MXPA01012092A

MXPA01012092A - Adipocyte complement related protein homolog zacrp5.

Info

Publication number: MXPA01012092A
Application number: MXPA01012092A
Authority: MX
Inventors: Christopher S Piddington
Original assignee: Zymogenetics Inc
Priority date: 1999-05-27
Filing date: 2000-05-18
Publication date: 2002-07-22
Also published as: WO2000073444A1; CA2378737A1; AU4855800A; JP2003501027A; ZA200109295B; EP1185643A1

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zacrp5, a novel member of the family of proteins bearing a collagen-like domain and a C1q domain. The polypeptides and polynucleotides encoding them, are involved in trimerization and be used in the study thereof. The present invention also includes antibodies to the zacrp5 polypeptides.

Description

PROTEIN HOMOLOGA ZACRP5 RELATED TO THE COMPLEMENT OF ADIPOCITS BACKGROUND OF THE INVENTION Cell-cell and extracellular matrix-cell interactions allow the exchange of information between, and coordination among, several cells of a multicellular organism and are fundamental to most biological processes. These interactions play a role in all matters 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, the interaction between the cell and its environment is necessary to initiate and have a mediating role in tissue remodeling. Tissue remodeling can be initiated, for example, in response to many factors including physical injuries, cytotoxic lesions, metabolic stress or developmental stimuli. The modulation between the pathology and the healed (or metabolic optimization) can be done, in part, by the interaction of the stimulated cells with the extracellular matrix as well as with the local solvent. Ref .134386 A family of proteins that plays a role in the interaction of cells with their environment, and seems to act as the interface of the extracellular matrix and the cell, are the proteins related to the adipocyte complement. These proteins include, Acrp30, a 247 amino acid polypeptide that is expressed exclusively by adipocytes. The Acrp30 polypeptide is composed of a sequence of the amino-terminal signal, a 27 amino acid extension of unknown homology, 22 repeats of perfect Gly-Xaa-Pro collagen or of imperfect Gly-Xaa-Xaa 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 a structural similarity, particularly in the carboxy-terminal globular domain, with respect to the complement factor CIq and with a summer serum protein of the Siberian squirrels in hibernation (Hib27). The expression of Acrp30 is induced up to 100 times during the differentiation of adipocytes. Acrp30 is suggested for use in the modulation of energy balance and in the identification of adipocytes in test samples. .? aai .. * - & i - ^ .- ^ fc - ^ - ^? a --------- á -? ^ á - Kf & ^ I. -fe- Additional elements include zsig37, a protein of 281 amino acid residues predominantly expressed in the heart, aorta and placenta, which has 14 collagen repeats and a globular CIq domain similar to ACRP30 (WO 99/04000). Zsig37 has been shown to inhibit complement activity, bind to SK5 fibroblasts and stimulate proliferation at known concentrations to initiate cell-Clq responses. Zsig37 also specifically inhibits the activation of platelet collagen in whole human blood and platelet-rich plasma in a dose-dependent manner (co-pending US Patent Application, 09 / 253,604). Also included is zsig39, a protein of 243 amino acid residues predominantly expressed in the heart and small intestine, which has 22 to 23 repeats of collagen and one CIq domain similar to ACRP30 and zsig37 (99/10492). These proteins all share a CIq domain. The CIq factor of the complement consists of six copies of three related polypeptides (A, B and C chains), with each polypeptide being approximately 225 amino acids in length with a domain of nearby amino-terminal collagen and a carboxy-terminal globular region. Six triple helical regions are formed by the domains of the collagen of the six A chains, six B chains and six C chains, forming a central region and six stems. A globular head portion is formed by the association of the globular carboxy terminal domain of an A chain, a B chain and a C chain. The CIq is therefore composed of six globular heads linked by means of six stems similar to collagen 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 CIq globular domain of ACRP30 has been determined to have a "jelly-filled sponge cake topology" of 10 beta strands (Shapiro and Scherer, Curr. Biol. 8: 335-8, 1998). Structural elements such as folding topologies, conserved residues and similar trimmed interfaces and intron positions are homologous with respect to the tumor necrosis factor family, suggesting a link between the TNF and CIq families. Zsig39 and zsig37 share this structure and also the homology. Proteins that play a role in cellular interaction, such as transcription factors and hormones, are useful as therapeutic and diagnostic agents. The proteins that have a mediating role in • I > ? .? ijfcj-ti - * ^ J, -, > a "a -it t¡r [, 1. | í | H-miM- rtiiit .t t- -mi ni, | The specific interactions, such as remodeling, could 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.

Brief Description of the Invention Within one aspect, the invention provides an isolated polypeptide comprising an amino acid residue sequence that is at least 80% identical in amino acid sequence to residues 70-252 of SEQ ID NO: 2, wherein the sequence comprises: the collagen repeats of Gly-Xaa-Xaa and Gly-Xaa-Pro that form a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal CIq domain. Wherein one embodiment of the polypeptide is at least 90% identical in the amino acid sequence to residues 18-252 of SEQ ID NO: 2. Within a related embodiment, any differences between the polypeptide and SEQ ID NO: 2 are they owe to the substitutions of preservative amino acids. Within another embodiment, the collagen-like domain consists of 14 repeats of Gly-Xaa-Xaa collagen and 1 repetition of Gly-Xaa-Pro collagen. Within yet another embodiment, the polypeptide comprises: an amino terminal region; 14 repeats of Gly-Xaa-Xaa collagen and 1 repetition of Gly-Xaa-Pro collagen that form a collagen-like domain, where Xaa is any amino acid residue; and a carboxyl-terminal CIq 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. Within a further embodiment, the polypeptide specifically binds to an antibody that specifically binds to a polypeptide of SEQ ID NO: 2. Within another embodiment, the collagen-like domain comprises amino acid residues 70-111 of the SEC ID NO: 2. Within another embodiment the CIq 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 converted into a complex by the intermolecular disulfide bonds to form a homotrimer. Still in another embodiment, the polypeptide is converted into a complex by intermolecular disulfide bonds, to one or more polypeptides having a domain similar to collagen, to form a heterotrimer. Within an additional embodiment the polypeptide is covalently linked in the amino or carboxyl termination to a portion 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 and a second portion. portion bound by a peptide bond, the first portion consists of a polypeptide selected from the group consisting of: a) 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 the sequence comprises: the collagen repeats of Gly-Xaa-Xaa and Gly-Xaa-Pro that form a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal CIq domain; b) a polypeptide comprising: a terminal amino region; 14 repeats of collagen of Gly-Xaa-Xaa and 1 repetition of collagen of Gly-Xaa-Pro that forms a domain similar to collagen, where Xaa is any amino acid residue; and a carboxyl-terminal CIq 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 the SEC 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 CIq domain or an active portion of the t > of CIq; or e) a portion of the zacrp2 polypeptide as shown in SEQ ID NO: 2 comprising the collagen-like domain and the CIq domain; and the second portion comprises another polypeptide. Within a related embodiment, the first portion is selected from the group consisting of: a) a polypeptide consisting of the sequence from 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 amino acid residue 252 of SEQ ID NO: 2; d) a polypeptide consisting of the residue sequence of amino acids 18 to amino acid residue 252 of SEQ ID NO: 2; and e) a polypeptide consisting of the sequence from amino acid residue 1 to amino acid residue 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 for a polypeptide comprising; inoculating an animal with a polypeptide selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 80% identical in the amino acid sequence to residues 70-252 of SEQ ID NO: 2, wherein the sequence comprises: the collagen repeats of Gly-Xaa-Xaa and Gly-Xaa-Pro that form a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clq domain; b) a polypeptide comprising: a terminal amino region; 14 repeats of Gly-Xaa-Xaa collagen and 1 repetition of Gly-Xaa-Pro collagen that forms a collagen-like domain, where Xaa is any amino acid residue; and a carboxyl-terminal Clq domain comprising 10 beta strands corresponding to amino acid residues 119-123, 141-143, 149-152, 156-158, Xt?, -i-_i-stefei ^. * W - ^ 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 Clq domain or an active portion of the Clq domain; or e) a portion of the zacrp5 polypeptide as shown in SEQ ID NO: 2 comprising the collagen-like domain and the Clq domain; and wherein the polypeptide produces an immune response in the animal to produce the antibody; and isolate the animal's antibody. Antibodies or antibody fragments that specifically bind to a polypeptide as described above are also provided. Within one embodiment, the antibody is selected from the group consisting of: a) a monoclonal antibody; b) a murine monoclonal antibody; c) a humanized antibody derived from b); and d) a human monoclonal antibody. Within another embodiment the antibody fragment is selected from the group consisting of F (ab ') / F (ab), Fab1, Fab, Fv, scFv, and the minimal recognition unit. Within another embodiment, an anti-idiotype antibody that specifically binds to the antibody described above is provided. As well ! * rlHfttfff.? fc? - & ia «.« - Bt4 - «- tii" fi'- ^ «3É¡ü ^^ i '^ ií -' ^ '- ^ ftff ^ ^! fF .. The invention provides an agglutination protein that binds agglutin to an epitope of a polypeptide as described above In another aspect the invention provides an isolated polynucleotide encodes a polypeptide as described above.An isolated polynucleotide selected from the group consisting of: a) a nucleotide sequence from nucleotide 1 to nucleotide 759 of SEQ ID NO: 1; b) a nucleotide sequence is also provided from nucleotide 52 to nucleotide 759 of SEQ ID NO: 1, c) a nucleotide sequence from nucleotide 208 to nucleotide 333 of SEQ ID NO: 1, d) a nucleotide sequence from nucleotide 334 to nucleotide 759 of SEQ ID NO: 1; e) a nucleotide sequence from nucleotide 208 to nucleotide 759 of SEQ ID NO: 1; NO: l; f) a nucleotide sequence 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 NO: 2; i) a polynucleotide that remains hybridized, following stringent washing conditions, to a polynucleotide consisting of - ¿- ÍAáÉ -.- t--? . nucleotide sequence of SEQ ID NO: 1, or the complement of SEQ ID NO: 1; j) complementary nucleotide sequences with a), b), c), d), e), f), g), h), or i) and k) of the degenerate nucleotide sequences of g) or h). An isolated polynucleotide encoding a fusion protein as described above is also provided. The invention also provides an isolated polynucleotide consisting of the sequence from 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 promoter of transcription; an element of DNA encoding a polypeptide as described above; and a transcription terminator. Within one embodiment, the DNA segment further encodes a sequence of the secretory signal operably linked to the polypeptide. Within a related embodiment, the sequence of the secretory signal comprises residues 1-17 of SEQ ID NO: 2. The invention also provides a cultured cell in which an expression vector has been introduced as described above, wherein the cell expresses the polypeptide encoded by the DNA segment. Within In one embodiment, the cultured cell further includes one or more expression vectors comprising the DNA segments encoding the polypeptides having collagen-like domains. Within another aspect the invention provides a method of producing a protein comprising: culturing a cell in which an expression vector has been introduced as described above; whereby the cell expresses the protein encoded by the DNA segment; and recover the expressed protein. Within one modality the expressed protein is a homotrimero. In another embodiment, the expressed protein is a heterotrimer. Within another aspect the invention provides a method of detecting the presence of the expression of the zacrp5 gene in a biological sample, comprising: (a) contacting a zacrp5 nucleic acid probe under hybridization conditions with either ( i) the 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 sequence of nucleotides of the nucleic acid molecule as described above, or complements thereof, and (b) detecting hybrid formation of the acid probe nucleic acid and either the test RNA molecules or the nucleic acid molecules synthesized, wherein the presence of the hybrids indicates the presence of zacrp5 RNA in the biological sample. Within another aspect there is provided 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, as described above, wherein the contacting is effected under conditions that allow agglutination of the antibody or antibody fragment to the biological sample, and (b) detecting either the bound antibody or the bound antibody fragment.

Brief Description of the Drawing Figure 1 illustrates a multiple alignment of the zacrp5 polypeptide of the present invention and the zsig37 homologous protein related to the adipocyte complement (SEQ ID NO: 3, WO 99/04000), the human ACRP30 (ACR3_HUMAN) (SEQ ID NO: 4, Maeda et al., Biochem. Biophys., Res. Commun. 221: 286-9, 1996), the protein homologue zs? g39 related to complement of adipocytes (SEQ ID NO: 5, WO 99/10492) and human Clq C (SEQ ID NO: 6, Sellar et al., Biochem J. 274: 481-90, 1991 and Reid, Biochem J. 179: 361-71, 1979). Multiple alignment performed using a Clustalx multiple alignment tool with the default settings: Weight Matrices of the Blosum Series, Penalty for Gap Opening: 10.0, Penalty for Gap Extension: 0.05. The multiple alignments were adjusted by hand additionally before calculating the percentage identity.

DETAILED DESCRIPTION OF THE INVENTION Prior to the description of the invention in detail, the definition of the following terms may be useful for understanding it. The term "affinity tag" is used herein to denote a segment of the peptide that can be attached to a polypeptide to provide for purification or detection of the polypeptide or provide sites for binding of the polypeptide to a substrate. In the beginning, any peptide or protein for which an antibody or other specific binding agent is available, such as an affinity tag, can be used. 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), the substance P, the peptide ------ Flag ™ (Hopp et al., Biotechnology 6: 1204-10, 1988; available from Eastman Kodak Co., New Haven, CT), streptavidin binding peptide, or other antigenic epitope or Union. See, in general Ford et al., Protein Expression and Purification 2: 95-107, 1991. The DNAs encoding the affinity tags are available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ). The term "allelic variant" denotes any of two or more alternative forms of a gene occupying the same chromosomal site. Allelic variation arises naturally through mutation, and can lead to phenotypic polymorphism within populations. Genetic mutations can be inactive (without change in the encoded polypeptide) or can encode polypeptides having the 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 and proteins. Wherever the context permits, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to denote pmity or relative position. For example, a certain sequence placed carboxyl-terminal with respect to a reference sequence within a protein, is located close to the carboxyl terminus of the reference sequence, but not necessarily in the carboxyl termination of the complete protein. The term "complement / anti-complement pair" denotes non-identical portions that form a stable pair, associated non-covalently, under the appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypic members of a complement / anticomplement pair. Other exemplary complement / anti-complement pairs include the receptor / ligand pairs, the antibody / antigen (or hapten or epitope) pairs, the sense / antisense polynucleotide pairs, and the like. Where the subsequent dissociation of the complement / anti-complement pair is desirable, the complement / anti-complement pair preferably has an agglutination affinity of <109 M "1. The term" complement of a polynucleotide molecule "is a polynucleotide molecule having a complementary base sequence and the reverse orientation when compared to a reference sequence. example, the 5 'sequence ATGCACGGG 3' is complementary with 5 'CCCGTGCAT 3'. The term "contiguous" denotes a polynucleotide having a contiguous extension of an identical or complementary sequence with another polynucleotide. The contiguous sequences are said to "overlap" over a given extension of the polynucleotide sequence either in its entirety or along a partial extension of the polynucleotide. For example, the representative elements contiguous with the sequence of polynucleotides 5 '-ATGGCTTAGCTT-3' are 5'-TAGCTTgagtct-3 'and 3' -gtcgacTACCGA-5 '. The term "degenerate nucletide sequence" denotes a nucleotide sequence that includes one or more degenerate codons (when compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but they encode the same amino acid residue (ie, the GAU and GAC triplets each encode Asp). The term "expression vector" denotes a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to provide its transcription. Such additional segments may include the promoter and the sequences terminators, and optionally may 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 the 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 environment and therefore is free of other foreign or undesirable coding sequences, and is in a form suitable for use within the genetically engineered production systems of the protein. Such isolated molecules are those that are separated from their natural environment and include the cDNA and the genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include the 5 'and 3' untranslated regions that are naturally present, such as promoters and terminators. The identification of the associated regions will be evident to a person with ordinary experience in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985). An "isolated" polypeptide or protein is a protein or polypeptide that is found in a condition other than ÍÍÍ-.írá,? _ ^ ¡^ ^ ^., Íi. ^^ t¿ ^ t ^ -, í ^^. ^? ^ Íjt3.1¡F ^ FF.¿Í? .. -atofeai- -.-. i - i ------- l-í ---- > -their natural environment, such as away from the blood and tissue of the animal. 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, ie with a purity greater than 95%, more preferably of a purity greater than 99%. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as trimers or alternatively derived or glycosylated forms. The term "operably linked", when referring to the DNA segments, denotes that the segments are distributed so that they function cooperatively for their intended purposes, for example the transcription initiated in the promoter and proceeds through the coding segment until the terminator. The term "ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The sequence differences between orthologs are the result of speciation. The "paralogs" are different but structurally related proteins, made by an organism. Paralogs are believed to arise through gene duplication. For example, a-globin, ß-globin and myoglobin are paralogs among themselves. The term "polynucleotide" denotes a single-stranded or double-stranded polymer of the deoxyribonucleotide or ribonucleotide bases read from the 5 'to 3' end. The polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The sizes of the polynucleotides are expressed as the base pairs "abbreviated" pb "), the nucleotides (" nt "), or kilobases (" kb "). Where the content allows, these last two terms can describe the polynucleotides The term is applied to the double-stranded molecules, it is used to denote the total length and it will be understood that it will be equivalent to the term "base pairs". 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; therefore all nucleotides within a double-stranded polynucleotide molecule are not necessarily grouped by couples. Such extremes that are not even in general will not exceed 20 nt in length. A "polypeptide" is a polymer of amino acid residues linked by peptide bonds, produced either naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides". The "probes and / or primers" as used herein may be RNA or DNA. The DNA can be any cDNA or genomic DNA. The probes and the polynucleotide primers are the single-stranded or double-stranded DNA or RNA, generally the synthetic oligonucleotides, but can be generated from the genomic or cDNA sequences or their complements. Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) may be used. The PCR primers are at least 5 nucleotides in length, preferably 15 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 the total analysis of the genes, a polynucleotide probe can comprise a complete exon or more. The probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, flurophore, chemiluminescent agent, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene , OR, and Amersham Corp., Arlington Heigts, IL, using techniques that are well known in the art. The term "promoter" denotes a portion of a gene that contains the DNA sequences that are provided for the binding of the RNA polymerase and the initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'non-coding regions of the genes. The term "receptor" denotes a protein associated with the cell that binds to a bioactive molecule (i.e., a ligand) and has a mediating effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-domain structure comprising an agglutination domain of the extracellular ligand and an intracellular effector domain that are typically involved in signal transduction. Agglutination of the ligand to the receptor leads to a conformational change in the receptor that causes an interaction between the effector domain and another (s) molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. The metabolic events that are linked to the ., «&Jfc8a ------------ a --- receptor-ligand interactions include transcription of genes, phosphorylation, dephosphorylation, increases in cyclic AMP production , mobilization of cellular calcium, mobilization of membrane lipids, adhesion of cells, hydrolysis of inositol lipids and hydrolysis of phospholipids. Most nuclear receptors also exhibit a structure of multiple domains, including a transactivation, amino-terminal domain, a DNA agglutination domain and a ligand agglutination domain. In general, the receptors may be cytosolic or nuclear, membrane-bound, monomeric receptors (eg, thyroid-stimulating hormone receptor, beta-adrenergic receptor) or multimeric (eg, the PDGF receptor, the growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor, and IL-6 receptor). The term "secretory signal sequence" denotes a DNA sequence encoding a polypeptide (a "secretory peptide" which, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in the which is synthesized, the largest peptide is segmented .-.- -t-tt---yi---.fr? r ff .. ^ .-., .---- Li¡ ».. ^ .. ---- .---- - ---. A .- ,. ^^ .- ^ ... J -.-. -J -. - It is commonly used to remove the secretory peptide during transit through the secretory route. A "soluble receptor" is a receptor polypeptide that is not bound to a membrane of the cell. Soluble receptors are more commonly ligand agglutination receptor polypeptides that lack the transmembrane and cytoplasmic domains. Soluble receptors may comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for binding of the polypeptide to a substrate, or sequences of the constant region of the immunoglobulin. Many receptors on the surface of the cell have soluble counterparts, which are naturally present, which are produced by proteolysis or translated from the alternatively spliced mRNAs. The receptor polypeptides are said to be substantially free of the transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane binding or signal transduction, respectively. The term "splice variant" is used here to denote alternative forms of RNA transcribed from a gene. The variation of the splice arises in a natural by means of the use of alternative splicing sites within a transcribed RNA molecule, or less commonly between the separately transcribed RNA molecules, and can lead to several mRNAs transcribed from the same gene. The splice variants can encode polypeptides having an altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splicing variant of a mRNA transcribed from a gene. The weights and molecular lengths of the polymers determined by imprecise analytical methods (for example, gel electrophoresis) will be understood to be approximate values. When such value is expressed as "almost" X or "approximately" X, the established value of X will be understood to be accurate to + 10%. The present invention is based in part on the discovery of a novel DNA sequence encoding a polypeptide having homology with a zsig37 protein related to adipocyte complement (WO 99/04000). The novel DNA sequence encodes a polypeptide having an amino-terminal signal sequence, an adjacent N-terminal region of no homology, a collagen domain composed of 14 collagen repeats, and a globular, Clq-like carboxyl domain. terminal. The ..---- > ia ---- t-ti «t > t «-« > --.:-J-i.--h,.-i±ttE. ».--- aA'-lTii-a > lli ^ jfe ^ g »j; -» -? l, V-il.: - ..: - -í-. -F?, ^ FJf-FfM..lÍ¡Üu structure of the general polypeptide described above is shared by zsig37, zsig379, Acrp30 and Clq C (see Figure). Other regions of homology, found in the carboxy-terminal globular Clq domain in the aligned proteins, are here identified as useful primers for investigating other members of the family. For example, zsig37, zsig39, Acrp30 and Clq C, could be identified in a search using the primers. The intra-chain disulfide bond can involve the cysteines at residues 26, 29, 30, 112 and 158 of SEQ ID NO: 2. The novel zacrpd polypeptides of the present invention were initially identified in an unfinished genomic sequence. The genomic sequence is located on the site HS349E11 which is derived from chromosome 16. SEQ ID NO: 7 provides the identified exon 1 of the zacrpd starting at the start codon, nucleotides 1-208, intron 1, nucleotides 209-870 and exon 2 ending with the stop codon, nucleotides 871-1421. With the predictions of the exon strictly judged, the factor related to the novel adipocyte complement was found to be homologous with respect to the other factor related to the adipocyte complement, zsig37 (WO 99/04000). The percentage identity at the level of the amino acids on the total molecule between the zacrp5 and the -8--. -.--- i .--- i.i --- --- jf ???? irrTÉ? lÉÉtf-, at-A --- > * -a '* a:' ^ - ~ > .- ~ * ¿«--- > -fa .-- J-.iJ-- other members of the family is shown in Table 1A. The percentage identity over the Clq domain is only shown in Table IB. The alignments were made using a Clustalx multiple alignment tool with the default settings: Weight Matrices of the Blosum Series, Penalty for Gap Opening: 10.0, Gap Extension Penalty: 0.05. Multiple alignments were adjusted by hand in addition before the percentage identity calculation. The percentage identity is the total number of identical residues over the length of the overlap.

