ZA200109295B

ZA200109295B - Adipocyte complement related protein homolog zacrp5.

Info

Publication number: ZA200109295B
Application number: ZA200109295A
Authority: ZA
Inventors: Christopher S Piddington; Paul O Sheppard
Original assignee: Zymogenetics Inc
Priority date: 1999-05-27
Filing date: 2001-11-12
Publication date: 2002-06-26
Also published as: EP1185643A1; AU4855800A; MXPA01012092A; JP2003501027A; CA2378737A1; WO2000073444A1

Description

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’ . .

Description

ADIPOCYTE COMPLEMENT RELATED PROTEIN HOMOLOG ZACRP5

BACKGROUND OF THE INVENTION

Cell-cell and cell-extracellular matrix interactions allow for exchange of information between, and coordination among, various cells of a multi-cellular organism and are fundamental for most biological processes. These interactions play a role in everything from fertilization to death. Such interactions are essential during development and differentiation and are critical for the function and protection of the organism.

For example, interaction between the «cell and its environment 1s necessary to initiate and mediate tissue - remodeling. Tissue remodeling may be initiated, for example, in response to many factors including physical injury, cytotoxic injury, metabolic stress or developmental stimuli. Modulation between pathology and healing (or metabolic optimization) may be done, in part, by the interaction of stimulated cells with the extracellular matrix as well as the local solvent.

A family of proteins that plays a role in the : interaction of cells with their environment, and appear to act at the interface of the extracellular matrix and the . cell, are the adipocyte complement related proteins.

These proteins include, Acrp30, a 247 amino acid polypeptide that is expressed exclusively by adipocytes.

The Acrp30 polypeptide is composed of a amino-terminal signal sequ=ance, a 27 amino acid stretch of no known homology, 22 perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa collagen repeats and a carboxy terminal globular domain.

See, Scherer et al., J. Biol. Chem. 270(45): 26746-9, 1995 and International Patent Application No. WO 96/39429.

Acrp30, an abundant human serum protein regulated by insulin, shares structural similarity, particularly in the

: ; carboxy-terminal globular domain, to complement factor Clg and to a summer serum protein of hibernating Siberian chipmunks (Hib27). Expression of Acrp30 is induced over 100-fold during adipocyte differentiation. Acrp30 is suggested for use in modulating energy balance and in identifying adipocytes in test samples.

Additional members include =zsig37, a 281 amino acid residue protein expressed predominantly in heart, aorta and placenta, having 14 collagen repeats and a Clg globular domain similar to ACRP30 (WO 99/04000). Zsig37 has been shown to inhibit complement activity, binds to

SK5 fibroblasts and stimulates proliferation at concentrations known to initiate Clg-cell responses.

Zsigl37 also specifically inhibits collagen activation of platelets in human whole blood and platelet rich plasma in a dose dependent manner (copending US Patent Application, 09/253,604) . Also included is zsig39, a 243 amino acid residue protein expressed predominantly in heart and small intestine, having 22 or 23 collagen repeats and a Clg domain similar to ACRP30 and zsig37 (99/10492).

These proteins all share a Clg domain.

Complement factor Clg consists of six copies of three related polypeptides (A, B and C chains), with each . polypeptide being about 225 amino acids long with a near amino-terminal collagen domain and a carboxy-terminal globular region. Six triple helical regions are formed by the collagen domains of the six A, six B and six C chains, forming a central region and six stalks. A globular head portion is formed by association of the globular carboxy terminal domain of an A, a B and a C chain. Clg is therefore composed of six globular heads linked via six collagen-like stalks to a central fibril region. Sellar et al., Biochem. J. 274: 481-90, 1991. This configuration is often referred to as a bouquet of flowers. Acrp30 has a similar bouquet structure formed from a single type of polypeptide chain. The Clg globular domain of ACRP30 has been determined to have a 10 beta strand ~“jelly roll"

- - 3- I" ) , . . “topology (Shapiro and Scherer, Curr. Biol. 8:335-8, 1998).

The structural elements such as folding topologies, ‘conserved residues and ‘similar trimer interfaces and intron positions are homologous to the tumor necrosis : 5 factor family suggesting a link between the TNF and Clg families. Zsig39 and 2zsig37 share this structure and homology as well.

Proteins that play a role in cellular : interaction, such as transcription factors and hormones are useful diagnostic and therapeutic agents. Proteins ‘that mediate specific interactions, such a remodeling, would be particularly useful. The present invention : provides such polypeptides for these and other uses that " should be apparent to those skilled in the art from the teachings herein. “SUMMARY OF THE INVENTION

Within one aspect, the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 70-252 of SEQ ID NO:2, wherein said “sequence comprises: Gly-Xaa-Xaa and Gly-Xaa-Pro collagen repeats forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain. ;

Within one embodiment the polypeptide is at least 90% identical in amino acid sequence to residues 18-252 of SEQ .

ID NO:2. Within a related embodiment any differences between said polypeptide and SEQ ID NO:2 are due to conservative amino acid substitutions. Within another embodiment the collagen-like domain consists of 14 Gly-

Xaa-Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeat. Within yet another embodiment the polypeptide comprises: an amino terminal region; 14 Gly-Xaa-Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeat forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain comprising 10 beta strands corresponding to amino acid residues 119-123,

+ 4 ; y “ 141-143, 149-152, 156-158, 162-173, 178-184, 189-196, 200- 211, 216-221 and 240-244 of SEQ ID NO:2. Within a further embodiment the polypeptide specifically binds with an antibody that specifically binds with a polypeptide of SEQ

ID NO:2. Within another embodiment the collagen-like domain comprises amino acid residues 70-111 of SEQ ID

NO:2. Within another embodiment the Clg domain comprises amino acid residues 112-252 of SEQ ID NO:2. Within other embodiments the polypeptide comprises residues 70-252 of

SEQ ID NO:2, residues 18-252 of SEQ ID NO:2 or 1-252 of

SEQ ID NO:2. Within another embodiment the polypeptide is complexed by intermolecular disulfide bonds to form a homotrimer. Within yet another embodiment the polypeptide is complexed by intermolecular disulfide bonds, to one or more polypeptides having a collagen-like domain, to form a heterotrimer. Within a further embodiment the polypeptide is covalently linked at the amino or carboxyl terminus to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores.

