substitutions that may be
introduced into the amino acid sequences of the present
invention. As used herein, the language ~~conservative
amino acid substitution" refers to a substitution
represented by a BLOSUM62 value of greater than -1. For
example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of
0,1,2, or 3. Preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at
least 1 (e. g., 1,2 or 3), while more preferred
conservative substitutions are characterized by a BLOSUM62
value of at least 2 (e.g., 2 or 3). Accordingly the
present invention claims those polypeptides which are at
least 90$, preferably 95o and most preferably 99~
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29
identical to SEQ ID N0:3 and which are able to stimulatE~
antibody production in a mammal, and said antibodies arf~
able to bind the native sequence of SEQ ID N0:3.
Variant Zdscl polypeptides or substantially
identical Zdscl polypeptides arf=_ characterized as having
one or more amino acid substitutions, deletions or
additions. These changes are preferably of a minor
nature, that is conservative amino acid substitutions (:gee
Table 3) and other substitution: that do not significantly
affect the folding or activity of the polypeptide; small.
deletions, typically of one to about 30 amino acids; and
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide
of up to about 20-25 residues, or an affinity tag.
Polypeptides comprising affinity tags can further comprise
a proteolytic cleavage site between the Zdsc polypeptide~
and the affinity tag. Preferred such sites include
thrombin cleavage sites and factor Xa cleavage sites.
Table 3
Conservative amino acid substitutions
Basic: arginine
lysine
histidine
Acidic: glutamic acid
Table 3 cont.
aspartic acid
Polar: glutamine
asparagine
Hydrophobic: leucine
isoleucine
valine
Aromatic: phenylalanine
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tryptophan
tyrosine
Small: glycine
alanine
serine
threonine
methionine
10 Different species can exhibit "preferential
colon usage." In general, see, Grantham et al., Nuc.
Acids Res. 8:1893 (1980), Haas er_ a1. Curr. Biol. 6:315
(1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean
and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids Res.
15 19:3075 (1986), Ikemura, ~l. Mol. Biol. 158:573 (1982),
Sharp and Matassi, Curr. Opin. Genet. Dev. Q:851 (1994),
Kane, Curr. Opin. Biotechnol. 6:494 (1995), and Makr.ides,
Microbiol. Rev. 60:512 (1996). As used herein, the term
"preferential colon usage" or "preferential colons" is a
20 term of art referring to protein translation colons that
are most frequently used in cells of a certain species,
thus favoring one or a few repre~;entatives of the possible
colons encoding each amino acid. For example, the amino
acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or
25 ACT, but in mammalian cells, ACC is the most commonly used
colon; in other species, for example, insect cells, yeast,
viruses or bacteria, different Th,r colons may be
preferential. Preferential colons for a particular
species can be introduced into the polynucleotides of the
30 present invention by a variety of methods known in the
art. Introduction of preferential colon sequences into
recombinant DNA can, for example, enhance production of
the protein by making protein translation more efficient
within a particular cell type or species. Sequences
containing preferential colons can be tested and optimized
for expression in various species, and tested for
functionality as disclosed herein.
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31
The present invention further provides variant
polypeptides and nucleic acid molecules that represent
counterparts from other species (orthologs). These
species include, but are not limited to mammalian, avian,
amphibian, reptile, fish, insect and other vertebrate and
invertebrate species. Of particular interest are Zdscl
polypeptides from other mammalian species, including
murine, porcine, ovine, bovine, canine, feline, equine,
and other primate polypeptides. Orthologs of human Zdscl
can be cloned using information and compositions providE~d
by the present invention in combination with conventional
cloning techniques. For example, a cDNA can be cloned
using mRNA obtained from a tissue or cell type that
expresses Zdscl as disclosed herein. Suitable sources c>f
mRNA can be identified by probing northern blots with
probes designed from the sequences disclosed herein. A
library is then prepared from mRNA of a positive tissue or
cell line.
An Zdsc1-encoding cDNA can then be isolated by a
variety of methods, such as by probing with a complete cr
partial human cDNA or with one or more sets of degenerate
probes based on the disclosed sequences. A cDNA can also
be cloned using the polymerase chain reaction with primers
designed from the representative human Zdscl sequences
disclosed herein. Within an additional method, the cDNA
library can be used to transform or transfect host cells,
and expression of the cDNA of interest can be detected
with an antibody to Zdscl polypeptide. Similar techniques
can also be applied to the isolation of genomic clones.
Those skilled in the art will recognize that the
sequence disclosed in SEQ ID N0:1 represents a single
allele of human Zdscl, and that allelic variation and
alternative splicing are expected to occur. Allelic
variants of this sequence can be cloned by probing cDNA or
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32
genomic libraries from different individuals according t=o
standard procedures. Allelic variants of the nucleotide
sequences shown in SEQ ID NO:l or SEQ ID N0:4, including
those containing silent mutatio:zs and those in which
mutations result in amino acid sequence changes, are
within the scope of the present invention, as are prote~_ns
which are allelic variants of SEQ ID N0:2, SEQ ID N0:3 or
SEQ ID N0:5. cDNA molecules generated from alternative7_y
spliced mRNAs, which retain the properties of the Zdscl
polypeptide are included within the scope of the present.
invention, as are polypeptides encoded by such cDNAs and
mRNAs. Allelic variants and splice variants of these
sequences can be cloned by probing cDNA or genomic
libraries from different individuals or tissues according
to standard procedures known i.n the art.
In general, stringent conditions are selected to
be about 5°C lower than the thermal melting point ('~'my for
the specific sequence at a defined ionic strength and pI-I.
The Tm is the temperature (under defined ionic strength
and pH~ at which 50% of the target sequence hybridizes t:o
a perfectly matched probe.
A pair of nucleic acid molecules, such as DNA--
DNA, RNA-RNA and DNA-RNA, can hybridize if the nucleotide
sequences have some degree of complementarity. Hybrids c:an
tolerate mismatched base pairs ~n the double helix, but
the stability of the hybrid is influenced by the degree of
mismatch. The Tm of the mismatched hybrid decreases by 1°C
for every 1-1.5% base pair mismatch. Varying the
stringency of the hybridization conditions allows control
over the degree of mismatch that. will be present in the
hybrid. The degree of stringency increases as the
hybridization temperature increases and the ionic strength
of the hybridization buffer decreases. Stringent
hybridization conditions encompass temperatures of about:
5-25°C below the Tm of the hybrid and a hybridization
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33
buffer having up to 1 M Na+. Higher degrees of str:ingen.cy
at lower temperatures can be achieved with the addition of
formamide which reduces the Tm c.>f the hybrid about 1°C for
each to formamide in the buffer solution. Generally, such
stringent conditions include ternperatures of 20-70°'' anc~ a
hybridization buffer containing up to 6x SSC and 0-500
formamide. A higher degree of stringency can be achieved
at temperatures of_ from 40-70°C with a hybridization
buffer having up to 4x SSC and from 0-50% formamide.
Highly stringent conditions typically encompass
temperatures of 42-70°C with a hybridization buffer having
up to lx SSC and 0-50o formamide. Different degrees of
stringency can be used during hybridization and washing to
achieve maximum specific binding to the target sequence.
Typically, the washes following hybridization are
performed at increasing degrees of stringency to remove
non-hybridized pol~ynucleotide probes from hybridized
complexes.
The above conditions are meant to serve as a
guide and it is well within the abilities of one skilled
in the art to adapt these conditions for use with a
particular polypeptide hybrid. The Tm for a specific
target sequence is the temperature (under defined
conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those
conditions which influence the Tm include, the size and
base pair content of the polynucleotide probe, the ionic
strength of the hybridization solution, and the presence
of destabilizing agents in the hybridization solution.
Numerous equations for calculating Tm are known in the
art, and are specific for DNA, RNA and DNA-RNA hybrids and
polynucleotide probe sequences of varying length (see, for
example, Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition (Cold Spring Harbor Press 19$9);
Ausubel et al., (eds.), Current Protocols in Molecular
Biology (John Wiley and Sons, Inc. 1987); Berger and
CA 02330187 2000-12-04
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34
Kimmel (eds.), Guide to Molecul~3r Cloning Techniques,
(Academic Press, Inc. 1987); and Wetmur, Crit. Rev.
Biochem. Mol. Bial. 26:227 (199C)}). Sequence analysis
software such as OLIGO 6.0 (LSR; Long Lake, MN) and Prirr!er
Premier 9.0 (Premier Biosoft International; Palo Alto,
CA}, as well as sites on the Internet, are available tools
for analyzing a given sequence and calculating Tm based on
user defined criteria. Such programs can also analyze a
given sequence under defined conditions and identify
suitable probe sequences. Typically, hybridization of
longer polynucleotide sequences, >50 base pairs, is
performed at temperatures of about 20-25°C below the
calculated Tm. For smaller probes, <50 base pairs,
hybridization is typically carried out at the Tm or 5-10°C
below. This allows for the maximum rate of hybridization
for DNA-DNA and DNA-RNA hybrids.
The length of the polynucleotide sequence
influences the rate and stability of hybrid formation.
Smaller probe sequences, <SO base pairs, reach equilibrium
with complementary sequences rapidly, but may form less
stable hybrids. Incubation times of anywhere from minutes
to hours can be used to achieve hybrid formation. Longer
probe sequences come to equilibrium more slowly, but: form
more stable complexes even at lower temperatures.
Incubations are allowed to proceed overnight or longer.
Generally, incubations are carried out for a period equal
to three times the calculated Cot time. Cot time, t:he
time it takes for the polynucleotide sequences to
reassociate, can be calculated for a particular sequence
by methods known in the art.
The base pair composition of polynucleotide
sequence will effect the thermal stability of the hybrid
complex, thereby influencing the choice of hybridization
temperature and the ionic strength of the hybridization
buffer. A-T pairs are less stable than G-C pairs in
CA 02330187 2000-12-04
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aqueous solutions containing sodium chloride. Therefore,
the higher the G-C content, the more stable the hybrid.
