Human cardiac/brain tolloid-like protein

This application is a division of U.S. application Ser. No. 08/991,408, filed Dec. 16, 1997, now U.S. Pat. No. 6,008,017 which claims the benefit of U.S. Provisional Application No. 60/034,471, filed Jan. 2, 1997, both of whose contents are incorporated herein by reference in their entireties.

This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the astacin protein family, hereinafter referred to as human cardiac/brain tolloid-like protein (hC/BTLP). The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

The hC/BTLP gene appears to possess all of the important protein domains present in the bone morphogenetic protein (BMP)-1/procollagen C-proteinase (PCP) protein. Members of the astacin family of metalloproteinases, such as BMP-1, have previously been linked to cell differentiation and pattern formation during development through a proposed role in the activation of latent growth factors of the TGF-β superfamily. In addition, recent findings indicate that BMP-1 is identical to PCP, which is a metalloproteinase involved in the synthesis of matrix collagen. This observation suggests that a functional link may exist between astacin metalloproteinases, growth factors and cell differentiation and pattern formation during development, as well as fibrotic processes characterized by the accumulation of matrix collagen.

Nucleotide and amino acid sequence homologues suggest that hC/BTLP, like BMP-1, possesses PCP activity. PCP activity is one of the essential enzymatic steps required for the extracellular production of insoluble collagen fibrils from soluble procollagen. However, mouse mammalian tolloid-like protein is the most closely related homologues of hC/BTlP. Mouse mammalian tolloid-like protein and BMP-1 are distinct gene products with differential tissue distribution. Based on cross-species comparisons, the regulation and distribution of hC/BTlP would be expected to be distinct from BMP-1. Indeed, mouse mammalian tolloid-like protein exhibits a unique tissue distribution when compared to BMP-1. Thus, the selective inhibition of matrix collagen accumulation is important in highly localized fibrotic disorders, e.g., gliosis associated with neurotrauma and ventricular fibrosis associated with congestive heart failure. This indicates that the astacin protein family has an established, proven history as therapeutic targets.

Clearly there is a need for identification and characterization of further members of the astacin protein family which can play a role in preventing, ameliorating or con g dysfunctions or diseases, including, but not limited to, restenosis, atherosclerosis, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hypertrophy (BPH), nephritis, fibrosis, glomeruloneplritis, gliosis, cirrhosis and anomalies of wound healing, such as keloids, among others.

In one aspect, the invention relates to hC/BTLP polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such hC/BTLP polypeptides and polynucleotides. Such uses include the treatment of restenosis, atherosclerosis, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hypertrophy (BPH), nephritis, fibrosis, glomenulonephritis, gliosis, cirrhosis and anomalies of wound healing, such as keloids, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with hC/BTLP imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate hC/BTLP activity or levels.

The following definitions are provided to facilitate understanding of certain terms used frequently herein.

“HC/BTLP” refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or an allelic variant thereof.

“HC/BTLP activity or hC/BTLP polypeptide activity” or “biological activity of the hC/BTLP or hC/BTLP polypeptide” refers to the metabolic or physiologic function of said hC/BTLP including similar activities or improved activities or these activities with decreased undesirable sideffects. Also included are antigenic and immunogenic activities of said hC/BTLP.

“HC/BTLP gene” refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:1 or allelic variants thereof and/or their complements.

“Antibodies” as used he rein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

“Isolated” means altered “by the hand of man” from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RENA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. “Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. “Polypeptides” include amino acid sequences modified either by natural processes, such as post translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-lining, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Post translational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POST TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al, “Analysis for protein modifications and nonprotein cofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “Protein Synthesis: Post translational Modifications and Aging”, Ann NY Acad Sci (1992) 663:48-62.

“Variant” as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

“Identity” is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques. See, e.g. (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATION AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER. Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al, Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J Molec Biol (1990) 215:403).

As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% “identity” to a reference nucleotide sequence of SEQ ID NO: 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% “identity” to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

Polypeptides of the Invention

In one aspect, the present invention relates to hC/BTLP polypeptides (or hC/BTLP proteins). The hC/BTLP polypeptides include the polypeptide of SEQ ID NOS:2 and 4; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO:2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly preferred. Also included within hC/BTLP polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO.2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO:2. Furthermore, those with at least 97-99% are highly preferred. Preferably hC/BTLP polypeptide exhibit at least one biological activity of hC/BTLP.

The hC/BTLP polypeptides may be in the form of the “mature” protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

Fragments of the hC/BTLP polypeptides are also included in the invention. A fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned hC/BTLP polypeptides. As with hC/BTLP polypeptides, fragments may be “free-standing,” or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid numbers 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of hC/BTLP polypeptide. In this context “about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of hC/BTLP polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and tum-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, suit-forming regions, substrate binding region, and high antigenic index regions. Other preferred fragments are biologically active fragments. Biologically active fragents are those that mediate hC/BTLP activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.

