Human osteoclast-derived cathepsin

Abstract

Disclosed is a human osteoclast-derived cathepsin (Cathepsin O) polypeptide and DNA(RNA) encoding such cathepsin O polypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques. The present invention also discloses antibodies, antagonists and inhibitors of such polypeptide which may be used to prevent the action of such polypeptide and therefore may be used therapeutically to treat bone diseases such as osteoporosis and cancers, such as tumor metastases.

Description

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a human osteoclast-derived cathepsin (Cathepsin O). The invention also relates to inhibiting the action of such polypeptide and to assays for identifying inhibitors of the polypeptide.

Bone resorption involves the simultaneous removal of both the mineral and the organic constituents of the extracellular matrix. This occurs mainly in an acidic phagolysosome-like extracellular compartment covered by the ruffled border of osteoclasts. Barron, et al., J. Cell Biol., 101:2210-22, (1985). Osteoclasts are multinucleate giant cells that play key roles in bone resorption. Attached to the bone surface, osteoclasts produce an acidic microenvironment between osteoclasts and bone matrix. In this acidic microenvironment, bone minerals and organic components are solubilized. Organic components, mainly type-I collagen, are thought to be solubilized by protease digestion. There is evidence that cysteine proteinases may play an important role in the degradation of organic components of bone. Among cysteine proteinases, cathepsins B, L, N, and S can degrade type-I collagen in the acidic condition. Etherington, D. J. Biochem. J., 127, 685-692 (1972). Cathepsin L is the most active of the lysosomal cysteine proteases with regard to its ability to hydrolyze azocasein, elastin, and collagen.

Cathepsins are proteases that function in the normal physiological as well as pathological degradation of connective tissue. Cathepsins play a major role in intracellular protein degradation and turnover, bone remodeling, and prohormone activation. Marx, J. L., Science. 235:285-286 (1987). Cathepsin B, H, L and S are ubiquitously expressed lysosomal cysteine proteinases that belong to the papain superfamily. They are found at constitutive levels in many tissues in the human including kidney, liver, lung and spleen. Some pathological roles of cathepsins include an involvement in glomerulonephritis, arthritis, and cancer metastasis. Sloan, B. F., and Honn, K. V., Cancer Metastasis Rev., 3:249-263 (1984). Greatly elevated levels of cathepsin L and B mRNA and protein are seen in tumor cells. Cathepsin L mRNA is also induced in fibroblasts treated with tumor promoting agents and growth factors. Kane, S. E. and Gottesman, M. M. Cancer Biology, 1:127-136 (1990).

In vitro studies on bone resorption have shown that cathepsins L and B may be involved in the remodelling of this tissue. These lysosomal cysteine proteases digest extracellular matrix proteins such as elastin, laminin, and type I collagen under acidic conditions. Osteoclast cells require this activity to degrade the organic matrix prior to bone regeneration accomplished by osteoblasts. Several natural and synthetic inhibitors of cysteine proteinases have been effective in inhibiting the degradation of this matrix.

The isolation of cathepsins and their role in bone resorption has been the subject of an intensive study. OC-2 has recently been isolated from pure osteoclasts from rabbit bones. The OC-2 was found to encode a possible cysteine proteinase structurally related to cathepsins L and S. Tezuka, K., et al., J. Biol. Chem., 269:1106-1109, (1994).

An inhibitor of cysteine proteinases and collagenase, Z-Phe-Ala-CHN 2 has been studied for its effect on the resorptive activity of isolated osteoclasts and has been found to inhibit resorption pits in dentine. Delaisse, J. M. et al., Bone, 8:305-313 (1987). Also, the effect of human recombinant cystatin C, a cysteine proteinase inhibitor, on bone resorption in vitro has been evaluated, and has been shown to significantly inhibit bone resorption which has been stimulated by parathyroid hormone. Lerner, U. H. and Grubb Anders, Journal of Bone and Mineral Research, 7:433-439, (1989). Further, a cDNA clone encoding the human cysteine protease cathepsin L has been recombinantly manufactured and expressed at high levels in E. coli in a T7 expression system. Recombinant human procathepsin L was successfully expressed at high levels and purified as both procathepsin L and active processed cathepsin L forms. Information about the possible function of the propeptide in cathepsin L folding and/or processing and about the necessity for the light chain of the enzyme for protease activity was obtained by expressing and purifying mutant enzymes carrying structural alterations in these regions. Smith, S. M. and Gottesman, M. M., J. Bio Chem., 264:20487-20495, (1989). There has also been reported the expression of a functional human cathepsin S in Saccharomyces cerevisiae and the characterization of the recombinant enzyme. Bromme, D. et al., J. Bio Chem., 268:4832-4838 (1993).

In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is a osteoclast-derived cathepsin as well as fragments, analogs and derivatives thereof. The human osteoclast-derived cathepsin of the present invention is of human origin.

In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with still another aspect of the present invention, there is provided a procedure for producing such polypeptide by recombinant techniques.

In accordance with yet a further aspect of the present invention, there is provided an antibody which inhibits the action of such polypeptide.

In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, e.g., a small molecule inhibitor which may be used to inhibit the action of such polypeptide, for example, in the treatment of metastatic tumors and osteoporosis.

In accordance with still another aspect of the present invention, there is provided a procedure for developing assay systems to identify inhibitors of the polypeptide of the present invention.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are meant only as illustrations of specific embodiments of the present invention and are not meant as limitations in any manner.

FIGS. 1A-B show the polynucleotide sequence (SEQ ID NO:1) and corresponding deduced amino acid sequence (SEQ ID NO:2) for cathepsin O. The cathepsin O shown is the predicted precursor form of the protein where approximately the first 15 amino acids represent the leader sequence and the first 115 amino acids are the prosequence. The standard three letter abbreviation has been used for the amino acid sequence.

FIGS. 2A-C are illustrations of the amino acid homology of cathepsin O to other human cathepsins (SEQ ID NO:8-14) and rabbit OC-2 (SEQ ID NO:7).

In accordance with one aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of FIGS. 1A-B (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75671 on Feb. 9, 1994.

The ATCC number referred to above is directed to a biological deposit with the ATCC (American Type Culture Collection), 10801 University Boulevard, Manassas, Va. 20110-2209. Since the strains referred to are being maintained under the terms of the Budapest Treaty, they will be made available to a patent office signatory to the Budapest Treaty.

A polynucleotide encoding a polypeptide of the present invention may be obtained from a cDNA library derived from human osteoclastoma cells, placenta, kidney or lung. The polynucleotide described herein was isolated from a cDNA library derived from human osteoclastoma cells. The cDNA insert is 1619 base pairs (bp) in length and contains an open reading frame encoding a protein 329 amino acids in length of which approximately the first 15 amino acids represent the leader sequence and first 115 amino acids represent the prosequence. Thus, the mature form of the polypeptide of the present invention consists of 214 amino acids after the 115 amino acid prosequence (which includes the approximately 15 amino acid leader sequence) is cleaved. The polypeptide encoded by the polynucleotide is structurally related to human cathepsin S with 56% identical amino acids and 71% similarity over the entire coding region. It is also structurally related to rabbit OC-2 cathepsin with 94% identical amino acids and 97% similarity over the entire coding region. The polypeptide may be found in lysosomes of, or extracellularly near, osteoclasts.

The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in FIGS. 1A-B (SEQ ID NO:1) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of FIGS. 1A-B (SEQ ID NO:1) or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIGS. 1A-B (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs, and derivatives of the polypeptide having the deduced amino acid sequence of FIGS. 1A-B (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide. The present invention also relates to polynucleotide probes constructed from the polynucleotide sequence of FIGS. 1A-B (SEQ ID NO:1) or a segment of the sequence of FIGS. 1A-B (SEQ ID NO:1) amplified by the PCR method, which could be utilized to screen an osteoclast cDNA library to deduce the polypeptide of the present invention.

Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in FIGS. 1A-B (SEQ ID NO:2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of FIGS. 1A-B (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in FIGS. 1A-B (SEQ ID NO:1) or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5′ amino acid residues. A mature protein having a prosequence is a proprotein and may in some cases be an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a presequence (leader sequence) and a prosequence.

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term “stringent conditions” means hybridization will occur only if there is as least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of FIGS. 1A-B or the deposited cDNA.