Table 1A Table IB 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. , which varies 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 varies 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 unknown homology is found, which varies between amino acid residue 18 (Trp) and 69 (Lys) of SEQ ID NO: 2, nucleotides 52-207 of SEQ ID NO: 2. NO: l. 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, a repetition of perfect collagen of Gly-Xaa-Pro and 13 imperfect repeats of the collagen Gly-Xaa-Xaa is observed.The acrp30 contains 22 perfect or imperfect collagen repeats, the zsig37 has 14 repetitions of collagen and the zsig39 has 22 or 23 Collagen repeats The proline residues found in this domain at amino acid residues 90 and 108 of SEQ ID NO: 2 can be hydroxylated The zacrp5 polypeptide also includes a carboxy-terminal Clq domain, varying from about amino acid 112 (Cys) to 252 (Leu) of SEQ ID NO: 2, nucleotides 334 to 759 of SEQ ID NO: 1. There is an adequate amount of the structure conserved within the Clq domain to enable a folding appropriate.An aromatic portion a of Clq imperfect (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 which does not correspond perfectly with the portion. X represents any amino acid residue and the number in parentheses () indicates the number of amino acids in the residues. The amino acid residues contained within the square brackets [] restrict the choice of amino acid residues in this particular position. The final residue of this portion is Leu instead of Phe or Tyr.

The polypeptide of zacrp5, human zsig37, human zsig39, human Clq C and Acrp30 appears to be homologous within the collagen domain and in the Clq domain, but not in the N-terminal portion of the mature polypeptide. Another aspect of the present invention includes the fragments of the zacrp5 polypeptide. Preferred fragments include those that contain the collagen-like domain of zacrp5 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 zacrp5 polypeptide that contains the collagen-like domain or a portion of the collagen-like domain capable of trimerization or oligomerization. When used herein the term "collagen" or "collagen-like domain" refers to a series of amino acid sequences of repeating triplets, "repeats, or" collagen repeats "represented by the Gly-Xaa-Pro or Gly- Xaa-Xaa, where Xaa is any amino acid residue, such domains may contain as many as 14 repeats of collagen or more, and such fragments or proteins containing such collagen-like domains may form heteromeric constructs, usually trimers. and the homology with respect to other collagen-like domains that contain proteins, ^ .a ^ naaij indicate that the zacrp5 polypeptides, fragments or fusions comprising the collagen-like domain, can be converted into a complex with other collagen domains containing the polypeptides to form the homotrimers and the heterotrimers. These fragments containing the collagen-like domain are particularly useful in the study of collagen trimerization or oligomerization or in the formation of fusion proteins as described in greater detail below. Polynucleotides encoding such fragments are also encompassed by the present invention, including the group consisting of (A) the polynucleotide molecule comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 1, 52 or 208 to nucleotide 333; (b) polynucleotide molecules that encode a fragment of the zacrp5 polypeptide 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 ( To); (c) the complementary molecules with respect to (a) or (b); and (d) degenerate nucleotide sequences encoding a fragment of the collagen-like domain of the zacrp5 polypeptide.

Another collagen-like domain containing the polypeptides includes members of the adipocyte-related protein family, such as zsig37, zsig39 and ACRP30, for example. The trimeric proteins of the present invention are formed by the intermolecular disulfide bonds formed between the cysteine residues conserved within the polypeptides. The present invention therefore provides the zacrp6 polypeptides converted into a complex by the intermolecular disulfide bonds to form the homotrimers. The invention further provides the zacrp5 polypeptides converted into a complex by the intermolecular disulfide bonds with other polypeptides having a collagen-like domain, to form the heterotrimers. Other preferred fragments include the globular Clq domain of the zacrp5 polypeptides, which vary from amino acid 112 (Cys) to 252 (Leu) of SEQ ID NO: 2, a portion of the zacrp5 polypeptide that contains the Clq domain or an active portion of the Clq domain. Another Clq domain containing the proteins, includes zsig37 (WO 99/04000), zsig39 (WO 99/10492), Clq A, B and C (Sellar et al., Ibid., Reid, ibid., And Reid et al. al., Biochem. J. 203: 559-69, 1982), the HP-20 plasma proteins associated with the i - ^ - f.-i - LÍ > -fe ----------- tt --------- ^^ -. * * Fm ????? Hibernation of the squirrel, 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 ), the human preerebellen (Urade et al., Proc. Nati, Acad. Sci. USA 88 ^: 1069-73, 1991), the multimerin of human endothelial cells (Hayward et al., J. Biol. Chem. 270 : 18246-51, 1995) and the vertebrate type VIII and X collagens (Muragaki et al., Eur. J. Biochem. 197: 615-22, 1991). The globular Clq domain of ACRP30 has been determined to have a topology of "rolled jelly-filled sponge cake" of 10 hrbeas beta (Shapiro and Scherer, Curr. Biol. 8 ^: 335-5, 1998) showing significant homology with 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 threads have been designated "A", "A", "B", "B", "C", "D", "E", "F", "G" and "H" respectively. Zacrp5 has two receptor agglutination loops, at amino acid residues 125-151 and 183-196. The amino acid residues 162 (Gly), 164 (Tyr), 211 (Leu) and 241 (Phe) seem to be conserved through the superfamily that includes the CD40, TNFa, TNFß, ACRP30 and zacrp5. These fragments are particularly useful in the study or modulation of the cell-cell or extracellular-cell matrix interaction. The antimicrobial activity may also be present in such fragments. The homology with respect to TNF proteins suggests that such fragments could be useful in insulin resistance related to obesity, immune regulation, inflammatory response, apoptosis, and maturation of osteoclasts. Polynucleotides encoding such fragments are also encompassed by the present invention, including the group consisting of (a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 334 to nucleotide 252, (b) the 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 the amino acid residue 252 (Leu), (c) the complementary molecules with (a) or (b); and (d) the degenerate nucleotide sequences that encode a fragment of the Clq domain of the zacrp5 polypeptide.

Other fragments of the zacrp5 polypeptide of the present invention include both the collagen-like domain and the Clq 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 the nucleotides as shown in SEQ ID NO: 1 from nucleotide 208 to nucleotide 759; (b) polynucleotide molecules that encode a fragment of the zacrp5 polypeptide that is at least 80% identical with respect to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 70 (Gly) to the amino acid residue 252 (Leu); (c) the complementary molecules with respect to (a) or (b); and (d) the degenerate nucleotide sequences encoding a fragment of the Clq domain-like domain-collagen of the zacrp5 polypeptide. The highly conserved amino acids, particularly those in the carboxy-terminal Clq domain of the zacrpd polypeptide, can be used as a tool to identify new members of the family. For example, the polymerase chain reaction-reverse transcription (RT-PCR) can be used to amplify the sequences encoding the conserved portions of the RNA obtained from a variety of tissue sources. In particular, highly degenerate primers and their complements designed from the 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 WN 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 the complements of the polynucleotides described above are also encompassed. The present invention also provides polynucleotide molecules, including DNA and RNA molecules, ; fl ---j-----.?í? -afiiiiiii-fa itl-l l iiilp -t .-- »* -» ^ »* .-» te ---------. ..-- c-. -. ^ ..-- jt ^^ - -. ~ -. i-tí. ^ - atia -------. ^,., - i ^ á- -, ---..- "- --------? .------ fc-l-ii.d encoding the zacrp5 polypeptides described herein. To isolate the polynucleotide of SEQ ID NO: 1 from various tissues, the probes and / or primers are designed from the predicted regions of the exon of SEQ ID NO: 1 and SEQ ID NO: 7. The tissues that expressing the zacrp5 could be identified either by means of hybridization (Northerns Blots) or by the PCR of reverse transcriptase (RT). The libraries are then generated from the tissues that appear to show the expression of zacrp5. The unique clones of such libraries are then identified by means of hybridization with the probes and / or by PCR with the t > primers as described here. The conformation of the cDNA sequence of zacrp5 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 variation of the sequence between these polynucleotide molecules is possible. SEQ ID NO: 12 is a generated DNA sequence encompassing all of the DNAs encoding 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 the RNA sequences encoding SEQ ID NO: 2 by the substitution of U by T. Therefore, the -it-? 4 ------ --l- 'S ----- t? ai ^ Jt & i - »-.... ..., ia ---- it -.- .- • a - ", ^ j ^ .iéfa -, ¿-.tA« a - a «i.-Üft - * - f #? - n-1 _--- te - * - ii-? to--. polynucleotides encoding the zacrp5 polypeptide comprising nucleotide 1 to nucleotide 756 of SEQ ID NO: 12 and their RNA equivalents are contemplated by the present invention. Table 2 describes the one-letter codes used within SEQ ID NO: 12 to denote the positions of the degenerate nucleotides. "Resolutions" are the nucleotides denoted by a letter of the code. The "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 is complementary to T, and G is complementary to C. ; k ,. ft • ------- to * TABLE 2 Nucleotide Resolution Complement Resolution AATTCCGGGGCCTTAARA | GYC | TYC | TRA | GMA | CKG | TKG | TK | TMA | CSC | GSC | GWA | TWA | THA | C | TDA | G | TB CIGIT VA | C | GVA | C | GB CIGIT DA | G | THA | C | TNA | C | G | TNA | C | G | T The degenerate codons used in SEQ ID NO: 12, which encompass all possible codons for a given amino acid, are described in Table 3 TABLE 3 Amino Code - One Codon Codon Acid Degenerate Letter Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T AC AC ACC AC AC Pro P CCA CCC CCG CCT CCN Wing A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR 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 He 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 T and T TAT TAT T TRp W TGG TGG T TA T T T T TR TR Asp | Asp B RAY GluIGln Z SAR Any X NNN A person with ordinary experience in the art will appreciate that some ambiguity is introduced in the determination of a degenerate codon, representative of all possible codons that encode each amino acid. For example, the degenerate codon for serine (WSN), in some circumstances, can encode arginine (ARG), and the degenerate codon for arginine (MGN), in some circumstances, can encode serine (AGY). There is a similar relationship between the codons that encode phenylalanine and leucine. Accordingly, some polynucleotides encompassed by the degenerate sequence can encode the 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 easily tested to verify functionality as described here. A person with ordinary experience in the art will also appreciate that different species may exhibit a "preferential codon use". 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. L_4: 3075-87, 1986; Ikemura, J. Mol. Biol. 158: 573-97, 1982. When used here. The term "preferential codon usage" or "preferential codons" is a term of the a -.- t-t - ^ - M-? l - 8 «A < Ma ----- Jb art that refers to protein translation codons that are used more frequently in the cells of certain species, thus favoring one or a small amount of the representative elements of the possible codons that encode each amino acid (See Table 3). For example, the amino acid threonine (Thr) can 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, yeasts, viruses or bacteria, different Thr codons may be preferential. The preferential codons for particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferential codon sequences in recombinant DNA, for example, they can improve the production of the protein by making the translation of the protein more efficient within a particular type or type of cell. Thore, the sequence of the degenerate codon described in SEQ ID NO: 12 serves as a template for the optimization of the expression of polynucleotides in various types and species of cells commonly used in the art and described hn. The sequences containing the preftial codons can be tested and optimized for expression in several species and tested to verify functionality as described hn. The present invention also provides variant polypeptides and nucleic acid molecules that represent the counterparts of other species (orthologs). These species include, but are not limited to mammals, birds, amphibians, reptiles, fish, insects and other vertebrate and invertebrate species. Of particular intt are zacrp5 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. The orthologs of human zacrp5 can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using the mRNA obtained from a tissue or cell type expressing zacrp5 as described hn. Suitable sources of mRNA can be identified by probing with northern blots with probes designed from the sequences described h A library was then prepared from the mRNA of a positive tissue or cell line. A cDNA encoding zacrp5 can then be isolated by a variety of methods, such as by probing with a partial or complete human cDNA or with one or more sets of degenerate probes based on the described sequences. A cDNA can also be cloned using the polymerase chain reaction with primers designed from the zacrp5 sequences described hn. Within a further method, the cDNA library can be used to transform or transfect the host cells, and expression of the cDNA of intt can be detected with an antibody to the zacrp5 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequence described in SEQ ID NO: l represents a single allele of human zacrp5, and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries of difft individuals according to standard procedures. Allelic variants of the nucleotide sequence shown in SEQ ID NO: 1, including those containing the inactive mutations and those in which the mutations lead to changes in the amino acid sequence, are within the scope of the present invention, what are the proteins which are allelic variants of SEQ ID NO: 2. The cDNA molecules and generated from the alternatively spliced mRNAs, which retain the properties of the zacrp5 polypeptide are included within the scope of the present invention, as are the polypeptides encoded by such cDNAs and mRNAs. The allelic variants and splice variants of these sequences can be cloned by probing the cDNA or the genomic libraries of different individuals or tissues according to standard procedures known in the art. Within the preferred embodiments of the invention, the isolated nucleic acid molecules can hybridize under severe conditions to nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 1 or to nucleic acid molecules having a sequence of nucleotides complementary to SEQ ID NO: 1. In general, severe 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 the temperature (under defined ionic strength and pH) at which 50% of the target sequence is hybridized to a perfectly matched probe. A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA, can be hybridized if the i? mitit tfir'-t X 0 - • - '* • * • * - - - nucleotide sequences have some degree of complementarity. Hybrids can tolerate non-corresponding base pairs in the double helix, but the stability of the hybrid is influenced by the degree of inequality or mismatch. The Tm of the non-corresponding or mismatched hybrid is reduced by 1 ° C every 1-1.5% of base pair inequality. By varying the severity of the hybridization conditions, control over the degree of inequality that will be present in the hybrid is allowed. The degree of severity is increased when the hybridization temperature is increased and the ionic strength of the hybridization buffer is reduced. Severe hybridization conditions encompass temperatures of about 5-25 ° C below the Tm of the hybrid and a hybridization buffer having up to 1 M Na +. Higher degrees of severity can be achieved at lower temperatures with the addition of formamide which reduces the Tm of the hybrid by approximately 1 ° C for every 1% of formamide in the buffer solution. In general, such stringent conditions include temperatures of 20-70 ° C and a hybridization buffer containing up to 6x SSC and 0-50% formamide. A higher degree of severity can be achieved at temperatures from 40-70 ° C with a hybridization buffer that has up to 4x SSC and from 0-50% of formamide. Highly severe conditions typically encompass temperatures of 42-70 ° C with a hybridization buffer having up to lx SSC and 0-50% formamide. Different degrees of severity can be used during hybridization and washing to achieve maximum specific binding to the target or target sequence. Typically, the washings following the hybridization are performed at increasing degrees of severity to remove the probes of unhybridized polynucleotides from the hybridized complexes. The above conditions are understood to serve as a guide and are considered within the capabilities 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 the defined conditions) at which 50% of the target sequence will hybridize to a perfectly matched probe sequence. These conditions which influence the Tm include, the size and content of base pairs 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 the DNA, RNA and DNA-RNA hybrids and the variable length polynucleotide probe sequences (see, for example, Sambrook et al., Molecular Cloning : A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al., (Eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26: 227 (1990)). Sequence analysis programs, such as OLIGO 6.0 (LSR, Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International, Palo Alto, CA), as well as Internet sites, are tools available to analyze a given sequence and calculate the Tm based on the criteria defined by the user. Such programs can also analyze a given sequence under the defined conditions and identify the sequences of the appropriate probes. Typically, hybridization of the longer polynucleotide sequences, > 50 base pairs, is carried out at temperatures of approximately 20-25 ° C below the calculated Tm. For smaller probes, < 50 base pairs, the hybridization is typically carried out at Tm or 5-10 ° C below. This allows the maximum hybridization rate for the DNA-DNA and DNA-RNA hybrids. The length of the polynucleotide sequence has an influence on the speed and stability of the -.i.ilákA -l-i-i-ti ---..- .. t- formation of the hybrid. The smaller probe sequences, from < 50 base pairs, reach equilibrium with the complementary sequences quickly, but can form less stable hybrids. Incubation times anywhere from minutes to hours can be used to achieve hybrid formation. The sequences of the longer probes reach equilibrium more slowly, but they form more stable complexes even at lower temperatures. The incubations are allowed to proceed overnight or for a longer period. In general, the incubations are carried out for a period equal to three times the calculated Cot time. The time Cot, the 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 the polynucleotide sequence will affect the thermal stability of the hybrid complex, thereby influencing the choice of the hybridization temperature and the ionic strength of the hybridization buffer. The A-T pairs are less stable than the G-C pairs in aqueous solutions containing sodium chloride. Therefore, the higher the G-C content, the more stable the hybrid will be. The uniform distribution of waste G and C within the The sequence also contributes positively to the stability of the hybrid. In addition, the composition of base pairs can be manipulated to alter the Tm of a given sequence. For example, 5-methyl deoxycytidine can be replaced by deoxycytine and 5-bromodeoxyuridine can be replaced by thymidine to increase Tm, while 7-desazz-2 '-deoxyguanosine can be replaced by guanosine to reduce dependence of Tm. The ionic concentration of the hybridization buffer also affects the stability of the hybrid. Hybridization buffers generally contain agents Go blocking such as Denhardt's solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powder (BLOTTO), heparin or SDS, 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, 10 mM NaH2P04, 1 mM EDTA, pH 7.7). By reducing the ionic concentration of the buffer, the specificity of the hybridization is increased. Typically, hybridization buffers contain from 10 mM - 1 M Na +. The addition of destabilizing or denaturing agents such as formamide, tetraalkylammonium salts, guanidinium cations or thiocionate cations to the hybridization solution will alter the Tm of k? ti, a hybrid. Typically, formamide is used at a concentration of up to 50% to allow incubations to be carried out at lower and more convenient temperatures. Formamide also acts to reduce non-specific background when the RNA probes are used. As an illustration, a nucleic acid molecule encoding the variable zacrp5 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its 10 complement) at 42 ° C overnight in a solution comprising 50% formamide, 5x SSC (lx SSC: 0.15 M sodium chloride and 15 M sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (lOOx Denhardt's solution: 2% (w / v) Ficoll 400, 2% (w / v) polyvinylpyrrolidone, and 2% (w / v) 15 of bovine serum albumin), 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA. A person skilled in the art can contemplate variations of these hybridization conditions. For example, the hybridization mixture can be 20 incubated at a higher or lower temperature, such as about 65 ° C, in a solution that does not contain formamide. In addition, premixed hybridization solutions are available (eg, the EXPRESSHYB Hybridization Solution from Clontech Laboratories, Inc.), and hybridization It can be done according to the manufacturer's instructions. Following hybridization, the nucleic acid molecules can be washed to remove unhybridized nucleic acid molecules under severe conditions, or under highly stringent conditions. Typical, severe wash conditions include washing in a 0.5x-2x SSC solution with 0.1% sodium dodecyl sulfate (SDS) at 55-65 ° C. That is, the nucleic acid molecules encoding a variable zacrp5 polypeptide are hybridized to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) under severe wash conditions, in which the severity of the wash is equivalent to 0.5x-2x SSC with 0.1% SDS at 50-65 ° C, including 0.5x SSC with 0.1% SDS at 55 ° C, or 2x SSC with 0.1% SDS at 65 ° C. A person skilled in the art can easily contemplate equivalent conditions, for example, replacing the SSPE with the SSC in the wash solution. Typical, highly stringent washing conditions include washing in a 0.1x-0.2x SSC solution with 0.1% sodium dodecyl sulfate (SDS) at 50-65 ° C. In other words, the nucleic acid molecules encoding a variable zacrp5 polypeptide are hybridized to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) under highly stringent washing conditions, in which the severity of the wash is equivalent to 0.1x-0.2x SSC with 0.1% SDS at 50-65 ° C, including 0. lx SSC with 0.1% SDS at 50 ° C or 0.2x SSC with 0.1% SDS at 65 ° C. The present invention also provides isolated zacrp5 polypeptides having a sequence identity substantially similar to the polypeptides of SEQ ID NO: 2, or their orthologs. The term "substantially similar sequence identity" is used herein to denote polypeptides having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% identity of the sequence with the sequences shown in SEQ ID NO: 2, or their orthologs. The present invention also includes polypeptides comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% identity of the sequence with the residue sequence of amino acids 70-252 of SEQ ID NO: 2. The present invention further includes the nucleic acid molecules encoding such polypeptides. The methods for determining the percentage identity are described later. .-. i-i - »--..--» -i. i¿z *, tkif t._t - ^ - '? "^^ *« ^ Mfe' «^ - ^^ ° ^ fc '^^^^^ ---» - »? ate - i The present invention also contemplates the zacrp5 variable nucleic acid molecules that can be modified using two criteria: a determination of the similarity between the polypeptide encoded with the amino acid sequence of SEQ ID NO: 2, and a hybridization assay, such as As described above, such variants of zacrp5 include nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) under severe wash conditions, in which the severity of the wash is equivalent to 0.5X-2X SSC with 0.1% SDS at 50-65%, and (2) which encode a polypeptide that has at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity with the amino acid sequence of SEQ ID NO: 2. Alternatively, the zacrp5 variants may be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) under highly severe washing conditions, in which the severity of the washing is equivalent to 0.1X-0.2X SSC with 0.1% SDS at 50-65 ° C, and (2) which encode a polypeptide that has at least 70%, at least 80%, at least 90%, at least & T-A-fastoaLÁ-i- 1. ??, **. 95% or greater than 95% sequence identity with the amino acid sequence of SEQ ID NO: 2. The percent identity of the sequence is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603, 1986, and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 89: 10915, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment marks using a penalty gap opening 10, a penalty gap extension 1, and the evaluation matrix "BLOSUM62" from Henikoff and Henikoff (ibid.) as shown in Table 4 (amino acids are indicated by standard letter codes). The percentage identity is then calculated as: ([Total number of equalities or identical correspondences] / [length of the longest sequence plus the number of holes entered in the longest sequence to align the two sequences]) (100). r- ^ H C i ro 1 In Li. NJ or i • ^ rH ro CNJ CN] 1 1 1 00 CNJ ^ CNJ CNJ or ro CNi CNI CO PO OO tH CNJ iH CNl r-I CNl CNJ CNJ OO l l l l l l l l l I l l l l l l l l l l l w m CNJ or ro oo CNJ OO iH O tH OO CNl CNJ I I I l l l l l l l o Ln CNJ CNl O rO CNJ rH O rO tH O rH CNl rH CNl I I I I I l l l l o s. r 'C 00 C tH rH 00 H CNl f rH H CNl CNI hiiiiiiiiiiiiiii or (£ >??? ro or N, H tH r < 3 tH f H r- <?. 3 or I llllllllllll V rH OO OOO rH OO OO O CNJ OO CNJ tH O 'ír CNl OO I llllllll LO CNl OO rH O CNl O OO CNl CNJ rH OO CNJ rH iH OO CNJ OO IIIII llllllll Fí ^ HJ CM OHHONHHHHNHHO í. OJ O lll II lllllll II? (-i 2; p? S?? O? H j ii S [u cn o- -H g ^ > or CN i a --- a-Sfc. '&a ^^ JAa - i ^ - j --.- i.-t.Afe --.- fc. ^ - á.fe ---- i-¿ii - ^ -. a-MÍail - M > --Mi - »- t ------« pf ij Those skilled in the art will appreciate that there are many established algorithms available to align two amino acid sequences. The similarity search algorithm "FASTA" of Pearson and Lipman is a method of alignment suitable for examining the level of identity shared by an amino acid sequence described here and the amino acid sequence of variable zacrp5 course proteins. The FASTA algorithm is described by Pearson and Lipman, Proc. Nati Acad. Sci. USA 85: 2444, 1988, and by Pearson, Meth. Enzymol. 183: 63, 1990. Briefly, FASTA first characterizes the frequency similarity by identifying the sequences shared by the interrogation sequence (eg, SEQ ID NO: 2) and a test sequence having either the highest density of the identities (if the variable ktup is 1) or the pairs of identities (if ktup = 2), without considering substitutions, insertions, or deletions of conservative amino acids. The ten regions with the highest density of identities are then re-evaluated by comparing the similarity of all amino acids formed in pairs using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest evaluation. If there are several regions with Xl &A ^? Iá evaluations larger than the "cut" value (calculated by a predetermined formula based on the length of the sequence and the value of ktup), then the trimmed initial regions are examined to determine if the regions can be joined to form a close alignment with the gaps. Finally, the highest evaluation regions of the two amino acid sequences are aligned using a modification of the algorithm of Needleman-Wunsch-Sellers (Needle an and Wunsch, J. Mol. Biol. 48: 444, 1970; Sellers, SIAM J. Appl. Math. 26: 787, 1974), which allows the insertions and deletions of amino acids. The illustrative parameters for the analysis of FASTA are: ktup = l, penalty for gap opening = 10, penalty for gap extension = l, and substitution matrix = BLOSUM62. These parameters can be entered in a FASTA program by modifying the file of the evaluation matrix ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183: 63, 1990. FASTA can also be used to determine the identity of the sequence of the nucleic acid molecules using a ratio as described above. For comparisons of nucleotide sequences, the value of ktup can vary from one to six, preferably from four to six.