Preferably, all of these polypeptide fragments retain the biological activity of the hC/BTLP, including antigenic activity. Among the most preferred frEment is that having the amino acid sequence of SEQ ID NO: 4. Variants of the defined sequence and fragments also form part of the present invention. Preferred variants are those that vary from the referents by conservative amino acid substitutions—i.e., those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or arornatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.

The hC/BTLP polypeptides of the invention can be prepared in any suitable marmer. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

Polynucleotides of the Invention

Another aspect of the invention reltes to hC/BTLP polynucleotides. hC/BTLP polynucleotides include isolated polynucleotides which encode the hC/BTLP polypeptides and fragments, and polynucleotides closely related thereto. More specifically, hC/BTLP polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a hC/BTLP polypeptide of SEQ ID NO: 2, and polynucleotides having the particular sequences of SEQ ID NOS: 1 and 3. hC/BTLP polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the hC/BTLP polypeptide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to that of SEQ ID NO: 1 over its entire length. In this regard, polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred. Futhermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred with at least 99% being the most preferred. Also included under hC/BTLP polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID No: 1 to hybridize under conditions useable for amplification or for use as a probe or marker. The invention also provides polynucleotides which are complementary to such hC/BTLP polynucleotides.

HC/BTLP of the invention is structurally related to other proteins of the astacin protein family, by the results of sequencing the cDNA encoding hC/BTLP. The cDNA sequence of SEQ ID No:1 contains an open reading fme (nucleotide number 252 to 3293) encoding a polypeptide of 1013 amino acids of SEQ ID NO:2. The amino acid sequence of Table 2 (SEQ ID NO:2) has about 93.4% indentity (using BlastP) in 945 of 1012 amino acid residues with mus musculus (mouse) mammalian tolloid-like protein. GenBank Accession #U34042. The nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 88.4% identity (using BlastN) in 2731 of 3089 nuclecoide residues with mus musculus mammalian tolloid-like protein. GenBank Accession #U34042. Thus, hC/BTLP polypeptides and polynucleotides of the present inventon are expect to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their uilit is obvious to anyone skilled in the art.

One polynucleotide of the present invention encoding hC/BTLP may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in cells of human 8 week old human embryo using the expressed sequence tag (EST) analysis (Adams, M. D., et al. Science (1991) 252:1651-1656; Adams, M. D. et al., Nature , (1992) 355:632-634; Adams, M. D., et al., Nature (1995) 377 Supp:3-174). Polvnucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.

The nucleotide sequence encoding hC/BTLP polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 252 to 3293 of SEQ ID NO: 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.

When the polynucleotides of the invention are used for the recombinant production of hC/BTLP polypeptide, the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself, the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which filitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz etal., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5′ and 3′ sequences, such as transcribed, non-translated sequences, splicing and polvadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding hC/BTLP variants comprising the amino acid sequence of hC/BTLP polypeptide of Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination. Among the preferred polynucleotides of the present invention is contained in Table 3 (SEQ ID NO: 3) encoding the amino acid sequence of Table 4 (SEQ ID NO: 4).

The present invention further relates to polvnucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term “stringent conditions” means hvbridization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, vet even more preferably 97-99% identity between the sequences.

Polynucleotides of the invention, wvhich are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof (including that of SEQ ID NO:3), may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding hC/BTLP polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) taat have a high sequence similarity to the hC/BTLP gene. Such hybridization techniques are known to those of skill in the art. Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the rerent. The probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range betnveen 30 and 50 nucleotides.

In one embodiment, to obtain a polynucleotide encoding hC/BTLP polypeptide, including homologs and orthologs from species other than human, comprises the steps of screening an appropriate library under stingent hybridization conditions with a labeled probe having the SEQ ID NO: 1 or a fragment thereof (including that of SEQ ID NO: 3), and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridiztion techniques are well known to those of skill in the art. Thus in another aspect, hC/BTLP polynucleotides of the present invention further include a nucleotide sequence comprising a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having SEQ ID NO: 1 or a fragwent thereof (including that of SEQ ID NO:3). Also included with hC/BTLP polypeptides are pol peptide comprising amino acid sequence encoded by nucleotide sequence obtained by the above hybridization condition. Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 microgramrnil denatured, sheared salmon sperm DNA, followed by washing the filters in 0×SSC at about 65° C.