The deposits referred to herein will be maintained under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure. These deposits are provided merely as a convenience and are not an admission that a deposit is required under 35 U.S.C. § 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

The present invention further relates to a cathepsin O polypeptide which has the deduced amino acid sequence of FIGS. 1A-B (SEQ ID NO:2) or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to the polypeptide of FIGS. 1A-B (SEQ ID NO:2) or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIGS. 1A-B (SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the cathepsin O genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotide of the present invention may be employed for producing a polypeptide by recombinant techniques. Thus, for example, the polynucleotide sequence may be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other plasmid or vector may be used as long as it is replicable and viable in the host.

As hereinabove indicated, the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into appropriate restriction endonuclease sites by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli . lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Salmonella typhimurium ; Streptomyces; fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as it is replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P R , P L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described construct. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, 1986)).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), the disclosure of which is hereby incorporated by reference.

Transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), a cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, PKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., U.S.A.). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well-known to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

Cathepsin O is recovered and purified from recombinant cell cultures by methods used heretofore, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.1-5 mM) of calcium ion present during purification (Price, et al., J. Biol. Chem., 244:917 (1969)). Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be naturally purified products expressed from a high expressing cell line, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphism's) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clone from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques. Pergamon Press, N.Y. (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).

Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

The present invention is directed to inhibiting cathepsin O in vivo by the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes for the mature polypeptide of the present invention, is used to design an antisense RNA oligonucleotide of from 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al, Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al, Science, 251:1360 (1991), thereby preventing transcription and the production of cathepsin O. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of an mRNA molecule into the cathepsin O (antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).

Alternatively, the oligonucleotides described above can be delivered to cells by procedures in the art such that the anti-sense RNA or DNA may be expressed in vivo to inhibit production of cathepsin O in the manner described above.

Antisense constructs to cathepsin O, therefore, inhibit the action of cathepsin O and may be used for treating certain disorders, for example, osteoporosis, since bone resorption is slowed or prevented. These antisense constructs may also be used to treat tumor metastasis since elevated levels of cathepsins are found in some tumor cells, and cathepsin L mRNA and protein is increased in ras-transformed fibroblasts. Further, there is evidence that metastatic B16 melanomas all upregulate cathepsin B compared with non-metastatic tumors.

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present also includes chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptide corresponding to a sequence of the present invention or its in vivo receptor can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.

Antibodies specific to the cathepsin O may further be used to inhibit the biological action of the polypeptide by binding to the polypeptide. In this manner, the antibodies may be used in therapy, for example, to treat cancer since cathepsin L mRNA and protein is increased in ras-transformed fibroblasts and after addition of phorbol esters and growth factors. Also, osteoporosis may be treated with these antibodies since bone resorption by cathepsin O is prevented.

Further, such antibodies can detect the presence or absence of cathepsin O and the level of concentration of cathepsin O and, therefore, are useful as diagnostic markers for the diagnosis of disorders such as high turnover osteoporosis, Paget's disease, tumor osteolysis, or other metabolic bone disorders. Such antibodies may also function as a diagnostic marker for tumor metastases.

The present invention is also directed to antagonists and inhibitors of the polypeptides of the present invention. The antagonists and inhibitors are those which inhibit or eliminate the function of the polypeptide.

Thus, for example, an antagonist may bind to a polypeptide of the present invention and inhibit or eliminate its function. The antagonist, for example, could be an antibody against the polypeptide which eliminates the activity of cathepsin O by binding to cathepsin O, or in some cases the antagonist may be an oligonucleotide. An example of an inhibitor is a small molecule inhibitor which inactivates the polypeptide by binding to and occupying the catalytic site, thereby making the catalytic site inaccessible to a substrate, such that the biological activity of cathepsin O is prevented. Examples of small molecule inhibitors include but are not limited to small peptides or peptide-like molecules.

In these ways, the antagonists and inhibitors may be used to treat bone disease, such as osteoporosis by preventing cathepsin O from functioning to break down bone. The antagonists and inhibitors may also be used to treat metastatic tumors since cathepsins play a role in increasing metastatic tumor growth.

The antagonists and inhibitors may be employed in a composition with a pharmaceutically acceptable carrier, including but not limited to saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. Administration of cathepsin inhibitors are preferably systemic. Intraperitoneal injections of the cysteine proteinase inhibitor leupeptin (0.36 mg/kg body weight) and E-64 (0.18 mg/kg body weight) in rats were able to decrease serum calcium and urinary excretion of hydroxyproline. Delaisse et al., BBRC, 125:441-447 (1984). A direct application on areas of bone vulnerable to osteoporosis such as the proximal neck of the femur may also be employed.

The present invention also relates to an assay for identifying the above-mentioned small molecule inhibitors which are specific to Cathepsin O and prevent it from functioning. Either natural protein substrates or synthetic peptides would be used to assess proteolytic activity of cathepsin O, and the ability of inhibitors to prevent this activity could be the basis for a screen to identify compounds that have therapeutic activity in disorders of excessive bone resorption. Maciewicz, R. A. and Etheringtin, D. J., BioChem. J. 256:433-440 (1988).

A general example of such an assay for identifying inhibitors of cathepsin O utilizes peptide-based substrates which are conjugated with a chromogenic tag. An illustrative example of such a peptide substrate has the X-(Y)n-Z, wherein X represents an appropriate amino protecting group such as acetyl, acetate or amide, Y is any naturally or non-naturally occurring amino acid which in combination forms a substrate which cathepsin O recognizes and will cleave in the absence of an inhibitor, n represents an integer which may be any number, however, which is usually at least 20, and Z represents any chromogenic or flourogenic tag, for example, para-nitroanelide or n-methyl coumarin, which upon cleavage of the Y group by the cathepsin O can be monitored for color production. If the potential inhibitor does not inhibit cathepsin O and the substrate (Y group) is cleaved, Z has a corresponding change in configuration, which change allows fluorescence to be detected by a fluorimeter in the case of a flourogenic tag and color to be detected by a spectrophotometer in the case of a chromogenic tag. When the inhibitor successfully inhibits cathepsin O from cleaving the substrate, the Y group is not cleaved and Z does not have a change in configuration and no fluorescence or color is detectable which indicates that the inhibitor has inhibited the action of cathepsin O.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of the following examples, certain frequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).

“Oligonucleotides” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase (“ligase”) per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described in the methods of Graham, F. and Van Der Eb, A., Virology, 52:456-457 (1973).

EXAMPLE 1

Expression and Purification of the Osteoclast-Derived Cathepsin

The DNA sequence encoding for cathepsin O (ATCC #75671) is initially amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ end of the DNA sequence to synthesize insertion fragments. The 5′ oligonucleotide primer has the sequence 5′ GCTAAGGATCCTGGGGGCTCAAGGTT 3′ (SEQ ID NO:3) contains a Bam H1 restriction enzyme site followed by 15 nucleotides of cathepsin O coding sequence starting from the codon following the methionine start codon; the 3′ sequence, 5′ GCTAATCTAGATCACATCTTGGGGAA 3′ (SEQ ID NO:4) contains complementary sequences to XbaI site, and the last 12 nucleotides of cathepsin O coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen Inc., 9259 Eton Ave., Chatsworth, Calif. 91311). The plasmid vector encodes antibiotic resistance (Amp r ), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His) and restriction enzyme cloning sites. The pQE-9 vector was digested with Bam HI and XbaI and the insertion fragments were then ligated into the vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture was then used to transform the E. coli strain m15/rep4 (available from Qiagen under the trademark m15/rep4). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan r ). Transformants are identified by their ability to grow on LB plates containing both Amp and Kan. Clones containing the desired constructs were grown overnight (O/N) in liquid culture in either LB media supplemented with both Amp (100 μg/ml) and Kan (25 μg/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density of 600 (O.D. 600 ) between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a final concentration of 1 mM. IPTG induces by inactivating the laci repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3-4 hours. Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 molar guanidine-HCL and 50 mM NaPO 4 pH 8.0. After clarification, solubilized cathepsin O was purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag. (Hochuli, E. et al., Genetic Engineering, Principle & Methods, 12:87-98 Plenum Press, New York (1990)). Cathepsin O (95% pure) was eluted from the column in 6 molar guanidine-HCL, 150 mM NaPO 4 pH 5.0.