Di ** 'The present invention includes nucleic acid molecules that encode a polypeptide having one or more "conservative amino acid substitutions", compared to the amino acid sequence of SEQ ID NO: 2. substitutions of conservative amino acids they can be based on the chemical properties of amino acids. That is, variants can be obtained which contain one or more amino acid substitutions of SEQ ID NO: 2, in which an alkyl amino acid is substituted by an alkyl amino acid in an amino acid sequence of zacrp5, an aromatic amino acid is substituted by an aromatic amino acid in a zacrp5 amino acid sequence, a sulfur-containing amino acid is substituted by a sulfur-containing amino acid in a zacrp5 amino acid sequence, a hydroxy-containing amino acid is substituted by a hydroxy-containing amino acid in a sequence of amino acids of zacrpS, an acidic amino acid is substituted by an acidic amino acid in an amino acid sequence of zacrp5, a basic amino acid is substituted by a basic amino acid in a zacrpd amino acid sequence, or a monocarboxylic amino acid dibasic is substituted by a monocarboxylic amino acid dibasic in an amino acid sequence acids of zacrp5.

I --- ..-..-.- Un- -i f | lb || itt ¿-Ja), BAfc. ^,, ".

Among the common amino acids, for example, a "conservative amino acid substitution" is illustrated by a substitution between the amino acids within each of the following groups: (1) glycine, alanine, valine, leucine and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine, and histidine. The BLOSUM table 62 is an amino acid substitution matrix derived from approximately 2,000 local alignments of segments of the protein sequence, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nati. Acad. Sci. USA 89: 10915, 1992). Accordingly, substitution frequencies of BLOSUM62 can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based on chemical properties only (as described above), the language "substitution of conservative amino acids" preferably refers to a substitution represented by a BLOSUM62 value 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. According to this system, the preferred conservative amino acid substitutions are characterized by a value BLOSUM62 of at least 1 (eg, 1, 2 or 3), whereas substitutions of the most preferred conservative amino acids are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3). Changes of the preservative amino acids in a zacrp5 gene can be introduced by the substitution nucleotides for the nucleotides recited in SEQ ID NO: 1. Such "conservative amino acid" variants can be obtained, for example, by t> g directed mutagenesis. oligonucleotides, linker scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) on pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)). The ability of such variants to modulate cellular interactions or other properties of the wild-type protein as described herein, can be determined using standard methods, such as the assays described herein. Alternatively, a variant zacrp5 polypeptide can be identified by the ability to bind specifically to anti-zacrp5 antibodies.

The proteins of the present invention can also comprise the amino acid residues that are naturally present. Amino acids that are not naturally present include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine, allorthonine. , methyl-threonine, hydroxyethyl-cysteine, hydroxy-ethyl-homocysteine, nitroglutamine, homo-glutamine, pipecolic acid, thiazolidin carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethyl-proline, tert-leucine, norvaline, 2-azaphenylalanine , 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Various methods are known in the art to incorporate the amino acid residues that are not naturally present in the proteins. For example, an in vitro system can be used wherein the antisense mutations are suppressed using the chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are already known in the art. The transcription and translation of the plasmids containing the antisense mutations is typically carried out in a cell-free system comprising an extract of E. coli S30 and commercially available enzymes and other reagents. The 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., Sciences 259: 806, 1993, and Chung et al., Proc. Nati Acad. Sci. USA 90: 10145, 1993. In a second method, translation is carried out in Xenopus oocytes by microinjection of the mutated mRNA and the chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271: 19991, 1996). Within a third method, the E. coli cells are cultured in the absence of a natural amino acid that is going to be replaced '(for example, with phenylalanine) and in the presence of the amino acid (s) that are not naturally present (for example, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The amino acid that is not naturally present is incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem. 3J3: 7474, 1994. Amino acid residues that are naturally present can be converted to species that are not naturally present by chemical modification in vitro. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395, 1993). ., .. ¡, -. «--.- > - --- A-É- A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, amino acids that are not naturally present, and unnatural amino acids, can be substituted by the amino acid residues of zacrp5 . Multiple amino acid substitutions can be made and tested using the known methods of mutagenesis and selection, such as those described by Reidhaar-Olson and Sauer (Science 241: 53, 1998) or Bowie and Sauer (Proc. Nat. Acad. Sci. USA 86: 2152, 1989). Briefly, these authors describe methods for simultaneously randomly locating two or more positions in a polypeptide, selecting the functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (eg, Lowman et al., Biochem. 3 ^: 10832, 1991, Ladner et al., US Patent No. 5,223,409, Huse, international application No. WO 92 / 06204, and region-directed mutagenesis (Derbyshire et al., Gene _46: 145, 1986, and Ner et al., DNA 7: 127, 1988.) Described zacrp5 nucleotide variants and polypeptide sequences can also be generated by the messy DNA as described by Stemmer, Nature 370: 389, 1994, Stemmer, Proc. Nat. Acad. Sci. USA 91: 10747, 1994, and international application No. WO 97/20078. Briefly, variable DNA molecules are generated by homologous recombination in vitro by randomly fragmenting an original DNA followed by re-binding using PCR, leading to randomly introduced point mutations. This technique can be modified using a family of original DNA molecules, such as allelic variants or DNA molecules from different species, to introduce additional variability into the process. The selection or choice of the desired activity, followed by additional iterations of the mutagenesis and the assay, provides a rapid "evolution" of the sequences by selecting the desirable mutations while simultaneously selecting against the deleterious changes. Mutagenesis methods as described herein can be combined with automated, high throughput screening methods to detect the activity of mutagenized, cloned polypeptides in host cells. Mutagenized DNA molecules that encode biologically active polypeptides, or polypeptides that bind with anti-zacrp5 antibodies, can be recovered from host cells and sequenced ----- U-Ui.fc.MU-A- .. -tfcj -. ^.

Quickly using modern equipment. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. The essential amino acids in the polypeptides of the present invention can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081, 1989, Bass et al., Proc. Nat. Acad. Sci. USA 88: 4498, 1991, Coombs and Corey, "Site Directed Mutagenesis and Protein Engineering", in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 ( Academic Press, Inc. 1998)). In this latter technique, unique alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested to verify biological activity as described below to identify the amino acid residues that are critical for the activity of the molecule . See also, Hilton et al., J. Biol. Chem. 271: 699, 1996. The identities of the essential amino acids can also be inferred from the analyzes of homologies with zacrp5. ? & sh? * 1 ~ .-- »» «& _« .. 'wí «t'miX < - fF¿ *?; .

The location of the agglutination domains of the zacrp5 receptor can be identified by the physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with the mutation of the amino acids of the supposed contact site. See, for example, de Vos et al., Science 255: 306, 1992, Smith et al., J. Mol. Biol. 224: 899, 1992, and Wlodaver et al., FEBS Lett. 309: 59, 1992. In addition, zacrp5 labeled with biotin or FITC can be used for the expression of cloning of zacrp5 receptors. The present invention also provides fragments of polypeptides or peptides comprising an epitope-bearing portion of a zacrp5 polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope" which is a part of a protein that produces an antibody response when the entire protein is used as an immunogen. Peptides carrying the immunogenic epitope can be identified using standard methods (see, for example, Geysen et al., Proc. Nat. Acad. Sci. USA 81: 3998, 1983). In contrast, polypeptide fragments or peptides may comprise an "antigenic epitope" which is ? .. A A .-- fa_a, .i ..-..--- iiif) f ^ - - a- t- -. - ---- - a region of a protein molecule to which an antibody can bind specifically. Certain epitopes consist of a linear or contiguous extension of the amino acids, and the antigenicity of such epitope is not altered by the denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic the epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al., Science 219: 660, 1983). Accordingly, the peptides and polypeptides carrying the antigenic epitope of the present invention are useful for enhancing antibodies that bind with the polypeptides described herein. Peptides and polypeptides carrying the antigenic epitope preferably contain at least four to ten amino acids, at least ten to fifteen amino acids, or approximately 15 to approximately 30 amino acids of SEQ ID NO: 2. Such epitope-bearing peptides and polypeptides can be produced by fragmenting a zacrp5 polypeptide, or by the synthesis of the chemical peptide, as described herein. In addition, the epitopes can be selected by the phage display of the random peptide libraries (see, for example, Lane and Stephen, Curr Opin. Immunol., 5: 268, 1993, and Cortese et al., Curr. Opin. . -4? Uia afe aá - », L - tej --- *?,. - _ ... -.,., Biotechnol. 7: 616, 1996). Standard methods for identifying epitopes and producing antibodies from small peptides comprising an epitope are described, for example, by Mole, "Epitope Mapping", in Methods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-16 (The Humana Press, Inc. 1992), Price "Production and Characterization of Synthetic Peptide-Derived Antibodies", in Monoclonal Antibodies: Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages 60- 84 (Cambridge University Press 1995), and Coligan et al. (eds.), Current Protocols in Immunology, pages 9.3.1 - 9.3.5 and i * pages 9.4.1 - 9.4.11 (John Wiley and Sons 1997). Regardless of the particular nucleotide sequence of a variable zacrp5 gene, the gene encodes a polypeptide that is characterized by its ability to modulate cellular and extracellular interactions, or other activities of the wild-type protein as described herein, or by the ability to specifically bind to an anti-zacrp5 antibody. More specifically, the variable zacrp5 genes encode polypeptides that exhibit at least 50%, and preferably more than 70, 80, or 90%, of the activity of the polypeptide encoded by the human zacrp5 gene described herein. .-l-.iiÁ.fcfa * ^ '* -----------.-......- MÉHAMut -..-! ^ & > & T ^, i ^, h? Mit tmi * it ti, For any zacrp5 polypeptide, including variants and fusion proteins, a person with ordinary skill in the art can easily generate a completely degenerate polynucleotide sequence, which encodes this variant using the information described in Tables 2 and 3 above. In addition, those skilled in the art can use the standard programs to contemplate zacrp5 variants based on the nucleotide and amino acid sequences described herein. Accordingly, the present invention includes a computer-readable medium encoded with the data structure that provides at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11. Suitable means of the computer-readable medium include magnetic media and media that can be read optically. Examples of the magnetic media include a fixed or hard drive unit, an integrated random access memory (RAM) circuit, a flexible magnetic disk, a digital linear tape (DTL), an associated disk memory, and a disk of ZIP. The optically readable media is exemplified by compact discs (for example, memory only for CD reading (ROM), capable of writing to a CD (RW), and capable of recording on a CD), and the discs of -. Í? , video / versatile, digital (DVD) (for example, DVD-ROM, DVD-RAM, and DVD + RW). The present invention further provides a variety of mergers of related multimeric pins and polypeptides comprising one or more polypeptide fusions. For example, a zacrp5 polypeptide can be prepared as a fusion to a dimerization pin, such as immunoglobulin constant region domains, as described in U.S. Pat. Nos. 5,155,027 and 5,567,584. The zacrp5-immunoglobulin polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zacrp5 analogues. Auxiliary domains can be fused to zacrp5 polypeptides to target them with respect to specific cells, tissues, or macromolecules (e.g., collagen). For example, a zacrp5 polypeptide or pin could be targeted to a predetermined cell type by fusing a zacrp5 polypeptide to a ligand that is specifically used to a receptor on the surface of the target cell. In this way, polypeptides and pins can be targeted for therapeutic or diagnostic purposes. A zacrp5 polypeptide can be fused to two or more portions, such as an affinity tag for purification and a targeting domain as target. Fusions of the polypeptide may also comprise one or more cleavage sites, particularly between the domains. See, Tuan et al., Connective Tissue Research 34: 1-9, 1996. The zacrp5 fusion pins of the present invention encompass (1) a polypeptide selected from the group consisting of: (a) the polypeptide molecules comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 1 (Met), 18 (Trp) or 70 (Gly) to amino acid residue 252 (Leu); (b) polypeptide molecules ranging from amino acid 70 (Gly) to amino acid 111 (Pro) of SEQ ID NO: 2, a portion of the zacrp5 polypeptide that contains the collagen-like domain or a portion of the domain similar to collagen capable of dimerization or oligomerization; (c) the polypeptide molecules ranging from amino acid 112 (Cys) to 252 (Leu) of SEQ ID NO: 2, a portion of the zacrp5 polypeptide that contains the Clq domain or an active portion of the Clq domain; or (d) the polypeptide molecules ranging from amino acid 70 (Gly) to 252 (Leu), a portion of the zacrp5 polypeptide that includes the collagen-like domain and the Clq domain; and (2) another polypeptide. The other polypeptide i i. i 1 tíé-liíiiifefei may be the alternative or additional Clq domain, a domain similar to alternative or additional collagen, a signaling peptide to facilitate the secretion of the fusion pin or the like. Such domains can be obtained from other members of the pin family related to the complement of adipocytes, other pins having collagen and / or Clq domains as described herein. The globular domain of the complement agglutinates to IgG, therefore, the globular domain of the polypeptide, fragment or fusion of zacrp5 may have a similar role. The polypeptides of zacrp5, which vary from amino acid 1 (Met) to amino acid 252 (Leu); the mature zacrp5 polypeptides, which vary from amino acid 18 (Trp) to amino acid 252 (Leu); or the fragments directing the secretion thereof, such fragments ranging from amino acid 1 (Met) to amino acid 17 (Ala) can be used in the study of the secretion of pins from cells. In the preferred embodiments of this aspect of the present invention, the mature polypeptides are formed as fusion pins with the sequences of the putative secretory signal; the plasmids carrying regulatory regions capable of directing the expression of the fusion pin are introduced into the test cells; and the secretion of the mature protein is verified. Verification can be done by techniques known in the art, such as CLAR and the like. The polypeptides of the present invention, including full-length proteins, fragments thereof and fusion proteins, can be produced in host cells genetically engineered according to conventional techniques. Suitable host cells are those types of cells that can be transformed or transfected with the exogenous DNA and grown in the culture, and include the highest cultivated bacteria, fungal cells, and eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for the manipulation of cloned DNA molecules and the introduction of exogenous DNA into a variety of host cells are described by Sambrook et al., Ibid. , and Ausubel et al. ibid. In general, a DNA sequence encoding a zacrp5 polypeptide of the present invention is linked - operatively to other genetic elements required for their expression, which generally include a transcription promoter and a terminator within an expression vector. The vector will also commonly contain one or more i? ñAá Lh &? r? Mi ^ .. * ..._ M ^^ ri¡a8k? t. --w. a-jtaj »-. *, ik- & -iaaÉ- ^ selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems the selectable markers can be provided on separate vectors, and the replication of the exogenous DNA can be provided by the integration into the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary experience in the art. Many such elements are described in the literature and are available through commercial providers. To direct a polypeptide of zacrp5 towards the secretory pathway of a host cell, a sequence of the secretory signal (also known as a leader sequence, the signaling sequence, the prepro sequence or the presequence), is provided in the vector of the expression . The sequence of the secretory signal may be that of the zacrp5 polypeptide, or it may be derived from another protein secreted (eg, t-PA) or synthesized de novo. The sequence of the secretory signal is linked to the DNA sequence of the zacrp5 polypeptide in the correct reading structure. The sequences of the secretory signal are located 5 'commonly with respect to the sequence of the DNA encoding the polypeptide of interest, although certain Vamp; .AiA., Í! ki.i * h.mi & - tf- '- -t "- - i_., .. Mr &, more? i. £ A -'y ^^^' - ^ '^ ^ r »^? v? r? irf» ^ ?? «&I signal sequences can be placed elsewhere in the DNA sequence of interest (see, for example, Welch et al., US Patent No 5,037,743, Holland et al., US Patent No. 5,143,830.) In contrast, the portion of the signal sequence of the zacrp5 polypeptide (amino acid residues 1-17 of SEQ ID NO: 2) can be used to directing the secretion of an alternative protein by analogous methods The sequence of the secretory signal contained in the polypeptides of the present invention can be used to direct other polypeptides towards the secretory path The present invention provides such fusion polypeptides. Fusion of the signal can be done where a sequence of the secretory signal derived from amino acid residues 1-17 of SEQ ID NO: 2 is operably linked to another polypeptide using methods known in the art and described herein. The sequence of the secretory signal contained in the polypeptides of the fusion of the present invention is preferably amino-terminally fused to an additional peptide to direct the additional peptide towards the secretory pathway. Such constructions have numerous applications known in the art. For example, these fusion constructs of the novel secretory signal sequence can direct the secretion of a active component of a protein not normally secreted, such as a receptor. Such fusions can be used in vivo or in vitro to direct the 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 14 ^: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981: Graham and Van der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-5, 1982), transfection mediated by DEAE-dextran (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 et al. Rosman, BioTechniques: 980_90 '1989; Wang and Finer, Nature Med. 2: 714-6, 1996). The production of recombinant polypeptides in cultured mammalian cells is described, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. 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 COS-1 cell lines (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 (eg CHO-K1, ATCC No. CCL 61 and DG44 CHO , Chasin et al., Som.Cell.Molec.Genet.12: 555-666, 1986). Additional suitable cell lines are already known in the art and are available from public depositories such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters, such as the SV-40 or cytomegalovirus promoters, are preferred. See, for example, U.S. Pat. No. 4,956,288. Other suitable promoters include those of the metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the major final promoter of the adenovirus. The selection of the drug is generally used to select the cultured mammalian cells within which the 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 capable of passing the gene of interest to their progeny are referred to as "stable transfectants". A preferred selectable marker is a gene that encodes antibiotic neomycin resistance. The selection is carried out in the presence of a drug of the neomycin type, such as G-418 or the like. Selection systems can also JL? .A.tLAjk? Aía »*.« Ki. used to increase the level of expression of the gene of interest, a process referred to as "amplification". The amplification is carried out by culturing the transfectants in the presence of a low level of the selective agent and then increasing the amount of the selective agent to select the cells that produce high levels of the products of the introduced genes. A selectable, amplifiable, preferred marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, resistance to multiple drugs, puromycin acetyltransferase), may also be used. Alternate markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, MHC Class I, alkaline phosphatase of the placenta, can be used to select transfected cells of the cells not transfected by means such as the selection of FACS or the separation technology of the magnetic beads. Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and bird cells. The use of Agrobacterium um rhizogenes as a vector for the expression genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11: 47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is described by Guarino et al., U.S. Pat. No. 5,162,222 and the publication WIPO 94/06463. Insect cells can be infected with the recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See, King and Possee, The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. A second method for the manufacture of recombinant zacrp5 baculovirus utilizes a transposon-based system described by Luckow (Luckow et al., J. Virol. 67: 4566-79 , 1993). This system, which uses the transfer vectors, is sold in the Bac-to-Bac ™ suite (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl ™ (Life Technologies) which contains a Tn7 transposon to move the DNA encoding the zacrp5 polypeptide into a baculovirus genome maintained in E. coli co or a large plasmid called a **.? * áhiJA?. *? ií ~.