Even distribution of_ G and C residues within the sequence
also contribute positively to hybrid stability. In
5 addition, the base pair composition can be manipulated t:o
alter the Tm of a given sequence. For example, 5-
methyldeoxycytidine can be substituted for deoxycytidine
and 5-bromodeoxuridine can be substituted for thymidine to
increase the Tm, whereas 7-deazz--2'-deoxyguanosine can be
10 substituted for guanosine to reduce dependence on Tm .
The ionic concentration of the hybridization
buffer also affects the stability of the hybrid.
Hybridization buffers generally contain blocking agents
15 such as Denhardt's solution (Sigma Chemical Co., St.
Louis, Mo.), denatured salmon sperm DNA, tRNA, milk
powders (BLOTTO), heparin or SD~i, and a Na+ source, such as
SSC (lx SSC: 0.15 M sodium chloride, 15 mM sodium citrate)
or SSPE (lx SSPE: 1.8 M NaCl, 1C1 mM NaH2P04, 1 mM EDTA, pH
20 7.7). By decreasing the ionic concentration of the
buffer, the stability of the hybrid is increased.
Typically, hybridization buffers contain from between 1C
mM - 1 M Na+. The addition of destabilizing or denaturing
agents such as formamide, tetralkylammonium salts,
25 guanidinium cations or thiocyanate cations to the
hybridization solution will alter the Tm of a hybrid.
Typically, formamide is used at a concentration of up to
50o to allow incubations to be carried out at more
convenient and lower temperatures. Formamide also acts to
30 reduce non-specific background when using RNA probes.
As an illustration, a nucleic acid molecule
encoding a variant Zdscl polypeptide can be hybridized
with a nucleic acid molecule having the nucleotide
35 sequence of SEQ ID NO:l (or its complement) at 42°C
overnight in a solution comprising 50$ formamide, SxSSC
(lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate),
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36
50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution
(100x Denhardt's solution: 20 (w/v) Ficoll 400, 20 (w/v)
polyvinylpyrrolidone, and 20 (w/v) bovine serum albumin),
loo dextran sulfate, and 20 ~g/ml denatured, sheared
salmon sperm DNA. One of skill in the art can devise
variations of these hybridization conditions. For
example, the hybridization mixture can be incubated at a
higher temperature, such as about 65°C, in a solution that
does not contain formamide. Moreover, premixed
hybridization solutions are available (e. g., EXPRESSHYB
Hybridization Solution from CLONTECH Laboratories, lnc.),
and hybridization can be performed according to the
manufacturer's instructions.
Following hybridization, the nucleic acid
molecules can be washed to remove non-hybridized nucleic
acid molecules under stringent conditions, or under highly
stringent conditions. Typical stringent washing
conditions include washing in a solution of C.5x - 2x SSC
with 0.1o sodium dodecyl sulfate (SDS) at 55 - 65°C. That.
is, nucleic acid molecules encoding a variant Zdscl
polypeptide hybridize with a nucleic acid molecule having
the nucleotide sequence of SEQ ID NO:1 (or its complement)
under stringent washing conditions, in which the wash
stringency is equivalent to 0.5x - 2x SSC with 0.1o SDS at
55 - 65°C, including 0.5x SSC with O.lo SDS at 55°C, or
2xSSC with O.la SDS at 65°C. One of skill in the art can
readily devise equivalent conditions, for example, by
substituting SSPE for SSC in the wash solution.
Typical highly stringent washing conditions
include washing in a solution of O.lx - 0.2x SSC with 0._Lo
sodium dodecyl sulfate (SDS) at 50 - 65°C. In other
words, nucleic acid molecules encoding a variant Zdscl
polypeptide hybridize with a nucleic acid molecule having
the nucleotide sequence of SEQ ID NO:1 (or its complement:)
under highly stringent washing conditions, in which the
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37
wash stringency is equivalent to O.lx - 0.2x SSC with 0"l0
SDS at 50 - 65°C, including O.l:~c SSC with 0.1~ SDS at
50°C, or 0.2xSSC with C.lo SDS <~t 65°C.
The present invention also provides isolated
Zdscl polypeptides that have a substantially similar
sequence identity to the polypeptides of SEQ ID N0:2, ox-
their orthologs. The term "substantially similar sequence
identity" is used herein to denote polypeptides having at
least 700, at least 80°, at lea:~t 90%, at least 950 or
greater than 95o sequence identity to the sequences shown
in SEQ ID N0:2, or their orthologs. The present invention
also includes polypeptides that comprise an amino acid
sequence having at least 700, ar. least 800, at least 90%,
at least 950 or greater than 95~> sequence identity to th.e
sequence of amino acid residues of SEQ ID N0:3. The
present invention further includes nucleic acid molecules
that encode such polypeptides. Methods for determining
percent identity are described below.
The present invention also contemplates Zdscl
variant nucleic acid molecules that can be identified
using two criteria: a determination of the similarity
between the encoded polypeptide with the amino acid
sequence of SEQ ID N0:3, and a hybridization assay, as
described above. Such Zdscl variants include nucleic acid
molecules (1) that hybridize with a nucleic acid mo~.ecule
having the nucleotide sequence of SEQ ID N0:1 (or it:s
complement) under stringent washing conditions, in which
the wash stringency is equivalent to 0.5x - 2x SSC with
O.lo SDS at 55 - 65°C, and (2) that encode a polypeptide
having at least 70~, at least 80%, at least 900, at least
950 or greater than 95o sequence identity to the amino
acid sequence of SEQ ID N0:3. Alternatively, Zdscl
variants can be characterized as nucleic acid molecules
(1) that hybridize with a nucleic acid molecule having t:he
nucleotide sequence of SEQ ID NO:l (or its complement)
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38
under highly stringent washing conditions, in which the
wash stringency is equivalent to 0.1x. - 0.2x SSC with 0.1%
SDS,at 50 - 65°C, and (2) that encode a polypeptide having
at least 700, at least 800, at least 90°, at least 950 or
S greater than 95o sequence identity to the amino acid
sequence of SEQ ID N0:2.
The present invention further provides a variety
of other polypeptide fusions [and related multimeric
proteins comprising one or more polypeptide fusions]. For
example, a Zdsc polypeptide can be prepared as a fusion to
a dimerizing protein as disclosed in U.S. Patents Nos.
5,155,027 and 5,567,584. Preferred dimerizing proteins in
this regard include immunoglobulin constant region
domains. Immunoglobulin-Zdscl polypeptide fusions can be
expressed in genetically engineered cells [to produce a
variety of multimeric Zdscl analogs]. Auxiliary domains
can be fused to Zdscl polypeptides to target them to
specific cells, tissues, or macromolecules (e. g.,
collagen). For example, a Zdsc~L polypeptide or protein
could be targeted to a predetermined cell type by fusing a
Zdscl polypeptide to a ligand that specifically binds to a
receptor on the surface of the target cell. In this way,
polypeptides and proteins can be targeted for therapeutic
or diagnostic purposes. A Zdsc_1 polypeptide can be fused
to two or more moieties, such as an affinity tag for
purification and a targeting domain. Polypeptide fusions
can also comprise one or more c'~.eavage sites, particularly
between domains. See, Tuan et al., Connective Tissue
Research 34:1-9 (1996).
The proteins of the present invention can alsc>
comprise non-naturally occurring amino acid residues.
Non-naturally occurring amino acids include, without
limitation, traps-3-methylproline, 2,4-methanoproline,
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39
cis-4-hydroxyproline, trans-4-hydroxyproline, N-
methylglycine, allo-threonine, methylthreonine,
hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid,
thiazolidine carboxylic acid, dehydroproline, 3- and 4-
methylproline, 3,3-dimethy:Lprol:ine, tert-leucine,
norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-
azaphenylalanine, and 4-fluoroplzenylalanine. Several
methods are known in the art for incorporating non-
naturally occurring amino acid residues into proteins.
For example, an in vitro system can be employed wherein
nonsense mutations are suppressed using chemically
aminoacylated suppressor tRNAs.
Methods for synthesizing amino acids and
aminoacylating tRNA are known in the art. Transcription
and translation of plasmids containing nonsense mutations
is carried out in a cell-free s~~stem comprising an E. coli
S30 extract and commercially available enzymes and other
reagents. Proteins are purified by chromatography. See,
for example, Robertson et al., J. Am. Chem. Soc. 113:2722
(1991); Ellman et al., Methods Enzymol. 202:301 (1991);
Chung et al., Science 259:806-809 (1993); and Chung et
al., Proc. Natl. Acad. Sci. USA 90:10145-10149 (1993). In
a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs, Turcatti et al., J. Biol.
Chem. 271:19991-19998 (1996). Within a third method, E.
coli cells are cultured in the absence of a natural amino
acid that is to be replaced (e.g., phenylalanine) and in
the presence of the desired non-naturally occurring amino
acids) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-
azaphenylalanine, or 4-fluorophenylalanine). The non-
naturally occurring amino acid is incorporated into the
CA 02330187 2000-12-04
WO 99/63091 PCT/U599I12545
protein in place of its natural counterpart. See, Koide
et al., Biochem. 33:7470-7476 (1994). Naturally occurring
amino acid residues can be converted to non-naturally
occurring species by in vitro chemical modification.
5 Chemical modification can be combined with site-directed
mutagenesis to further expand the range of substitutions,
Wynn and Richards, Protein Sci. 2:395-403 (1993}.