The invention also provided an isolated polypeptide selected from the group consisting of: a) a polypeptide consisting of a sequence of amino acid residues from residue 70 to residue 111 of SEQ ID NO:2; ’ and b) a polypeptide consisting of a sequence of amino acid residues from residue 112 to residue 252 of SEQ ID . NO:2.

Within another aspect the invention provides a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion consisting of a polypeptide selected from the group consisting of: a) polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 70-252 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa and Gly-Xaa-

Pro collagen repeats forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl- terminal Clg domain; b) polypeptide comprising: an amino

» — 5 I JE - IRR [ES . . . terminal region; 14 Gly-Xaa-Xaa collagen repeats and 1

Gly-Xaa-Pro collagen repeat forming a collagen-like : : "domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain comprising 10 beta strands - 5 corresponding to amino acid residues 119-123, 141-143, - 149-152, 156-158, 162-173, 178-184, 189-196, 200-211, 216- 221 and 240-244 of SEQ ID NO:2; c) a portion of the zacrps polypeptide as shown in SEQ ID NO:2, comprising the collagen-like domain or a portion of the collagen-like domain capable of trimerization or oligomerization; d) a portion of the zacrpb polypeptide as shown in SEQ ID NO:2, comprising the Clg domain or an active portion of the Clg domain; or e) a portion of the zacrp2 polypeptide as shown in SEQ ID NO:2 comprising of the collagen-like domain and the Clg domain; and said second portion comprising another polypeptide. Within a related embodiment the first - portion is selected from the group consisting of: a) a y polypeptide consisting of the sequence of amino acid residue 70 to amino acid residue 111 of SEQ ID NO:2; b) a polypeptide consisting of the sequence of amino acid residue 112 to amino acid residue 252 of SEQ ID NO:2; c) a polypeptide consisting of the sequence of amino acid residue 70 to 252 of SEQ ID NO:2; d) a polypeptide consisting of the sequence of amino acid residue 18 to 252 ’ of SEQ ID NO:2; and e) a polypeptide consisting of the sequence of amino acid residue 1 to 252 of SEQ ID NO:2. .

The invention also provides a polypeptide as described above; in combination with a pharmaceutically acceptable vehicle.

Within another aspect the invention provides a method of producing an antibody to a polypeptide comprising: inoculating an animal with a polypeptide selected from the group consisting of: a) polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 70- 252 of SEQ ID NO:2, wherein said sequence comprises: Gly-

Xaa-Xaa and Gly-Xaa-Pro collagen repeats forming a

° 6 4 ] hl collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain; b) polypeptide comprising: an amino terminal region; 14 Gly-

Xaa-Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeat forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain comprising 10 beta strands corresponding to amino acid residues 119-123, 141-143, 149-152, 156-158, 162-173, 178- 184, 189-196, 200-211, 216-221 and 240-244 of SEQ ID NO:2; c¢) a portion of the zacrp5 polypeptide as shown in SEQ ID

NO:2, comprising the collagen-like domain or a portion of the collagen-like domain capable of trimerization or oligomerization; d) a portion of the zacrp5 polypeptide as shown in SEQ ID NO:2, comprising the Clg domain or an active portion of the Clg domain; or e) a portion of the zacrp5 polypeptide as shown in SEQ ID NO:2 comprising of ‘the collagen-like domain and the Clg domain; and wherein said polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from : 20 the animal.

Also provides are antibodies or antibody fragments that specifically binds to a polypeptide as described above. Within one embodiment the antibody is ; selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; «c¢) humanized ; antibody derived from Db); and d) human monoclonal antibody. Within another embodiment the antibody fragment is selected from the group consisting of F(ab'), F(ab),

Fab', Fab, Fv, scFv, and minimal recognition unit. Within another embodiment is provided an anti-idiotype antibody that specifically binds to the antibody described above.

Also provided by the invention is a binding protein that specifically binds to an epitope of a polypeptide as described above.

Within another aspect the invention provides an isolated polynucleotide encoding a polypeptide as described above. Also provided herein is an isolated

I Ce . 7 ee - Ce h le me . “ . polynucleotide selected from the group consisting of: a) a sequence of nucleotides from nucleotide 1 to nucleotide

LL 759 of SEQ ID NO:1; b) a sequence of nucleotides from nucleotide 52 to nucleotide 759 of SEQ ID NO:1; c¢) a - 5 :sequence of nucleotides from nucleotide 208 to nucleotide - 333 of SEQ ID NO:1; 4d) a sequence of nucleotides from nucleotide 334 to nucleotide 759 of SEQ ID NO:1; e) a : sequence of nucleotides from nucleotide 208 to nucleotide 759 of SEQ ID NO:1; ff) a sequence of nucleotides from nucleotide 52 to nucleotide 111 of SEQ ID NO:1; g) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of residues 70 to 111 of SEQ ID NO:2; h) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of residues 112 to 252 of SEQ ID : 15 NO:2; i) a polynucleotide that remains hybridized, following stringent wash conditions, to a polynucleotide ; consisting of the nucleotide sequence of SEQ ID NO:1, or : the complement of SEQ ID NO:1; 3j) nucleotide sequences

B complementary to a), b), ¢), 4), e), £), g), h) or i) and k) degenerate nucleotide sequences of g) or h).

Also provided is an isolated polynucleotide ‘encoding a fusion protein as described above.

The invention also provided an isolated polynucleotide consisting of the sequence of nucleotide 1 to nucleotide 756 of SEQ ID NO:12.