The polynucleotides and polymeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise a polynucleotde or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polvnucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL , 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate trnsfection DEAE-dextran mediated tfansection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli , Streptomyces and Bacillus subtilis cells; fingal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great variety of expression svstems can be used. Such systems include, among others, chromosomal, episomal and virus-derived svstns, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from veast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any systern or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

If the hC/BTLP polypeptide is to be expressed for use in screening assays, generally, it is preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If hC/BTLP polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered. hC/BTLP polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including armoniun sum late or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinit chromatography, hydroxylapatite chromatophy and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of hC/BTLP polynucleotides for use as diagnostic reagents. Detection of a mutated form of hC/BTLP gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, overexpression or altered expression of hC/BTLP. Individuals carrying mutations in the hC/BTLP gene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the nomwal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled hC/BTLP nucleotide sequences. Perfctly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denug agents, or by direct DNA sequencing. See, e.g., Myers etal., Science (1985)230:1242. Sequence changes at specific locations mayalso be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method. See Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401. In another embodiment, an array of oligonucleotides probes comprising hC/BTLP nucleotide sequence or fiagents thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicabilitv and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al., Science , Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or deternining a susceptibility to restenosis, atherosclerosis, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hypertrophy (BPH), nephritis, fibrosis, glomerulonephritis, gliosis, cirrhosis and anomalies of wound healing, such as keloidsthrough detection of mutation in the hC/BTLP gene by the methods described.

In addition, restenosis, atherosclerosis, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hnpetrophy (BPH), nephritis, fibrosis, glomerulonephritis, gliosis, cirrhosis and anomalies of wound healing, such as keloids, can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of hC/BTLP polypeptide or hC/BTLP mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as an hC/BTLP polypeptide, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assavs.

Thus in another aspect, the present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly restenosis, atherosclerosis, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hypertrophy (BPH), nephritis, fibrosis, glomerulonephritis, gliosis, cirrhosis and anomalies of wound healing, such as keloids, which comprises:

(a) a hC/BTLP polynucleotide, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof,

(b) a nucleotide sequence complementary to that of (a);

(c) a hC/BTLP polypeptide, preferably the polypeptide of SEQ ID NO: 2, or a fgment thereof; or

(d) an antibody to a hC/BTLP polypeptide, preferably to the polypeptide of SEQ ID NO: 2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specificallv tailed to and can hybridize with a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. 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). The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. 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.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the hC/BTLP polypeptides.

The term “immunospecific” means that the antibodies have substantial greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.

Antibodies generated against the hC/BTLP polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an anirral, preferably a nonhuman, using routine protocols. 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, G. and Milstein, C, Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, trarsngenic mice, or other organisms including other mannials, may be used to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.

Antibodies against hC/BTLP polypeptides may also be employed to treat restenosis, atherosclerosis, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hypertrophy (BPH), nephritis, fibrosis, glomerulonephiitis, gliosis, cirrhosis and anomalies of wound healing, such as keloids, among others.

Vaccines

Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with hC/BTLP polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from restenosis, atherosclerosis, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hypertrophy (BPH), nephritis, fibrosis, glomerulonephritis, gliosis, cirrhosis and anomalies of wound healing, such as keloids, among others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering hC/BTLP polypeptide via a vector directing expression of hC/BTLP polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.

Further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a hC/BTLP polypeptide wherein the composition comprises a hC/BTLP polypeptide or hC/BTLP gene. The vaccine formulation may further comprise a suitable carrier. Since hC/BTLP polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

Screening Assays

The hC/BTLP polypeptide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the hC/BTLP polypeptide of the present invention. Thus, polypeptides of the invention may also be used to assess identify agonist or antagonists from, fbr example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors, etc., as the case may be, of the polypeptide of the present invention; or may be structural or functional mimetics of the polypeptide of the present invention. See Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).

HC/BTLP polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate hC/BTLP polypeptide on the one hand and which can inhibit the function of hC/BTLP polypeptide on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as restenosis, atherosclerosis, congestive heart filure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hypertrophy (BPH), nephritis, fibrosis, glomerulonephritis, gliosis, cirrhosis and anomalies of wound healing, such as keloids. Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as restenosis, atherosclerosis, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hypertrophy (BPH), nephritis, fibrosis, glomerulonephritis, gliosis, cirrhosis and anomalies of wound healing, such as keloids

In general, such screening procedures may involve using appropriate cells which express the hC/BTLP polypeptide or respond to hC/BTLP polypeptide of the present invention. Such cells include cells from mammals, yeast, Drosophila or E coli . Cells which express the hC/BTIP polypeptide (or cell membrane containing the expressed polypeptide) or respond to hC/BTLP polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for hC/BTLP activity.

The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the hC/BTLP polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the hC/BTLP polypeptide, using detection systems appropriate to the cells bearing the hC/BTLP polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.

Further, the assays may simplv comprise the steps of mixing a candidate compound with a solution containing a hC/BTLP polypeptide to form a mixture, measuring hC/BTLP activity in the mixture, and comparing the hC/BTLP activity of the mixture to a standard.