A limited number of non-conservative amino
10 acids, amino acids that are not encoded by the genetic
code, non-naturally occurring amino acids, and unnatural
amino acids may be substituted for Zdscl amino acid
residues. Essential amino acids in the polypeptides of the
present invention can be identified according to
15 procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis, Cunningham
and Wells, Science 244: 1081-1085 (1989); Bass et al.,
Proc. Natl. Acad. Sci. USA 88:4498-4502 (1991). In the
latter technique, single alanine mutations are introducE:d
20 at every residue in the molecule, and the resultant mutant
molecules are tested for biological activity as disclosed
below to identify amino acid re:~idues that are critical to
the activity of the molecule. See also, Hilton et al., ,T.
Biol. Chem. 271:4699-4708 (1996). Sites of ligand-
25 receptor or other biological interaction can also be
determined by physical analysis of structure, as
determined by such techniques a:~ nuclear magnetic
resonance, crystallography, electron diffraction or
photoaffinity labeling, in conjunction with mutation of
30 putative contact site amino acids. See, for example, de
Vos et al., Science 255:306-12 (1992); Smith et al., J.
Mol. Biol. 224:899-904 (1992); Wlodaver et al., FEBS Let:t.
309:59-64, 1992.
PROTEIN PRODUCTION
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41
The Zdscl polypeptides of the present invention,
including full-length polypeptides, biologically active
fragments, and fusion polypeptides, can be produced in
genetically engineered host cells according to
conventional techniques. Suitable host cells are those
cell types that can be transformed or transfected with
exogenous DNA and grown in culture, and include bacteria,
fungal cells, and cultured higher eukaryotic cells.
Eukaryotic cells, particularly cultured cells of
multicellular organisms, are preferred. Techniques for
manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed
by Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1989), and Ausubel et al., eds.,
Current Protocols in Molecular Biology, (John Wiley and
Sons, Inc., NY, 1987).
In general, a DNA sequence encoding a Zdscl
polypeptide is operably linked to other genetic elements
required for its expression, generally including a
transcription promoter and terminator, within an
expression vector. The vector will also commonly contain
one or more selectable markers .and one or more origins of
replication, although those skilled in the art will
recognize that within certain systems selectable marker:
may be provided on separate vectors, and replication of
the exogenous DNA may be provided by integration into the
host cell genome. Selection of promoters, terminators,
selectable markers, vectors and other elements is a matt=er
of routine design within the level of ordinary skill in
the art. Many such elements arE=_ described in the
literature and are available through commercial suppliers.
To direct a Zdscl polypeptide into the secretary
pathway of a host cell, a secret~ory signal sequence (also
CA 02330187 2000-12-04
WO 99!63091 PCT/US99l1254:5
42
known as a leader sequence, prepro sequence or pre
sequence) is provided in the expression vector. The
secretory signal sequence may be that of Zdscl, or may be
derived from another secreted protein (e.g., t-PA) or
synthesized de novo. The secretory signal sequence is
operably linked to the Zdscl DNA sequence, .i.e., the two
sequences are joined in the correct reading frame and
positioned to direct the newly synthesized polypeptide
into the secretory pathway of the host cell. Secretory
signal sequences are commonly positioned 5' to the DNA
sequence encoding the polypeptide of interest, although
certain secretory signal sequences may be positioned
elsewhere in the DNA sequence of interest (see, e.g.,
Welsh et al., U.S. Patent No. 5,037,743; Holland et al.,,
U.S. Patent No. 5,143,830).
Alternatively, the secretory signal sequence
contained in the polypeptides o.f the present invention .Ls
used to direct other polypeptides into the secretory
pathway. The present invention provides for such fusion
polypeptides. The secretory signal sequence contained in
the fusion polypeptides of the present invention is
preferably fused amino-terminally to an additional peptide
to direct the additional peptide into the secretory
pathway. Such constructs have numerous applications known
in the art. For example, these novel secretory signal
sequence fusion constructs can direct the secretion of an
active component of a normally non-secreted protein, such
as a receptor. Such fusions may be used in vivo or in
vitxo to direct peptides through the secretory pathway.
Cultured mammalian cells are suitable hosts
within the present invention. Methods for introducing
exogenous DNA into mammalian host cells include calcium
phosphate-mediated transfection, Wigler et al., Cell
CA 02330187 2000-12-04
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43
14:725 (1978); Corsaro and Pearson, Somatic Cell Genetics
7:603 (1981): Graham and Van der Eb, Virology 52:456
(1973), electroporation, Neumar~n et al., EMBO J. 1:841-845
(1982), DEAE-dextran mediated transfection, Ausubel et
al., ibid., and liposome-mediated transfection, Hawley-
Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus
15:80 (1993), and viral vectors, Miller and Rosman,
BioTechniques 7:980 (1989); Wang and Finer, Nature Med.
2:714 (1996). The production of recombinant polypeptides
in cultured mammalian cells is disclosed, for example, by
Levinson et al., U.S. Patent No. 4,713,339; Hagen et al.,
U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent
No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134.
Suitable cultured mammalian cells include the COS-1 (ATCC
No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No.
CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL
1573; Graham et al., J. Gen. Virol. 36:59-72 (1977) and
Chinese hamster ovary (e. g. CHO-K1; ATCC No. CCL 61) cell
lines. Additional suitable cell lines are known in the
art and available from public depositories such as the
American Type Culture Collection, Rockville, Maryland. In
general, strong transcription promoters are preferred,
such as promoters from SV-40 or cytomegalovirus. See,
e.g., U.S. Patent No. 4,956,288. Other suitable promoters
include those from metallothionein genes (U. S. Patent Nos.
4,579,821 and 4,601,978) and the adenovirus major late
promoter.
Drug selection is generally used to select fo:r
cultured mammalian cells into which foreign DNA has been
inserted. Such cells are commonly referred to as
"transfectants". Cells that have been cultured in the
presence of the selective agent and are able to pass th~~
gene of interest to their progeny are referred to as
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44
"stable transfectants." A preferred selectable marker is
a gene encoding resistance to the antibiotic neomycin.
Selection is carried out in the presence of a neomycin-
type drug, such as G-418 or the like. Selection systems
can also be used to increase the expression level of the
gene of interest, a process referred to as
"amplification." Amplification is carried out by
culturing transfectants in the presence of a low level of
the selective agent and then increasing the amount of
selective agent to select for cells that produce high
levels of the products of the introduced genes. A
preferred amplifiable selectable marker is dihydrofolat~e
reductase, which confers resistance to methotrexate.
Other drug resistance genes (e. g. hygromycin resistance,
mufti-drug resistance, puromycin acetyltransferase) can
also be used. Alternative markers that introduce an
altered phenotype, such as green fluorescent protein, o:r
cell surface proteins such as CD4, CD8, Class I MHC,
placental alkaline phosphatase may be used to sort
transfected cells from untransfected cells by such mean:
as FRCS sorting or magnetic bead separation technology.
Other higher eukaryotic cells can also be used
as hosts, including plant cells, insect cells and avian
cells. The use of Agrobacteriurn rhizogenes as a vector
for expressing genes in plant cells has been reviewed b~r
Sinkar et al., J. Biosci. (Bangalore) 11:47-58 (1987).
Transformation of insect cells and production of foreign
polypeptides therein is disclose=d by Guarino et al., U.~~.
Patent No. 5,162,222 and WIPO publication WO 94/06463.
Insect cells can be infected wit=h recombinant baculovirus,
commonly derived from Autographs californica nuclear
polyhedrosis virus (AcNPV). DNA encoding the Zdscl
polypeptide is inserted into the baculoviral genome in
place of the AcNPV polyhedrin gene coding sequence by one
of two methods. The first is the traditional method of
CA 02330187 2000-12-04
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homologous DNA recombination between wild-type AcNPV and a
transfer vector containing the Zdscl flanked by AcNPV
sequences. Suitable insect cells, e.g. SF9 cells, are
infected with wild-type AcNPV and transfected with a
5 transfer vector comprising a Zdscl polynucleotide opera:bly
linked to an AcNPV polyhedrin gene promoter, terminator,
and flanking sequences. See, King, L.A. and Possee, R.~~.,
The Baculovirus Expression System: A Laboratory Guide,
Chapman & Hall, (London); O'Reilly, D.R. et al.,
10 Baculovirus Expression Vectors: A Laboratory Manual,
(Oxford University Press, New York, 1994); and,
Richardson, C. D., Ed., Baculovi.rus Expression Protocols.
Methods in Molecular Biology, (Humans Press, Totowa, NJ,,
1995). Natural recombination within an insect cell wil:L
15 result in a recombinant baculov:irus which contains Zdscl
driven by the polyhedrin promoter. Recombinant viral
stocks are made by methods commonly used in the art.
The second method of making recombinant
20 baculovirus utilizes a transposon-based system described
by Luckow, V.A, et al., J Viro1 67:4566-79 (1993). This
system is sold in the Bac-to-Bac kit (Life Technologies,
Rockville, MD). This system utilizes a transfer vector,
pFastBaclT"" (Life Technologies) containing a Tn7 transpoaon
25 to move the DNA encoding the Zdscl polypeptide into a
baculovirus genome maintained in E. coli as a large
plasmid called a "bacmid." The pFastBaclT"" transfer vector
utilizes the AcNPV polyhedrin promoter to drive the
expression of the gene of interest, in this case Zdscl.
30 However, pFastBaclT"" can be modified to a considerable
degree. The polyhedrin promoter- can be removed and
substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is
expressed earlier in the baculovirus infection, and has
35 been shown to be advantageous far expressing secreted
CA 02330187 2000-12-04
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46
proteins. See, Hill-Perkins, M.S. and Possee, R.D., J Gen
Viro1 71:971 (1990); Bonning, B.C. et al., J Gen Virol
75:1551 (1994); and, Chazenbalk, G.D., and Rapoport, B., J
Biol Chem 270:1543 (1995). In such transfer vector
constructs, a short or long version of the basic protein
promoter can be used.