Within another aspect the invention provides an } expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator. Within one embodiment the DNA segment further encodes a secretory signal sequence operably linked to said polypeptide. Within a related embodiment the secretory signal sequence comprises residues 1-17 of SEQ

ID NO:2.

The invention also provides a cultured cell into which has been introduced an expression vector as described above, wherein said cell expresses said

} WQO 00/73444 PCT/US00/13608 polypeptide encoded by said DNA segment. Within one embodiment the cultured cell further includes one or more expression vectors comprising DNA segments encoding polypeptides having collagen-like domains.

Within another aspect the invention provides a method of producing a protein comprising: culturing a cell into which has been introduced an expression vector as described above; whereby said cell expresses said protein encoded by said DNA segment; and recovering said expressed protein. Within one embodiment the expressed protein is a homotrimer. Within another embodiment the expressed protein is a heterotrimer.

Within another aspect the invention provides a method of detecting the presence of zacrpS gene expression in a biological sample, comprising: (a)contacting a zacrpb nucleic acid probe under hybridizing conditions with either (i) test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe consists of a nucleotide sequence comprising a portion of the nucleotide sequence of the nucleic acid molecule as described above, or complements thereof, and (b) detecting . the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of zacrp5 RNA in the biological sample.

Within another aspect is provided a method of detecting the presence of zacrpS5 in a biological sample, comprising: (a) contacting the biological sample with an antibody, or an antibody fragment, as described above, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.

= 9 : oT

BRIEF DESCRIPTION OF THE DRAWING

The Figure illustrates a multiple alignment of

B “and zacrp5 polypeptide of the present invention and = adipocyte complement related protein homolog zsig37 (SEQ - 5 "ID NO:3, WO 99/04000), human ACRP30 (ACR3 HUMAN) (SEQ ID

NO: 4, Maeda et al., Biochem. Biophys. Res. Commun . - 221:286-9, 1996), adipocyte complement related protein

B homolog zsig39 (SEQ ID NO:5, WO 99/10492) and human Clg C - (SEQ ID NO:6, Sellar et al., Biochem J. 274:481-90, 1991 and Reid, Biochem J. 179:361-71, 1979). The multiple “alignment performed using a Clustalx multiple alignment tool with the default settings: Blosum Series Weight : Matricies, Gap Opening penalty:10.0, Gap Extension penalty:0.05. Multiple alignments were further hand tuned ) 15 before computing percent identity.

N DETAILED DESCRIPTION OF THE INVENTION

. . Prior to setting forth the invention in detail, : it may be helpful to the understanding thereof to define the following terms.

The term “Taffinity tag'' is used herein to denote a peptide segment that can be attached to a polypeptide to provide for purification or detection of the polypeptide or provide sites for attachment of the . polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent 1s available can be used as an affinity tag.

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), substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988; available from Eastman Kodak Co., New Haven, CT), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general Ford et al., Protein

Expression and Purification 2: 95-107, 1991. DNAs a 10 $ In hd encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).

The term "allelic variant" denotes 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 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 1s 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 and proteins. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a protein is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete protein.

The term ““complement/anti-complement pair® . denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For } 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 dissociation of the complement /anti-complement pair is desirable, the complement /anti-complement pair preferably has a binding affinity of <109 M™ .

The term “complements of a polynucleotide molecule!’ is a polynucleotide molecule having a complementary base sequence and reverse orientation as

Co 11 So Ce compared to a reference sequence. For example, the . sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT a 3. : ol The term ““contig'' denotes a polynucleotide that i 5 "has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to “Toverlap'' a given stretch of polynucleotide sequence either in their entirety or along a partial - stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-

ATGGCTTAGCTT-3' are 5'-TAGCTTgagtct-3"' and 3'- gtcgacTACCGA-5"'. - The term ~“degenerate nucleotide sequence!’ denotes a sequence of nucleotides that includes one or “ 15 more degenerate codons (as compared to a reference i” 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" denotes a DNA molecule, linear or circular, that comprises a segment . encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.

Such additional segments may include promoter and - terminator sequences, and may optionally include one or more origins of replication, one or more selectable } markers, an enhancer, a polyadenylation signal, and the like. 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 genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include <¢cDNA and genomic clones. Isolated DNA iy 12 molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated 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 316:774-78, 1985).

An “Tisolated!'' polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term ““isolated'' does not exclude the presence of the same

B polypeptide in alternative physical forms, such as trimers or alternatively glycosylated or derivatized forms.

The term "operably linked", when referring to

DNA segments, denotes that the segments are arranged so : 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 functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation. “Paralogs! are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a-globin, pB-globin, and myoglobin are paralogs of each other.

The term "polynucleotide" denotes a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the S5' to the 3' end.

BE 13. - - SL . - Cee

Polynucleotides include RNA and DNA, and may be isolated . © from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. i "Sizes of polynucleotides are expressed as base pairs (abbreviated ““bp''), nucleotides (“"nt''), or kilobases a {("kb''). Where the context allows, the latter two terms on may describe polynucleotides that are single-stranded or = .. double-stranded. When the term is applied to double- oy . 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

OL 15 cleavage; thus all nucleotides within a double-stranded — polynucleotide molecule may not be paired. Such unpaired gs . ends will in general not exceed 20 nt in length. wv A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced 20 naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as . "peptides". “Probes and/or primers'' as used herein can be

RNA or DNA. DNA can be either c¢DNA or genomic DNA. - 25 Polynucleotide probes and primers are single or double- stranded DNA or RNA, generally synthetic oligonucleotides, ) but may be generated from cloned cDNA or genomic sequences or its complements. Analytical probes will generally be at least 20 nucleotides in length, although somewhat 30 shorter probes (14-17 nucleotides) can be used. PCR primers are at least 5 nucleotides in length, preferably or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is targeted for analysis. For gross analysis of genes, a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide,

fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and

Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.

The term "promoter" denotes a portion of a gene containing DNA sequences that provide for the binding of

RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5° non-coding regions of genes. : The term "receptor" denotes a cell-associated protein that binds to a bicactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.