"Bacmido" The pFastbacl ™ transfer vector uses the AcNPV polyhedrin promoter to activate the expression of the gene of interest, in this case zacrp5. However, pFastbacl ™ can be modified to a considerable degree. The polyhedrin promoter can be removed and replaced with the baculovirus basic protein promoter (also known as the Peor vector, p6.9 or MP) which is previously expressed in the infection of the vaculovirus and has been shown to be advantageous to express the secreted proteins. See, Hill-Perkins and Possee, J. Gen. Virol. 71: 971-6, 1990; Bonning et al., J. Gen. Virol. 75: 1551-6, 1994; and, Chazenbalk, and Rapoport, J. Biol. Chem. 270: 1543-9, nineteen ninety five. In such constructions of the transfer vector, a short or long version of the basic protein promoter can be used. In addition, the transfer vectors can be constructed, which replace the sequences of the secretory signal of natural zacrp5 with the sequences of the secretory signal derived from insect proteins. For example, a sequence of the secretory signal of the Ecdysteroid Glucosyltransferase (EGT), Melitin of the honey bee (Invitrogen, Carlsbad, CA), or the gp67 baculovirus (PharMingen, San diego, CA) can be used in constructions to replace the sequence of the natural zacrp5 secretory signal. In addition, transfer vectors can include a fusion within the structure with the DNA encoding an epitope tag at the C or N terminus of the expressed zacrp5 polypeptide, eg, a Glu-Glu epitope tag (Grussenmeyer et al. ., Proc. Nati, Acad. Sci. 82: 7952-4, 1985). Using a technique known in the art, a transfer vector containing the zacrp5 is transformed into E. coli, and selected for the bacmides which contain an interrupted lacZ gene indicative of the recombinant baculovirus. Bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, for example Sf9 cells. The recombinant virus expressing zacrp5 is subsequently produced. Viral storage materials, recombinants, are made by methods commonly used in the art. The recombinant virus is used to infect the host cells, typically a cell line derived from the caterpillar of the autumn moth, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, DC, 1994. Another suitable cell line is the High FiveO ™ cell line (Invitrogen) derived from Trichopl usia ni (US Pat. No. 5,300,435). Commercially available free serum media are used to grow and maintain cells. Suitable media are Sf900 II ™ (Life Technologies) or ESF 921 ™ (Expression Systems) for Sf9 cells; and ExcellO405 ™ (JHR Biosciences, Lenexa, KS) or Express FiveO ™ (Life Techonologies) for T. Ni cells. The cells are grown from an inoculation density of about 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells, at which time they are added into recombinant viral storage material at a multiplicity of sites of infection. (MOI) from 0.1 to 10, more typically close to 3. The procedures used are generally described in the available laboratory manuals (King and Possee, ibid., O'Reilly et al., Ibid., Richardson, ibid.) . Subsequent purification of the zacrp5 polypeptide from the supernatant can be accomplished using the 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 Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. The methods to transform S. cerevisiae cells with exogenous DNA and produce iilrtf * ^? ift ^ j ^ - * '^^^^^ w. «-« fa ^ i-- -A-, ^ •' ^. ^ Mj tJu? ^ ía? j ^ iJ ^ fii ^^ ^^^^ The recombinant polypeptides from them are described, for example, by Kawasaki, US Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are selected by the 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 described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows the transformed cells to be selected for growth in the glucose-containing medium. Suitable promoters and terminators for use in yeast include those from glycolytic genes enzyme (see, eg, Kawasaki, US Patent No. 4,599,311; Kingsman et al., US Patent No. 4,615,974, and Bitter, US Patent No. 4,977,092) and the alcohol dehydrogenase genes. See also U.S. Pat. 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, Kl uyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida bad cough are already known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-65, 1986 and Cregg, U.S. Pat.

No. 4,882,279. Aspergillus cells can be used according to the methods of McKnight et al., U.S. Patent No. 4,935,349. The methods to transform Acremoni um chrysogenum are described by Sumino et al., U.S. Patent No. 5,162,228. The methods to transform Neurospora are described by Lambowitz, U.S. Pat. No. 4,486,533. The use of Pichia methanolica as the host for the production of recombinant proteins is described in Publications WIPO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in the transformation of P. Methanolica will be commonly prepared as circular, double-stranded plasmids, which are preferably linearized prior to transformation. For the production of polypeptides in P. methanolica, it is preferred that the promoter and terminator in the plasmid be those of a P. methanolica gene, such as a gene utilization Alcohol P. methanolica (AUG1 or AUG2) . Other useful promoters include those of the dihydroxyacetone synthase (DHAS) genes, formate dehydrogenase (FMD), and catalase (CAT). To facilitate the integration of A áM.at-i -...-. I- --.- Aa ^ _ --- .-- a. ? - ~ M ----?. »- tm« --- F ÜFÍ. In the DNA in the host chromosome, it is preferred to have the complete expression segment of the plasmid flanked at both ends by the host DNA sequences. A preferred for use in Pichia methanolica ADE2 selectable marker is a P. methanolica gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in absence of adenine. For large-scale industrial processes, where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both of the methanol utilization genes (AUG1 and AUG2) are deleted. For the production of the secreted proteins, host cells deficient in the vacuolar protease genes (PEP4 and PREB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing the DNA, which encodes a polypeptide of interest in P. methanolica cells. It is preferred to transform the P. methanolica cells by electroporation using an exponentially decaying pulsing electric field, having a field strength of 2.5 to 4.5 kV / cm, preferably about 3. 75 kV / cm, and a time constant (t) from 1 to 40 milliseconds, more preferably approximately 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria of Escherichia coli, Bacillus and other genera, are also useful host cells within the present invention. Techniques for the transformation of these hosts and the expression of the foreign DNA sequences cloned there are well known in the art (see, for example, Sambrook et al., Ibid.). When a polypeptide of zacrp5 is expressed in bacteria such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or can be directed to the periplasmic space by a sequence of bacterial secretion. In the first case, the cells are used, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be re-folded and dimensioned by eluting the denaturant, such as by 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 altering or breaking the cells (for example, by the application of sound or by osmotic shock) to release the contents of the periplasmic space and recover the protein, whereby it is eliminated the need for denaturation and re-folding. The transformed or transfected host cells are cultured according to the conventional procedures in a culture medium containing the nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a source of carbon, a source of nitrogen, essential amino acids, vitamins and minerals. The media may also contain components such as growth factors or serum, when required. The growth medium will generally be selected for cells containing the exogenously added DNA, for example, by drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or cotransfected in the host cell. The recombinant zacrp5 polypeptides expressed (or the chimeric zacrp5 polypeptides) can be purified using conventional fractionation and / or purification methods and media. Precipitation with ammonium sulfate and extraction with acid or a chaotropic substance can be used for the fractionation of the samples. Exemplary purification steps may include hydroxyapatite treatment, size exclusion chromatography, FPLC, and reverse phase high resolution liquid chromatography. Suitable chromatographic media include the dextrans derivatives, agarose, cellulose, polyacrylamide, special silicas, and the like. The derivatives of PEI, DEAE, QAE and Q are preferred. Exemplary chromatographic media include those media derived with the phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeriville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Hass) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like which are insoluble under the conditions in which they will be used. These supports can be modified with the reactive groups that allow the fixation of the proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties. Examples of chemical procedures binding include activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl, activation with hydrazide, and carboxyl and amino derivatives for the chemical carbodiimide binding processes. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for agglutination of the receptor polypeptides to the support media are well known in the art. The selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen medium. See, for example, Affinity Chromatography: Principies & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988. The polypeptides of the present invention can be isolated by exploitation of their structural or agglutination properties. For example, adsorption chromatography of immobilized metal ions (IMAC) can be used to purify histidine-rich proteins or proteins that have a His tag. Briefly, a gel is charged first with the divalent metal ions to form a chelate (Sulkowski, Trends in Biochem.3: 1-7, 1985). The proteins rich in histidine will be adsorbed to this matrix with different affinities, depending on the metal ion used, and will be eluted by competitive elution, reducing the pH, or using strong chelating agents. Other purification methods include the purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", Deutscher, (ed.), Acad. Press, San Diego, 1990, pp. 529-39). Within the further preferred embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (for example, the maltose agglutination protein, FLAG, Glu-Glu, an immunoglobulin domain) can be constructed to facilitate purification as described in greater detail in the sections of the Examples below. The procedures to re-fold the protein (and optionally, reoxidation) can be used advantageously. It is preferred to purify the protein to a purity > 80%, more preferably up to a purity > 90%, even more preferably > 95%, and a pharmaceutically pure state is particularly preferred, ie greater than 99.9% pure with respect to the contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin. The polypeptides of zacrp5 or fragments thereof can also be prepared through chemical synthesis by methods well known in the art, such as exclusive solid phase synthesis, partial solid phase methods, condensation synthesis of fragments or in classical solution, see for example, Merrifield, J. Am. Chem. Soc. 85: 2149, 1963. Such zacrp5 polypeptides can be monomers or multimers; glycosylated or non-glycosylated, treated with PEG or not treated with PEG; and may or may not include an initial methionine amino acid residue. A ligand agglutination polypeptide, such as a zacrp5 agglutination polypeptide, can also be used for the purification of the ligand. The polypeptide is immobilized on a solid support, such as agarose beads, crosslinked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or similar materials that are stable under the conditions of use. Methods for linking the polypeptides to the solid supports are already known in the art, and include the chemistry of the amines, the activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl, and activation with hydrazide. The resulting medium will generally be configured in the form of a column, and the fluids containing the ligand are passed through the column one or more times to allow the ligands to bind to the ligand agglutination polypeptide. The ligand is then eluted using changes in the concentration of the salt, the chaotropic agents (guanidine HCl), or the pH to alter the agglutination of the receptor-ligand. A test system using a ligand agglutination receptor (or an antibody, an element of a complement / anti-complement pair) or an agglutination fragment thereof, and a commercially available biosensor instrument (BIAcore ™, Pharmacia Biosensor, Piscataway, NJ) can be used advantageously. Such receptor, antibody, element of a complement / anti-complement pair or fragment, is immobilized on the surface of an integrated circuit of the receiver. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cumnningham and Wells, J. Mol. Biol. 234: 554-63, 1993. A receptor, antibody, element or fragment is covalently bound, using the chemical procedure of the amine or sulfhydryl, to the fibers "" "'? .tH? - * - ** LJ¡ ***» * - + - J ^ of dextran that are fixed to the gold film inside the flow cell.A test sample is passed to If a ligand, epitope, or opposite member of the complement / anti-complement pair is present in the sample, it will agglutinate with the receptor, antibody or element, immobilized, respectively, causing a change in the refractive index of the medium, which is detected as a change in the resonance of the surface plasmon of the gold film.This system allows the determination of the activation and deactivation rates, from which the affinity of agglutination can be calculated, and the t evaluation of the agglutination stoichiometry Ligand agglutination polypeptides can also be used within other assay systems known in the art, such systems include Scatchard analysis for the determination of agglutination affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253: 545-48, 1991; Cunningham et al., Science 245: 821-25, 1991). The invention also provides anti-zacrp5 antibodies. Antibodies to zacrp5 can be obtained, for example, by using as an antigen the product of a zacrp5 expression vector, or zacrpd isolated from a source natural. Particularly useful anti-zacrp5 antibodies "specifically agglutinate" with zacrp5. Antibodies are considered to bind specifically if the antibodies are bound with a polypeptide, peptide or zaprpd epitope with an agglutination affinity (Ka) of 106 M "1 or larger, preferably 107 M" 1 or larger, more preferably of 108 M "1 or larger, and even more preferably 109 M" 1 or larger. The affinity of the binding of an antibody can easily be determined by a person of ordinary skill in the art, for example, by the Scatchard analysis (Scartchard, Ann. NY Acad. Sci. 5: 1, 660, 1949). Suitable antibodies include antibodies that bind with zacrp5 in the particular domains. Anti-zacrp5 antibodies can be produced using the peptides and polypeptides carrying the antigenic zacrp5 epitope. The zacrp5 peptides and polypeptides carrying the antigenic epitope of the present invention contain a sequence of at least nine, preferably from 15 to about 30 amino acids contained within SEQ ID NO: 2. However, the peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length -------- - «^.« .-. *? ----- t'llÉ? Ét < μi - ^ * '«* ^ ------ l -« - tt --- át-aa-t-ai -sl- up to and including the complete amino acid sequence of a polypeptide of the invention, they are also useful for inducing antibodies that bind with zacrp5. It is desirable that the amino acid sequence of the epitope-bearing peptide be selected to provide substantial solubility in aqueous solvents (ie, the sequence includes relatively hydrophilic residues, whereas hydrophobic residues are preferably avoided). Hydrophilic peptides can be predicted by a person skilled in the art from a graph of hydrophobicity, see for example, Hopo and Woods (Proc. Nat. Acad. Sci. USA 78: 3824-8, 1981) and Kyte and Doolittle (J. Mol. Biol. 157: 105-142, 1982). In addition, amino acid sequences containing the proline residues may also be desirable for the production of the antibodies. Within one embodiment 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) a polypeptide comprising a sequence of amino acid residues that is minus 80% identical in the amino acid sequence for residues 70-252 of SEQ ID NO: 2, wherein the sequence comprises; the repeats of Gly-Xaa-Xaa and Gly-Xaa-Pro that form a domain similar to collagen, in j where Xaa is any amino acid residue; and a carboxyl-terminal Clq domain; b) a polypeptide comprising; an amino terminal region, 14 repeats of Gly-Xaa-Xaa and 1 repeat of Gly-Xaa-Pro that forms a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clq 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; 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 Clq domain or an active portion of the Clq domain; or e) a portion of the zacrp5 polypeptide as shown in SEQ ID NO: 2 comprising the collagen-like domain and the Clq domain; and wherein the polypeptide produces an immune response in the animal to produce the antibody; and isolate the animal's antibody. Polyclonal antibodies to the recombinant zacrp5 protein or to zacrp5 isolated from native sources can be prepared using methods well known to those skilled in the art. See, for example, Green et al., "Production of Polyclonal antisera", in I munochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al., "Expression of foreign proteins in E.coli using plasmid vectors and purification of specific polyclonal antibodies", in DNA Cloning 2: Expression Systems, 2 / a. Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995). The immunogenicity of a zacrp5 polypeptide can be increased by the use of an auxiliary, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for tr immunization also include fusion polypeptides, such as zacrp5 fusions or a portion thereof with an immunoglobulin polypeptide or with the maltose agglutination protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the portion of the polypeptide is "hapten-like", such a portion can be advantageously linked or bound to a macromolecular carrier (such as hawthorn haemacyanine (KHL), bovine serum albumin (BSA) or toxoid). tetanus) for immunization. Although polyclonal antibodies are typically elevated in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, hamsters, guinea pigs of Indian, goat, or sheep, an anti-zacrp5 antibody of the present invention can also be derived from a subhuman primate antibody. General techniques for enhancing the diagnostically and therapeutically useful antibodies in mandrels can be found, for example, in Goldenberg et al., International Patent Publication No.

WO 91/11465, and Losman et al., Int. J. Cancer 46: 310, 1990.

The antibodies can also be enhanced in transgenic animals such as sheep, cattle, goats or transgenic pigs, and can also be expressed in yeast and fungi in the modified forms as well as in the cells of mammals and insects. Alternatively, monoclonal anti-zacrp5 antibodies can be generated. Monoclonal antibodies of rodents for specific antigens can be obtained by methods known to those skilled in the art. (see, for example, Kohler et al., Nature 256: 495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, vol. 1, pages 2.2.1-2.6.7 (John Wiley &Sons 1991), Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli", in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)).