EXAMPLE 2

Expression Pattern of Cathepsin O in Human Tissue

[ 35 S]-labeled sense or antisense riboprobes generated from a partial cDNA clone of Cathepsin O were used as part of a Northern blot analysis to probe cryosections of osteoclastoma tissue, which demonstrated a single mRNA species, and spleen tissue. Current Protocols in Molecular Biology, Vol. 2, Ausubel et al., editors, section 14.3. Total RNA was isolated from osteoclastoma tissue and spleen. The RNA was electrophoresed on a formaldehyde agarose gel, and transferred to nitrocellulose. Following pre-hybridization, the blot was hybridized overnight with either sense or antisense [ 32 P]-labeled riboprobe at 2×10 6 cpm/ml at 42° C. Following stringent washes (0.2×SSC at 65° C.), the blots were exposed to x-ray film. When used in in situ hybridization on sections of osteoclastoma tissue, specific, high level expression was observed in the osteoclasts; some expression was observed in mononuclear cells, but the stromal cells and osteoblasts did not express the mRNA for Cathepsin O at detectable levels. When sections of spleen tissue were used for in situ hybridization, no expression of Cathepsin O was observed. These data indicate that the mRNA for Cathepsin O is expressed at high levels in osteoclasts, and appears to be selectively expressed in these cells.

EXAMPLE 3

Analysis of Cathepsin O Using Antibodies

Antibodies were prepared against synthetic peptides from the Cathepsin O sequence, from regions sufficiently dissimilar to other members of the cathepsin family to allow specific analysis of Cathepsin O in Western blots. The antibodies were affinity purified and used to probe Western blots of osteoclastoma tissue. Synthetic peptides (AIDASLTSFQFYSK (SEQ ID NO:5) and YDESCNSDNLN (SEQ ID NO:6)) were prepared based upon the predicted sequence of Cathepsin O (corresponding to amino acids 248-261 and 265-275 in FIG. 1 ). The regions were chosen because of lowest identity to other members of the cathepsin family. The peptides were conjugated to Keyhole Limpet Hemocyanin with glutaraldehyde, mixed with adjuvant, and injected into rabbits. Immune sera was affinity purified using the immobilized peptide. Drake et al., Biochemistry, 28:8154-8160 (1989).

Tissue samples were homogenized in SDS-PAGE sample buffer and run on a 14% SDS-PAGE. The proteins were transferred to nitrocellulose, followed by blocking in bovine serum albumin. Immunoblotting was carried out with affinity purified anti-peptide antibodies, followed by alkaline phosphatase conjugated second antibody and visualization with a chromogenic substrate. Molecular mass determination was made based upon the mobility of pre-stained molecular weight standards (Rainbow markers, Amersham). Antibodies to two different peptides recognized a major band of approximately 29 kDa and a minor band of approximately 27 kDa. The immunoreactivity could be competed by the peptides used to generate the antibodies, confirming the specificity of the signal. This indicates that the mRNA for Cathepsin O is actually expressed in the tissue, and produces a protein with a size consistent with that of a fully processed Cathepsin O (assuming processing similar to related cathepsins).