Membrane-bound receptors are characterized by a multi- domain structure comprising an extracellular 1ligand- " 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 : 20 effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked 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 inositol lipids and hydrolysis of phospholipids. Most nuclear receptors also exhibit a multi-domain structure, including an amino-terminal, transactivating domain, a DNA binding domain and a ligand binding domain. 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,

IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).

. 15 . - - LE “ v }

The term "secrétory signal sequence" denotes a

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 5° of a cell in which it is synthesized. The larger peptide = is commonly cleaved to remove the secretory peptide during - transit through the secretory pathway. + ) A "soluble receptor" is a receptor polypeptide a that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains. Soluble receptors can comprise additional amino acid residues, a. such as affinity tags that provide for purification of the

B polypeptide or provide sites for attachment of the

N 15 polypeptide to a substrate, or immunoglobulin constant - region sequences. Many cell-surface receptors have d ~ naturally occurring, soluble counterparts that are + N produced by proteolysis or translated from alternatively spliced mRNAs. ‘Receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.

The term ““splice variant'' is 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 1s 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 “Tapproximately'' X, the stated value of X will be understood to be accurate to +10%.

The present invention is based in part upon the discovery of a novel DNA sequence that encodes a polypeptide having homology to an adipocyte complement related protein zsig37 (WO 99/04000). The novel DNA sequence encodes a polypeptide having an amino-terminal signal sequence, an adjacent N-terminal region of non- homology, a collagen domain composed of 14 collagen repeats and a carboxy-terminal globular-like Clg domain.

The general polypeptide structure set forth above is shared by 2zsig37, 2sig39, Acrp30 and Clg C (see Figure).

Other regions of homology, found in the carboxy-terminal globular Clg domain in the aligned proteins, are identified herein as useful primers for searching for other family members. Zsig37, zsig39, Acrp30 and Clg C, for example, would be identified in a search using the primers. Intra-chain disulfide bonding may involve the cysteines at residues 26, 29, 30, 112 and 158 of SEQ ID

NO:2. : The novel zacrp5 polypeptides of the present invention were initially identified in an unfinished . genomic sequence. The genomic sequence is located on locus HS349El1l which is derived from chromosome 16. SEQ

ID NO:7 provides the identified exon 1 of zacrp5 beginning at the start codon, nucleotides 1-208, intron 1, nucleotides 209-870 and exon 2 ending with the stop codon, nucleotides 871-1421. With stringently called exon predictions, the novel adipocyte complement related factor was found to be homologous to another adipocyte complement related factor, zsig37 (WO 99/04000). Percent identity at the amino acid level over the whole molecule between : zacrp5 and other family members is shown in Table 1A. The percent identity over the Clg domain only is shown in

Table 1B. The alignments were performed using a Clustalx multiple alignment tool with the default settings: Blosum

~ 17 i. oo " Series Weight Matricies, Gap Opening penalty:10.0, Gap

Extension penalty:0.05. Multiple alignments were further hand tuned before computing percent identity. Percent identity is the total number of identical residues over 5" the length of the overlap.

The nucleotide sequence of zacrp5 is described . in SEQ ID NO:1, and its deduced amino acid sequence is described in SEQ ID NO:2. As described generally above, the zacrp5 polypeptide includes a signal sequence, ranging from amino acid 1 (Met) to amino acid residue 17 (Ala) of

SEQ ID NO:2, nucleotides 1-51 of SEQ ID NO:1. The mature polypeptide therefore ranges from amino acid 18 (Trp) to amino acid 252 (Leu) of SEQ ID NO:2, nucleotides 52 to 759 of SEQ ID NO:1. Within the mature polypeptide, an N- terminal region of no known homology is found, ranging between amino acid residue 18 (Trp) and 69 (Lys) of SEQ ID

NO:2, nucleotides 52-207 of SEQ ID NO:1. In addition, a collagen-like domain is found between amino acid 70 (Gly) and 111 (Ala) of SEQ ID NO:2, nucleotides 208 to 333 of

SEQ ID NO:1. In the collagen-like domain, 1 perfect Gly-

Xaa-Pro and 13 imperfect Gly-Xaa-Xaa collagen repeats are observed. Acrp30 contains 22 perfect or imperfect collagen repeats, 2zsig37 has 14 collagen repeats and zsig39 has 22 or 23 collagen repeats. Proline residues found in this domain at amino acid residue 90 and 108 of

SEQ ID NO:2 may be hydroxylated. The =zacrp5 polypeptide also includes a carboxy-terminal Clg domain, ranging from about amino acid 112 (Cys) to 252 (Leu) of SEQ ID NO:2Z, nucleotides 334 to 759 of SEQ ID NO:1. There is a fair amount of conserved structure within the Clg domain to enable proper folding. An imperfect Clg aromatic motif (F-X (5) - [ND] -X (4) - [FYWL] -X(6) -F-X (5) -G-X-Y-X-F-X- [FY] {SEQ

ID NO:8) is found between residues 138 (Phe) and 168 (Leu) of SEQ ID NO:2 that does not match the motif perfectly. X : represents any amino acid residue and the number in parentheses () indicates the amino acid number of residues. The amino acid residues contained within the square parentheses [] restrict the choice of amino acid residues at that particular position. The final residue of this motif is Leu instead of Phe or Tyr. Zacrp5s . polypeptide, human 2zsig37, human zsig39, human Clg C and

Acrp30 appear to be homologous within the collagen domain and in the Clg domain, but not in the N-terminal portion of the mature polypeptide.