- ^^^ ^^^!? J ^ Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a gene product zacrp5, verifying the presence of antibody production by removing a sample of serum, removing the spleen to obtain the B lymphocytes, fusing the B lymphocytes with the myeloma cells to produce hybridomas, cloning the hybridomas, selecting the positive clones which produce antibodies for the antigen, cultivating the clones that produce the antibodies for the antigen, and isolating the antibodies from the hybridoma cultures. In addition, an anti-zacrp5 antibody of the present invention can be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been designed to produce specific human antibodies in response to antigenic stimulation. In this technique, the elements of the human light and heavy chain site are introduced into the strains of the mice derived from the embryonic stem cell lines containing the labeled alterations of the light chain and heavy chain sites, endogenous Transgenic mice can synthesize human antibodies specific for human antigens, and mice can be used to produce -jfcA «?. A? --- ¿- ** --- * - i .. -t_-i - a - i .. -v-k-1. the hybridomas that secrete human antibodies. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al., Nature Genet. 7:13, 1994, Lonberg et al., Nature! 368: 856, 1994, and Taylor et al., Int. Immun. 6: 579, 1994. Monoclonal antibodies can be isolated and purified from the hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose chromatography, size exclusion chromatography, and ion exchange chromatography. (see, for example, Coligan on pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of Immunoglubulin G (IgG)", in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)). For particular uses, it may be desirable to prepare the fragments of anti-zacrp5 antibodies. Such antibody fragments can be obtained, for example, by the proteolytic hydrolysis of the antibodies. The fragments of the antibodies can be obtained by dissolving the pepsin or papain of the total antibodies by conventional methods. As an illustration, antibody fragments can be produced by enzymatic cleavage of the antibodies with pepsin to provide a 5S fragment denoted F (ab ') 2. This fragment can be further cleaved using a thiol reducing agent to produce the 3.5B Fab 'monovalent fragments. Optionally, the cleavage reaction can be effected using a blocking group for the sulfhydryl groups resulting from the cleavage of the disulfide bonds. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and one Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al., Arch. Biochem. Biophys. 89: 230, 1960, Porter, Biochem. J. 73: 119, 1959, Edelman et al., In Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan, ibid. Other methods of cleavage of antibodies, such as the separation of heavy chains to form monovalent light-heavy chain fragments, the further fragmentation of fragments, or other enzymatic, chemical or genetic techniques can also be used, provided that the fragments agglutinate the antigen that is recognized by the intact antibody. For example, the Fv fragments comprise an association of the VH and V_ chains. This association may be non-covalent, as described by Inbar et al., Proc. Nati Acad. Sci. USA 69: 2659, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or crosslinked by chemical substances such as glutaraldehyde (see, eg, Sandhu, Crit. Rev. Biotech 12: 437, 1992). The Fv fragments can comprise the VH and VL chains which are connected by a peptide linker. These single chain antigen agglutination proteins (scFv) are prepared by constructing a structural gene comprising the DNA sequences encoding the VH and V_ domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide that bridges the two V domains. Methods for producing scFvs are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97, 1991, also see, Bird et al., Science 242: 423, 1988, Ladner et al., US Patent. No. 4,946,778, Pack et al., Bio / Technology 11: 1271, 1993, and Sandhu, ibid. As an illustration, a scFv can be obtained by exposing the lymphocytes to the zacrp5 polypeptide in vitro, and selecting antibody display libraries in the phage or similar vectors (eg, through the use of immobilized or tagged zacrp5 peptide or protein). The genes encoding the polypeptides having the potential zacrp5 polypeptide agglutination domains can be obtained by selecting the libraries of the random peptides displayed on the phage (phage display) or on the bacteria, such as E. coli. The nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as by means of random mutagenesis and synthesis of the random polynucleotides. These display libraries of the random peptides can be used to select the peptides which interact with a known target, which can be a protein or a polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic substances or inorganic Techniques for creating and selecting such random peptide display libraries are already 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., Patent US No. 5,403,484, Ladner et al., US Patent No. 5,571,698, and Kay et al., Phage Display of Peptides and Proteins (Academic Press, Inc. 1996)) and libraries ^ ja tüite u-- of display of the random peptides and sets to select such libraries are commercially available, for example 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 selected using the zacrp5 sequences described herein to identify proteins which bind to zacrp5. Another form of an antibody fragment is a peptide that encodes a unique β-complementarity (CDR) determination region. The CDR peptides ("minimum recognition units") can be obtained by constructing the genes encoding the CDRs of an antibody of interest. Such genes are prepared, for example, by using the poly-eraser chain reaction to synthesize the variable region of the RNA of the cells that produce antibodies (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106, 1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies", in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press, 1995), and Ward et al., "Genetic Manipulation and Expression of Antibodies", in Monoclonal Antibodies: Principies and Applications, Birch et al., (Eds.), Page 137 (Wiley-Liss, Inc. 1995)). Alternatively, an anti-zacrp5 antibody can be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring the regions of complementary determination of the mouse from the light and heavy variable chains of the mouse immunoglobulin into a human variable domain. The typical residues of the human antibodies are then substituted in the structure regions of the murine counterparts. The use of the antibody components derived from the humanized monoclonal antibodies eliminates the potential problems associated with the immunogenicity of the murine constant regions. General techniques for the cloning of the variable domains of murine immunoglobulin are described, for example, by Orlandi et al., Proc. Nati Acad. Sci. USA 8_6: 3833, 1989. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321: 522, 1986, Carter et al., Proc. Nat. Acad. Sci. USA 89: 4285, 1992, Sandhu, Crit. Rev. Biotech. 12: 437, 1992, Singer et al., J. Immun. 150: 2844, 1993, Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, "Engineering Therapeutic Antibodies", in Protein kA &AÁAs ?? Engineering: Principies and Practice, Cleland et al. (eds.), pages 399-434 (John Wiley &Sons, Inc. 1996), and by Queen et al., U.S. Pat. No. 5,693,762 (1997). Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-zacrp5 antibodies or antibody fragments, using standard techniques. See, for example, Green et al., "Production of Polyclonal Antisera," in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), Pages 1-12 (Humana Press 1992). Also, see Coligan, ibid. On pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can be prepared using anti-zacrp5 antibodies or antibody fragments as immunogens with the techniques described above. As another alternative, anti-humanized, alternative anti-idiotype antibodies or sub-human primate anti-idiotype antibodies can be prepared using the techniques described above. Methods for producing anti-idiotype antibodies are described, for example, by Irie, U.S. Pat. No. 5, 208, 146, Greene, et al., U.S. Pat. No. 5,637,677, and Verthakavi and Minocha, J. Gen. Virol. 77: 1875, 1996. Genes encoding the polypeptides having the agglutination domains of the potential zacrp5 polypeptide, the "agglutination proteins", can be obtained by screening the random or targeted peptide libraries displayed on the phage (phage display) or about bacteria, such as E. coli. The nucleotide sequences encoding the polypeptides can be obtained in numerous ways, such as through random mutagenesis and random polynucleotide synthesis. Alternatively, restricted phage display libraries can also be produced. These peptide display libraries can be used to select the peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and selecting such peptide display libraries are already 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 the display libraries of the peptides and kits for selecting such libraries are commercially available, for example from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). The peptide display libraries can be selected using the zacrp5 sequences described herein to identify the proteins which bind to zacrp5. These "agglutination proteins" which interact with zacrp5 polypeptides can be used essentially in an antibody-like manner. A variety of assays known to those skilled in the art can be used to detect antibodies and / or agglutination proteins which agglutinate specifically to zacrp5 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 immunoelectrophoresis, radioimmunoassay, radioimmunoassay, the assay enzyme-linked immunosorbent (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich or sandwich assay. In addition, the antibodies can be selected for agglutination to the mutant zacrp5 protein or polypeptide against the wild type. Antibodies and agglutination proteins to zacrp5 can be used to label cells expressing zacrp5; to isolate the zacrpd by purifying Mintáfr-. * < * - • * - • - • ^ jXfú ^^^ affinity; for diagnostic assays to determine circulation levels of zacrp5 polypeptides; to detect or quantify soluble zacrp5 as a marker of the underlying disease or disease; in the analytical methods that use FACS; to select the expression libraries; to generate the anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block modulation of the zacrp5 polypeptide of spermatogenesis or similar activity in vitro and in vivo. Suitable labels or direct labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect tags or labels can characterize the use of avidin-biotin or other complement / anti-complement pairs as intermediates. In addition, antibodies to zacrp5 or fragments thereof can be used in vitro to detect denatured zacrp5 or fragments thereof in assays, eg, Western Blots and other assays known in the art. The antibodies or polypeptides herein can also be conjugated directly or indirectly with drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnosis or other therapeutic applications. For example, the polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anticomplementary molecule (receptor or antigen, respectively, for example). More specifically, zacrp5 polypeptides or anti-zacrp5 antibodies, or bioactive fragments or portions thereof, can be attached to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-DNA molecule. -complementary. A further aspect of the present invention provides methods for identifying the agonists or antagonists of the zacrp5 polypeptides described above, such agonists or antagonists may have valuable properties as described herein further. Within another embodiment, there is provided a method of identifying agonists of the zacrp5 polypeptide, which comprises providing cells that are activated in response to them, culturing the cells in the presence of a test compound and comparing the cellular response with the cell cultured in the presence of the zacrp5 polypeptide, and selecting the test compounds from which the cell response is of the same type.

Within another embodiment, there is provided a method of identifying the zacrp5 polypeptide antagonists, which comprises providing cells in response to a zacrp5 polypeptide, culturing a first portion of the cells in the presence of the zacrp5 polypeptide, culturing a second portion. of the cells in the presence of the zacrp5 polypeptide and a test compound, and detect a reduction in a cellular response of the second portion of the cells when compared to the first portion of the cells. In addition to these assays described herein, samples can be tested for the inhibition of zacrp5 activity within a variety of assays designed to measure receptor agglutination or stimulation / inhibition of zacrp5-dependent cellular responses. For example, the zacrp5 response cell lines can be transfected with a reporter gene construct that functions in response to a cell path stimulated by zacrp5. Constructs of the reporter gene of this type are already known in the art, and will generally comprise a zacrp5-responsive element operably linked to a gene encoding a testable protein, such as luciferase. DNA response elements may include, but are not limited to, cyclic AMP response elements (CRE), the hormone response elements (HRE), the insulin response elements (IRE) (Nasrin et al., Proc. Nati. Acad. Sci. USA 87: 5273-7, 1990) and the serum response elements (SRE) (Shaw et al., Cell 56: 563-72, 1989). The cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063-6, 1988 and Habener, Molec. Endocrinol 4 (8): 1087-94, 1990. The elements of response to hormones are reviewed in Beato, Cell 56: 335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested to verify their ability to inhibit zacrp5 activity on target cells as evidenced by a reduction in zacrp5 stimulation of reporter gene expression. Tests of this type will detect compounds that directly block the agglutination of zacrp5 to cell-surface receptors, as well as compounds that block processes in the cell pathway subsequent to agglutination of the receptor-ligand. In the alternative, compounds or other samples can be tested for direct blocking of agglutination of zacrp5 to the receptor using zacrp5 labeled with a detectable label (eg, 125I, biotin, horseradish peroxidase, FITC and the like). Within assays of this type, the ability of a test sample to inhibit the tá¡? to.? -L-t-t-itl - .. «-, i * 4 ^ -------». ».--. _-- A.-t.j-t .--. fc. > n j »i-U-_ ---? ----- * .-- M- > »» I ----. »< - «-. ami-fa i? %? agglutination of zacrp5 labeled to the receptor is indicative of inhibitory activity, which can be confirmed by secondary assays. The receptors used within the agglutination assays may be cellular receptors or immobilized receptors, isolated. Proteins related to adipocyte complement are involved in extracellular-cell or cell-cell matrix interactions, particularly those that involve the modulation of tissue remodeling. The phenotypic manifestation of many autoimmune diseases and related to remodeling is the extensive activation of tissue remodeling and / or inflammatory processes. The result is often that the tissue of the functional organ or sub-organ is replaced by a variety of components of the extracellular matrix (ECM) unable to perform the function of the replaced biological structure. There is incomplete understanding of the initiation events in these diseases, and the deposition of excessive extracellular matrix. The initiation events are hypothesized to involve an alteration or initial perturbation of the regulation of the optimal biological structure. Interestingly, sometimes the intracellular components are JI- M-M --- '«ni- - --tf - 8a f.jáfÁ. t ^ f .- ^ F ^ ^^ found as self-antigens, indicative of particular diseases. It could be that the production of antibodies by the immune system, after excessive exposure to these intracellular proteins, is a result of excessive or inappropriate remodeling. By targeting remodeling processes, it may be possible to reduce the effect of autoantigens. For this, polypeptides, fragments, fusions, agonists, zacrp5 antagonists and the like, could be beneficial in mediating a variety of autoimmune and remodeling diseases. It is possible that an inappropriate remodeling response to connective tissue or joint muscle injuries leads to sensitivity for excessive release of cellular components at the site of injury. Polypeptides, fragments, zacrp5 fusions and the like could be useful in determining if there is an association between such response and the inflammation associated with arthritis. Such indicators include a reduction in inflammation and relief of pain or stiffness, in the animal models, the indications could be derived from a macroscopic inspection of the joints and the change in the swelling of the hind legs. In animal models, the indications could be derived from the gross inspection of the joints and the change in the swelling of the hind legs. Polypeptides, fragments, zacrp5 fusions and the like, can be administered to animal models of osteoarthritis (Kikuchi et al., Osteoarthritis Cartilage 6: 177-86, 1998 and Lohmander et al., Arthritis Rheum. 42 !: 534-44, 1999) to seek the inhibition of tissue destruction resulting from inflammation stimulated by the action of collagenase. Recent discoveries have shown that the diagnosis of scleroderma autoantigens is what could be considered cytoplasmic proteins. The t > proteins, fragments, fusions of zacrp5 and the like, as provided herein, could be useful in determining whether antibodies to such proteins are enhanced or elevated as a response to inflation due to inadequate or incomplete repair of local tissue when it is mediated by a protein related to the complement of adipocytes. Polypeptides, fragments, zacrp5 fusions and the like, as provided herein, could be useful in determining whether excessive and / or inappropriate arterial remodeling plays a role in the formation of plaque in arterial sclerosis and arterial lesions. , such as arterial occlusion, using the methods provided here. The treatment of a vascular lesion (and the underlying extracellular matrix) with the zsig37 protein of the adipocyte complement appears to alter the process of vascular remodeling at a very early stage (co-pending US Patent No. 09 / 253,604). Treatment with a complement protein of adipocytes can act to keep platelets relatively inactive after injury, eliminating excessive recruitment of proteins and pro-inflammatory and pro-inflammatory cells. Other members of the family can modulate the remodeling induced by the presence for example of fat, or cholesterol. Excessive amounts of cholesterol and fat in the blood could activate remodeling, in the absence of the correct member of the adipocyte complement protein family. ACRO30 is expressed only in the active proliferation of adipose tissue. The remodeling of the connective tissue is closely related to this activation of the fat cells. There is clearly a relationship between excessive weight gain (fat) and diabetes. Therefore, it is likely that ARCP30 is involved in the remodeling of fat and this process is overwhelming in obese individuals. As a result, the effects of inadequate and inappropriate fat storage contribute to the onset of Type II diabetes. The energy balance (which involves energy metabolism, nutritional status, storage of lipids and the like) is an important criterion for health. This energy hemostasis involves the admission of food and the metabolism of carbohydrates and lipids to generate the energy necessary for voluntary and involuntary functions. The metabolism of proteins can lead to the generation of energy, but preferably leads to the formation or repair of muscles. Among other consequences, a lack of energy hemostasis leads to an envelope or sub-formation of adipose tissue. The formation and storage of fat is modulated by insulin. For example, insulin stimulates the transport of glucose to cells, where it is metabolized in a-glycerophosphate which is used in the esterification of fatty acids to allow the storage of them as triglycerides. In addition, adipocytes (fat cells) express a specific transport protein that improves the transfer of free fatty acids to adipocytes. , to. fc -.-- Adipocytes also secrete several proteins that are thought to modulate the hemostatic control of glucose and lipid metabolism. These additional secreted-adipocyte proteins include adipsin, complement factors C3 and B, tumor necrosis factor a, the product of the ob gene, Acrp30. There is also evidence suggesting the existence of a secretory pathway regulated by insulin in adipocytes. Scherer et al., J. Biol. Chem. 270 (45): 26746-9, 1995. The over or undercrease of these portions, affected in part by the over or underdevelopment of the adipose tissue, can lead to the pathological conditions directly or indirectly associated with the obesity or anorexia. Based on the homology on adipocyte-related proteins, such as ACRP30, polypeptides, fragments, fusions, agonists or zacrp5 antagonist, can be used to modulate energy balance in mammals or to protect cells endothelial lesions. With regard to the modulation of the energy balance, zacrp5 polypeptides modulate the cellular metabolic reactions. Such metabolic reactions include adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, J-ta. «--- ** - i.t > -.---- g »» ^? s ^ ------ t ^ Affla-ji-te-ej »^ &i- j -? --...-».-.- a- áU-A i .L? ' thermogenesis, the use of oxygen and the like. Zacrp5 polypeptides can also find use as neurotransmitters or as modulators of neurotransmission, as indicated by the expression of the polypeptide in tissues associated with the parasympathetic or sympathetic nervous system. In this regard, zacrp5 polypeptides can find utility in the modulation of nutrient uptake, as demonstrated, for example, by the absorption of 2-deoxy glucose in the brain and the like. Among other methods known in the art or described herein, the energy balance of mammals can be evaluated by checking one or more of the following metabolic functions: adipogenesis, gluconeogenesis, glycogenolysis, lipogenolysis, glucose uptake, protein synthesis , thermogenesis, the use of oxygen or the like. These metabolic functions are verified by techniques (tests or animal models) known to a person with ordinary experience in the art, as described more fully below. For example, the glucurregulatory effects of insulin are predominantly exerted in the liver, musculoskeletal tissue and adipose tissue. Insulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that lead, by & Af > A & üibÁ l «.F- * - --- ... . "-, .. -: -. I. , - ¿, -, »,; --..- .- i ----? example, to the inhibition of the production of glucose and to the stimulation of the use of the gummy. In the liver, insulin stimulates the absorption of glucose and inhibits gluconeogenesis and glycogenolysis. In musculoskeletal and adipose tissue, insulin acts to stimulate the absorption, storage and utilization of glucose. There are self-recognized methods to verify the totality of the metabolic functions mentioned above. Accordingly, a person skilled in the art is able to evaluate the polypeptides, fragments, fusion proteins, antibodies, agonists and antagonists of zacrp5 for the functions of metabolic modulation. Exemplary modulation techniques are described below. Adipogenesis, gluconeogenesis and glycogenolysis are interrelated components of the energy balance of the mammal, which can be evaluated by known techniques using, for example, ob / ob mice or db / db mice. Ob / ob mice are consanguineous mice that are homozygous for a mutation of inactivation at the ob (obese) site. Such ob / ob mice are hyperphagic and hypometabolic, and it is believed that they will be deficient in the production of the circulating OB protein. The mice of . •• «• v db / db are consanguineous mice that are homozygous for an inactivation mutation in the db site (diabetes). The db / db mice exhibit a phenotype similar to that of the ob / ob mice, except that the db / db mice also exhibit a diabetic phenotype. Such db / db mice are believed to be resistant to the effects of the circulating OB protein. Also, various methods of in vitro evaluation of these parameters are known in the art. Insulin-stimulated lipogenesis, for example, can be verified by measuring the incorporation of 14C-acetate into triglycerides (Mackall et al., J. Biol. Chem. 251: 6462-4, 1976) or the accumulation of triglycerides ( Kletzien et al., Mol.Pharmacol. 41: 393-8, 1992). The absorption of glucose can be evaluated, for example, in a test for the transport of glucose stimulated by insulin. The differentiated, nontransfected L6 myotubes (maintained in the absence of G418) are placed in DMEM containing 1 g / 1 glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM glutamine. After two to five hours of culture, the medium is replaced with fresh glucose-free DMEM, containing 0.5 to 1.0% BSA, 20 mM Hepes, 1 mM pyruvate, and 2 mM glutamine. The appropriate concentrations of insulin or IGF-1, or a series of dilution of the test substance, are added, and the cells are incubated for 20-30 minutes. The deoxyglucose labeled with 3H or 14C is added to "50" μM of the final concentration, and the cells are incubated for about 10-30 minutes.The cells are quickly rinsed with the buffer cold (for example PBS), then it is lysed with a suitable lysis agent (for example 1% SDS or 1 N NaOH) The cell lysate is then evaluated by counting in a scintillation counter. 10 associated with the cells is taken as a measure of glucose transport after subtraction of non-specific agglutination as determined by incubation of the cells in the presence of cytokinesin b, an inhibitor of glucose transport. Other methods 15 include those described, for example, by Manchester et al., Am. J. Physiol. 266 (Endocrinol Metab 29): E326-E333, 1994 (glucose transport stimulated with insulin). The synthesis of proteins can be evaluated, for example, by comparing the precipitation of proteins 20 labeled with 35S-methionine following the incubation of the test cells with 35S-methionine and 35S-methionine and a putative modulator of the synthesis of the protein. Thermogenesis can be evaluated as described by B. Stanley in The Biology of Neuropeptide Y and «& -s-ss *»!. * Í? AÁ &?. A¡Ááq * ij? jJÜá-fc ... ,"",.-"to you ?.? j Related Peptides, W. Colmers and C. Wahlestedt (eds.), Humana Press, Ottawa, 1993, p. 457-509; C. Billington et al., Am. J. Physiol. 260: R321, 1991; N. Zarjevski et al., Endicronology 133: 1753, 1993; C. Billington et al., Am. J. Physiol. 266: R1765, 1994; Heller et al., Am. J. Physiol. 252 (4 Pt 2): R661-7, 1987; and Heller et al., Am. J. Physiol. 245: R321-8, 1983. Also, the metabolic rate, which can be measured by a variety of techniques, is an indirect measure of thermogenesis. Oxygen utilization can be evaluated as described by Heller et al., Pflugers Arch 369: 55-9, 1977. This method also involved an analysis of the temperature of the hypothalamus and the production of metabolic heat. Oxygen utilization and thermoregulation have also been evaluated in humans as described by Haskell et al., J. Appl. Physiol. 51: 948-54, 1981. The neurotransmission functions can be evaluated by verifying the absorption of 2-deoxy-glucose in the brain. This parameter is verified by the techniques (tests or animal models) known by a person experienced in the art, for example, autoradiography. Useful verification techniques are described, for example, by Kilduff et al., J. Neurosci. 10 2463-75, 1990, with the related techniques used to evaluate the "heart in hibernation" as described in Gerber et al. Circulation 94: 651-8, 1996, and Fallavollita et al., Circulation 95: 1900-9, 1997. In addition, the polypeptides, fragments, agonists or antagonists of the fusions thereof, may be therapeutically useful for antimicrobial applications. For example, the Clq of the complement component plays a role in the defense of the host against infectious agents, such as bacteria and viruses. Clq is known to exhibit several specialized functions. For example, Clq triggers the complement cascade through interaction with bound antibody or C-reactive protein (CRP). Also, Clq interacts directly with certain bacteria, RNA viruses, mycoplasma, uric acid crystals, the lipid A component of bacterial endotoxin and the membranes of certain intracellular organelles. Agglutination of Clq to the Clq receptor is believed to promote phagocytosis. Clq also appears to improve the appearance of antibody formation of the host defense system. See, for example, Johnston, Pediatr. Infect. Dis. J. 12 (11): 933-41, 1993. Thus, soluble Clq-like molecules can be useful as antimicrobial agents, which promote lysis or phagocytosis of infectious agents. The fragments of zacrp5 as well as the polypeptides, fusion proteins, agonists, antagonists or antibodies of zacrp5, can be evaluated with respect to their antimicrobial properties according to the procedures known in the art. See, for example, Barsum et al., Eur. Respir. J. 8 (5): 709-14, 1995; Sandosvsky-Losica et al., J. Med. Vet. Mycol (England) 28 (4): 279-87, 1990; Mehentee et al., J. Gen. Microbiol. (England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage, (J. Med. Vet. Mycol., 24: 477-9, 1986 and the like.) If desired, the functioning of zacrp5 in this respect can be compared with the proteins known to be functional in this respect. In addition, fragments, polypeptides, fusion proteins, agonists, antagonists or antibodies of zacrp5 can be evaluated in combination with one or more antimicrobial agents to identify the proteins, such as proteins rich in proline, lysozyme, histatins, lactoperoxidase or similar. synergistic effects A person with ordinary skill in the art will recognize that the antimicrobial properties of polypeptides, fragments, fusion proteins, agonists, antagonists and zacrp5 antibodies can be evaluated in a similar way.