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

#             SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 14
<210> SEQ ID NO 1
<211> LENGTH: 1619
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (21)..(1009)
<220> FEATURE:
<221> NAME/KEY: mat_peptide
<222> LOCATION: (365)..(1009)
<220> FEATURE:
<221> NAME/KEY: sig_peptide
<222> LOCATION: (21)..(1009)
<400> SEQUENCE: 1
tcagatttcc atcagcagg atg tgg ggg ctc aag gtt ctg
# ctg cta cct gtg      52
#   Met Trp Gly Leu Lys Val Leu Leu Leu
# Pro Val
#
#    -110
#   -105
gtg agc ttt gct ctg tac cct gag gag ata ct
#g gac acc cac tgg gag      100
Val Ser Phe Ala Leu Tyr Pro Glu Glu Ile Le
#u Asp Thr His Trp Glu
-100
#               -95
#               -90
cta tgg aag aag acc cac agg aag caa tat aa
#c aac aag gtg gat gaa      148
Leu Trp Lys Lys Thr His Arg Lys Gln Tyr As
#n Asn Lys Val Asp Glu
-85
#           -80
#           -75
atc tct cgg cgt tta att tgg gaa aaa aac ct
#g aag tat att tcc atc      196
Ile Ser Arg Arg Leu Ile Trp Glu Lys Asn Le
#u Lys Tyr Ile Ser Ile
-70
#       -65
#       -60
cat aac ctt gag gct tct ctt ggt gtc cat ac
#a tat gaa ctg gct atg      244
His Asn Leu Glu Ala Ser Leu Gly Val His Th
#r Tyr Glu Leu Ala Met
-55
#   -50
#   -45
aac cac ctg ggg gac atg acc agt gaa gag gt
#g gtt cag aag atg act      292
Asn His Leu Gly Asp Met Thr Ser Glu Glu Va
#l Val Gln Lys Met Thr
-40                 -
#35                 -
#30                 -
#25
gga ctc aaa gta ccc ctg tct cat tcc cgc ag
#t aat gac acc ctt tat      340
Gly Leu Lys Val Pro Leu Ser His Ser Arg Se
#r Asn Asp Thr Leu Tyr
-20
#               -15
#               -10
atc cca gaa tgg gaa ggt aga gcc cca gac tc
#t gtc gac tat cga aag      388
Ile Pro Glu Trp Glu Gly Arg Ala Pro Asp Se
#r Val Asp Tyr Arg Lys
-5
#         -1   1
#          5
aaa gga tat gtt act cct gtc aaa aat cag gg
#t cag tgt ggt tcc tgt      436
Lys Gly Tyr Val Thr Pro Val Lys Asn Gln Gl
#y Gln Cys Gly Ser Cys
10
#     15
#     20
tgg gct ttt agc tct gtg ggt gcc ctg gag gg
#c caa ctc aag aag aaa      484
Trp Ala Phe Ser Ser Val Gly Ala Leu Glu Gl
#y Gln Leu Lys Lys Lys
25
# 30
# 35
# 40
act ggc aaa ctc tta aat ctg agt ccc cag aa
#c cta gtg gat tgt gtg      532
Thr Gly Lys Leu Leu Asn Leu Ser Pro Gln As
#n Leu Val Asp Cys Val
45
#                 50
#                 55
tct gag aat gat ggc tgt gga ggg ggc tac at
#g acc aat gcc ttc caa      580
Ser Glu Asn Asp Gly Cys Gly Gly Gly Tyr Me
#t Thr Asn Ala Phe Gln
60
#             65
#             70
tat gtg cag aag aac cgg ggt att gac tct ga
#a gat gcc tac cca tat      628
Tyr Val Gln Lys Asn Arg Gly Ile Asp Ser Gl
#u Asp Ala Tyr Pro Tyr
75
#         80
#         85
gtg gga cag gaa gag agt tgt atg tac aac cc
#a aca ggc aag gca gct      676
Val Gly Gln Glu Glu Ser Cys Met Tyr Asn Pr
#o Thr Gly Lys Ala Ala
90
#     95
#    100
aaa tgc aga ggg tac aga gag atc ccc gag gg
#g aat gag aaa gcc ctg      724
Lys Cys Arg Gly Tyr Arg Glu Ile Pro Glu Gl
#y Asn Glu Lys Ala Leu
105                 1
#10                 1
#15                 1
#20
aag agg gca gtg gcc cga gtg gga cct gtc tc
#t gtg gcc att gat gca      772
Lys Arg Ala Val Ala Arg Val Gly Pro Val Se
#r Val Ala Ile Asp Ala
125
#               130
#               135
agc ctg acc tcc ttc cag ttt tac agc aaa gg
#t gtg tat tat gat gaa      820
Ser Leu Thr Ser Phe Gln Phe Tyr Ser Lys Gl
#y Val Tyr Tyr Asp Glu
140
#           145
#           150
agc tgc aat agc gat aat ctg aac cat gcg gt
#t ttg gca gtg gga tat      868
Ser Cys Asn Ser Asp Asn Leu Asn His Ala Va
#l Leu Ala Val Gly Tyr
155
#       160
#       165
gga atc cag aag gga aac aag cac tgg ata at
#t aaa aac agc tgg gga      916
Gly Ile Gln Lys Gly Asn Lys His Trp Ile Il
#e Lys Asn Ser Trp Gly
170
#   175
#   180
gaa aac tgg gga aac aaa gga tat atc ctc at
#g gct cga aat aag aac      964
Glu Asn Trp Gly Asn Lys Gly Tyr Ile Leu Me
#t Ala Arg Asn Lys Asn
185                 1
#90                 1
#95                 2
#00
aac gcc tgt ggc att gcc aac ctg gcc agc tt
#c ccc aag atg tga         1009
Asn Ala Cys Gly Ile Ala Asn Leu Ala Ser Ph
#e Pro Lys Met
205
#               210
ctccagccag ccaaatccat cctgctcttc catttcttcc acgatggtgc ag
#tgtaacga   1069
tgcactttgg aagggagttg gtgtgctatt tttgaagcag atgtggtgat ac
#tgagattg   1129
tctgttcagt ttccccattt gtttgtgctt caaatgatcc ttcctacttt gc
#ttctctcc   1189
acccatgacc tttttcactg tggccatcag gactttcccc tgacagctgt gt
#actcttag   1249
gctaagagat gtgactacag cctgcccctg actgtgttgt cccagggctg at
#gctgtaca   1309
ggtacaggct ggagattttc acataggtta gattctcatt cacgggacta gt
#tagcttta   1369
agcaccctag aggactaggg taatctgact tctcacttcc taagttccct tc
#tatatcct   1429
caaggtagaa atgtctatgt tttctactcc aattcataaa tctattcata ag
#tctttggt   1489
acaagtttac atgataaaaa gaaatgtgat ttgtcttccc ttctttgcac tt
#ttgaaata   1549
aagtatttat ctcctgtcta cagtttaata aatagcatct agtacacatt ca
#aaaaaaaa   1609
aaaaaaaaaa
#
#
#      1619
<210> SEQ ID NO 2
<211> LENGTH: 329
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2
Met Trp Gly Leu Lys Val Leu Leu Leu Pro Va
#l Val Ser Phe Ala Leu
-115               -110
#               -105
#              -100
Tyr Pro Glu Glu Ile Leu Asp Thr His Trp Gl
#u Leu Trp Lys Lys Thr
-95
#               -90
#               -85
His Arg Lys Gln Tyr Asn Asn Lys Val Asp Gl
#u Ile Ser Arg Arg Leu
-80
#           -75
#           -70
Ile Trp Glu Lys Asn Leu Lys Tyr Ile Ser Il
#e His Asn Leu Glu Ala
-65
#       -60
#       -55
Ser Leu Gly Val His Thr Tyr Glu Leu Ala Me
#t Asn His Leu Gly Asp
-50
#   -45
#   -40
Met Thr Ser Glu Glu Val Val Gln Lys Met Th
#r Gly Leu Lys Val Pro
-35                 -
#30                 -
#25                 -
#20
Leu Ser His Ser Arg Ser Asn Asp Thr Leu Ty
#r Ile Pro Glu Trp Glu
-15
#               -10
#                -5
Gly Arg Ala Pro Asp Ser Val Asp Tyr Arg Ly
#s Lys Gly Tyr Val Thr
-1    1
#         5
#         10
Pro Val Lys Asn Gln Gly Gln Cys Gly Ser Cy
#s Trp Ala Phe Ser Ser
15
#     20
#     25
Val Gly Ala Leu Glu Gly Gln Leu Lys Lys Ly
#s Thr Gly Lys Leu Leu
30
# 35
# 40
# 45
Asn Leu Ser Pro Gln Asn Leu Val Asp Cys Va
#l Ser Glu Asn Asp Gly
50
#                 55
#                 60
Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gl
#n Tyr Val Gln Lys Asn
65
#             70
#             75
Arg Gly Ile Asp Ser Glu Asp Ala Tyr Pro Ty
#r Val Gly Gln Glu Glu
80
#         85
#         90
Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala Al
#a Lys Cys Arg Gly Tyr
95
#    100
#    105
Arg Glu Ile Pro Glu Gly Asn Glu Lys Ala Le
#u Lys Arg Ala Val Ala
110                 1
#15                 1
#20                 1
#25