Another aspect of the present invention includes zacrp5 polypeptide fragments. Preferred fragments include those containing the collagen-like domain of zacrps polypeptides, ranging from amino acid 1 (Met), 18 (Trp) or 70 (Gly) to amino acid 111 (Ala) of SEQ ID NO:2, a portion of the =zacrpb5 polypeptide containing the collagen-like domain or a portion of the collagen-like domain capable of trimerization or oligomerization. As used herein the term ~“collagen'' or ““collagen-like domain¥ refers to a series of repeating triplet amino acid sequences, ~~ repeats'' or

““collagen repeats'' represented by the motifs Gly-Xaa-Pro or Gly-Xaa-Xaa, where Xaa is any amino acid reside. Such © domains may contain as many as 14 collagen repeats or ° more. Moreover, such fragments or proteins containing such collagen-like domains may form heteromeric constructs, usually trimers. Structural analysis and homology to other collagen-like domain containing proteins indicates that zacrpS polypeptides, fragments or fusions comprising the collagen-like domain can complex with other collagen domain containing polypeptides to form homotrimers and : heterotrimers. . These collagen-like domain containing fragments are particularly useful in the study of collagen trimerization or oligomerization or in formation of fusion proteins as described more fully below. Polynucleotides encoding such fragments are also encompassed by the . present invention, including the group consisting of (a) polynucleotide molecule comprising a sequence of : nucleotides as shown in SEQ ID NO:1 from nucleotide 1, 52 or 208 to nucleotide 333; (b) polynucleotide molecules : that encode a zacrp5 polypeptide fragment that is at least © 80% identical to the amino acid sequence of SEQ ID NO:2 : from amino acid residue 70 (Gly) to amino acid residue 111 (Ala); (c) molecules complementary to (a) or (b); and (4d) . degenerate nucleotide sequences encoding a zacrp5s polypeptide collagen-like domain fragment.

Other collagen-like domain containing polypeptides include members of the adipocyte complement related protein family, such as zsig37, zsig39 and ACRP30, for example. The trimeric proteins of the present invention are formed by intermolecular disulfide bonds formed between conserved cysteine residues within the polypeptides. The present invention therefore provides zacrp6é polypeptides complexed by intermolecular disulfide bonds to form homotrimers. The invention further provides zacrp5 polypeptides complexed by intermolecular disulfide bonds to other polypeptides having a collagen-like domain, to form heterotrimers.

Other preferred fragments include the globular

Clg domain of zacrp5 polypeptides, ranging from amino acid 112 (Cys) to 252 (Leu) of SEQ ID NO:2, a portion of the zacrp5 polypeptide containing the Clg domain or an active portion of the Clg domain. Other Clg domain containing proteins include zsig37 (WO 99/04000), zsig39 (WO 99/10492), Clg A, B and C (Sellar et al., ibid., Reid, ibid., and Reid et al., Biochem. J. 203: 559-69, 1982), chipmunk hibernation-associated plasma proteins HP-20, HP- 25 and HP-27 (Takamatsu et al., Mol. Cell. Biol. 13: 1516- 21, 1993 and Kondo & Kondo, J. Biol. Chem. 267: 473-8, 1992), human precerebellin (Urade et al., Proc. Natl.

Acad. Sci. USA 88:1069-73, 1991), human endothelial cell multimerin (Hayward et al., J. Biol. Chem. 270:18246-51, 1995) and vertebrate collagens type VIII and X (Muragaki : et al., Bur. J. Biochem. 197:615-22, 1991).

The globular Clg domain of ACRP30 has been determined to have a 10 beta strand ““jelly roll'' topology (Shapiro and Scherer, Curr. Biol. 8:335-8, 1998) that 2 shows significant homology to the TNF family and the zacrp5 sequence as represented by SEQ ID NO:2 contains all : 10 beta-strands of this structure (amino acid residues 119-123, 141-143, 149-152, 156-158, 162-173, 178-184, 189- . 196, 200-211, 216-221 and 240-244 of SEQ ID NO:2). These strands have been designated ~TA'', “TA''"', TB'', “TB'!'!, ~~¢c'', “tp'', “SEY, TTF'', “°G'' and "TH'' respectively.

Zacrp5 has two receptor binding loops, at amino acid residues 125-151 and 183-196. Amino acid residues 162 (Gly), 164 (Tyr), 211 (Leu) and 241 (Phe) appear to be conserved across the superfamily including CD40, TNFa,

TNFf3, ACRP30 and zacrpsS.

These fragments are particularly useful in the study or modulation of cell-cell or cell-extracellular matrix interaction. Anti-microbial activity may also be present in such fragments. The homology to TNF proteins

-. suggests such fragments would be useful in obesity-related insulin resistance, immune regulation, inflammatory response, apoptosis and osteoclast maturation.

Polynucleotides encoding such fragments are also 5: encompassed by the present invention, including the group consisting of (a) polynucleotide molecules comprising a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 334 to nucleotide 252; (b) polynucleotide - molecules that encode a zacrp5 polypeptide fragment that is at least 80% identical to the amino acid sequence of

SEQ ID NO:2 from amino acid residue 112 (Phe) to amino : acid residue 252 (Leu); (c) molecules complementary to (a) or (b); and (d) degenerate nucleotide sequences encoding a zacrp5 polypeptide Clg domain fragment.

Other zacrp5 polypeptide fragments of the present invention include both the collagen-like domain and the Clg domain ranging from amino acid residue 70 (Gly) to 252 (Leu) of SEQ ID NO:2. Polynucleotides encoding such fragments are also encompassed by the present invention, including the group consisting of (a) polynucleotide molecules comprising a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 208 to "nucleotide 759; (b) polynucleotide molecules that encode a zacrp5 polypeptide fragment that is at least 80% identical - to the amino acid sequence of SEQ ID NO:2 from amino acid residue 70 (Gly) to amino acid residue 252 (Leu); (c) } molecules ccmplementary to (a) or (b); and (d) degenerate nucleotide sequences encoding a zacrpSs polypeptide collagen-like domain-Clg domain fragment.