Like neurotransmitters or modulators of neurotransmission, fragments of the zacrp5 polypeptide as well as the polypeptides, fusion proteins, agonists, antagonists or zacrp5 antibodies of the present invention can also modulate the calcium ion concentration, the contraction of muscles, secretion of hormones, growth of cells or DNA synthesis, renewal or rotation of inositol phosphate, release of arachidonate, activation of phospholipase C, gastric emptying, activation with human neutrophils or the capacity of ADCC, the production of superoxide anions and the like. The evaluation of these properties can be carried out by known methods, such as those described herein. The effect of the zacrp5 polypeptide, fragment, fusion, antibody, agonist or antagonist on the intracellular calcium level can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341-52 , 1993, and the like. The effect of the zacrp5 polypeptide, fragment, fusion, agonist or antagonist on contraction of muscles can be evaluated by methods known in the art, such as those described by S & amp; Lebebvre, J ^ _ Auton. Pharmacol. 14: 383-92, 1994, Belloli et al., J. Vet.

Pharmacol. Therap. 17: 379-83, 1994, Maggi et al., Regulatory Peptides 53: 259-74, 1994, and the like. The effect of the zacrp5 polypeptide, fragment, fusion, agonist or antagonist on the secretion of hormones can be evaluated by methods known in the art, such as those described for the release of prolactin described by Henriksen et al., J. Recep. Next Transd. Res. 15 (1-4): 529-41, 1995, and the like. The effect of the zacrp5 polypeptide, fragment, fusion, agonist or antagonist on DNA synthesis or cell growth can be evaluated by methods known in the art, such as those described t > by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993, and the like. The effect of the polypeptide, fragment, fusion, agonist or antagonist of zacrp5 on the renewal or return of inositol phosphate can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341- 52, 1993, and the like. Also, the effect of the zacrp5 polypeptide, fragment, fusion, agonist or antagonist on the release of the arachidonate can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993, and similar. The effect of the polypeptide, fragment, fusion, agonist or -----.- »« Atl Altit / &and itrSAiÍ? Ut * .., _ .. - ---. ----- jé- . - - - -to-- - . . .- - > . -. < - «-. ?, t - i »?, i .. < ? v. -_ > ~ n «r-S -.- A t-O-i antagonist of zacrp5 on the activation of phospholipase C can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993, and the like. The effect of the zacrp5 polypeptide, fragment, fusion, agonist or antagonist on gastric emptying can be evaluated by methods known in the art, such as those described by Varga et al., Eur. J. Pharmacol. 286: 109-112, 1995, and the like. The effect of the zacrp5 polypeptide, fragment, fusion, agonist or antagonist on the activation of human neutrophils and the ability of ADCC can be evaluated tt by methods known in the art, such as those described by Wosniak et al., Immunology 78 : 629-34, 1993, and the like. The effect of the zacrp5 polypeptide, fragment, fusion, agonist or antagonist on the production of the superoxide anion can be evaluated by methods known in the art, such as those described by Wosniak et al., Immunology 78: 629-34, 1993 and similar Collagen is a potent inducer of platelet aggregation. This poses a risk for patients recovering from vascular injuries. Platelet aggregation inhibitors induced by collagen could be useful to block platelet agglutination to collagen-coated surfaces and reduce collagen-induced aggregation of platelets, associated. Clq is a component of the complement pathway and has been found to stimulate defense mechanisms as well as trigger the generation of toxic oxygen species that can cause tissue damage (Tenner, Behring Inst. Mitt. 93: 241-53 , 1993). Clq agglutination sites are found on platelets. Clq, independent of an associate by immune agglutination, has been found to inhibit platelet aggregation but not platelet adhesion or shape change. The amino terminal region of Clq shares the homology with collagen (Peerschke and Ghebrehiwet, J. Immunol., 145: 2984-88, 1990). The inhibition of Clq and the complement pathway can be determined using the methods described herein or known in the art, such as those described in Suba and Csako, J. Immunol. 117: 304-9, 1976. The effect of zacrp5 polypeptides, fragments, fusions, agonists or antagonists on complement inhibition can be evaluated by methods known in the art. The effect of the zacrp5 polypeptide, fragment, fusion, agonist or antagonist on Clq agglutination activity can be evaluated by methods known in the art.

The effect of the polypeptide, fragments, fusions, agonists or antagonists of zacrp5 on the adhesion, activation or aggregation of the platelets mediated by collagen can be evaluated using the methods described here or known in the art, such as the aggregation assay of the platelets (Chiang et al., Thrombosis Res. _37: 605-12, 1985) and platelet adhesion assays (Peerschke and Ghebrehiwet, J. Immunol., 144: 221-25, 1990). Tests for the adhesion of platelets to collagen and the inhibition of platelet aggregation induced by collagen can be measured using the methods described in Keller et al., J. Biol. Chem. 268: 5450-6, 1993; Waxman and Connolly, J. Biol. Chem. 268: 5445-9, 1993; Noeske-Jungblut et al., J. Biol. Chem. 269: 5050-3 or 1994 Deckmyn et al., Blood 5: 712-9, 1995. The effect of the polypeptide, fragments, fusions, agonists or antagonists of zacrp5 on the vasodilatation of the aortic rings can be measured according to the methods of Dainty et al., J. Pharmacol. 100: 767, 1990 and Rhee et al., Neurotox. 1_6: 179, 1995. Several in vitro and in vivo models are available for evaluation of the effects of polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists of zacrp5 on ischemia and reperfusion injury. See, for example, Shandelya et al., Circulation 88: 2812-26, 1993; Weisman et al., Science 249: 146-151, 1991; Buerke et al., Circulation 91: 393-402, nineteen ninety five; Horstick et al., Circulation 95: 701-8, 1997 and Burke et al., J. Phar. | Exp. Therp. 286: 429-38, 1998. An ex vivo hamster platelet aggregation assay is described by Deckmyn et al., Ibid. Bleeding times in hamsters and baboons can be measured following the injection of zacrp5 polypeptides using the model described by Deckmyn et al., Ibid. The formation of thrombi in response to the administration of the proteins of the present t > invention can be measured using the thrombosis model of the hamster femoral vein which is provided by Deckmyn et al., ibid. Changes in platelet adhesion under flow conditions following the administration of zacrp5 can be measured using the method described in Harsfalvi et al., Blood 85: 705-11, 1995. Inhibition of complement and healing of wounds by polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists of zacrp5 can be evaluated alone or in combination with other known inhibitors of the activation and aggregation of platelets induced by collagen, such as palldipine, moubatin or haze , for example.

The polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists of zacrp5 can be evaluated using the methods described herein or known in the art, such as the healing of dermal layers in pigs (Lynch et al., Proc. Nati. Acad. Sci. USA 84: 7696-700, 1987) and skin wounds of normal or natural thickness in genetically diabetic mice (Greenhalgh et al., Am. J. Pathol., 136: 1235-46, 1990). , for example. The polypeptides of the present invention can be tested alone or in combination with other complement inhibitors known as described above. The hybrid's mapping by radiation. is a genetic technique of somatic cells developed for the construction of contiguous, high-resolution maps of the chromosomes of mammals (Cox et al., Science 250: 245-50, 1990). The partial or total knowledge of a gene sequence allows the design of PCR primers suitable for use with the chromosomal radiation hybrid mapping panels. Commercially available radiation hybrid mapping panels, which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL), are available. These panels make possible chromosomal locations, based on PCR, rapid, and ordering of genes, sites labeled with the sequence (STSs), and other non-polymorphic and polymorphic markers within a region of interest. This includes directly establishing the proportional physical distances between newly discovered genes of interest and previously mapped markers. Accurate knowledge of a position of the gene can be useful in a number of ways including: a) the determination that if a sequence is part of an existing contiguous element and obtain the additional surrounding genetic sequences in various forms such as clones of YAC-, BAC- or cDNA, 2) provide a possible candidate gene for an inheritable disease which shows a link to the same chromosomal region, and 3) for cross-reference of model organisms such as the mouse which can be beneficial to help determine what function a particular gene might have. The radiation hybrid mapping can be used to confirm the location of zacrp5 on human chromosone 16. The present invention also provides reagents which will find use in diagnostic applications. For example, the zacrp5 gene, a probe comprising the DNA or RNA of zacrp5, or a substance thereof, can be used to determine whether the zacrp5 gene is present on chromosome 16 or whether a mutation has occurred. Chromosomal aberrations detectable at the zacrp5 gene site include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, changes and rearrangements of the restriction site. These aberrations may occur within the coding sequence, within the introns, or within the flanking sequence, including the upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or as changes in the level of expression of the gene. In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with an oligonucleotide probe or primer as described above, under the conditions wherein the polynucleotide will hybridize to the complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first product of the reaction with a product of the control reaction. A difference between the first product of the reaction and the product of the control reaction is indicative of a genetic abnormality in the patient. Genetic examples for use within the present invention include genomic DNA, cDNA and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO: 1, the complement of SEQ ID NO: 1, or an equivalent RNA thereof. Appropriate assay methods in this regard include molecular genetic techniques known to those skilled in the art, such as restriction fragment length polymorphism (RFLP) analysis, short series repeat (STR) analysis employing PCR techniques, the chain reaction for binding or ligation (Barany, PCR Methods and Applications 1: 5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al., Ibid., Ausubel et al., Ibid., Marian, Chest 108: 255-65, 1995). The ribonuclease protection assays (see, for example, Ausubel et al., Ibid., Chapter 4) comprise the hybridization of an RNA probe to a sample of the patient's RNA, after which the product of the reaction (RNA-RNA hybrid) is exposed to RNase. The hybridized regions of the RNA are protected from dissolution. Within PCR assays, a genetic sample from the patient is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in the size or quantity of the recovered product are indicative of mutations in the patient. Another PCR-based technique that can be employed is the analysis of single-strand conformation polymorphism (SSCP) (Hayashi, PCR Methods and Applications 1: 34-8, 1991). The present invention also contemplates kits for performing a diagnostic assay for the expression of the zacrp5 gene or for examining the zacrp5 site. Such sets comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 1, or a portion thereof, as well as single-stranded nucleic acid molecules. strand having the complement of the nucleotide sequence of SEQ ID NO: 1, or a portion thereof. The molecules of the probe can be DNA, RNA, oligonucleotides, and the like. The pools may comprise the nucleic acid primers for carrying out the PCR. Such a set may contain all the elements necessary to perform a diagnostic assay of the nucleic acid described above. A set will comprise at least one container comprising a zacrp5 probe or primer. The assembly may also comprise a second container comprising one or more reagents capable of indicating the presence of the zacrp5 sequences. Examples of such indicator reagents include detectable labels such as radioactive labels, fluorochromes, chemiluminescent agents, and the like. A set may also comprise a means of transport for the user so that probes and zacrp5 primers are used to detect the expression of the zacrp5 gene. For example, written instructions can establish that the nucleic acid molecules enclosed can be used to detect either a nucleic acid molecule encoding zacrp5, or a nucleic acid molecule having a nucleotide sequence that is complementary to a sequence. of nucleotides encoding zacrp5. The written material can be applied directly to a container, or the written material may be provided in the form of a packaging insert. Also contemplated is a method of detecting the presence of the expression of the zacrp5 gene in a biological sample, comprising: (a) contacting a nucleic acid probe of zacrp5 under the conditions of hybridization with either (i) the test RNA molecules isolated from the biological sample, or (ii) the 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 i -? - á-j -, ¿ii- _fc¿ * l .. nucleic acid molecule as described herein, or the complements thereof, and (b) detect the formation of the nucleic acid probe hybrids and either the test RNA molecules or the synthesized nucleic acid molecules, in where the presence of the hybrids indicates the presence of zacrp5 RNA in the biological sample. Additionally, a method of detecting the presence of zacrp5 in a biological sample is provided, comprising: (a) contacting the biological sample with an antibody, or an antibody fragment, as described herein, wherein the contacting it is carried out under conditions that allow the agglutination of the antibody or the antibody fragment to the biological sample, and (b) detect any of the agglutinated antibody or the agglutinated antibody fragment. The polypeptides of zacrp5 can be used in the analysis of energy efficiency in a mammal. The zacrp5 polypeptides found in serum or tissue samples may be indicative of an ability of mammals to store food, with more highly efficient mammals tending towards obesity. More specifically, the present invention contemplates methods for detecting zacrp5 polypeptide comprising: Is it possible to expose a sample possibly containing the polypeptide of zacrp5 to an antibody bound or bound to a solid support, wherein the antibody binds to an epitope of a polypeptide of zacrp5; wash the immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized polypeptide-antibody to a second antibody directed to a second epitope of a zacrp5 polypeptide, wherein the second antibody is associated with a detectable tag; and detect the detectable label. The concentration t > of the zacrp5 polypeptide in the test sample appears to be indicative of the energy efficiency of a mammal. This information can help the nutritional analysis of a mammal. Potentially, this information may be useful in the identification and / or location as target of the energy-deficient tissue. A further aspect of the invention provides a method for studying insulin. Such methods of the present invention comprise the incubation of the adipocytes in a culture medium comprising the polypeptide of zacrp5, the monoclonal antibodies, the agonist or antagonist thereof, and the observation of changes. in the secretion or differentiation of adipocyte proteins. The antimicrobial protective agents can be direct acting or indirect acting. Such agents operate by mechanisms of membrane association action or pore formation for direct fixation to the offending microbe. The antimicrobial agents can also act by means of an enzymatic mechanism, breaking the microbial protective substances or the membrane / cell wall thereof. Antimicrobial agents, capable of inhibiting the proliferation or action of microorganisms or of altering the integrity of the microorganism by any mechanism described above, are useful in methods for the prevention of contamination in cell culture by microbes susceptible to this activity antimicrobial Such techniques involve culturing the cells in the presence of an effective amount of the zacrp5 polypeptide or an agonist or antagonist thereof. Also, polypeptides of zacrp5 or agonists thereof can be used as reagents of the cell culture in in vitro studies of the infection of the exogenous microorganism, such as bacterial, viral or fungal infection. Such portions can also i - i a, .-- - -. i --- The. . --to------.--". . »^ --- > ^ .. • -.a - * .- -4kn. * a & r-f -. - 4. * ^. -----. lx &rx -jfatjut} J., & - be used in in vivo animal models of infection. The present invention also provides methods for studying the cellular metabolism of mammals. Such methods of the present invention comprise incubating the cells to be studied, for example, human vascular endothelial cells, + zacrp5 polypeptide, monoclonal antibody, agonist or antagonist thereof and observe changes in adipogenesis, gluconeogenesis, glycogenolysis, glucose uptake, or the like. The polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists of zacrp5 of the present invention can be used in methods to promote blood flow within the vasculature of a mammal by reducing the number of platelets that are Adherence and activation and the size of platelet aggregates Used for this purpose, zacrp5 can be administered prior to, during or following an acute vascular injury in the mammal.The vascular lesion may be due to vascular reconstruction including, but not limited to, angioplasty, coronary artery bypass graft, microvascular repair or anastomosis of a vascular graft, vascular injuries due to trauma, access or aneurysm are also contemplated.

In other preferred methods vascular injury is due to rupture of the plaque, degradation of the vasculature, complications associated with diabetes and atherosclerosis. The rupture of the plaque in the coronary artery induces the attack to the heart and in the cerebral artery it induces the accesses. The use of polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists of zacrp5 in such methods could also be useful to ameliorate the diseases of the entire system of the vasculature associated with the immune system, such as disseminated intravascular coagulation (DIC). ) and SIDS. Additionally, the activity that inhibits the complement could be useful for the treatment of immune diseases that are not of the vasculature, such as arteriolosclerosis. If desired, the functioning of the polypeptide, fragment, fusion protein, agonist, antagonist or zacrp5 antibody in this respect can be compared with proteins that are known to be functional in this respect, such as zsig37 or the like. In addition, polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists of zacrp5 can be evaluated in combination with one or more platelet aggregation or activation inhibiting agents to identify the synergistic effects. aá¿l-¿-t.- »-Jati-É ----- & i - i - i-k! ---. »T. , i-i > .- > - < -n - »- .. i». * «att .. * .-- < »-m» - »? - ».- > .--, - ».,. • -.-« - .. *. *. ------ -s - fa ,, - i Polypeptides, fragments, fusion proteins, antibodies, agonists or antagonists may also be useful in the treatment of acute vascular injury. Acute vascular lesions are those that occur rapidly (that is, for days to months), in contrast to chronic vascular lesions (for example, atherosclerosis) which develop over a lifetime. Acute vascular injuries frequently result from surgical procedures such as vascular reconstruction, where the techniques of angioplasty, endarterectomy, atherectomy of reduction, placement of small endovascular tubes, ablation with endovascular laser, vascular graft anastomosis or similar, are employees. Hyperplasia can also occur as a delayed response in response to, for example, the placement of a vascular graft or organ transplant. A correlation has been found between the presence of Clq in the localized ischemic myocardium and the accumulation of leukocytes following coronary occlusion and reperfusion. The release of cellular components following tissue damage complements the activation which leads to toxic oxygen products that may be the main cause of myocardial damage (Rossen »4 ^ -a - t-A ^ - - et al., Circ. Res. 62: 572-84, 1998 and Tenner, ibid.). Blocking the complement pathway was found to protect the ischemic myocardium from the reperfusion injury (Buerke et al., J. Pharm. Exp. Therp. 286: 429-38, 1998). Proteins that have complement inhibition and Clq agglutination activity could be useful for such purposes. The collagen and Clq agglutination capacities of adipocyte-related protein homologs such as zacrp5 could be useful in reassuring damaged collagenous tissues by preventing adhesion, activation or aggregation of platelets, and activation of inflammatory processes which lead to the release of toxic oxygen products. By making the tissue inert to processes such as complement activity, thrombotic activity and immune activation, the detrimental effects of ischemia and reperfusion are reduced. In particular, such injuries could include ischemia due to traumatic injury, intestinal strangulation, and injuries associated with pre and post-establishment of blood flow. Such polypeptides could be useful in the treatment of cardiopulmonary bypass ischemia and the temporary suspension of cardiac functioning, the infarct.

"" U ...--; ------- t ~ ---- ---------------. --- »-------- *. "J- S - t -» - »-. «...., - ^^ - faith? Mi_a - l-i« «? - i--? > l > --.- ». 4 -? - .., i j -. A -.--, -t ?? and the myocardium and vasospasm after trauma, such as access or percutaneous transluminal angioplasty as well as accidental vascular trauma or induced by surgery. Additionally such collagen and Clq agglutination polypeptides could be useful to treat the prosthetic and surgical biomaterials to make the surface of the materials inert toward complement activation, thrombotic activity or immune activation. Such materials include, but are not limited to, collagen or biomaterials coated with fragments of 'collagen, gelatin-coated biomaterials, fibrin-coated biomaterials, fibronectin-coated biomaterials, heparin-coated biomaterials, small tubes coated with collagen and gel, arterial grafts, synthetic heart valves, artificial organs or any prosthetic application exposed to blood that is it will agglutinate zacrp5 to a value greater than 1 x 108. The coating of such materials can be done using methods known in the art, see for example, Rubens, US Patent No. 5,272,074. Complement and Clq play a role in inflammation. The activation of complement is initiated by Clq agglutination to immunoglobulins (Johnston, Pediatr. Infect. Dis. J. 12: 933-41, 1993; Ward and Ghetie, i i ^ j j _.-- - «fe-t. Í-I ~ * Í.

Therap. Immunol. 2: 77-94, 1995). Clq inhibitors and complement may be useful as anti-inflammatory agents. Such an application can be made to prevent infection. Additionally, such inhibitors can be administered to an individual suffering from inflammation mediated by complement activation and agglutination of immune complexes to Clq. Clq inhibitors and complement may be useful in methods of mediating wound repair, improving the progression of wound healing by overcoming the healing of altered wounds. The progression in wound healing could include, for example, elements such as a reduction in inflammation, recruitment of fibroblasts, retraction of wounds and reduction of infection. The ability of tumor cells to bind to collagen may contribute to the metastasis of tumors. Collagen agglutination inhibitors are also useful for mediating adhesive interactions and the metastatic spread of tumors (Noeske-Jungbult et al., US Patent No. 5,723,312). In addition, the zacrp5 polypeptides, fragments, fusions, agonists or antagonists thereof may be therapeutically useful for antimicrobial applications. iifaia-il ítat ,, ?? £ Man. .-- i-J-: A, .. », ... -, .- -j > -i -i- -.¡¿i'.¿-J- --.---- I -.-. a..a-aJ. .1- .- -,, - --- ..: r.F. . ... Srtj. For example, the Clq of the complement component plays a role in the defense of the host against infectious agents, such as bacteria and viruses. Clq is known to exhibit several specialized functions. For example, Clq triggers the complement cascade through interaction with bound antibody or C-reactive protein (CRP). Also, Clq interacts directly with certain bacteria, RNA viruses, mycoplasma, uric acid crystals, the lipid A component of bacterial endotoxin and the membranes of certain intracellular organelles. Agglutination of Clq to the Clq receptor is believed to promote phagocytosis. Clq also appears to improve the appearance of antibody formation of the host defense system. See, for example, Johnston, Pediatr. Infect. Dis. J. 12 (11): 933-41, 1993. Thus, soluble Clq-like molecules can be useful as antimicrobial agents, promoting lysis or phagocytosis of infectious agents. The collagen, triple helix, extracellular, positively charged domains of Clq and the macrophage scavenger receptor were determined to play a role in the agglutination of the ligand and were shown to have a broad agglutination specificity towards the polyanions. (Acton et al., J. Biol. Chem. 268: 3530-37, : & ¿, ^ # £ XL # ± &% ^ & r - ^^ * ¡¡¡!! * - ^ - - * "» _ »-» <.,.,. & * 1993. The growth factor of lysophospholipid (lysophosphatidic acid, LPA) and other mitogenic anions locate the site of damaged tissues and help repair wounds LPA exerts many biological effects including the activation of platelets and the superior regulation of matrix assembly.It is thought that LPA is synergized with other blood coagulation factors and has a mediating effect in the healing of wounds.The collagen domains of proteins such as Clq and the macrophage scavenger receptor are known to bind to acidic phospholipids such as LPA.tr A 9mer region of the collagen domain of zacrp5, amino acid residues 98-106 of SEQ ID NO: 2, share a sequence homology with the collagen domain found on Clq and the macrophage scavenger receptor.The interaction of polypeptides, fragments, fusions, agonists or antagonists of zacrp5 with mitogenic anions such as LPA can be determined using assays known in the art, see for example, Acton et al., ibid. The inhibition of inflammatory processes by the polypeptides and antibodies of the present invention could also be useful in the prevention of infection at the site of the wound.