Arg Val Gly Pro Val Ser Val Ala Ile Asp Al
#a Ser Leu Thr Ser Phe
130
#               135
#               140
Gln Phe Tyr Ser Lys Gly Val Tyr Tyr Asp Gl
#u Ser Cys Asn Ser Asp
145
#           150
#           155
Asn Leu Asn His Ala Val Leu Ala Val Gly Ty
#r Gly Ile Gln Lys Gly
160
#       165
#       170
Asn Lys His Trp Ile Ile Lys Asn Ser Trp Gl
#y Glu Asn Trp Gly Asn
175
#   180
#   185
Lys Gly Tyr Ile Leu Met Ala Arg Asn Lys As
#n Asn Ala Cys Gly Ile
190                 1
#95                 2
#00                 2
#05
Ala Asn Leu Ala Ser Phe Pro Lys Met
210
<210> SEQ ID NO 3
<211> LENGTH: 26
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 3
gctaaggatc ctgggggctc aaggtt
#
#              26
<210> SEQ ID NO 4
<211> LENGTH: 26
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 4
gctaatctag atcacatctt ggggaa
#
#              26
<210> SEQ ID NO 5
<211> LENGTH: 14
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 5
Ala Ile Asp Ala Ser Leu Thr Ser Phe Gln Ph
#e Tyr Ser Lys
1               5
#                 10
<210> SEQ ID NO 6
<211> LENGTH: 11
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6
Tyr Asp Glu Ser Cys Asn Ser Asp Asn Leu As
#n
1               5
#                 10
<210> SEQ ID NO 7
<211> LENGTH: 329
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 7
Met Trp Gly Leu Lys Val Leu Leu Leu Pro Va
#l Val Ser Phe Ala Leu
1               5
#                 10
#                 15
His Pro Glu Glu Ile Leu Asp Thr Gln Trp Gl
#u Leu Trp Lys Lys Thr
20
#             25
#             30
Tyr Ser Lys Gln Tyr Asn Ser Lys Val Asp Gl
#u Ile Ser Arg Arg Leu
35
#         40
#         45
Ile Trp Glu Lys Asn Leu Lys His Ile Ser Il
#e His Asn Leu Glu Ala
50
#     55
#     60
Ser Leu Gly Val His Thr Tyr Glu Leu Ala Me
#t Asn His Leu Gly Asp
65
# 70
# 75
# 80
Met Thr Ser Glu Glu Val Val Gln Lys Met Th
#r Gly Leu Lys Val Pro
85
#                 90
#                 95
Pro Ser Arg Ser His Ser Asn Asp Thr Leu Ty
#r Ile Pro Asp Trp Glu
100
#           105
#           110
Gly Arg Thr Pro Asp Ser Ile Asp Tyr Arg Ly
#s Lys Gly Tyr Val Thr
115
#       120
#       125
Pro Val Lys Asn Gln Gly Gln Cys Gly Ser Cy
#s Trp Ala Phe Ser Ser
130
#   135
#   140
Val Gly Ala Leu Glu Gly Gln Leu Lys Lys Ly
#s Thr Gly Lys Leu Leu
145                 1
#50                 1
#55                 1
#60
Asn Leu Ser Pro Gln Asn Leu Val Asp Cys Va
#l Ser Glu Asn Tyr Gly
165
#               170
#               175
Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gl
#n Tyr Val Gln Arg Asn
180
#           185
#           190
Arg Gly Ile Asp Ser Glu Asp Ala Tyr Pro Ty
#r Val Gly Gln Asp Glu
195
#       200
#       205
Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala Al
#a Lys Cys Arg Gly Tyr
210
#   215
#   220
Arg Glu Ile Pro Glu Gly Asn Glu Lys Ala Le
#u Lys Arg Ala Val Ala
225                 2
#30                 2
#35                 2
#40
Arg Val Gly Pro Val Ser Val Ala Ile Asp Al
#a Ser Leu Thr Ser Phe
245
#               250
#               255
Gln Phe Tyr Ser Lys Gly Val Tyr Tyr Asp Gl
#u Asn Cys Ser Ser Asp
260
#           265
#           270
Asn Val Asn His Ala Val Leu Ala Val Gly Ty
#r Gly Ile Gln Lys Gly
275
#       280
#       285
Asn Lys His Trp Ile Ile Lys Asn Ser Trp Gl
#y Glu Ser Trp Gly Asn
290
#   295
#   300
Lys Gly Tyr Ile Leu Met Ala Arg Asn Lys As
#n Asn Ala Cys Gly Ile
305                 3
#10                 3
#15                 3
#20
Ala Asn Leu Ala Ser Phe Pro Lys Met
325
<210> SEQ ID NO 8
<211> LENGTH: 331
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 8
Met Lys Arg Leu Val Cys Val Leu Leu Val Cy
#s Ser Ser Ala Val Ala
1               5
#                 10
#                 15
Gln Leu His Lys Asp Pro Thr Leu Asp His Hi
#s Trp His Leu Trp Lys
20
#             25
#             30
Lys Thr Tyr Gly Lys Gln Tyr Lys Glu Lys As
#n Glu Glu Ala Val Arg
35
#         40
#         45
Arg Leu Ile Trp Glu Lys Asn Leu Lys Phe Va
#l Met Leu His Asn Leu
50
#     55
#     60
Glu His Ser Met Gly Met His Ser Tyr Asp Le
#u Gly Met Asn His Leu
65
# 70
# 75
# 80
Gly Asp Met Thr Ser Glu Glu Val Met Ser Le
#u Thr Ser Ser Leu Arg
85
#                 90
#                 95
Val Pro Ser Gln Trp Gln Arg Asn Ile Thr Ty
#r Lys Ser Asn Pro Asn
100
#           105
#           110
Arg Ile Leu Pro Asp Ser Val Asp Trp Arg Gl
#u Lys Gly Cys Val Thr
115
#       120
#       125
Glu Val Lys Tyr Gln Gly Ser Cys Gly Ala Cy
#s Trp Ala Phe Ser Ala
130
#   135
#   140
Val Gly Ala Leu Glu Ala Gln Leu Lys Leu Ly
#s Thr Gly Lys Leu Val
145                 1
#50                 1
#55                 1
#60
Thr Leu Ser Ala Gln Asn Leu Val Asp Cys Se
#r Thr Glu Lys Tyr Gly
165
#               170
#               175
Asn Lys Gly Cys Asn Gly Gly Phe Met Thr Th
#r Ala Phe Gln Tyr Ile
180
#           185
#           190
Ile Asp Asn Lys Gly Ile Asp Ser Asp Ala Se
#r Tyr Pro Tyr Lys Ala
195
#       200
#       205
Met Asp Gln Lys Cys Gln Tyr Asp Ser Lys Ty
#r Arg Ala Ala Thr Cys
210
#   215
#   220
Ser Lys Tyr Thr Glu Leu Pro Tyr Gly Arg Gl
#u Asp Val Leu Lys Glu
225                 2
#30                 2
#35                 2
#40
Ala Val Ala Asn Lys Gly Pro Val Ser Val Gl
#y Val Asp Ala Arg His
245
#               250
#               255
Pro Ser Phe Phe Leu Tyr Arg Ser Gly Val Ty
#r Tyr Glu Pro Ser Cys
260
#           265
#           270
Thr Gln Asn Val Asn His Gly Val Leu Val Va
#l Gly Tyr Gly Asp Leu
275
#       280
#       285
Asn Gly Lys Glu Tyr Trp Leu Val Lys Asn Se
#r Trp Gly His Asn Phe
290
#   295
#   300
Gly Glu Glu Gly Tyr Ile Arg Met Ala Arg As
#n Lys Gly Asn His Cys
305                 3
#10                 3
#15                 3
#20
Gly Ile Ala Ser Phe Pro Ser Tyr Pro Glu Il
#e
325
#               330
<210> SEQ ID NO 9
<211> LENGTH: 333
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 9
Met Asn Pro Thr Leu Ile Leu Ala Ala Phe Cy
#s Leu Gly Ile Ala Ser
1               5
#                 10
#                 15
Ala Thr Leu Thr Phe Asp His Ser Leu Glu Al
#a Gln Trp Thr Lys Trp
20
#             25
#             30
Lys Ala Met His Asn Arg Leu Tyr Gly Met As
#n Glu Glu Gly Trp Arg
35
#         40
#         45
Arg Ala Val Trp Glu Lys Asn Met Lys Met Il
#e Glu Leu His Asn Gln
50
#     55
#     60
Glu Tyr Arg Glu Gly Lys His Ser Phe Thr Me
#t Ala Met Asn Ala Phe
65
# 70
# 75
# 80
Gly Asp Met Thr Ser Glu Glu Phe Arg Gln Va
#l Met Asn Gly Phe Gln
85
#                 90
#                 95
Asn Arg Lys Pro Arg Lys Gly Lys Val Phe Gl
#n Glu Pro Leu Phe Tyr
100
#           105
#           110
Glu Ala Pro Arg Ser Val Asp Trp Arg Glu Ly
#s Gly Tyr Val Thr Pro
115