The highly conserved amino acids, particularly those in the carboxy-terminal Clg domain of the =zacrps polypeptide, can be used as a tool to identify new family members. For instance, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved motifs from RNA obtained from a variety of tissue sources. In particular, highly degenerate primers and their complements designed from conserved sequences are useful for this purpose. In particular, the following primers are useful for this purpose:

Degenerate primer sequence encoding amino acid residues 161-166 of SEQ ID NO:2

MSN GGN NTN TAY TWY YT (SEQ ID NO:9)

Degenerate primer sequence encoding amino acid residues 214-219 of SEQ ID NO:2

SRN GAN VVN GTN TGG BT (SEQ ID NO:10)

Degenerate primer sequence encoding amino acid residues 240-245 of SEQ ID NO:2

RYN TTY WSN GGN YWY YT (SEQ ID NO:11)

Probes corresponding to complements of the polynucleotides . set forth above are also encompassed.

The present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zacrp5 polypeptides disclosed herein. In order to isolate the polynucleotide of SEQ ID NO:1 from various tissues, probes and/or primers are designed from . the exon predicted regions of SEQ ID NO:1 and SEQ ID NO:7.

Tissues expressing zacrp5 could be identified either through hybridization (Northern Blots) or by reverse transcriptase (RT) PCR. Libraries are then generated from tissues which appear to show expression of zacrp5. Single clones from such libraries are then identified through hybridization with the probes and/or by PCR with the primers as described herein. Conformation of the zacrps cDNA sequence can be verified using the sequences provided herein.

Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:12 is a degenerate

DNA sequence that encompasses all DNAs that encode the zacrp5 polypeptide of SEQ ID NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID

.NO:11 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T.

Thus, zacrp5 polypeptide- encoding polynucleotides comprising nucleotide 1 to nucleotide 756 of SEQ ID NO:12 and their RNA equivalents are contemplated by the present invention.

Table 2 sets forth the one-letter codes used within SEQ ID NO:12 to denote degenerate nucleotide positions. ""Resolutions'' are the nucleotides denoted by a code letter. ““Complement'' indicates the code for the complementary nucleotide (s) . For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.

oo TABLE 2

Nucleotide Resolution Complement Resolution

A A T T

C C G G

G G C C

T T A A

R A|G Y CIT

Y CIT R A|G

M A|C K G|T

K G|T M A|C

S C|G S Cla

W AT W AIT

H AlC|T D AlG)T

B C|G|T J) A|C|G

V A|CI|G B CIG|T

D AIG|T H AICIT

N A|CIG|T N AlCIGIT

The degenerate codons used in SEQ ID NO:12, encompassing all possible codons for a given amino acid, are set forth in Table 3.

. : 25 Co - RE.

TABLE 3

One

Amino Letter Codons Degenerate

Acid Code Codon

Cys C TGC TGT TGY

Ser S AGC AGT TCA TCC TCG TCT WSN

Thr T ACA ACC ACG ACT ACN

Gly G GGA GGC GGG GGT GGN

Asn N AAC AAT AAY

Gln Q CAA CAG CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAA AAG AAR

Met M ATG ATG

Ile I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

Val V GTA GTC GTG GTT GTN

Phe F TTC TTT TTY

Tyr Y TAC TAT TAY

Trp W TGG TGG :

Ter } TAA TAG TGA TRR

Asn|Asp B RAY }

Glu|GIn Z SAR

Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.

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 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J.

Mol. Biol. 158:573-97, 1982. As used herein, the term “preferential 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 (See Table 3). 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, yeast, viruses or bacteria, different Thr 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. Therefore, the degenerate codon sequence

B disclosed in SEQ ID NO:12 serves as a template for optimizing expression of polynucleotides in various cell ‘types and species commonly used in the art and disclosed : herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.

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 . 15 invertebrate species. Of particular interest are zacrp5 a polypeptides from other mammalian species, including : murine, porcine, ovine, bovine, canine, feline, equine, : and other primate polypeptides. Orthologs of human zacrpS : 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 mRNA obtained from a tissue or cell type that expresses zacrp5 as disclosed herein. Suitable sources of 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 zacrp5-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 probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction with primers designed from the representative human zacrp5 sequences disclosed herein. Within an additional method, the <¢DNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to

Claims

95 ee oo a CLAIMS What is claimed is: on 1. An isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 70-252 of SEQ ID NO:2, wherein said sequence comprises: : Gly-Xaa-Xaa and Gly-Xaa-Pro collagen repeats forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain.

- 2. An isolated polypeptide according to claim 1, wherein said polypeptide is at least 90% identical in amino acid sequence to residues 18-252 of SEQ ID NO:2. n - 3. An isolated polypeptide according to claim 2, wherein any differences between said polypeptide and SEQ 1D NO:2 are due to conservative amino acid substitutions.

4. An isolated polypeptide according to claim 2, whérein said collagen-like domain consists of 14 Gly-Xaa-Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeats.

5. An isolated polypeptide according to claim 2, . wherein said polypeptide comprises: an amino terminal region; 14 Gly-Xaa-Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeats forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain comprising 10 beta strands corresponding to amino acid residues 119-123, 141-143, 149-152, 156-158, 162-173, 178-184, 189-196, 200-211, 216-221 and 240-244 of SEQ ID NO:2.

+ 96

6. An isolated polypeptide according to claim 2, wherein said polypeptide specifically binds with an antibody that specifically binds with a polypeptide of SEQ ID NO:2.

7. An isolated polypeptide according to claim 2, wherein said collagen-like domain comprises amino acid residues 70-111 of SEQ ID NO:2.

8. An isolated polypeptide according to claim 2, wherein said Clg domain comprises amino acid residues 112-252 of SEQ ID NO:2.

9. An isolated polypeptide according to claim 1, wherein said polypeptide comprises residues 70-252 of SEQ ID NO: 2.

10. An isolated polypeptide according to claim 2, wherein said polypeptide comprises residues 18-252 of SEQ ID NO:2.

11. An isolated polypeptide according to claim 2, wherein said polypeptide comprises residues 1-252 of SEQ ID NO: 2,

12. An isolated polypeptide according to claim 1, wherein said polypeptide is complexed by intermolecular disulfide bonds to form a homotrimer.