Moreover, transfer vectors can be constructed
which replace the native Zdscl secretory signal sequences
with secretory signal sequences derived from insect
proteins. For example, a secretory signal sequence from
Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin
(Invitrogen, Carlsbad, CA), or baculovirus gp67
(PharMingen, San Diego, CA) can be used in constructs to
replace the native Zdsc1 secretory signal sequence. In
addition, transfer vectors can include an in-frame fusion
with DNA encoding an epitope tag at the C- or N-terminus
of the expressed Zdscl polypeptide, for example, a Glu-Glu
epitope tag (Grussenmeyer, T. et al., Proc Nat1 Acad Sci.
82:7952-4, 1985). Using a technique known in the art, a
transfer vector containing Zdscl is transformed into
E. coli, and screened for bacmids which contain an
interrupted lacZ gene indicative of recombinant
baculovirus. The bacmid DNA containing the recombinant
baculovirus genome is isolated, using common techniques,
and used to transfect Spodoptera frugiperda cells, e.q.
Sf9 cells. Recombinant virus that expresses Zdscl is
subsequently produced. Recombinant viral stocks are made
by methods commonly used the art.
The recombinant virus is used to infect host
cells, typically a cell line derived from the fall
armyworm, Spodoptera frugiperda. See, in general, Glick
and Pasternak, Molecular Biotechnology: Principles and
Applications of Recombinant DNA (ASM Press, Washington,,
CA 02330187 2000-12-04
WO 99/63091 PCT/US99/12545
47
D.C., 1994). Another suitable cell line is the High
FiveOT"" cell line (Invitrogen) derived from Trichoplusia. ni
(U. S. Patent #5,300,435). Commercially available serum-
free media are used to grow and maintain the cells.
Suitable media are Sf900 IIr"" (Life Technologies) or ESF
921T"" (Expression Systems) for the Sf9 cells; and Ex-
ce110405T"" (JRH Biosciences, Lenexa, KS) or Express FiveOT""
(Life Technologies) for the T. ni cells. The cells are
grown up from an inoculation density of approximately 2-5
x 105 cells to a density of 1-2 x 106 cells at which time a
recombinant viral stock is added at a multiplicity of
infection (MOI) of 0.1 to 10, more typically near 3. The
recombinant virus-infected cells typically produce the
recombinant Zdscl polypeptide at 12-72 hours post-
infection and secrete it with varying efficiency into the
medium. The culture is usually harvested 48 hours post--
infection. Centrifugation is used to separate the cells
from the medium (supernatant). The supernatant contain_Lng
the Zdscl polypeptide is filtered through micropore
filters, usually 0.45 ~m pore size. Procedures used are
generally described in available laboratory manuals (King,
L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et al.,
ibid.; Richardson, C. D., ibid.). Subsequent purification
of the Zdscl polypeptide from the supernatant can be
achieved using methods described herein.
Fungal cells, including yeast cells, can also be
used within the present invention. Yeast species of
particular interest in this regard include Saccharomyce~~
cerevisiae, Pichia pastoris, and Pichia methanolica.
Methods for transforming S. cerevisiae cells with
exogenous DNA and producing recombinant polypeptides
therefrom are disclosed by, for example, Kawasaki, U.S.
Patent No. 4,599,311; Kawasaki et al., U.S. Patent No.
4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al.,
CA 02330187 2000-12-04
WO 99/63091 PCT/US99/1254:5
48
U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent
No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly
drug resistance or the ability to grow in the absence of a
particular nutrient (e. g., leucine). A preferred vector
system for use in Saccharomyces cerevisiae is the POT1
vector system disclosed by Kawasaki et al. (U. S. Patent
No. 4,931,373), which allows transformed cells to be
selected by growth in glucose-containing media.
Suitable promoters and terminators for use in
yeast include those from glycolytic enzyme genes (see,
e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et
al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent
No. 4,977,092) and alcohol dehydrogenase genes. See al:~o
U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and
4,661,454. Transformation systems for other yeasts,
including Hansenula polymorpha, Schizosaccharomyces pombe,
Kluyveromyces lactis, Kluyveromyces f.ragilis, Ustilago
maydis, Pichia pastoris, Pichia methanolica, Pichia
guillermondii and Candida maltosa are known in the art.
See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459 (1986) and Cregg, U.S. Patent No. 4,882,279.
Aspergillus cells may be utilized according to the methods
of McKnight et al., U.S. Patent No. 4,935,349. Methods
for transforming Acremonium chrysogenum are disclosed by
Sumino et al., U.S. Patent No. ri,162,228. Methods for
transforming Neurospora are disclosed by Lambowitz, U.S.
Patent No. 4,486,533.
The use of Pichia methanolica as host for the
production of recombinant proteins is disclosed in WIPO
Publications WO 97/17450, WO 97f17451, WO 98/02536, and WO
98/02565. DNA molecules for usE~ in transforming P.
CA 02330187 2000-12-04
WO 99/63091 PCT/US99i12545
49
methanolica will commonly be prepared as double-stranded,
circular plasmids, which are preferably linearized prior
to transformation. For polypeptide production in P.
methanolica, it is preferred that the promoter and
terminator in the plasmid be that of a P. methanolica
gene, such as a P. methanolica alcohol utilization gene
(AUG1 or AUG2). Other useful promoters include those o:E
the dihydroxyacetone synthase (DHAS), formate
dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host
chromosome, it is preferred to have the entire expression
segment of the plasmid flanked at both ends by host DNA
sequences.
A preferred selectable marker for use in Pich:ia
methanolica is a P. methanolica ADE2 gene, which encoder
phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), which allows ade2 ho:~t cells to grow in the
absence of adenine. For large-scale, industrial processes
where it is desirable to minimize the use of methanol, ~.t
is preferred to use host cells in which both methanol
utilization genes (AUG1 and AUG?) are deleted. For
production of secreted proteins, host cells deficient in
vacuolar proteinase genes (PEP4 and PR81) are preferred.
Electroporation is used to facilitate the introduction of
a plasmid containing DNA encoding a polypeptide of
interest into P. methanolica cells. It is preferred to
transform P, methanolica cells by electroporation using
an exponentially decaying, pulsE:d electric field having a
field strength of from 2.5 to 4.5 kV/cm, preferably about
3.75 kV/cm, and a time constant (T) of from 1 to 40
milliseconds, most preferably about 20 milliseconds.
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Prokaryotic host cells, including strains of the
bacteria Escherichia coli, Bacillus and other genera are
also useful host cells within the present invention.
Techniques for transforming these hosts and expressing
5 foreign DNA sequences cloned therein are well known in the
art (see, e.g., Sambrook et al., ibid.). When expressing
a Zdscl polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasm_Lc
10 space by a bacterial secretion sequence. In the former
case, the cells are lysed, and the granules are recovered
and denatured using, for example, guanidine isothiocyanate
or urea. The denatured polypeptide can then be refolded
and dimerized by diluting the denaturant, such as by
15 dialysis against a solution of urea and a combination of.
reduced and oxidized glutathione, followed by dialysis
against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic
space in a soluble and functional form by disrupting the
20 cells (by, for example, sonication or osmotic shock) to
release the contents of the periplasmic space and
recovering the protein, thereby obviating the need for
denaturation and refolding.
25 Transformed or transfected host cells are
cultured according to conventional procedures in a culture
medium containing nutrients and other components required
for the growth of the chosen host cells. A variety of
suitable media, including defined media and complex media,
30 are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins
and minerals. Media may also contain such components as.
growth factors or serum, as required. The growth medium
will generally select for cells containing the exogenous;ly
35 added DNA by, for example, drug selection or deficiency in
an essential nutrient which is complemented by the
selectable marker carried on the expression vector or co-
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Sl
transfected into the host cell. P. methanolica cells are
cultured in a medium comprising adequate sources of-
carbon, nitrogen and trace nutrients at a temperature of
about 25°C to 35°C. Liquid cultures are provided with
sufficient aeration by conventional means, such as shaking
of small flasks or sparging of fermentors. A preferred
culture medium for P. methanol.ica is YEPD (2$~ D-glucose,
2a BactoTM Peptone (Difco Laboratories, Detroit, MII, 1%
BactoTM yeast extract (Difco Laboratories), 0.0040 adenine
and 0.0060 L-leucine).
Protein Isolation
It is preferred to purify the polypeptides of
the present invention to >_80o purity, more preferably to
>_90% purity, even more preferably >_95% purity, and
particularly preferred is a pharmaceutically pure state,.
that is greater than 99.9°s pure with respect to
contaminating macromolecules, particularly other proteins
and nucleic acids, and free of infectious and pyrogenic
agents. Preferably, a purified polypeptide is
substantially free of other polypeptides, particularly
other polypeptides of animal origin.
Expressed recombinant Zdscl polypeptides (or
chimeric Zdsc1 polypeptides) can be purified using
fractionation and/or conventional purification methods and
media. Ammonium sulfate precipitation and acid or
chaotrope extraction may be used for fractionation of
samples. Exemplary purification steps may include
hydroxyapatite, size exclusion, FPLC and reverse-phase
high performance liquid chromatography. Suitable
chromatographic media include derivatized dextrans,
agarose, cellulose, polyacrylamide, specialty silicas, and
the like. PEI, DEAE, QAE and Q derivatives are preferred.
Exemplary chromatographic media include those media
derivatized with phenyl, butyl, or octyl groups, such as
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Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso
Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia)
and the like; or polyacrylic resins, such as Amberchrom CG
71 (Toso Haas) and the like. Suitable solid supports
include glass beads, silica-based resins, cellulosi.c
resins, agarose beads, cross-linked agarose beads,
polystyrene beads, cross-linked polyacrylamide resins a:nd
the like that are insoluble under the conditions in which
they are to be used. These supports may be modified with
reactive groups that allow attachment of proteins by amino
groups, carboxyl groups, sulfhydryl groups, hydroxyl
groups and/or carbohydrate moieties. Examples of coupling
chemistries include cyanogen bromide activation, N-
hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl
and amino derivatives for carbodiimide coupling
chemistries. These and other solid media are well known
and widely used in the art, and are available from
commercial suppliers. Methods .for binding receptor
polypeptides to support media are well known in the art..