#       120
#       125
Val Lys Asn Gln Gly Gln Cys Gly Ser Cys Tr
#p Ala Phe Ser Ala Thr
130
#   135
#   140
Gly Ala Leu Glu Gly Gln Met Phe Arg Lys Th
#r Gly Arg Leu Ile Ser
145                 1
#50                 1
#55                 1
#60
Leu Ser Glu Gln Asn Leu Val Asp Cys Ser Gl
#y Pro Gln Gly Asn Glu
165
#               170
#               175
Gly Cys Asn Gly Gly Leu Met Asp Tyr Ala Ph
#e Gln Tyr Val Gln Asp
180
#           185
#           190
Asn Gly Gly Leu Asp Ser Glu Glu Ser Tyr Pr
#o Tyr Glu Ala Thr Glu
195
#       200
#       205
Glu Ser Cys Lys Tyr Asn Pro Lys Tyr Ser Va
#l Ala Asn Asp Thr Gly
210
#   215
#   220
Phe Val Asp Ile Pro Lys Gln Glu Lys Ala Le
#u Met Lys Ala Val Ala
225                 2
#30                 2
#35                 2
#40
Thr Val Gly Pro Ile Ser Val Ala Ile Asp Al
#a Gly His Glu Ser Phe
245
#               250
#               255
Leu Phe Tyr Lys Glu Gly Ile Tyr Phe Glu Pr
#o Asp Cys Ser Ser Glu
260
#           265
#           270
Asp Met Asp His Gly Val Leu Val Val Gly Ty
#r Gly Phe Glu Ser Thr
275
#       280
#       285
Glu Ser Asp Asn Asn Lys Tyr Trp Leu Val Ly
#s Asn Ser Trp Gly Glu
290
#   295
#   300
Glu Trp Gly Met Gly Gly Tyr Val Lys Met Al
#a Lys Asp Arg Arg Asn
305                 3
#10                 3
#15                 3
#20
His Cys Gly Ile Ala Ser Ala Ala Ser Tyr Pr
#o Thr Val
325
#               330
<210> SEQ ID NO 10
<211> LENGTH: 335
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10
Met Trp Ala Thr Leu Pro Leu Leu Cys Ala Gl
#y Ala Trp Leu Leu Gly
1               5
#                 10
#                 15
Val Pro Val Cys Gly Ala Ala Glu Leu Ser Va
#l Asn Ser Leu Glu Lys
20
#             25
#             30
Phe His Phe Lys Ser Trp Met Ser Lys His Ar
#g Lys Thr Tyr Ser Thr
35
#         40
#         45
Glu Glu Tyr His His Arg Leu Gln Thr Phe Al
#a Ser Asn Trp Arg Lys
50
#     55
#     60
Ile Asn Ala His Asn Asn Gly Asn His Thr Ph
#e Lys Met Ala Leu Asn
65
# 70
# 75
# 80
Gln Phe Ser Asp Met Ser Phe Ala Glu Ile Ly
#s His Lys Tyr Leu Trp
85
#                 90
#                 95
Ser Glu Pro Gln Asn Cys Ser Ala Thr Lys Se
#r Asn Tyr Leu Arg Gly
100
#           105
#           110
Thr Gly Pro Tyr Pro Pro Ser Val Asp Trp Ar
#g Lys Lys Gly Asn Phe
115
#       120
#       125
Val Ser Pro Val Lys Asn Gln Gly Ala Cys Gl
#y Ser Cys Trp Thr Phe
130
#   135
#   140
Ser Thr Thr Gly Ala Leu Glu Ser Ala Ile Al
#a Ile Ala Thr Gly Lys
145                 1
#50                 1
#55                 1
#60
Met Leu Ser Leu Ala Glu Gln Gln Leu Val As
#p Cys Ala Gln Asp Phe
165
#               170
#               175
Asn Asn Tyr Gly Cys Gln Gly Gly Leu Pro Se
#r Gln Ala Phe Glu Tyr
180
#           185
#           190
Ile Leu Tyr Asn Lys Gly Ile Met Gly Glu As
#p Thr Tyr Pro Tyr Gln
195
#       200
#       205
Gly Lys Asp Gly Tyr Cys Lys Phe Gln Pro Gl
#y Lys Ala Ile Gly Phe
210
#   215
#   220
Val Lys Asp Val Ala Asn Ile Thr Ile Tyr As
#p Glu Glu Ala Met Val
225                 2
#30                 2
#35                 2
#40
Glu Ala Val Ala Leu Tyr Asn Pro Val Ser Ph
#e Ala Phe Glu Val Thr
245
#               250
#               255
Gln Asp Phe Met Met Tyr Arg Thr Gly Ile Ty
#r Ser Ser Thr Ser Cys
260
#           265
#           270
His Lys Thr Pro Asp Lys Val Asn His Ala Va
#l Leu Ala Val Gly Tyr
275
#       280
#       285
Gly Glu Lys Asn Gly Ile Pro Tyr Trp Ile Va
#l Lys Asn Ser Trp Gly
290
#   295
#   300
Pro Gln Trp Gly Met Asn Gly Tyr Phe Leu Il
#e Glu Arg Gly Lys Asn
305                 3
#10                 3
#15                 3
#20
Met Cys Gly Leu Ala Ala Cys Ala Ser Tyr Pr
#o Ile Pro Leu Val
325
#               330
#               335
<210> SEQ ID NO 11
<211> LENGTH: 339
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 11
Met Trp Gln Leu Trp Ala Ser Leu Cys Cys Le
#u Leu Val Leu Ala Asn
1               5
#                 10
#                 15
Ala Arg Ser Arg Pro Ser Phe His Pro Leu Se
#r Asp Glu Leu Val Asn
20
#             25
#             30
Tyr Val Asn Lys Arg Asn Thr Thr Trp Gln Al
#a Gly His Asn Phe Tyr
35
#         40
#         45
Asn Val Asp Met Ser Tyr Leu Lys Arg Leu Cy
#s Gly Thr Phe Leu Gly
50
#     55
#     60
Gly Pro Lys Pro Pro Gln Arg Val Met Phe Th
#r Glu Asp Leu Lys Leu
65
# 70
# 75
# 80
Pro Ala Ser Phe Asp Ala Arg Glu Gln Trp Pr
#o Gln Cys Pro Thr Ile
85
#                 90
#                 95
Lys Glu Ile Arg Asp Gln Gly Ser Cys Gly Se
#r Cys Trp Ala Phe Gly
100
#           105
#           110
Ala Val Glu Ala Ile Ser Asp Arg Ile Cys Il
#e His Thr Asn Ala His
115
#       120
#       125
Val Ser Val Glu Val Ser Ala Glu Asp Leu Le
#u Thr Cys Cys Gly Ser
130
#   135
#   140
Met Cys Gly Asp Gly Cys Asn Gly Gly Tyr Pr
#o Ala Glu Ala Trp Asn
145                 1
#50                 1
#55                 1
#60
Phe Trp Thr Arg Lys Gly Leu Val Ser Gly Gl
#y Leu Tyr Glu Ser His
165
#               170
#               175
Val Gly Cys Arg Pro Tyr Ser Ile Pro Pro Cy
#s Glu His His Val Asn
180
#           185
#           190
Gly Ser Arg Pro Pro Cys Thr Gly Glu Gly As
#p Thr Pro Lys Cys Ser
195
#       200
#       205
Lys Ile Cys Glu Pro Gly Tyr Ser Pro Thr Ty
#r Lys Gln Asp Lys His
210
#   215
#   220
Tyr Gly Tyr Asn Ser Tyr Ser Val Ser Asn Se
#r Glu Lys Asp Ile Met
225                 2
#30                 2
#35                 2
#40
Ala Glu Ile Tyr Lys Asn Gly Pro Val Glu Gl
#y Ala Phe Ser Val Tyr
245
#               250
#               255
Ser Asp Phe Leu Leu Tyr Lys Ser Gly Val Ty
#r Gln His Val Thr Gly
260
#           265
#           270
Glu Met Met Gly Gly His Ala Ile Arg Ile Le
#u Gly Trp Gly Val Glu
275
#       280
#       285
Asn Gly Thr Pro Tyr Trp Leu Val Ala Asn Se
#r Trp Asn Thr Asp Trp
290
#   295
#   300
Gly Asp Asn Gly Phe Phe Lys Ile Leu Arg Gl
#y Gln Asp His Cys Gly
305                 3
#10                 3
#15                 3
#20
Ile Glu Ser Glu Val Val Ala Gly Ile Pro Ar
#g Thr Asp Gln Tyr Trp
325
#               330
#               335
Glu Lys Ile
<210> SEQ ID NO 12
<211> LENGTH: 412
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12
Met Gln Pro Ser Ser Leu Leu Pro Leu Ala Le
#u Cys Leu Leu Ala Ala
1               5
#                 10
#                 15
Pro Ala Ser Ala Leu Val Arg Ile Pro Leu Hi
#s Lys Phe Thr Ser Ile
20
#             25
#             30
Arg Arg Thr Met Ser Glu Val Gly Gly Ser Va
#l Glu Asp Leu Ile Ala
35
#         40
#         45
Lys Gly Pro Val Ser Lys Tyr Ser Gln Ala Va
#l Pro Ala Val Thr Glu
50
#     55
#     60
Gly Pro Ile Pro Glu Val Leu Lys Asn Tyr Me
#t Asp Ala Gln Tyr Tyr
65
# 70
# 75
# 80
Gly Glu Ile Gly Ile Gly Thr Pro Pro Gln Cy
#s Phe Thr Val Val Phe