For pharmaceutical use, the proteins of the present invention can be formulated with pharmaceutically acceptable carriers for parenteral, orai, nasal, rectal, topical, transdermal or similar administration, according to conventional methods. In a preferred embodiment, administration is done at or near the site of the vascular injury. In general, the pharmaceutical formulations will include a zacrp5 protein in combination with a pharmaceutically acceptable carrier, such as brine, buffered brine, 5% dextrose in water or the like. The formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent the loss of proteins on the surfaces of the small vial, etc. Formulation methods are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton PA, 19 / a. edition, 1995. Therapeutic doses will generally be determined by the physician in accordance with accepted standards, taking into account the nature and severity of the condition to be treated, the characteristics of the patient, etc. The determination of the dose is within the level of ordinary experience in the art. .? Í Í aj aj aj aj aj Cuando Cuando Cuando Cuando Cuando Cuando Cuando aj aj aj aj aj aj Cuando Cuando Cuando Cuando Cuando Cuando Cuando Cuando Cuando Cuando Cuando Cuando ----. When a "pharmaceutically effective amount" of a zacrp5 polypeptide, fragment, fusion protein, agonist or antagonist is used herein, it is sufficient desired biological The result can be the relief of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an effective amount of a zacrp5 polypeptide is one that provides either subjective relief of the symptoms or an objectively identifiable improvement as may be observed by the physician or other qualified observer. Such an effective amount of the zacrp5 polypeptide could provide, for example, the inhibition of platelet activation activated by collagen and the complement pathway, including Clq, the increased blood flow located within the vasculature of a patient. and / or the reduction in the detrimental effects of ischemia and reperfusion. The modulation of inflammation associated with arthritis could include a reduction in inflammation and pain relief or stiffness, in animal models, indications could be derived from the gross inspection of the joints and the change in the swelling of the joints. hind legs. The effective amounts of zacrp5 polypeptides can vary widely depending on the disease ~ * 4 £ «* - or the symptom that is going to be treated. The amount of the polypeptide to be administered and its concentration in the formulations depends on the selected vehicle, the route of administration, the potency of the particular polypeptide, the clinical condition of the patient, the side effects and the stability of the compound in the formulation. Thus, the physician will use the appropriate preparation containing the appropriate concentration in the formulation, as well as the amount of the formulation administered, depending on the clinical experience with the patient in question or with similar patients. Such amounts will depend, in part, on the particular condition to be treated, the age, weight, and general health of the patient, and other factors evident to those skilled in the art. Typically, a dose will be in the range of 0.01-100 mg / kg of the subject. In applications such as balloon catheters, the typical dosage range could be 0.05-5 mg / kg of the subject. Doses for specific compounds can be determined from in vitro or ex vivo studies in combination with studies on experimental animals. The concentrations of the compounds that have been found to be effective in vi tro or ex vivo provide a guide for animal studies, where - -. . «1-.J. < U > - I -4j¡fct t * »doses are calculated to provide similar concentrations at the site of action. The polynucleotides encoding zacrp5 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit the activity of zacrp5. If a mammal has a mutated or absent zacrp5 gene, the zacrp5 gene can be introduced into the animal's cells. In one embodiment, a gene encoding a zacrpS polypeptide is introduced in vivo into a viral vector. Such vectors include a defective or attenuated 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 are completely or almost completely free of viral genes, are preferred. A defective virus is not infectious after introduction into a cell. The use of defective viral vectors allows administration to cells in a specific localized area, without interest if this vector can infect other cells. Examples of the particular vectors include, but are not limited to, a defective herpes simplex virus 1 vector (HSV1) (Kaplitt et al., Molec. Cell Neurosci.2: 320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-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; Saulski et al-, J. Virol. 63: 3822-8, 1989). In another embodiment, a zacrp5 gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell 33: 153, 1983; Temin et al., U.S. Pat.

No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 62: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; WIPO publication WO 95/07358; Y Kuo et al., Blood 2: 845, 1993. Alternatively, the vector can be introduced by lipofection in vivo using the liposomes. Synthetic cationic lipids can be used to prepare lysosomes for in vivo transfection of a gene encoding a marker (Felgner et al., Proc. Nati, Acad. Sci. USA 84: 7413-7, 1987; Mackey et al., Proc. Nati, Acad. Sci. USA £ 5: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. The molecular location as a target of liposomes to specific cells represents an area of benefit. More particularly, directing transfection to particular cells represents an area of benefit. For example, --É --- .. J- »JkJ f ^ &rJ. directing the transfection to the particular cell types could be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. The lipids can be applied chemically to other molecules for the purposes of location as a target. Target polypeptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or molecules other than peptides can be chemically linked to liposomes. It is possible to remove target cells from the body; introduce the vector as a plasmid of pure DNA; and then reimplant the transformed cells in the body. Pure DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation. , the use of a gene gun or the use of a DNA vector transporter. See, for example, Wu et al., J. Biol. Chem. 267: 963-7, 1992; Wu et al., J. Biol. Chem. 263: 14621-4, 1988. The antisense methodology can be used to inhibit the transcription of the zacrp5 gene, such as to inhibit the proliferation of cells in vivo. The '.Ai-aJh fcrt - .. afa. -, '- ».--. --- .---- i-- eles | fe ^^^^^^^ g polynucleotides that are complementary to a segment of a polynucleotide encoding zacrp5 (eg, a polynucleotide as described in SEC) ID N0: 1) are designed to bind to the mRNA encoding zacrp5 and to inhibit the translation of such mRNA. Such antisense polynucleotides are used to inhibit the expression of the genes encoding the zacrp5 polypeptide in the culture of cells or in a subject. Transgenic mice, designed to express the zacrp5 gene, and mice that exhibit a complete absence of the function of the zacrp5 gene, referred to as "knockout mice" (Snouwaert et al., Science 257: 1083, 1992), can also be generated (Lowell et al., Nature 366: 740-42, 1993). These mice can be used to study the zacrp5 gene and the protein encoded by it in an in vivo system. The invention is further illustrated by the following non-limiting examples.

Example 1 Identification of a Zacrp5 Sequence The novel zacrp5 polypeptide encoding the polynucleotide of the present invention was initially identified in an unfinished genomic sequence while that the homologs of the adipocyte-related protein, zsig37 (WO 99/04000), are investigated, characterized by a signal sequence, a collagen-like domain and a Clq domain. The genomic sequence is located on the HS349E11 site which is derived from chromosone 16. SEQ ID NO: 7 provides the identified exon 1 of zacrp5 starting at the start codon, nucleotides 1-208, intron 1, nucleotides 209-870 and exon 2 ending with the stop codon, nucleotides 871-1421. The resulting 1169 bp cDNA sequence is described in SEQ ID NO: 1. To isolate the polynucleotide of SEQ ID NO: 1 from various tissues, the probes and / or primers are designed from the predicted regions of the exon of SEQ ID NO: 1 and SEQ ID NO: 7. The tissues expressing the zacrp5 could be identified either by hybridization (Northern Blots) or by reverse transcriptase (RT) PCR. The libraries are then generated from tissues which appear to show the expression of zacrp5. The unique clones of such libraries are then identified by means of hybridization with the probes and / or by PCR with the primers as described herein. The conformation of the cDNA sequence of zacrp5 can be verified using the sequences provided herein.

JÚAX ^? I & eiÁmXk LISTING & THE SEQUENCES < 110 > ZymoGenetics, Inc. < 120 > HOMOLOGO ZACRP5 OF THE PROTEIN RELATED TO THE COMPLEMENT OF ADIPOCITS < 130 > 99-10 < 150 > 09 / 321,372 < 151 > 1999-05-27 < 160 > 12 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 759 < 212 > DNA < 213 > Homo sapiens p < 220 > < 221 > CDS < 222 > (1) ... (759) < 400 > 1 atg gca gcc ccc gcc ctg ctg etc cta gca ctg ctg ctg ccc gtg ggg 48 Met Ala Ala Pro Ala Leu Leu Leu Leu Ala Leu Leu Pro Val Gly 1 5 10 15 gcc tgg ccc ggg ctg ccc agg agg ccc tgt gtg cae tgc tgc cgc ccg 96 Wing Trp Pro Gly Leu Pro Arg Arg Pro Cys Val His Cys Cys Arg Pro 20 25 30 gcc tgg ccc cct gga ccc tat gcc cgg gtg agt gac agg gac ctg tgg 144 Wing Trp Pro Pro Gly Pro Tyr Wing Arg Val Be Asp Arg Asp Leu Trp 35 40 45 agg ggg gac ctg tgg agg ggg ctg cct cg cg ggg cgg ccc act ata aac 192 Arg Gly Asp Leu Trp Arg Gly Leu Pro Arg Val Arg Pro Thr He Asn 50 55 60 atc gaa atc etc aaa ggt gag aag ggt gag gcc ggc gtc cga ggt cgg 240 He Glu He Leu Lys Gly Glu Lys Gly Glu Wing Gly Val Arg Gly Arg 65 70 75 80 ? áA? ííx * .lfif.ía¡i? ± t .: il ... -. < fa-É í - j .. gcc ggc agg age ggg aaa gag ggg ccg cea ggc gcc cgg ggc ctg cag 288 Wing Gly Arg Ser Gly Lys Glu Gly Pro Pro Gly Wing Arg Gly Leu Gln 85 90 95 ggc cgc aga ggc cag aag ggg cag gtg ggg ccg ccg ggc gcc gcg tgc 336 Gly Arg Arg Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Wing Cys 100 105 110 cga cgt gcc tac gcc gcc ttc tec gtg ggc cgg cgc gag ggc ctg drops 384 Arg Arg Ala Tyr Ala Ala Phe Ser Val Gly Arg Arg Glu Gly Leu His 115 120 125 age tec gac falls tcc cag gcg gtg ccc tcc gac acg gag ctg gtg aac 432 Ser Ser Asp His Phe Gln Wing Val Pro Phe Asp Thr Glu Leu Val Asn 130 135 140 ctg gac ggc gcc ttc gac ctg gcc gcg ggc cgc ttc etc tgc acg gtg 480 Leu Asp Gly Ala Phe Asp Leu Ala Ala Gly Arg Phe Leu Cys Thr Val 145 150. 155 160 ccc ggc gtc tac ttc etc age etc aac gtg falls acc tgg aac tac aag 528 Pro Gly Val Tyr Phe Leu Ser Leu Asn Val His Thr Trp Asn Tyr Lys 165 170 175 gag acc tac ctg falls atc ctg aac cgg cgg cgg cgg gcg gcc gtg etc 576 Glu Thr Tyr Leu His He Met Leu Asn Arg Arg Pro Wing Wing Val Leu 180 185 190 tac gcg cag ccc age gag cgc age gtc atg cag gcc cag age ctg atg 624 Tyr Ala Gln Pro Ser Glu Arg Ser Val Met Gln Wing Gln Ser Leu Met 195 200 205 ctg ctg ctg gcg gcg ggc gac gcc gtc tgg gtg cgc atg ttc cag cgc 672 Leu Leu Leu Wing Wing Gly Asp Wing Val Trp Val Arg Met Phe Gln Arg 210 215 220 gac cgg gac aac gcc atc tac ggc gag falls gga gac etc tac atc acc 720 Asp Arg Asp Asn Ala He Tyr Gly Glu His Gly Asp Leu Tyr He Thr 225 230 235 240 ttc age ggc falls ctg gtc aag ccg gcc gcc gag ctg tag 759 Phe Ser Gly His Leu Val Lys Pro Ala Ala Glu Leu * 245 250 < 210 > 2 < 211 > 252 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Ala Ala Pro Ala Leu Leu Leu Leu Ala Leu Leu Leu Pro Val Gly 1 5. 10 15 Wing Trp Pro Gly Leu Pro Arg Arg Pro Cys Val His Cys Cys Arg Pro 20 25 30 Wing Trp Pro Pro Gly Pro Tyr Wing Arg Val Ser Asp Arg Asp Leu Trp 35 40 45 Arg Gly Asp Leu Trp Arg Gly Leu Pro Arg Val Arg Pro Thr He Asn 50 55 60 He Glu He Leu Lys Gly Glu Lys Gly Glu Wing Gly Val Arg Gly Arg 65 70 75 80 Wing Gly Arg Gly Lys Glu Gly Pro Pro Gly Wing Arg Gly Leu Gln 85 90 95 Gly Arg Arg Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Wing Cys 100 105 110 Arg Arg Ala Tyr Ala Wing Phe Ser Val Gly Arg Arg Glu Gly Leu His 115 120 125 Ser Ser'Asp His Phe Gln Ala Val Pro Phe Asp Thr Glu Leu Val Asn 130 135 140 Leu Asp Gly Ala Phe Asp Leu Ala Ala Gly Arg Phe Leu Cys Thr Val 145 150 155 160 Pro Gly Val Tyr Phe Leu Ser Leu Asn Val His Thr Trp Asn Tyr Lys 165 170 175 Glu Thr Tyr Leu His He Met-Leu Asn Arg Arg Pro Wing Wing Val Leu 180 185 190 Tyr Wing Gln Pro Ser Glu Arg Ser Val Met Gln Wing Gln Ser Leu Met 195 200 205 Leu Leu Leu Wing Wing Gly Asp Wing Val Trp Val Arg Met Phe Gln Arg 210 215 220 Asp Arg Asp Asn Wing He Tyr Gly Glu His Gly Asp Leu Tyr He Thr 225 230 235 240 Phe Ser Gly His Leu Val Lys Pro Wing Ala Glu Leu 245 250 < 210 > 3 < 211 > 281 < 212 > PRT < 213 > Homo sapiens ÍA, Xñ.¿Á, ¿, k? Ítiry: -. yil., < 400 > 3 Met Gly Ser Arg Gly Gln Gly Leu Leu Leu Wing Tyr Cys Leu Leu Leu 1 5 10 15 Wing Phe Wing Being Gly Leu Val Leu Being Arg Val Pro His Val Gln Gly 20 25 30 Glu Gln Gln qiu Trp Glu Gly Thr Glu Glu Leu Pro Ser Pro Pro Asp 35 40 45 His Wing Glu Arg Wing Glu Glu Gln His Glu Lys Tyr Arg Pro Ser Gln 50 55 60 Asp Gln Gly Leu Pro Wing Being Arg Cys Leu Arg Cys Cys Asp Pro Gly 65 70 75 80 Thr Ser Met Tyr Pro Wing Thr Wing Val Pro Gln He Asn lie Thr He 85 90 95 Leu Lys Gly Glu Lys Gly Asp Arg Gly Asp Arg Gly Leu Gln Gly Lys 100 105 110 Tyr Gly Lys Thr Gly Ser Wing Gly Wing Arg Gly His Thr Gly Pro Lys 115 120 125 Gly Gln L-ys Gly Ser Met Gly Wing Pro Gly Glu Arg Cys Lys Ser His 130 135 140 Tyr Wing Wing Phe Ser Val Gly Arg Lys Lys Pro Met His Ser Asn His 145 150 155 160 Tyr Tyr Gln Thr Val He Phe Asp Thr Glu Phe Val Asn Leu Tyr Asp 165 170 175 His Phe Asn Met Phe Thr Gly Lys Phe Tyr Cys Tyr Val Pro Gly Leu 180 '185 190 Tyr Phe Phe Ser Leu Asn Val His .Thr Trp Asn Gln Lys Glu Thr Tyr 195 200 205 Leu His He Met Met Lys Asn Glu Glu Glu Val Val He Leu Phe Wing Gln 210 215 220 Val Gly Asp Arg Ser He Met-Gln Ser Gln Ser Leu Met Leu Glu Leu 225 230 235 240 Arg Glu Gln Asp Gln Val T rp Val Arg Leu Tyr Lys Gly Glu Arg Glu 245 250 255 Asn Wing He Phe Ser Glu Glu Leu Asp Thr Tyr He Thr Phe Ser Gly 260 265 270 Tyr Leu Val Lys His Wing Thr Glu Pro 275 280 <; 210 > 4 < 211 > 244 < 212 > PRT < 213 > Homo sapiens < 400 > 4 Met Leu Leu Leu Gly Ala Leu Val Leu Leu Leu Ala Leu Pro Gly His _-ii ---. i- i - A-.i --- t - í -.lM * 4é s *; fcA ^ t? a -S ^ i- ^ 10 15 Asp Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Pro Leu Pro 20 25 30 Lys Gly Wing Cys Thr Gly Trp Met Wing Gly He Pro Gly His Pro Gly 35 40 45 His Asn Gly Wing Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu 50 55 60 Lys Gly Glu Lys Gly Asp Pro Gly Leu He Gly Pro Lys Gly Asp He 65 70 75 80 Gly Glu Thr Gly Val Pro Gly Wing Glu Gly Pro Arg Gly Phe Pro Gly 85 90 95 He Gln Gly Arg Lys Gly Glu Pro Gly Glu Gly Wing Tyr Val Tyr Arg 100 105 110 Be Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr He Pro Asn Met 115 120 125 Pro He Arg Phe Thr Lys He Phe Tyr Asn Gln Gln Asn His Tyr Asp 130 135 140 Gly Ser Thr Gly Lys Phe His Cys Asn He Pro Gly Leu Tyr Tyr Phe 145 150 155 160 Wing Tyr His He Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe 165 170 175 Lys Lys Asp Lys Wing Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn 180 185 190 Asn Val Asp Gln Wing Ser Gly Ser Val Leu Leu His Leu Glu Val Gly 195 200 '205 Asp Gln Val Trp Leu Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu 210 215 220 Tyr Wing Asp Asn Asp Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr 225 230 235 240 His Asp Thr Asn < 210 > 5 < 211 > 243 < 212 > PRT < 213 > Homo sapiens < 400 > 5 Met Arg Pro Leu Leu Val Leu Leu Leu Leu Gly Leu Ala Wing Gly Ser 1 5 10 15 Pro Pro Leu Asp Asp Asn Lys He Pro Be Leu Cys Pro Gly His Pro 20 25 30 Gly Leu Pro Gly Thr Pro Gly His His Gly Ser Gln Gly Leu Pro Gly 35 40. 45 Arg Asp Gly Arg Asp Gly Arg Asp Gly Wing Pro Gly Wing Pro Gly Glu 5 * and . { , X ±? -.? .. F ± ¿&? -i-i.-Í ... -. ----- ._ & • ~? --- »t - .A-c-- asAa-JEig. - *? 2 &e & * hMf ¿a - ^ ¿K ^ Ai --- .-- ¿-t - ^ ------.-- 50 55 60 Lys Gly Glu Gly Gly Arg Pro Gly Leu Pro Gly Pro Arg Gly Asp Pro 65 70 75 80 Gly Pro Arg Gly Glu Wing Gly Pro Wing Gly Pro Thr Gly Pro Wing Gly 85 90 95 Glu Cys Ser Val Pro Pro Arg Ser Wing Phe Ser Wing Lys Arg Ser Glu 100 105 110 Ser Arg Val Pro Pro Pro Ser Asp Wing Pro Leu Pro Phe Asp Arg Val 115 120 125 Leu Val Asn Glu Gln Gly His Tyr Asp Wing Val Thr Gly Lys Phe Thr 130 135 140 Cys Gln Val Pro Gly Val Tyr Tyr Phe Ala Val His Wing Thr Val Tyr 145 150 155 160 Arg Ala Ser Leu Gln Phe Asp Leu Val Lys Asn Gly Glu Ser He Wing 165 170 175 Ser Phe Phe Gln Phe Phe Gly Gly Trp Pro Lys Pro Wing Ser Leu Ser 180 185 190 Gly Gly Wing Met Val Arg Leu Glu Pro Glu Asp Gln Val Trp Val Gln 195 200 205 Val Gly Val Gly Asp Tyr He Gly He Tyr Ala Ser He Lys Thr Asp 210 215 • '220 Ser Thr Phe Ser Gly Phe Leu Val Tyr Ser Asp Trp His Ser Ser Pro 225 230 235 240 Val Phe Ala < 210 > 6 < 211 > 245 < 212 PRT < 213 > Homo sapiens < 400 > 6 Met Asp Val Gly Pro Be Ser Leu Pro His Leu Gly Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Arg Gly Gln Wing Asn Thr Gly Cys 20 25 30 Tyr Gly He Pro Gly Met Pro Gly Leu Pro Gly Wing Pro Gly Lys Asp 35 40 45 Gly Tyr Asp Gly Leu Pro Gly Pro Lys Gly Glu Pro Gly He Pro Wing 50 55 60 He Pro Gl He Arg Gly Pro Lys Gly Gln Lys Gly Glu Pro Gly Leu 65 70 75 80 Pro Gly His Pro Gly Lys Asn Gly Pro Met Gly Pro Pro Gly Met Pro 85 90 95 Gly Val Pro Gly Pro Met Gly He Pro Gly Glu Pro Gly Glu Glu Gly j -fÁÁ -------..-. - J-t --- U-tAtÍ - 3 X > a X i 100 105 110 Arg Tyr Lys Gln Lys Phe Gln Ser Val Phe Thr Val Thr Arg Gln Thr 115 120 125 His Gln Pro Pro Ala Pro Asn Ser Leu He Arg Phe Asn Ala Val Leu 130 135 140 Thr Asn Pro Gln Gly Asp Tyr Asp Thr Ser Thr Gly Lys Phe Thr Cys 145 150 155 160 Lys Val Pro Gly Leu Tyr Tyr Phe Val Tyr His Ala Ser His Thr Wing 165 170 175 Asn Leu Cys Val Leu Leu Tyr Arg Ser Gly Val Lys Val Val Thr Phe 180 185 190 Cys Gly His Thr Ser Lys Thr Asn Gln Val Asn Ser Gly Gly Val Leu 195 200 205 Leu Arg Leu Gln Val Gly Glu Glu Val Trp Leu Ala Val Asn Asp Tyr 210 215 220 Tyr Asp Met Val Gly He Gln Gly Ser Asp Ser Val Phe Ser Gly Phe 225 230 235 240 Leu Leu Phe Pro Asp 245 < 210 > 7 < 211 > 1421 < 212 > DNA < 213 > Homo sapiens < 220 > <; 221 > exon < 222 > (1) ... (208) < 223 > Exon 1 from zacrp5 < 221 > intron < 222 > (209) ... (870) < 223 > Intron 1 from zacrp5 < 221 > exon < 222 > (871) ... (1421) < 223 > Exon 2 from zacrp5 < 400 > 7 atggcagccc ccgccctgct gctcctagca ctgctgctgc ccgtgggggc ctggcccggg 60 ctgcccagga ggccctgtgt gcactgctgc cgcccggcct ggccccctgg accctatgcc 120 cgggtgagtg acagggacct gtggaggggg gacctgtgga gggggctgcc tcgagtacgg 180 cccactataa acatcgaaat cctcaaaggt gaggcccgtg ggtgctgcct gcatgctccc 240 ccaccaggac ccaacagccc acagggagtg gggagcacct ggggcttggg aggaggggcg 300 ggaggggcac tctgagcacc agtgtctgcc ctggcagcgc tccctgcaca gggacccctg 360 ggctcccccc gcaagcagga agaagcctgg cttggggagg gacccatgaa ccaaagtggg 420 tcctggggaa gtgctgagga gggggccagg gttcctgaga tccccaaagt agcagcccct 480 tgggaaggga gcctgggcag cccgcttgct ctgagacccc ttgacacggc tgcgctgtcc 540 cgaagctgta ctaaggttag gcttgggtag gaccttccca gccttctcat tctttaacac 600 ccaacgcaga ccgtagctgg ccctgaccac ccatgtcccc accctctcgg tggggacggc 660 cttgccacag gccttggtct agccacacct ttggagaacg gcttctcccc atcttacaga 720 cgtggacgcg gagáctggga gaggggccgt cacttgcaaa gagtgtgtcc ttacagtgac 780 ccttgagccc cagcccctgg cctgggtgct ggaatggggg aggcctgccc agcccggccc 840 cgacccgtgg ctttcgtttg tcccctgcag.gtgagaaggg tgaggccggc gtccgaggtc 900 gggccggcag gagcgggaaa gaggggccgc caggcgcccg gggcctgcag ggccgcagag 960 gccagaaggg gcaggtgggg ccgccgggcg ccgcgtgccg acgtgcctac gccgccttct 1020 ccgtgggccg gcgcgagggc ctgcacagct ccgaccactt ccaggcggtg cccttcgaca 1080 cggagctggt gaacctggac ggcgccttcg acctggccgc gggccgcttc ctctgcacgg 1140 tgcccggcgt ctacttcctc agcctcaacg tgcacacctg gaactacaag gagacctacc 1200 tgcacatcat gctg aaccgg cggcccgcgg ccgtgctcta cgcgcagccc agcgagcgca 1260 gcgtcatgca ggcccagagc ctgatgctgc tgctggcggc gggcgacgcc gtctgggtgc 1320 gcatgttcca gcgcgaccgg gacaacgcca tctacggcga gcacggagac ctctacatca 1380 ccttcagcgg ccacctggtc aagccggccg ccgagctgta g 1421 ^, - Ít¿¿- .. t > A- -áS- ¿--.- - i-j-. --------- < 210 > 8 < 211 > 31 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Aromatic portion of Clq < 221 > VARIANT < 222 > (2) ... (6) < 223 > Each Xaa is independently any amino acid residue "< 221 > VARIANTE < 222 > (7). (7) < 223 > Each Xaa is asparagine or aspartic acid < 221 > VARIANT < 222 > (8) ... (11) < 223 > Each Xaa is independently any amino acid residue < 221 > VARIANT < 222 > (12) ... (12) < 223 > Xaa is phenylalanine, tyrosine, tryptophan, or leucine < 221 > VARIANT < 222 > (13) ... (18) < 223 > Each Xaa is independently any amino acid residue < 221 > VARIANT < 222 > (20) ... (24) < 223 > Each Xaa is independently any amino acid residue < 221 > VARIANT < 222 > (26) ... (26) < 223 > Xaa is any amino acid residue < 221 > VARIANT < 222 > (28) ... (28) < 223 > Xaa is any amino acid residue < 221 > VARIANT < 222 > (30) ... (30) < 223 > Xaa is any amino acid residue < 221 > VARIANT < 222 > (31) ... (31) < 223 > Xaa is phenylalanine or tyrosine < 400 > 8 Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 - 10 15 Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Phe Xaa Xaa 20 25 30 < 210 > 9 < 211 > 17 < 212 > DNA < 223 > Artificial Sequence < 220 > < 223 > Degenerate oligonucleotide primer < 221 > variation < 222 > (1) ... (17) < 223 > Each N is independently A, T, G or C < 400 > 9 msnggnntnt ayt yyt 17 < 210 > 10 < 211 > 17 < 212 > DNA < 223 > Artificial Sequence < 220 > < 223 > Degenerate oligonucleotide sequence < 221 > variation < 222 > (1) ... (17) < 223 > Each N is independently A, T, G or C < 400 > 10 srnganvvng tntggbt 17 < 210 > 11 < 211 > 17 < 212 > DNA < 223 > Artificial Sequence < 220 > < 223 > Degenerate oligonucleotide primer < 221 > variation < 222 > (1) ... (17) < 223 > Each N is independently A, T, G or C < 400 > 11 ryntty sng gnywyyt 17 < 210 > 12 < 211 > 756 < 212 > DNA < 223 > Artificial Sequence < 220 > < 223 > Degenerate nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 < 221 > variation < 222 > (1) ... (756) < 223 > Each N is independently A, T, G or C < 400 > 12 < 400 > 12 atggcngcnc cngcnytnyt nytnytngcn ytnyt? Ytnc cngtnggngc ntggccnggn 60 ytnccnmgnm gnccntgygt rie-tftgaftgy ingnccngcnt ggccnccngg nccntaygcn 120 »-« .- £ - • -. a ^ -. AlA '^' - a «» iffi ^ -. JE ----; mgngtnwsng aymgngayyt ntgggnggn gayytntggm gnggnytncc nmgngtnmgn 180 ccnacnatha ayathgarat hytnaarggn garaarggng argcnggngt nmgnggnmgn 240 gcnggnmgnw sng ^ gnaarga rggnccnccn ggngcnmgng gnytncargg nmgnmgnggn 300 caraarggnc argtnggncc nccnggngcn gcntgygnm gngcntaygc ngcnttywsn 360 gtnggngnm gngarggnyt ncaywsnwsn gaycayttyc argcngtncc nttygayacn 420 garytngtna ayytngaygg ngcnttygay ytngcngcng gnmgnttyyt ntgyacngtn 480 ccnggngtnt ayttyytnws nytnaaygtp cayacntgga aytayaarga racntayytn 540 cayathatgy tnaaymgnmg nccngcngcn gtnytntayg cncarccnws ngarmgnwsn 600 gtnatgcarg cncarwsnyt natgytnyt? ytngcngcng gngaygcngt ntgggtnmgn 660 atgttycarm gngaygnga yaaygcnath tayggngarc ayggngayyt ntayathacn 720 ttywsnggnc ayytngtnaa rccngcngcn garytn 756 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims

Having described the invention as above, the content of the following claims is claimed as property: 1. An isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical in sequence to amino acids with residues 70-252 of SEQ ID NO: 2, characterized in that the sequence comprises: the collagen repeats of Gly-Xaa-Xaa and Gly-Xaa-Pro which form a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clq domain. 2. An isolated polypeptide according to claim 1, characterized in that the polypeptide is at least 90% identical in the amino acid sequence with residues 18-252 of SEQ ID NO: 2.
3. A polypeptide isolated in accordance with claim 2, characterized in that any differences between the polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.
4. An isolated polypeptide according to claim 2, characterized in that the collagen-like domain consists of 14 repeats of the Gly-Xaa-Xaa collagen and 1 repetition of Gly-Xaa-Pro collagen. i-t-A-l - - J - li - l_-t - Í-- < & ---- »_---- a,. .j -u - ji »< . < . .-.- faith-- ».» iff? «-» at-IÍ > -ÉSi--, .-; ^. -4-J - A. ^. - «- < - »aau * f * > /) t iJbií
5. An isolated polypeptide according to claim 2, characterized in that the polypeptide comprises: an amino terminal region; 14 repeats of Gly-Xaa-Xaa collagen and 1 repetition of Gly-Xaa-Pro collagen that form a collagen-like domain, where Xaa is any amino acid residue; and a carboxyl-terminal Clq 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.
6. An isolated polypeptide according to claim 2, characterized in that the polypeptide is specifically agglutinated with an antibody that specifically binds with a polypeptide of SEQ ID NO: 2.
7. An isolated polypeptide according to claim 2, characterized because the collagen-like domain comprises amino acid residues 70-111 of SEQ ID NO: 2.
8. An isolated polypeptide according to claim 2, characterized in that the Clq domain comprises amino acid residues 112-252 of the SEC ID NO: 2 . «I. -, - ^ t; ^ - --- & tg & at - iia8 ^ it '--'- * - * - * --- --- ---- 3¿ - i ^ THE.
9. An isolated polypeptide according to claim 1, characterized in that the polypeptide comprises residues 70-252 of SEQ ID NO: 2.
10. An isolated polypeptide according to claim 2, characterized in that the polypeptide comprises residues 18-252 of SEQ ID NO: 2.
11. An isolated polypeptide according to claim 2, characterized in that the polypeptide comprises residues 1-252 of SEQ ID NO: 2.
12. An isolated polypeptide according to claim 1, characterized in that the polypeptide is converted into a complex by the intermolecular disulfide bonds to form an omotrimer.
13. An isolated polypeptide according to claim 1, characterized in that the polypeptide is converted into a complex by the 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, characterized in that it is covalently linked at the amino or carboxyl terminus to a selected portion of the group consisting of the affinity tags, toxins, radionucleotides, enzymes and fluorophores.
15. An isolated polypeptide, characterized in that it is 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, characterized in that it consists essentially of a first portion and a second portion bound by a peptide linkage, the first portion consisting of a polypeptide selected from the group consisting of: a) a polypeptide according to claim 1; b) a polypeptide comprising: a terminal amino region; 14 repeats of Gly-Xaa-Xaa collagen and 1 repetition of Gly-Xaa-Pro collagen that form a collagen-like domain, where Xaa is any amino acid residue; and a carboxyl-terminal Clq 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 Clq domain or an active portion of the Clq domain.; or e) a portion of the zacrp2 polypeptide as shown in SEQ ID NO: 2 comprising the collagen-like domain and the Clq domain; and the second portion comprises another polypeptide.
17. A fusion protein according to claim 16, characterized in that the first portion is selected from the group consisting of: a) a polypeptide consisting of the sequence from residue 70 of the amino acids to residue 111 of the amino acids of the SEC ID NO: 2; b) a polypeptide consisting of the sequence of residue 112 of the amino acids to residue 252 of the amino acids of SEQ ID NO: 2; c) a polypeptide consisting of the sequence of amino acid residues 70 to 252 of SEQ ID NO: 2; d) a polypeptide consisting of the sequence of amino acid residues 18 to 252 of SEQ ID NO: 2; and e) a polypeptide consisting of the sequence of amino acid residues 1 to 252 of SEQ ID NO: 2.
18. A polypeptide according to claim 1, characterized in that it is used in combination with a pharmaceutically acceptable carrier.
19. A method of producing an antibody to a polypeptide, characterized in that it comprises: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide according to claim 1; b) a polypeptide comprising: an amino terminal region: 14 collagen repeats of Gly-Xaa-Xaa and 1 repetition of Gly-Xaa-Pro collagen that forms a collagen-like domain, wherein Xaa is any amino acid residue; and a carboxyl-terminal Clq 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 Clq domain or an active portion of the Clq domain; or e) a portion of the zacrp5 polypeptide as shown in SEQ ID NO: 2 comprising the collagen-like domain and the Clq domain; Y
* »Wherein the polypeptide produces an immune response in the animal to produce the antibody; and isolate the animal's antibody. 20. An antibody or antibody fragment, characterized in that it binds specifically to a polypeptide according to claim 1.
21. An antibody according to claim 20, characterized in that the antibody is selected from the group consisting of: a) a polyclonal antibody; b) a murine monoclonal antibody; c) a humanized antibody derived from b); and d) a human monoclonal antibody.
22. An antibody fragment according to claim 20, characterized in that the antibody fragment is selected from the group consisting of F (ab ') / F (ab), Fab', Fab, Fv, scFv, and the unit of minimal recognition
23. An anti-idiotype antibody, characterized in that it binds specifically to the antibody of claim 20.
24. An agglutination protein, characterized in that it agglutinates specifically to an epitope of a polypeptide according to claim 1. "___ & ? -j.- _-. * • »< -. .-_? -.- a..A &- »» .BJ - .-,, a -.- 'i --- to XX; k * i. »F
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, characterized in that the sequence comprises: repeats of collagen of Gly-Xaa-Xaa and Gly-Xaa-Pro that form a domain similar to collagen, where Xaa is any amino acid residue; and a carboxyl-terminal Clq domain.
26. An isolated polynucleotide according to claim 25, characterized in that the polypeptide is at least 90% identical in the amino acid sequence to residues 18-252 of SEQ ID NO: 2.
27. A polynucleotide isolated in accordance with claim 25, characterized in that the collagen-like domain consists of 14 repeats of the Gly-Xaa-Xaa collagen and 1 repetition of the Gly-Xaa-Pro collagen.
28. An isolated polynucleotide according to claim 25, characterized in that the polypeptide comprises: an amino terminal region; 14 repeats of collagen of Gly-Xaa-Xaa and 1 repetition of collagen of Gly-Xaa-Pro that form a domain similar to collagen, where Xaa is any amino acid residue; and a carboxyl-terminal Clq 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.
29. An isolated polynucleotide according to claim 25, characterized in that any differences between the polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.
30. An isolated polynucleotide according to claim 25, characterized in that the polypeptide is specifically agglutinated with an antibody that agglutinates specifically with a polypeptide of SEQ ID NO: 2.
31. An isolated polynucleotide according to claim 25, characterized because the collagen-like domain comprises amino acid residues 70-111 of SEQ ID NO: 2.
32. An isolated polynucleotide according to claim 25, characterized in that the polypeptide comprises residues 70-252 of SEQ ID NO: 2.
33. An isolated polynucleotide according to claim 25, characterized in that the polypeptide comprises residues 18 -252 of SEQ ID NO: 2.
34. An isolated polynucleotide according to claim 25, characterized in that the polypeptide comprises residues 1-252 of SEQ ID NO: 2.
35. An isolated polynucleotide according to claim 25, characterized in that the polypeptide is covalently bound at the termination. of amino or carboxyl to a portion selected from the group consisting of the affinity tags, toxins, radionucleotides, enzymes and fluorophores.
36. An isolated polynucleotide, characterized in that it is selected from the group consisting of: a) a nucleotide sequence 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 nucleotide sequence from nucleotide 208 to nucleotide 333 of SEQ ID NO: 1; d) a nucleotide sequence from nucleotide 334 to nucleotide 759 of SEQ ID NO: 1; e) a nucleotide sequence from nucleotide 208 to nucleotide 759 of SEQ ID NO: 1; f) a nucleotide sequence 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 NO: 2; i) a polynucleotide that remains hybridized, following the severe washing conditions, to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, or the complement of SEQ ID NO: 1; j) the complementary nucleotide sequences with a), b), c), d), e), f), g), h) or i) and k) the degenerate nucleotide sequences of g) or h).
37. An isolated polynucleotide encoding a fusion protein, characterized in that it consists essentially of a first portion and a second portion linked by a peptide bond, the first portion consists of a polypeptide selected from the group consisting of: a) a polypeptide of according to claim 1; b) a polypeptide comprising: an amino terminal region: 14 repeats of collagen of Gly-Xaa-Xaa and 1 repetition of collagen of Gly-Xaa-Pro forming a domain J? LJ i li-A- U similar to collagen, where Xaa is any amino acid residue; and a carboxyl-terminal Clq 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 Clq domain or an active portion of the Clq domain; or e) a portion of the zacrp5 polypeptide as shown in SEQ ID NO: 2 comprising the collagen-like domain and the Clq domain; and the second portion comprising another polypeptide.
38. An isolated polynucleotide, characterized in that it consists of the sequence from nucleotide 1 to nucleotide 756 of SEQ ID NO: 12.
39. An expression vector, characterized in that it comprises 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, characterized in that the DNA segment encodes a polypeptide that is at least 90% identical in the amino acid sequence to residues 18-252 of SEQ ID NO: 2.
41. A vector of expression according to claim 39, characterized in that the collagen-like domain consists of 14 repeats of Gly-Xaa-Xaa collagen and 1 repetition of Gly-Xaa-Pro collagen.
42. A vector of the expression according to claim 39, characterized in that the DNA segment encodes a polypeptide comprising: comprises: a terminal amino region; 14 repeats of Gly-Xaa-Xaa collagen and 1 repetition of Gly-Xaa-Pro collagen that form a collagen-like domain, where Xaa is any amino acid residue; and a carboxyl-terminal Clq 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. A vector of the expression according to claim 39, characterized in that the domain Collagen-like comprises amino acid residues 70-111 of SEQ ID NO: 2.
44. An expression vector according to claim 39, characterized in that any differences between the polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.
45. A vector of the expression according to claim 39, characterized in that the polypeptide is specifically agglutinated with an antibody that binds specifically to a polypeptide of SEQ ID NO: 2.
46. A vector of expression in accordance with claim 39, characterized in that the DNA encodes a polypeptide comprising residues 70-252 of SEQ ID NO: 2.
47. An expression vector according to claim 39, characterized in that the DNA segment encodes a polypeptide comprising residues 18-252 of SEQ ID NO: 2.
48. An expression vector according to claim 39, characterized in that the DNA segment encodes a polypeptide comprising residues 1-252 of SEQ ID NO: 2
49. A vector of the expression according to claim 39, characterized in that the DNA segment also encodes a sequence of the secretory signal operably linked to the polypeptide. ptido
50. A vector of the expression according to claim 39, characterized in that the sequence of the secretory signal comprises residues 1-17 of SEQ ID NO: 2.
51. A cultured cell into which a vector has been introduced. of the expression according to claim 39, characterized in that the cell expresses the polypeptide encoded by the DNA segment.
52. A cell cultured according to claim 51, characterized in that it also includes one or more expression vectors comprising the DNA segments encoding the polypeptides having the collagen-like domains.
53. A method of producing a protein, characterized in that it comprises: culturing a cell into which an expression vector according to claim 39 has been introduced; whereby the cell expresses the protein encoded by the DNA segment; and recover the expressed protein.
54. A method of producing a protein according to claim 53, characterized in that the expressed protein is a homotrimer.
55. A method of producing a protein according to claim 53, characterized in that the expressed protein is a heterotrimer.
56. A method for detecting the presence of zacrp5 gene expression in a biological sample, characterized in that it comprises: (a) contacting a zacrp5 nucleic acid probe under hybridization conditions with either (i) the RNA test isolated from the biological sample, or (ii) the 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 the complements thereof, and (b) detect the formation of the 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.
57. A method for the detection of the presence of zacrp5 in a biological sample, characterized in that it comprises: (a) contacting the biological sample with an antibody, or an antibody fragment, according to claim 20, wherein the contact is effected under conditions that allow the agglutination of the antibody or antibody fragment to the biological sample, and (b) detect any of the agglutinated antibody or agglutinated antibody fragment. ^^ j ^ ^ j ^^^ üSia