13. An isolated polypeptide according to claim 1, wherein said polypeptide is complexed by intermolecular disulfide bonds, to one or more polypeptides having a collagen-like domain, to form a heterotrimer.

14. An isolated polypeptide according to claim 1, covalently linked at the amino or carboxyl terminus to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores.

EE. S . . 97 . Ce wo 2 15. An isolated polypeptide selected from the group consisting of:

- . a) a polypeptide consisting of a sequence of amino - acid. residues from residue 70 to residue 111 of SEQ ID NO:2; and” b) a polypeptide consisting of a sequence of amino acid residues from residue 112 to residue 252 of SEQ ID NO:2.

16. A fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion consisting of a polypeptide selected from the group consisting of: : “ a) polypeptide according to claim 1; b) polypeptide comprising: an amino terminal region; = 14 Gly-Xaa-Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeats forming a collagen-like domain, wherein Xaa is any - amino acid residue; and a carboxyl-terminal Clg domain ut comprising 10 beta strands corresponding to amino acid residues 119-123, 141-143, 149-152, 156-158, 162-173, 178-184, 189-196, 200-211, 216-221 and 240-244 of SEQ ID NO:2; c) a portion of the zacrp5 polypeptide as shown in SEQ: ID NO:2, comprising the collagen-like domain or a portion of the collagen-like domain capable of trimerization or oligomerization; d) a portion of the zacrp5 polypeptide as shown in SEQ ID NO:2, comprising the Clg domain or an active portion of - the Clg domain; or e) a portion of the =zacrp2 polypeptide as shown in SEQ ID NO:2 comprising of the collagen-like domain and the Clg domain; and said second portion comprising another polypeptide.

17. A fusion protein according to claim 16, wherein said first portion is selected from the group consisting of: a) a polypeptide consisting of the sequence of amino acid residue 70 to amino acid residue 111 of SEQ ID NO:2;

. ® 98 b) a polypeptide consisting of the sequence of amino acid residue 112 to amino acid residue 252 of SEQ ID NO:2; c) a polypeptide consisting of the sequence of amino acid residue 70 to 252 of SEQ ID NO:2; d) a polypeptide consisting of the sequence of amino acid residue 18 to 252 of SEQ ID NO:2; and e) a polypeptide consisting of the sequence of amino acid residue 1 to 252 of SEQ ID NO:2.

18. A polypeptide according to Claim 1; in combination with a pharmaceutically acceptable vehicle.

19. A method of producing an antibody to a polypeptide comprising: inoculating an animal with a polypeptide selected from the group consisting of: a) a polypeptide according to claim 1; b) polypeptide comprising: an amino terminal region; 14 Gly-Xaa-Xaa ccllagen repeats and 1 Gly-Xaa-Pro collagen repeats forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain comprising 10 beta strands corresponding to amino acid residues 119-123, 141-143, 149-152, 156-158, 162-173, 178-184, 189-196, 200-211, 216-221 and 240-244 of SEQ ID NO:2; c) a portion of the zacrpS polypeptide as shown in : SEQ ID NO:2, comprising the collagen-like domain or a portion of the <collagen-like domain capable of trimerization or oligomerization; d) a portion of the zacrp5 polypeptide as shown in SEQ ID NO:2, comprising the Clg domain or an active portion of the Clg domain; or e) a portion of the zacrp5 polypeptide as shown in SEQ ID NO:2 comprising of the collagen-like domain and the Clq domain; and wherein said polypeptide elicits an immune response in the animal to produce the antibody; and

- . » he oo ol 99 } . — Lo. Ce 4 " , isolating the antibody from the animal. : -- 20. An antibody or antibody fragment that specifically binds to a polypeptide according to claim 1.

21. An antibody according to claim 20, wherein said antibody is selected from the group consisting of:

. a) polyclonal antibody; ol b) murine monoclonal antibody; c) humanized antibody derived from b); and d) human monoclonal antibody.

. 22. An antibody fragment according to claim 20, wherein said antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit.

23. An anti-idiotype antibody that specifically binds to said antibody of claim 20. } 24. A binding protein that specifically binds to an epitope of a polypeptide according the claim 1.

25. An isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 70-252 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa and Gly-Xaa-Pro collagen repeats forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain.

26. An isolated polynucleotide according to claim 25, wherein said polypeptide is at least 90% identical in amino acid sequence to residues 18-252 of SEQ ID NO:2.

g 100 o .

27. An isolated polynucleotide according to claim 25, wherein said collagen-like domain consists of 14 Gly-Xaa- Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeats.

28. An isolated polynucleotide according tc claim 25, wherein said polypeptide comprises: an amino terminal region; 14 Gly-Xaa-Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeats forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain comprising 10 beta strands corresponding to amino acid residues 119-123, 141-143, 148-152, 156-158, 162-173, 178-184, 189-196, 200-211, 216-221 and 240-244 of SEQ ID NO:2.

29. An isolated polynucleotide according to claim 25, wherein any differences between said polypeptide and SEQ ID NO:2 are due to conservative amino acid substitutions.

30. An isclated polynucleotide according to claim 25, wherein said polypeptide specifically binds with an antibody that specifically binds with a polypeptide of SEQ ID NO: 2.

31. An isolated polynucleotide according to claim 25, wherein said collagen-like domain comprises amino acid residues 70-111 of SEQ ID NO:2.