85
#                 90
#                 95
Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Se
#r Ile His Cys Lys Leu
100
#           105
#           110
Leu Asp Ile Ala Cys Trp Ile His His Lys Ty
#r Asn Ser Asp Lys Ser
115
#       120
#       125
Ser Thr Tyr Val Lys Asn Gly Thr Ser Phe As
#p Ile His Tyr Gly Ser
130
#   135
#   140
Gly Ser Leu Ser Gly Tyr Leu Ser Gln Asp Th
#r Val Ser Val Pro Cys
145                 1
#50                 1
#55                 1
#60
Gln Ser Ala Ser Ser Ala Ser Ala Leu Gly Gl
#y Val Lys Val Glu Arg
165
#               170
#               175
Gln Val Phe Gly Glu Ala Thr Lys Gln Pro Gl
#y Ile Thr Phe Ile Ala
180
#           185
#           190
Ala Lys Phe Asp Gly Ile Leu Gly Met Ala Ty
#r Pro Arg Ile Ser Val
195
#       200
#       205
Asn Asn Val Leu Pro Val Phe Asp Asn Leu Me
#t Gln Gln Lys Leu Val
210
#   215
#   220
Asp Gln Asn Ile Phe Ser Phe Tyr Leu Ser Ar
#g Asp Pro Asp Ala Gln
225                 2
#30                 2
#35                 2
#40
Pro Gly Gly Glu Leu Met Leu Gly Gly Thr As
#p Ser Lys Tyr Tyr Lys
245
#               250
#               255
Gly Ser Leu Ser Tyr Leu Asn Val Thr Arg Ly
#s Ala Tyr Trp Gln Val
260
#           265
#           270
His Leu Asp Gln Val Glu Val Ala Ser Gly Le
#u Thr Leu Cys Lys Glu
275
#       280
#       285
Gly Cys Glu Ala Ile Val Asp Thr Gly Thr Se
#r Leu Met Val Gly Pro
290
#   295
#   300
Val Asp Glu Val Arg Glu Leu Gln Lys Ala Il
#e Gly Ala Val Pro Leu
305                 3
#10                 3
#15                 3
#20
Ile Gln Gly Glu Tyr Met Ile Pro Cys Glu Ly
#s Val Ser Thr Leu Pro
325
#               330
#               335
Ala Ile Thr Leu Lys Leu Gly Gly Lys Gly Ty
#r Lys Leu Ser Pro Glu
340
#           345
#           350
Asp Tyr Thr Leu Lys Val Ser Gln Ala Gly Ly
#s Thr Leu Cys Leu Ser
355
#       360
#       365
Gly Phe Met Gly Met Asp Ile Pro Pro Pro Se
#r Gly Pro Leu Trp Ile
370
#   375
#   380
Leu Gly Asp Val Phe Ile Gly Arg Tyr Tyr Th
#r Val Phe Asp Arg Asp
385                 3
#90                 3
#95                 4
#00
Asn Asn Arg Val Gly Phe Ala Glu Ala Ala Ar
#g Leu
405
#               410
<210> SEQ ID NO 13
<211> LENGTH: 396
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 13
Met Lys Thr Leu Leu Leu Leu Leu Leu Val Le
#u Leu Glu Leu Gly Glu
1               5
#                 10
#                 15
Ala Gln Gly Ser Leu His Arg Val Pro Leu Ar
#g Arg His Pro Ser Leu
20
#             25
#             30
Lys Lys Lys Leu Arg Ala Arg Ser Gln Leu Se
#r Glu Phe Trp Lys Ser
35
#         40
#         45
His Asn Leu Asp Met Ile Gln Phe Thr Glu Se
#r Cys Ser Met Asp Gln
50
#     55
#     60
Ser Ala Lys Glu Pro Leu Ile Asn Tyr Leu As
#p Met Glu Tyr Phe Gly
65
# 70
# 75
# 80
Thr Ile Ser Ile Gly Ser Pro Pro Gln Asn Ph
#e Thr Val Ile Phe Asp
85
#                 90
#                 95
Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Va
#l Tyr Cys Thr Ser Pro
100
#           105
#           110
Ala Cys Lys Thr His Ser Arg Phe Gln Pro Se
#r Gln Ser Ser Thr Tyr
115
#       120
#       125
Ser Gln Pro Gly Gln Ser Phe Ser Ile Gln Ty
#r Gly Thr Gly Ser Leu
130
#   135
#   140
Ser Gly Ile Ile Gly Ala Asp Gln Val Ser Va
#l Glu Gly Leu Thr Val
145                 1
#50                 1
#55                 1
#60
Val Gly Gln Gln Phe Gly Glu Ser Val Thr Gl
#u Pro Gly Gln Thr Phe
165
#               170
#               175
Val Asp Ala Glu Phe Asp Gly Ile Leu Gly Le
#u Gly Tyr Pro Ser Leu
180
#           185
#           190
Ala Val Gly Gly Val Thr Pro Val Phe Asp As
#n Met Met Ala Gln Asn
195
#       200
#       205
Leu Val Asp Leu Pro Met Phe Ser Val Tyr Me
#t Ser Ser Asn Pro Glu
210
#   215
#   220
Gly Gly Ala Gly Ser Glu Leu Ile Phe Gly Gl
#y Tyr Asp His Ser His
225                 2
#30                 2
#35                 2
#40
Phe Ser Gly Ser Leu Asn Trp Val Pro Val Th
#r Lys Gln Ala Tyr Trp
245
#               250
#               255
Gln Ile Ala Leu Asp Asn Ile Gln Val Gly Gl
#y Thr Val Met Phe Cys
260
#           265
#           270
Ser Glu Gly Cys Gln Ala Ile Val Asp Thr Gl
#y Thr Ser Leu Ile Thr
275
#       280
#       285
Gly Pro Ser Asp Lys Ile Lys Gln Leu Gln As
#n Ala Ile Gly Ala Ala
290
#   295
#   300
Pro Val Asp Gly Glu Tyr Ala Val Glu Cys Al
#a Asn Leu Asn Val Met
305                 3
#10                 3
#15                 3
#20
Pro Asp Val Thr Phe Thr Ile Asn Gly Val Pr
#o Tyr Thr Leu Ser Pro
325
#               330
#               335
Thr Ala Tyr Thr Leu Leu Asp Phe Val Asp Gl
#y Met Gln Phe Cys Ser
340
#           345
#           350
Ser Gly Phe Gln Gly Leu Asp Ile His Pro Pr
#o Ala Gly Pro Leu Trp
355
#       360
#       365
Ile Leu Gly Asp Val Phe Ile Arg Gln Phe Ty
#r Ser Val Phe Asp Arg
370
#   375
#   380
Gly Asn Asn Arg Val Gly Leu Ala Pro Ala Va
#l Pro
385                 3
#90                 3
#95
<210> SEQ ID NO 14
<211> LENGTH: 255
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 14
Met Gln Pro Leu Leu Leu Leu Leu Ala Phe Le
#u Leu Pro Thr Gly Ala
1               5
#                 10
#                 15
Glu Ala Gly Glu Ile Ile Gly Gly Arg Glu Se
#r Arg Pro His Ser Arg
20
#             25
#             30
Pro Tyr Met Ala Tyr Leu Gln Ile Gln Ser Pr
#o Ala Gly Gln Ser Arg
35
#         40
#         45
Cys Gly Gly Phe Leu Val Arg Glu Asp Phe Va
#l Leu Thr Ala Ala His
50
#     55
#     60
Cys Trp Gly Ser Asn Ile Asn Val Thr Leu Gl
#y Ala His Asn Ile Gln
65
# 70
# 75
# 80
Arg Arg Glu Asn Thr Gln Gln His Ile Thr Al
#a Arg Arg Ala Ile Arg
85
#                 90
#                 95
His Pro Gln Tyr Asn Gln Arg Thr Ile Gln As
#n Asp Ile Met Leu Leu
100
#           105
#           110
Gln Leu Ser Arg Arg Val Arg Arg Asn Arg As
#n Val Asn Pro Val Ala
115
#       120
#       125
Leu Pro Arg Ala Gln Glu Gly Leu Arg Pro Gl
#y Thr Leu Cys Thr Val
130
#   135
#   140
Ala Gly Trp Gly Arg Val Ser Met Arg Arg Gl
#y Thr Asp Thr Leu Arg
145                 1
#50                 1
#55                 1
#60
Glu Val Gln Leu Arg Val Gln Arg Asp Arg Gl
#n Cys Leu Arg Ile Phe
165
#               170
#               175
Gly Ser Tyr Asp Pro Arg Arg Gln Ile Cys Va
#l Gly Asp Arg Arg Glu
180
#           185
#           190
Arg Lys Ala Ala Phe Lys Gly Asp Ser Gly Gl
#y Pro Leu Leu Cys Asn
195
#       200
#       205
Asn Val Ala His Gly Ile Val Ser Tyr Gly Ly
#s Ser Ser Gly Val Pro
210
#   215
#   220
Pro Glu Val Phe Thr Arg Val Ser Ser Phe Le
#u Pro Trp Ile Arg Thr
225                 2
#30                 2
#35                 2
#40
Thr Met Arg Ser Phe Lys Leu Leu Asp Gln Me
#t Glu Thr Pro Leu
245
#               250
#               255