Selection of a particular method is a matter of routine
design and is determined in pari~ by the properties of tree
chosen support. See, for example, Affinity
Chromatography: Principles & Methods, (Pharmacia LKB
Biotechnology, Uppsala, Sweden, 1988).
The polypeptides of the present invention can be
isolated by exploitation of their properties. For
example, immobilized metal ion adsorption (IMAC)
chromatography can be used to purify histidine-rich
proteins, including those comprising polyhistidine tags.
Briefly, a gel is first charged with divalent metal ions.
to form a chelate, Sulkowski, Trends in Biochem. 3:1-7
(1985). Histidine-rich proteins will be adsorbed to this
matrix with differing affinities, depending upon the metal
ion used, and will be eluted by competitive elution,
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lowering the pH, or use of strong chelating agents. Other
methods of purification include purification of
glycosylated proteins by lectin affinity chromatography
and ion exchange chromatography, Methods in Enzyrnol., V~~l.
182, "Guide to Protein Purification", M. Deutscher,
(ed.),pp.529-539 (Acad. Press, San Diego, 1990). Within
additional embodiments of the invention, a fusion of th~~
polypeptide of interest and an affinity tag (e. g.,
maltose-binding protein, an immunoglobulin domain) may be
constructed to facilitate purification. To direct the
export of a receptor polypeptid~=_ from the host cell, the
receptor DNA is linked to a second DNA segment encoding a
secretory peptide, such as a t-PA secretory peptide.
Fusion proteins can be prepared by methods known
to those skilled in the art by preparing each component of
the fusion protein and chemically conjugating them.
Alternatively, a polynucleotide encoding both components
of the fusion protein in the proper reading frame can be
generated using known techniques and expressed by the
methods described herein. For Example, part or all of a
domains) conferring a biological function may be swapped
between Zdscl of the present invention with the
functionally equivalent domain(:) from another family
member. Such domains include, but are not limited to, the
secretory signal sequence, cons~:rved motifs [provide lieu
if possible], and [significant domains or regions in this
family]. Such fusion proteins would be expected to have a
biological functional profile that is the same or similar
to polypeptides of the present invention depending on th.e
fusion constructed. Moreover, such fusion proteins may
exhibit other properties as disclosed herein. Zdscl
polypeptides or fragments thereof may also be prepared
through chemical synthesis. Zdsc1 polypeptides may be
monomers or multimers; glycosylated or non-glycosylated;
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pegylated or non-pegylated; and may or may not include an
initial methionine amino acid residue.
Antagonists are also useful as research reagents
for characterizing sites of ligand-receptor interaction.
Inhibitors of Zdscl activity (Zdscl antagonists) include'
anti-Zdsc1 antibodies and soluble Zdsc1 receptors, as well
as other peptidic and non-peptidic agents (including
ribozymes).
A Zdscl polypeptide can be expressed as a fusion
with an immunoglobulin heavy chain constant region,
typically an Fc fragment, which contains two constant
region domains and lacks the variable region. Methods for
preparing such fusions are disclosed in U.S. Patents Nos.
5,155,027 and 5,567,584. Such fusions are typically
secreted as multimeric molecules wherein the Fc portions
are disulfide bonded to each other and two non-Ig
polypeptides are arrayed in closed proximity to each
other. Fusions of this type can be used to affinity
purify ligand, as an in vitro assay tool, antagonist.).
For use in assays, the chimeras are bound to a support via
the Fc region and used in an ELISA format.
Ligand-binding receptor polypeptides can also :be
used within other assay systems known in the art. Such
systems include Scatchard analysis for determination of
binding affinity, see Scatchard, Ann. NY Acad. Sci. 51:
660 (1949) and calorimetric assays (Cunningham et al.,
Science 253:545 (1991); Cunningham et al., Science 245:8:21
(1991) .
Zdscl polypeptides can also be used to prepare
antibodies that specifically bind to Zdscl epitopes,
peptides or polypeptides. The Zdscl polypeptide or a
fragment thereof serves as an anr_igen (immunogen) to
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inoculate an animal and elicit an immune response. A
suitable antigen would be the Zdscl polypeptide encoded by
5 from amino acid number 36 to amino acid number 5l, also
defined by SEQ ID N0:18, or a contiguous 9 amino acid
5 residues or a fragment thereof. Antibodies generated from
this immune response can be isolated and purified as
described herein. Methods for preparing and isolating
polyclonal and monoclonal antibodies are well known in t=he
art. See, for example,_Current Protocols in Immunology,.
10 Cooligan, et al. (eds.), National Institutes of Health
(John Wiley and Sons, Inc., 1995); Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Second Edition
(Cold Spring Harbor, NY, 1989); and Hurrell, J. G. R.,
Ed., Monoclonal Hybridoma Antibodies: Techniques and
15 Applications (CRC Press, Inc., E3oca Raton, FL, 1982).
Polyclonal antibodies can be generated from
inoculating a variety of warm-b7.ooded animals such as
horses, cows, goats, sheep, dogs, chickens, rabbits, mice,
20 and rats with a Zdscl polypeptide or a fragment thereof.
The immunogenicity of a Zdscl polypeptide may be increased
through the use of an adjuvant, such as alum (aluminum
hydroxide) or Freund's complete or incomplete adjuvant.
Polypeptides useful for immunization also include fusion
25 polypeptides, such as fusions of Zdscl or a portion
thereof with an immunoglobulin polypeptide or with maltose
binding protein. The polypeptide immunogen may be a full-
length molecule or a portion thereof. If the polypeptide
portion is "hapten-like", such portion may be
30 advantageously joined or linked to a macromolecular
carrier (such as keyhole limpet hemocyanin (KLH), bovine
serum albumin (BSA) or tetanus toxoid) for immunization.
As used herein, the term "antibodies" includes
35 polyclonal antibodies, affinity-purified polyclonal
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antibodies, monoclonal antibod_~es, and antigen-binding
fragments, such as F(ab')2 and Fab proteolytic fragments.
Genetically engineered intact antibodies or fragments,
such as chimeric antibodies, Fv fragments, single chair,
antibodies and the like, as we~1 as synthetic antigen-
binding peptides and polypeptides, are also included.
Non-human antibodies may be humanized by grafting IlOn-
human CDRs onto human framework. and constant regions, cr
by incorporating the entire norn-human variable domains
(optionally "cloaking" them with a human-like surface by
replacement of exposed residue::, wherein the result. is a
"veneered" antibody). In some instances, humanized
antibodies may retain non-human residues within the human
variable region framework domains to enhance proper
binding characteristics. Through humanizing antibodies,
biological half-life may be increased, and the potential
for adverse immune reactions upon administration tc> humans
is reduced.
Alternative techniques for generating or
selecting antibodies useful herein include in vitro
exposure of lymphocytes to Zdscl protein or peptide, and
selection of antibody display libraries in phage or
similar vectors (for instance, through use of immobilized
or labeled Zdscl protein or peptide). Genes encoding
polypeptides having potential Zdscl polypeptide binding
domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on
bacteria, such as E. coli. Nucleotide sequences encoding
the polypeptides can be obtained in a number of ways, such
as through random mutagenesis and random polynucleotide
synthesis. These random peptide=_ display libraries can be
used to screen for peptides which interact with a known
target which can be a protein or polypeptide, such as a
ligand or receptor, a biologica_L or synthetic
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macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide
display libraries are known in the art (Ladner et al., US
Patent NO. 5,223,409; Ladner et al., US Patent NO.
4,946,778; Ladner et al., US Patent NO. 5,403,484 and
Ladner et al., US Patent NO. 5,571,698) and random peptide
display libraries and kits for screening such libraries
are available commercially, for instance from Clontech
(Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New
England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB
Biotechnology Inc. (Piscataway, NJ). Random peptide
display libraries can be screened using the Zdscl
sequences disclosed herein to identify proteins which bind
to Zdscl. These "binding proteins" which interact with
Zdscl polypeptides can be used for tagging cells; for
isolating homolog polypeptides by affinity purification;
they can be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like. These binding
proteins can also be used in analytical methods such as
for screening expression libraries and neutralizing
activity. The binding proteins can also be used for
diagnostic assays for determining circulating levels of
polypeptides; for detecting or quantitating soluble
polypeptides as marker of under:Lying pathology or diseaae.
These binding proteins can also act as Zdscl "antagonist: s"
to block Zdscl binding and sign<~l transduction in vitro
and in vivo.
Antibodies are determined to be specifically
binding if: 1) they exhibit a threshold level of binding
activity, and 2) they do not cross-react with related
prior art polypeptide molecules. First, antibodies herE~in
specifically bind if they bind t;o a Zdscl polypeptide,
peptide or epitope with a binding affinity (Ka) of 106 Nf 1
or greater, preferably 10~ M 1 or greater, more preferably
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108 M 1 or greater, and most preferably 109 M 1 or greater.
The binding affinity of an antibody can be readily
determined by one of ordinary skill in the art, fo.r
example, by Scatchard analysis, Scatchard, G., Ann. NY
Acad. Sci. 51: 660-672 (1949).
Second, antibodies are determined to
specifically bind if they do not significantly cross-react
with related polypeptides. Antibodies do not significantly
crass-react with related polypeptide molecules, for
example, if they detect Zdscl but not known related
polypeptides using a standard Western blot analysis
(Ausubel et al., ibid.). Examples of known related
polypeptides are orthologs, proteins from the same species
that are members of a protein family (e. g. IL-16), Zdscl
polypeptides, and non-human Zd~~cl. Moreover, antibodies
may be "screened against" known related polypeptides to
isolate a population that specifically binds to the
inventive polypeptides. For example, antibodies raised to
Zdsc1 are adsorbed to related polypeptides adhered to
insoluble matrix; antibodies specific to Zdscl will flow
through the matrix under the proper buffer conditions.