32. An isolated polynucleotide according to claim 25, wherein said polypeptide comprises residues 70-252 of SEQ ID NO:2.

33. An isolated polynucleotide according to claim 25, wherein said polypeptide comprises residues 18-252 of SEQ ID NO:2.

34. Air isolated polynucleotide according to claim 25, wherein said polypeptide comprises residues 1-252 of SEQ ID NO:2.

; : 35. An isolated polynucleotide according to * oo claim 25, wherein said polypeptide is covalently linked * at the amino or carboxyl terminus to a moiety selected : from the group consisting of affinity tags, toxins, a radionucleotides, enzymes and fluorophores. - 36. An isolated polynucleotide selected from the group consisting of, a) a sequence of nucleotides from nucleotide 1 to nucleotide 759% of SEQ ID NO:1; b) a sequence of nucleotides from nucleotide 52 to nucleotide 759 of SEQ ID NO:1; - c) a sequence of nucleotides from nucleotide : 208 to nucleotide 333 of SEQ ID NO:1; : : d) a sequence of nucleotides from nucleotide 334 to nucleotide 759 of SEQ ID NO:1; : : e) a sequence of nucleotides from nucleotide 208 to nucleotide 759 of SEQ ID NO:1; - - f) a sequence of nucleotides from nucleotide oe 52 ‘to nucleotide 111 of SEQ ID NO:1; g) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of residues 70 to 111 of SEQ ID NO:2; h) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of residues 112 to 252 of SEQ ID NO:2; ) i) a polynucleotide that remains hybridized, following stringent wash conditions, to a polynucleotide - consisting of the nucleotide sequence of SEQ ID NO:1, or the complement of SEQ ID NO:1; j) nucleotide sequences complementary to a), b), ¢), 4), e), £), g), h) or i) and k) degenerate nucleotide sequences of g) or h).

37. An isolated polynucleotide encoding a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, SUBSHTUTE SHEET (RULE 26)

i . said first portion consisting of a polypeptide ‘selected from the group consisting of: a) polypeptide according to claim 1; b) polypeptide comprising: an amino terminal region; 14 Gly-Xaa-Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeats forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain comprising 10 beta strands corresponding to amino acid residues 119-123, 141-143, 149-152, 156-158, 162-173, 178-184, 189-196, 200-211, 216-221 and 240-244 of SEQ ID NO:2; c) a portion of the zacrp5 polypeptide as shown in SEQ ID NO:2, comprising the collagen-like domain or a portion of the collagen-like domain capable of trimerization or oligomerization; d) a portion of the zacrp5 polypeptide as shown in SEQ ID NO:2, comprising the Clg domain or an active portion of the Clg domain; or e) a portion of the zacrp2 polypeptide as shown in SEQ ID NO:2 comprising of the collagen-1like domain and the Clg domain; and said second portion comprising another polypeptide.

38. An isolated polynucleotide consisting of ) the sequence of nucleotide 1 to nucleotide 756 of SEQ ID NO:12.

39. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide according to claim 1; and a transcription terminator.

40. An expression vector according to claim 39, wherein said DNA segment encodes a polypeptide that : SUBSTITUTE SHEET (RULE 26)

oo } 103 ’ “ is at least 90% identical in amino acid sequence to Co . * . residues 18-252 of SEQ ID NO:2.

41. An expression vector according to claim 39," wherein said collagen-like domain consists of 14 Gly- Xaa-Xaa collagen repeats and 1 Gly-Xaa-Pro collagen repeats.

42. An expression vector according to claim 39, wherein said DNA segment encodes a polypeptide comprising: an amino terminal region; a 14 Gly-Xaa-Xaa collagen repeats and 1 Gly-Xaa- Pror collagen repeats forming a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clg domain comprising 10 beta strands corresponding to amino acid residues 119- 123, 141-143, 149-152, 156-158, 162-173, 178-184, 189- 196, 200-211, 216-221 and 240-244 of SEQ ID NO:2.

43. An expression vector according to claim 39, wherein said collagen-like domain comprises amino acid residues 70-111 of SEQ ID NO:2. : 44. An expression vector according to claim 39, wherein any differences between said polypeptide and ] SEQ ID NO:2 are due to conservative amino acid substitutions.

45. An expression vector according to claim 39, wherein said polypeptide specifically binds with an antibody that specifically binds with a polypeptide of SEQ ID NO:2.

46. An expression vector according to claim 39, wherein said DNA encodes a polypeptide comprising residues 70-252 of SEQ ID NO:2. SUBSTITUTE SHEET (RULE 26)

] 5 o 47. An expression vector according to claim 39, wherein said DNA segment encodes a polypeptide comprising residues 18-252 of SEQ ID NO:2.

48. An expression vector according to claim 39, wherein said DNA segment encodes a polypeptide comprising residues 1-252 of SEQ ID NO:2.

49. An expression vector according to claim 39, wherein said DNA segment further encodes a secretory signal sequence operably linked to said polypeptide.

50. An expression vector according the claim 39, wherein said secretory signal sequence comprises residues 1-17 of SEQ ID NO:2.

51. A cultured cell into which has been introduced an expression vector according to claim 39, wherein said cell expresses said polypeptide encoded by : said DNA segment.

52. A cultured cell according to claim 51, which further includes one or more expression vectors comprising DNA segments encoding polypeptides having collagen-like domains.

53. A method of producing a protein ) comprising: culturing a cell into which has been introduced an expression vector according to claim 39; whereby said cell expresses said protein encoded by said DNA segment; and recovering said expressed protein.

54. A method of producing a protein according to claim 53, wherein said expressed protein is a homotrimer. SUBSTITUTE SHEET (RULE 26)

105 to

55. A method of producing a protein according . 3 -

to . claim 53, wherein said expressed protein is a heterotrimer. : : 56. A method of detecting the presence of zacrp5 gene expression in a biological sample, comprising: (a) contacting a zacrp5 nucleic acid probe under hybridizing conditions with either (i) test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe consists of a nucleotide sequence comprising a portion of the nucleotide sequence of the nucleic acid molecule of claim 25, or complements thereof, and : (b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or 8 the synthesized nucleic acid molecules, ~ wherein the presence of the hybrids indicates the presence of zacrp5 RNA in the biological sample.

57. A method of detecting the presence of zacrp5 in a biological sample, comprising: = (a) contacting the biological sample with an antibody, or an antibody fragment, of claim 20, wherein the contacting is performed under conditions that allow } the binding of the antibody or antibody fragment to the biological sample, and - (b) detecting any of the bound antibody or bound antibody fragment. SUBSTITUTE SHEET (RULE 26)