Claims

1. An isolated antibody or fragment thereof that specifically binds to a protein selected from the group consisting of:(a) a protein consisting of amino acid residues −115 to 214 of SEQ ID NO:2; (b) a protein consisting of amino acid residues −100 to 214 of SEQ ID NO:2; (c) a protein consisting of amino acid residues 1 to 214 of SEQ ID NO:2; (d) a protein consisting of amino acid residues 133 to 146 of SEQ ID NO:2; and (e) a protein consisting of amino acid residues 150 to 160 of SEQ ID NO:2.

2. The antibody or fragment thereof of claim 1 that specifically binds protein (a).

3. The antibody or fragment thereof of claim 1 that specifically binds protein (b).

4. The antibody or fragment thereof of claim 1 that specifically binds protein (c).

5. The antibody or fragment thereof of claim 1 that specifically binds protein (d).

6. The antibody or fragment thereof of claim 1 that specifically binds protein (e).

7. The antibody or fragment thereof of claim 2 that specifically binds protein (b).

8. The antibody or fragment thereof of claim 4 wherein said protein bound by said antibody or fragment thereof is glycosylated.

9. The antibody or fragment thereof of claim 4 which is a human antibody.

10. The antibody or fragment thereof of claim 4 which is a polyclonal antibody.

11. The antibody or fragment thereof of claim 4 which is selected from the group consisting of:(a) a chimeric antibody; (b) a humanized antibody; (c) a single chain antibody; and (d) a Fab fragment.

12. The antibody or fragment thereof of claim 4 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot.

13. An isolated cell that produces the antibody or fragment thereof of claim 4.

14. A hybridoma that produces the antibody of claim 4.

15. An isolated antibody or fragment thereof wherein said antibody is obtained from an animal that has been immunized with a protein selected from the group consisting of:(a) a protein consisting of the amino acid sequence of amino acid residues −115 to 214 of SEQ ID NO:2; (b) a protein consisting of the amino acid sequence of amino acid residues −100 to 214 of SEQ ID NO:2; (c) a protein consisting of the amino acid sequence of amino acid residues 1 to 214 of SEQ ID NO:2; (d) a protein consisting of the amino acid sequence of amino acid residues 133 to 146 of SEQ ID NO:2; and (e) a protein consisting of the amino acid sequence of amino acid residues 150 to 160 of SEQ ID NO:2; wherein said antibody or fragment thereof specifically binds to said amino acid sequence.

16. The antibody or fragment thereof of claim 15 obtained from an animal immunized with protein (a).

17. The antibody or fragment thereof of claim 15 obtained from an animal immunized with protein (b).

18. The antibody or fragment thereof of claim 15 obtained from an animal immunized with protein (c).

19. The antibody or fragment thereof of claim 15 obtained from an animal immunized with protein (d).

20. The antibody or fragment thereof of claim 15 obtained from an animal immunized with protein (e).

21. The antibody or fragment thereof of claim 15 which is selected from the group consisting of:(a) a chimeric antibody; (b) a polyclonal antibody; (c) a humanized antibody; (d) a single chain antibody; and (e) a Fab fragment.

22. An isolated monoclonal antibody or fragment thereof that specifically binds to a protein selected from the group consisting of:(a) a protein consisting of amino acid residues −115 to 214 of SEQ ID NO:2; (b) a protein consisting of amino acid residues −100 to 214 of SEQ ID NO:2; (c) a protein consisting of amino acid residues 1 to 214 of SEQ ID NO:2; (d) a protein consisting of amino acid residues 133 to 146 of SEQ ID NO:2; and (e) a protein consisting of amino acid residues 150 to 160 of SEQ ID NO:2.

23. The antibody or fragment thereof of claim 22 that specifically binds protein (a).

24. The antibody or fragment thereof of claim 22 that specifically binds protein (b).

25. The antibody or fragment thereof of claim 22 that specifically binds protein (c).

26. The antibody or fragment thereof of claim 22 that specifically binds protein (d).

27. The antibody or fragment thereof of claim 22 that specifically binds protein (e).

28. The antibody or fragment thereof of claim 23 that specifically binds protein (b).

29. The antibody or fragment thereof of claim 25 wherein said protein bound by said antibody or fragment thereof is glycosylated.

30. The antibody or fragment thereof of claim 25 which is a human antibody.

31. The antibody or fragment thereof of claim 25 which is selected from the group consisting of:(a) a chimeric antibody; (b) a humanized antibody; (c) a single chain antibody; and (d) a Fab fragment.

32. The antibody or fragment thereof of claim 25 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot.

33. An isolated cell that produces the antibody or fragment thereof of claim 25.

34. A hybridoma that produces the antibody of claim 25.

35. An isolated antibody or fragment thereof that specifically binds to a protein selected from the group consisting of:(a) a protein consisting of the full length polypeptide encoded by the cDNA contained in ATCC Deposit Number 75671; (b) a protein consisting of the proprotein of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 75671; and (c) a protein consisting of the mature polypeptide encoded by the cDNA contained in ATCC Deposit Number 75671.

36. The antibody or fragment thereof of claim 35 that specifically binds protein (a).

37. The antibody or fragment thereof of claim 35 that specifically binds protein (b).

38. The antibody or fragment thereof of claim 35 that specifically binds protein (c).

39. The antibody or fragment thereof of claim 36 that specifically binds protein (b).

40. The antibody or fragment thereof of claim 38 wherein said protein bound by said antibody or fragment thereof is glycosylated.

41. The antibody or fragment thereof of claim 38 which is a human antibody.

42. The antibody or fragment thereof of claim 38 which is a polyclonal antibody.

43. The antibody or fragment thereof of claim 38 which is selected from the group consisting of:(a) a chimeric antibody; (b) a humanized antibody; (c) a single chain antibody; and (d) a Fab fragment.

44. The antibody or fragment thereof of claim 38 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot.

45. An isolated cell that produces the antibody or fragment thereof of claim 38.

46. A hybridoma that produces the antibody of claim 38.

47. An isolated antibody or fragment thereof wherein said antibody is obtained from an animal that has been immunized with a protein selected from the group consisting of:(a) a protein comprising the amino acid sequence of the full length polypeptide encoded by the cDNA contained in ATCC Deposit Number 75671; (b) a protein comprising the amino acid sequence of the proprotein of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 75671; and (c) a protein comprising the amino acid sequence of the mature polypeptide encoded by the cDNA contained in ATCC Deposit Number 75671; wherein said antibody or fragment thereof specifically binds to said amino acid sequence.

48. The antibody or fragment thereof of claim 47 obtained from an animal immunized with protein (a).

49. The antibody or fragment thereof of claim 47 obtained from an animal immunized with protein (b).

50. The antibody or fragment thereof of claim 47 obtained from an animal immunized with protein (c).

51. The antibody or fragment thereof of claim 47 which is selected from the group consisting of:(a) a chimeric antibody; (b) a polyclonal antibody; (c) a humanized antibody; (d) a single chain antibody; and (e) a Fab fragment.

52. An isolated monoclonal antibody or fragment thereof that specifically binds to a protein selected from the group consisting of:(a) a protein consisting of the full length polypeptide encoded by the cDNA contained in ATCC Deposit Number 75671; (b) a protein consisting of the proprotein of the polypeptide encoded by the cDNA contained in ATCC Deposit Number 75671; and (c) a protein consisting of the mature polypeptide encoded by the cDNA contained in ATCC Deposit Number 75671.

53. The antibody or fragment thereof of claim 52 that specifically binds protein (a).

54. The antibody or fragment thereof of claim 52 that specifically binds protein (b).

55. The antibody or fragment thereof of claim 52 that specifically binds protein (c).

56. The antibody or fragment thereof of claim 53 that specifically binds protein (b).

57. The antibody or fragment thereof of claim 55 wherein said protein bound by said antibody or fragment thereof is glycosylated.

58. The antibody or fragment thereof of claim 55 which is a human antibody.

59. The antibody or fragment thereof of claim 55 which is selected from the group consisting of:(a) a chimeric antibody; (b) a humanized antibody; (c) a single chain antibody; and (d) a Fab fragment.

60. The antibody or fragment thereof of claim 55 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot.

61. An isolated cell that produces the antibody or fragment thereof of claim 55.

62. A hybridoma that produces the antibody of claim 55.

63. An isolated antibody or fragment thereof that specifically binds a protein purified from a cell culture wherein said protein is expressed from a polynucleotide encoding amino acids 1 to 214 of SEQ ID NO:2 operably associated with a regulatory sequence that controls gene expression.

64. The antibody or fragment thereof of claim 63 which is a monoclonal antibody.

65. The antibody or fragment thereof of claim 63 which is a human antibody.

66. The antibody or fragment thereof of claim 63 which is selected from the group consisting of:(a) a chimeric antibody; (b) a polyclonal antibody; (c) a humanized antibody; (d) a single chain antibody; and (e) a Fab fragment.

67. The antibody or fragment thereof of claim 63 wherein said antibody or fragment thereof specifically binds to said protein in a Western blot.