Such screening allows isolatiot: of polyclonal and
monoclonal antibodies non-crossreacti.ve to closely related
polypeptides (Antibodies: A Laboratory Manual, Harlow and
Lane (eds.),( Cold Spring Harbor Laboratory Press, 1988);
Current Protocols in Immunology, Cooligan, et al. (eds.),
National Institutes of Health, John Wiley and Sons, Inc.,
1995). Screening and isolation of specific antibodies is
well known in the art. See, Fundamental Immunology, Paul
(eds.), (Raven Press, 1993); C~etzoff et al., Adv. in
Immunol. 43: 1-98 (1988); Monoclonal Antibodies:
Principles and Practice, Goding, J.W. (eds.), (Academic
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Press Ltd., 1996); Benjamin et al., Ann. Rev. Immunol. 2:
67-101 (1984).
A variety of assays known to those skilled in
the art can be utilized to detect antibodies which
specifically bind to Zdscl proteins or peptides.
Exemplary assays are described in detail in Antibodies: A
Laboratory Manual, Harlow and Lane (Eds.) (Cold Spring
Harbor Laboratory Press, 1988). Representative examples
of such assays include: concurrent immunoelectrophoresi:~,
radioimmunoassay, radioimmuno-precipitation, enzyme-lin)ted
immunosorbent assay (ELISA), dot blot or Western blot
assay, inhibition or competition assay, and sandwich
assay. In addition, antibodies can be screened for
binding to wild-type versus mutant Zdscl protein or
polypeptide.
Antibodies to Zdscl may be used for tagging
cells that express Zdscl; for isolating Zdscl by affinity
purification; for diagnostic assays for determining
circulating levels of Zdsclpolypeptides; for detecting or
quantitating soluble Zdscl as marker of underlying
pathology or disease; in analytical methods employing
FRCS; for screening expression libraries; for generating
anti-idiotypic antibodies; and as neutralizing antibodies
or as antagonists to block Zdsc~. in vitro and in vivo.
Suitable direct tags or labels include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent
markers, chemiluminescent markers, magnetic particles and
the like; indirect tags or labels may feature use of
biotin-avidin or other complement/anti-complement pairs as
intermediates. Antibodies herein may also be directly or
indirectly conjugated to drugs, toxins, radionuclides and
the like, and these conjugates used for in vivo diagnostic
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or therapeutic applications. Moreover, antibodies to
Zdscl or fragments thereof may be used in vitro to detect
denatured Zdscl or fragments thereof in assays, for
example, Western Blots or other assays known in the art.
5
BIOACTIVE CONJUGATES:
Antibodies or po.lypeptides herein can also be
directly or indirectly conjugated to drugs, toxins,
radionuclides and the like, and these conjugates used for
10 in vivo diagnostic or therapeutic applications. For
instance, polypeptides or antibodies of the present
invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary
molecule (receptor or antigen, respectively, for
15 instance). More specifically, Zdscl polypeptides or ant~i-
Zdscl antibodies, or bioactive fragments or portions
thereof, can be coupled to detectable or cytotoxic
molecules and delivered to a mammal having cells, tissues
or organs that express the anti--complementary molecule.
Suitable detectable molecules may be directly or
indirectly attached to the polypeptide or antibody, and
include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent markers, chemiluminescent markers,
magnetic particles and the like. Suitable cytotoxic
molecules may be directly or indirectly attached to the
polypeptide or antibody, arid include bacterial or plant
toxins (for instance, diphtheria toxin, Pseudomonas
exotoxin, ricin, abrin and the 1-ike}, as well as
therapeutic radionuclides, such as iodine-131, rhenium-1.88
or yttrium-90 (either directly attached to the polypepti.de
or antibody, or indirectly attached through means of a
chelating moiety, for instance). Polypeptides or
antibodies may also be conjugated to cytotoxic drugs, such
as adriamycin. For indirect attachment of a detectable or
cytotoxic molecule, the detectable or cytotoxic molecule
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can be conjugated with a member of a complementary/
anticomplementary pair, where the other member is bound to
the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/
anticomplementary pair.
In another embodiment, polypeptide-toxin fusion
proteins or antibody-toxin fusion proteins can be used for
targeted cell or tissue inhibition or ablation (for
instance, to treat cancer cells or tissues).
Alternatively, if the polypeptide has multiple functional
domains (i.e., an activation domain or a ligand binding
domain, plus a targeting domain), a fusion protein
including only the targeting domain may be suitable for
directing a detectable molecule, a cytotoxic molecule o:r a
complementary molecule to a cell or tissue type of
interest. In instances where the domain only fusion
protein includes a camplementary molecule, the anti-
complementary molecule can be canjugated to a detectable
or cytotoxic molecule. Such domain-complementary molecule
fusion proteins thus represent a generic targeting vehicle
for cell/tissue-specific delivery of generic anti-
complementary-detectable/ cytotoxic molecule conjugates.
The bioactive polypeptide or antibody conjugates
described herein can be delivera_d intravenously,
intraarterially or intraductally, or may be introduced
locally at the intended site of action.
USES OF POLYNUCLEOTIDE/POLYPEPTIDE:
Molecules of the present invention can be used
to identify and isolate receptors. For example, proteins
and peptides of the present invention can be immobilized
on a column and membrane preparations run over the column
(Immobilized Affinity Ligand Techniques, Hermanson et a~:.,
eds., pp.195-202 (Academic Pres:~, San Diego, CA, 1992,).
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Proteins and peptides can also be radiolabeled (Methods in
Enzymol., vol. 162, "Guide to Protein Purification", M.
Deutscher, ed., 721-37 (Acad. Press, San Diego, 1990,) ~~r
photoaffinity labeled, Brunner et al., Ann. Rev. Bi:oche;m.
62:483-514 (1993) and Fedan et al., Biochem. Pharmacol.
33:1167-80 (1984) and specific cell-surface proteins can
be identified.
GENE THERAPY:
Polynucleotides encoding Zdscl polypeptides a:re
useful within gene therapy applications where it is
desired to increase or inhibit Zdscl activity. If a
mammal has a mutated or absent Zdscl gene, the Zdsc1 gene
can be introduced into the cells of the mammal In one
embodiment, a gene encoding a Zdsc polypeptide is
introduced in vivo in a viral vector. Such vectors
include an attenuated or defective DNA virus, such as, but
not limited to, herpes simplex virus (HSV),
papillomavirus, Epstein Barr virus (EBV), adenovirus,
adeno-associated virus (AAV), and the like. Defective
viruses, which entirely or almo:~t entirely lack viral
genes, are preferred. A defective virus is not infective
after introduction into a cell. Use of defective viral
vectors allows for administration to cells in a specific:,
localized area, without concern that the vector can infect
other cells. Examples of particular vectors include, but
are not limited to, a defective herpes simplex virus 1
(HSVl) vector, Kaplitt et al., Molec. Cell. Neurosci.
2:320-30 (1991); an attenuated adenovirus vector, such as
the vector described by Stratfoz-d-Perricaudet et al., J.
Clin. Invest. 90:626-30, 1992; and a defective adeno-
associated virus vector (Samulski et al., J. Virol.
61:3096-101 (1987); Samulski et al., J. Virol. 63:3822-
3828 (1989).
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In another embodiment, a Zdscl gene can be
introduced in a retroviral vector, e.g., as described in
Anderson et al., U.S. Patent No. 5,399,346; Mann et~ al.
Cell 33:153 (1983); Temin et al., U.S. Patent No.
4,650,764; Temin et al., U.S. Patent No. 4,980,289;
Markowitz et al., J. Virol. 62:1120 (1988); Temin et al.,
U.S. Patent No. 5,124,263; International Patent
Publication No. WO 95/07358, published March 16, 1995 by
Dougherty et al.; and Kuo et al., Blood 82:845 (1993).
Alternatively, the vector can be introduced by lipofect:ion
in vivo using liposomes. Synthetic cationic lipids can be
used to prepare liposomes for in vivo transfection of a
gene encoding a marker, Felgner et al., Proc. Natl. Aca~~.
Sci. USA 84:7413-7 (1987); Mackey et al., Proc. Natl.
Acad. Sci. USA 85:8027-31 (1988). The use of lipofection
to introduce exogenous genes into specific organs in vivo
has certain practical advantages. Molecular targeting of
liposomes to specific cells represents one area of
benefit. More particularly, directing transfection to
particular cells represents one area of benefit. For
instance, directing transfecti021 to particular cell typE~s
would be particularly advantageous in a tissue with
cellular heterogeneity, such as the pancreas, liver,
kidney, and brain. Lipids may be chemically coupled to
other molecules for the purpose of targeting. Targeted
peptides (e. g., hormones or neurotransmitters), protein;
such as antibodies, or non-peptide molecules can be
coupled to liposomes chemically,.
It is possible to remove the target cells from
the body; to introduce the vector as a naked DNA plasmid;
and then to re-implant the tran~~formed cells into the
body. Naked DNA vectors for gene therapy can be
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introduced into the desired host cells by methods known in
the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAF dextran,
calcium phosphate precipitation, use of a gene gun or use
of a DNA vector transporter. See, e.g., Wu et al., J.
Biol. Ch em. 267:963-7 (1992) ; Wu et a1. , J. Biol. C.hem.
263:14621-14624 (1988).
Antisense methodology can be used to inhibit
Zdsc gene transcription, such as to inhibit cell
proliferation in vivo. Polynucleotides that are
complementary to a segment of a Zdscl-encoding
polynucleotide (e.g., a polynucleotide as set froth in 3EQ
ID NO:1) are designed to bind to Zdscl-encoding mRNA and
to inhibit translation of such mRNA. Such antisense
polynucleotides are used to inhibit expression of Zdsc
polypeptide-encoding genes in cell culture or in a
subject.
The present invention also provides reagents
which will find use in diagnostic applications. For
example, the Zdscl gene, a probe comprising Zdsc1 DNA or
RNA or a subsequence thereof can be used to determine if-_
the Zdse gene is present or if a mutation has occurred.
Detectable chromosomal aberrations at the Zdsc1 gene locus
include, but are not limited to,, aneuploidy, gene copy
number changes, insertions, deletions, restriction site
changes and rearrangements. Such aberrations can be
detected using polynucleotides of the present invention by
employing molecular genetic techniques, such as
restriction fragment length polymorphism (RFLP) analysi:~,
short tandem repeat (STR) analysis employing PCR
techniques, and other genetic linkage analysis techniques
known in the art (Sambrook et a:L., ibid.; Ausubel et. a~:,
ibid.; Marian, Chest 108:255-65 (1995).
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Transgenic mice, engineered to express the Zd:~c
gene, and mice that exhibit a complete absence of Zdsc
gene function, referred to as "knockout mice", Snouwaert=
5 et al., Science 257:1083 {1992), may also be generated,
Lowell et al., Nature 366:740-42 (1993). These mice ma;r
be employed to study the Zdsc gene and the protein encoded
thereby in an in vivo system.
10 CHROMOSOMAL LOCALIZATION:
Radiation hybrid mapping is a somatic cell
genetic technique developed for constructing high-
resolution, contiguous maps of mammalian chromosomes, Cox
15 et al., Science 250:245-250 (1990). Partial or full
knowledge of a gene's sequence allows one to design PCR
primers suitable for use with chromosomal radiation hybrid
mapping panels. Radiation hybrid mapping panels are
commercially available which cover the entire human
20 genome, such as the Stanford G3 RH Panel and the
GeneBridge 4 RH Panel (Research Genetics, Inc.,
Huntsville, AL). These panels enable rapid, PCR-based
chromosomal localizations and ordering of genes, sequenc:e-
tagged sites {STSs), and other nonpolymorphic and
25 polymorphic markers within a region of interest. This
includes establishing directly proportional physical
distances between newly discovered genes of interest and
previously mapped markers. The precise knowledge of a
gene's position can be useful for a number of purposes,
30 including: 1) determining if a sequence is part of an
existing contig and obtaining additional surrounding
genetic sequences in various forms, such as YACs, BACs or
cDNA clones; 2) providing a possible candidate gene for an
inheritable disease which shows linkage to the same
35 chromosomal region; and 3) cross-referencing model
CA 02330187 2000-12-04
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66
organisms, such as mouse, which may aid in determining
what function a particular gene might have.
Sequence tagged sites (STSs) can also be used
independently for chromosomal localization. An STS is .a
DNA sequence that is unique in the human genome and can be
used as a reference point for a particular chromosome o:r
region of a chromosome. An STS is defined by a pair of
oligonucleotide primers that are used in a polymerase
chain reaction to specifically detect this site in the
presence of all other genomic sequences. Since STSs arE~
based solely on DNA sequence they can be completely
described within an electronic database, for example,
Database of Sequence Tagged Sites (dbSTS), GenBank,
(National Center for Biological Information, National
Institutes of Health, Bethesda, MD
http://www.ncbi.nlm.nih.gov), and can be searched with a
gene sequence of interest for the mapping data contained
within these short genomic landmark STS sequences.
For pharmaceutical use, the proteins of the
present invention are formulated for parenteral,
particularly intravenous or subcutaneous, delivery
according to conventional methods. Intravenous
administration will be by bolus injection or infusion over
a typical period of one to several hours. In general,
pharmaceutical formulations will include a Zdscl protein
in combination with a pharmaceutically acceptable vehicle,
such as saline, buffered saline,. 5% dextrose in water or
the like. Formulations may further include one or more
excipients, preservatives, solubilizers, buffering agents,
albumin to prevent protein loss on vial surfaces, etc.
Methods of formulation are well known in the art and area
disclosed, for example, in Remington: The Science and
Practice of Pharmacy, Gennaro, ed. (Mack Publishing Co.,
Easton, PA, 19th ed., 1995). Therapeutic doses will
CA 02330187 2000-12-04
WO 99/63091 PCTlUS99/12545
67
generally be in the range of 0.1 to 100 ~g/kg of patient.
weight per day, preferably 0.5-:?0 ~tg/kg per day, with tree
exact dose determined by the clinician according to
accepted standards, taking into account the nature and
severity of the condition to be treated, patient traits,
etc. Determination of dose is within the level of
ordinary skill in the art. The proteins may be
administered for acute treatment,, over one week or less,
often over a period of one to three days or may be used in
chronic treatment, over several months or years.
The invention is further illustrated by the
following non-limiting examples..
Example 1
Cloning of the Murine Zdscl Gene
SEQ ID NO:11, an Expressed Sequence Tag (EST) was
discovered in an EST data bank of an eosinophil cDNA
library. The cDNA clone corresponding to the EST was
discovered and sequenced to give' the DNA sequence of SEQ
ID NO:1. The mature protein is shown in SEQ ID NO: 3.
Example 2
Cloning of the Human Zdsc1 Gene
SEQ ID N0:12, an EST was discovered in an EST data
bank of a senescent human fibroblast cDNA library. The
cDNA clone corresponding to the EST was discovered, and
sequenced to give the DNA sequence of SEQ ID N0:4. The
mature protein is shown in SEQ ID NO: 5.
Example 3
Northern Blot Analysis of Zdscl
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68
Northern blot analysis was performed using mouse
multiple tissue blot and dot blot from Clontech (Palo
Alto, CA) and Mouse Multiple Tissue Blot from Origene
(Rockville, Maryland) using a 400 by DNA probe containing
the entire coding region of the Zdscl gene. The probe was
radioactively labeled using 32P using the MULTIPRIME° DNA
labeling system (Amersham, United Kingdom) according to
manufacturer s specifications. EXPRESSHYP~ solution
(Clontech) was used for prehybridization and as a
hybridizing solution for the Northern analysis.
Hybridization of the probe on the blots took place
overnight at 65° C, and the blot=s were than washed four
times in 2X standard sodium citrate (SCC) and O.lo sodium
dodecyl sulfate (SDS) at room temperature, followed by vwo
washes in O.1X SSC and 0.1% SDS at 50° C. The blots were
then exposed. Only one strong transcript was seen in liver
for both multiple tissue blots. The dot blot showed a
strong dot for liver. A faint dot for spleen and E. col.i
DNA was also seen.
From the foregoing, it will be appreciated that,
although specific embodiments of the invention have been
described herein for purposes of illustration, various
modifications may be made without deviating from the
spirit and scope of the invention. Accordingly, the
invention is not limited except as by the appended claims.
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1
SEQUENCE LIS~fING
<110> ZymoGenetics. Inc.
1201 Eastlake Avenue East
Seattle, Washington 98102
United States of America
<120> Disulfide Core Polypeptides
<130> 98-13PC
<150> 09/090.895
<151> 1998-06-04
<160> 23
<170> FastSEQ for Windows Version 3.0
<210>1
<211>365
<212>DNA
<213>Mus musculus
<220>
<221> CDS
<222> (18)...(206)
<400> 1
catccttcag cagcagc atg aag cta gga gcc ttc ctt ctg ttg gtg tcc ~0
Met Lys Leu Gly Ala Phe Leu Leu Leu Val Ser
1 5 10
ctc ate acc etc agc cta gag gta cag gag ctg cag get gca gtg aga 98
Leu Ile Thr Leu Ser Leu Glu Ual Gln Glu Leu Gln Ala Ala Val Arg
15 20 25
cct ctg cag ctt tta ggc acc tgt get gag etc tgc cgt ggt gac tgg 1~~6
Pro Leu Gln Leu Leu Gly Thr Cys Ala Glu Leu Cys Arg Gly Asp Trp
30 35 40
gac tgt ggg cca gag gaa caa tgt gtc agt att gga tgc agt cac atc 1!34
Asp Cys Gly Pro Glu Glu Gln Cys Val Ser Iie Gly Cys Ser His Ile
45 50 55
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2
tgt act aca aac taaaaacagc ttctacctgg aaaaaaaaat gtgtctgttt 246
Cys Thr Thr Asn
ggagctctgt gaccaagaaa acagttgaaa atggaggcca tgtatggaga ttacaagcag ?.06
cacagtggag tgggacaagg agttgtttct tttaataaat cattaatgta aaagtctca ?65
<210>2
<211>63
<212>PRT
<213>Mus musculus
<400> 2
Met Lys Leu Gly Ala Phe Leu Leu Leu Ual Ser Leu Ile Thr Leu Ser
1 5 10 15
Leu Glu Val Gln Glu Leu Gln Ala Ala Ual Arg Pro Leu Gln Leu Leu
20 25 30
Gly Thr Cys Ala Glu Leu Cys Arg Gly Asp Trp Asp Cys Gly Pro Glu
35 40 45
Glu Gln Cys Ual Ser Ile Gly Cys Ser His Ile Cys Thr Thr Asn
50 55 60
<210>3
<211>39
<212>PRT
<213>Mus musculus
<400> 3
AlaUal ProLeu Gln Leu Leu Gly Thr Cys Ala Glu Leu
Arg Cys Arg
1 5 10 15
GlyAsp AspCys Gly Pro Glu Glu Gln Cys Ual Ser Ile
Trp Gly Cys
20 25 30
SerHis CysThr Thr Asn
Ile
35
<210>4
<211>501
<212>DNA
<213>Homo sapiens
<220>
<221> CDS
<222> (94)...(204)
<400> 4
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3
gaattcggca cgaggcagca acatgaagtt ggcagccttc ctcctcctgt gatcctcatc 60
atcttcagcc tagaggtaca agagcttcag get gca gga gac cgg ctt ttg ggt 114
Ala Gly Asp Arg Leu Leu Gly
1 5
acc tgc gtc gag ctc tgc aca ggt gac tgg gac tgc aac ccc gga gac 162
Thr Cys Val Glu Leu Cys Thr Gly Asp Trp Asp Cys Asn Pro Gly Asp
15 20
cac tgt gtc agc aat ggg tgt ggc cat gag tgt gtt gca ggg 204
His Cys Val Ser Asn Gly Cys Gly His Glu C,ys Val Ala Gly
25 30 35
taaggacaggtaaaaacaccaggccctccctgctttctgaaacgttgttcagtctagatg 264
aagagttatcttaaggatcatctttccctaagatcgtcatcccttcctggagttcctatc 324
ttccaagatgtgactgtctggagttccttgactaggaagatggatgaaaacagcaagcct 384
gtggatggagactacaggggatatgggaggcagggaagaggggttgtttcttttaataaa 444
tcatcattgttaaaagcaaaaaaaaaaaaaaaaaaaaaaaaaaatggttgcggccgc 501
<210>5
<211>37
<212>PRT
<213>Homo Sapiens
<400> 5
AlaGly ArgLeu Leu Gly Thr Cys Val Glu Leu Cys Thr
Asp Gly Asp
1 5 10 15
TrpAsp AsnPro Gly Asp His Cys Val SE~r Asn Gly
Cys Cys Gly His
20 25 30
GluCys AlaGly
Val
35
<210>6
<211>39
<212>PRT
<213>Homo Sapiens
<220>
<221> VARIANT
<222> (0)...(0>
<223> Xaa at amino acid position 1 is Ala or is absent:
Xaa at amino acid position 2 is Val or is absent:
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WO 99/63091 PCT/US99/1254:5
4
Xaa at amino acid position 3 is Arg or Ala;
Xaa at amino acid position 4 is Pro or Gly;
Xaa at amino acid position 5 is Leu or Asp:
Xaa at amino acid position 6 is Gln. Arg, Lys or
Glu;
Xaa at amino acid position 12 is Val, Ala, Iie,
Leu. Met or Ser;
Xaa at amino acid position 16 is Thr. Arg. Ala,
Asn. Ser, Val. Gln, Glu, His or Lys;
Xaa at amino acid position 22 is Asn, Gly, Asp,
His or Ser;
Xaa at amino acid position 24 is Ala, Arg. Asn.
Asp, Glu, Gln, Gly, His, Lys, Pro, Ser, or Thr:
Xaa at amino acid position 25 is Asp or Glu
Xaa at amino acid position 26 is His. Gln Tyr or
Glu:
Xaa at amino acid position 30 is Ala, Arg, Asn,
Asp, Gln, Glu, Gly His, Ile. Leu, Lys, Met, Phe,
Ser, Thr, Tyr, or Val;
Xaa at amino acid position 33 is Gly. Ser, Ala,
Asn, Thr:
Xaa at amino acid position 35 is Ala, Arg. Asn,
Asp, Glu, Gln. Gly, His, Ile, Leu, Lys, Met Phe,
Pro, Ser. Thr, Trp, Tyr or Val;
Xaa at amino acid position 37 is Val or Thr;
Xaa at amino acid position 38 is Ala or Thr; and
Xaa at amino acid position 39 is Asn or Gly;
CA 02330187 2000-12-04
WO 99/63091 PCT/US99/1254:5
<400> 6
Xaa Xaa Xaa Xaa Xaa Xaa Leu Leu Gly Thr Cys Xaa Glu Leu Cys Xaa
1 5 10 15
Gly Asp Trp Asp Cys Xaa Pro Xaa Xaa Xaa Cys Val Ser Xaa Gly Cys
20 25 30
Xaa His Xaa Cys Xaa Xaa Xaa
<210>7
<211>40
<212>PRT
<213>Homo Sapiens
<400> 7
Ile Ile Leu Ile Arg Cys Ala Met Leu Asn Pro Pro Asn Arg Cys Leu
1 5 10 15
Lys Asp Thr Asp Cys Pro Gly Ile Lys Lys C,ys Cys Glu Gly Ser Cys
20 25 30
Gly Met Ala Cys Phe Val Pro Gln
35 40
<210>8
<211>24
<212>PRT
<213>Homo sapiens
<400> 8
Met Lys Leu Gly Ala Phe Leu Leu Leu Val Ser Leu Ile Thr Leu Ser
1 5 10 15
Leu Glu Val Gln Glu Leu Gln Ala
<210>9
<211>6
<212>PRT
<213>Homo Sapiens
<400> 9
Leu Gln Leu Leu Gly Thr
1 5
<210>10
<211>6
<212>PRT
<213>Homo sapiens
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WO 99/63091 PCTNS99/125d5
6
<400> 10
Asp Arg Leu Leu Gly Thr
1 5
<210>11
<211>371
<212>DNA
<213>Mus musculus
<400> 11
gcagcatgcaagctaggagccttccttctgttggtgtccctcatcaccctcagcctagag60
gtacaggagctgcaggctgcagtgagacctctgcagctattaggcacctgtgctgagctc120
tgccgtggtgactgggactgtgggccagaggaacaatgtgtcagtattggatgcagtcac180
atctgtactacaaactaaaaacagcttctacctggaaaaaaaaatgtgtctgtttggagc240
tctgtgaccaagaaaacagttgaaaatggaggccatgt:atggagattacaagcagcacag300
tggagtgggacaaggagttgtttcttttaataaatcat;taatgtaaaagtcaaaaaaaaa360
aaaaaaaattg 371
<210>12
<211>448
<212>DNA
<2I3>Homo Sapiens
<400>
12
cagcaacatgaagttggcagccttcctcctcctgtgatcctcatcatcttcagcctagag 60
gtacaagagcttcaggctgcaggagaccggcttttgggtacctgcgtcgagctctgcaca 120
ggtgactgggactgcaaccccggagaccactgtgtcagcaatgggtgtggccatgagtgt 180
gttgcagggtaaggacaggtaaaaacaccaggccctccctgctttctgaaacgttgttca 240
gtctagatgaagagttatcttaaggatcatctttccctaagatcgtcatcccttcctgga 300
gttcctatcttccaagatgtgactgtctggagttccttgactaggaagatggatgaaaac 360
agcaagcctgtggatggagactacaggggatatgggaggcagggaagaggggttgtttct 420
tttaataaatcatcattgttaaaaagca 448
<210>13
<211>569
<212>DNA
<213>Homo Sapiens
<400>
13
gaggacccagggtacacagggtgggtggctattctccagaaatgtcagtttctgggcagg 60
gcttaggtgtctgcagtccctagtcccacccctggccttgcattccagctcagcgagtgg 120
aaggtataaatttcagctgctctcagccctgctgtgtttttccaaagccttccaacagca 180
acatgaagttggcagccttcctcctcctgtgatcctcatcatcttcagcctagaggtaca 240
agagcttcaggctgcaggaagaccggcttttgggtacctgcgtcgagctctgcacaggtg 300
actgggactgcaaccccggagaccactgtgtcagcaatgggtgtggccatgagtgtgttg 360
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7
cagggtaagg acagatgaag agttatctta aggatcatct ttccctaaga tcgtcatccc 420
ttcctggagt tcctatcttc caagatgtga ctgtctggag ttccttgact aggaagatgg 480
atgaaaacag caagcctgtg gatggagact acagggggat attggaagca aggaagaggg :~40
gttgttcttt taataaatca tcattgtta X69
<210>14
<211>4
<212>PRT
<213>Homo Sapiens
<400> 14
Ala Ala Pro Val
1
<210>15
<211>4
<212>PRT
<213>Homo sapiens
<400> 15
Ala Ala Pro Phe
1
<210>16
<211>18
<212>PRT
<213>Homo Sapiens
<400> 16
Thr Cys Ala Glu Leu Cys Arg Gly Asp Trp Asp Cys Gly Pro Glu Glu
1 5 10 15
Gln Cys
<210>17
<211>24
<212>PRT
<213>Homo Sapiens
<220>
<221> VARIANT
<222> (0)...(0)
<223> Xaa can be any amino acid residue except for
cysteine
<400> 17
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8
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa
1 5 10 15
Xaa Cys Xaa Cys Xaa Xaa Xaa Cys
<210>18
<211>16
<212>PRT
<213>Homo Sapiens
<220>
<221> VARIANT
<222> (0)...(0)
<223> Xaa is any amino acid residue except for cysteine
<400> 18
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys
1 5 10 15
<210>19
<211>17
<212>PRT
<213>Homo Sapiens
<220>
<221> VARIANT
<222> (0)...(0)
<223> Xaa is any amino acid residue except for cysteine.
<400> 19
Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Cys
<210>20
<211>18
<212>PRT
<213>Homo Sapiens
<220>
<221> VARIANT
<222> (0)...(0)
<223> Xaa is any amino acid residue except for cysteine.
<400> 20
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9
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
1 5 10 15
Xaa Cys
<210>21
<211>14
<212>PRT
<213>Homo Sapiens
<220>
<221> VARIANT
<222> (0)...(0)
<223> Xaa is any amino acid residue except for cysteine.
<400> 21
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys
1 5 10
<210>22
<211>15
<212>PRT
<213>Homo sapiens
<220>
<221> VARIANT
<222> (0)...(0)
<223> Xaa is any amino acid residue except for cysteine.
<400> 22
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys
1 5 10 15
<210>23
<211>26
<212>PRT
<213>Homo sapiens
<220>
<221> VARIANT
<222> (0)...(0)
<223> Xaa is any amino acid residue except for cysteine.
<400> 23
Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
1 5 10 15
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io
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys
20 25