The hC/BTLP cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of hC/BTLP mRNA and protein in cells. For example, an ELISA may be constructed for measuring secreted or cell associated levels of hC/BTLP protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of hC/BTLP (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.

The hC/BTLP protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the hC/BTLP is labeled with a radioactive isotope (eg 125I), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or purification. and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. In addition to being used for purification and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of hC/BTLP wvhich compete with the binding of hC/BTLP to its receptors, if any. Standard methods for conducting screening assays are well understood in the art.

Examples of potential hC/BTLP polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins wlich are closely related to the ligands, substrates, enzymes, receptors, etc., as the case may be, of the hC/BTLP polypeptide. e.g., a fragment of the ligands, substraes, enzymes, receptors, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Thus in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for hC/BTLP polypeptides; or compounds which decrease or enhance the production of hC/BTLP polypeptides, which comprises:

(a) a hC/BTLP polypeptide, preferably that of SEQ ID NO:2;

(b) a recombinant cell expressing a hC/BTLP polypeptide, preferably that of SEQ ID NO:2;

(c) a cell membrane expressing a hC/BTLP polkpeptide; preferably that of SEQ ID NO: 2; or

(d) antibody to a hC/BTLP polypeptide, preferably that of SEQ ID NO: 2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.

Prophylactic and Therapeutic Methods

This invention provides methods of treating abnormal conditions such as, restenosis, atherosclerosis, congestive heart filure (CHF), chronic obstructive pulmonary disease (COPD), benign prostatic hypertrophy (BPH), nephritis, fibrosis, glomerulonephritis, gliosis, cirrhosis and anomalies of wound healing, such as keloids, related to both an excess of and insufficient amounts of hC/BTLP polypeptide activity.

If the activity of hC/BTLP polypeptide is in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmacutically acceptable carrier in an amount effective to inhibit the function of the hC/BTLP polypeptide, such as, for example by blocking the binding of ligands, substrates, enzymes, receptors, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of hC/BTLP polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous hC/BTLP polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the hC/BTLP polypeptide.

In another approach, soluble forms of hC/BTLP polypeptides still capable of binding the ligand in competition with endogenous hC/BTLP polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the hC/BTLP polypeptide.

In still another approach, expression of the gene encoding endogenous hC/BTLP polypeptide can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately adrinistered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Olizodeoxvnucleotides as Antisense Inhibitors of Gene Expression , CRC Press, Boca Raton, Fla. (1988). Alternatively, oligonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al, Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al., Science (1991) 251:1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an underxpression of hC/BTLP and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound vhich activates hC/BTLP polypeptide, i.e., an agonist as described above, in combination with a phalrmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of hC/BTLP by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defctive retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be admired to a subject for engineering cells in vivo and expression of the polypeptide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches , (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Another approach is to administer a therapeutic amount of hC/BTLP polypeptides in combination with a suitable phamiaceutical carrier.

Formulation and Administration

Peptides, such as the soluble form of hC/BTLP polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof Formulation should suit the mode of administration, and is well within the skill of the art. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositlons of the invention.

Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

Preferred forms of systemic admini on of the phaceutical compositions include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include trarsmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteric or encapsulated formulations, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0. 1-100 μg/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of adinu aion. For example, oral administration would be expected to require higher dosages than admmtion by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as “gene therapy” as described above. Thus, for example, cells fromn a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for exarnple, by the use of a retoviral plasmid vector. The cells are then introduced into the subject.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated bv reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

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cttacctgcc ctccgcccac ccgtgggccc ctagccaact tctccctgcg actgggggta 60

acaggcagtg cttgccctct ctactgtccc ggcggcatcc acatgtttcc ggacacctga 120

gcaccccggt cccgccgagg agcctccggg tggggagaag agcaccggtg cccctagccc 180

cgcacatcag cgcggaccgc ggctgcctaa cttctgggtc ccgtcccttc cttttcctcc 240

gggggaggag gatggggttg ggaacgcttt ccccgaggat gctcgtgtgg ctggtggcct 300

cggggattgt tttctacggg gagctatggg tctgcgctgg cctcgattat gattacactt 360

ttgatgggaa cgaagaggat aaaacagaga ctatagatta caaggacccg tgtaaagccg 420

ctgtattttg gggcgatatt gccttagatg atgaagactt aaatatcttt caaatagata 480

ggacaattga ccttacgcag aacccctttg gaaaccttgg acataccaca ggtggacttg 540

gagaccatgc tatgtcaaag aagcgagggg ccctctacca acttatagac aggataagaa 600

gaattggctt tggcttggag caaaacaaca cagttaaggg aaaagtacct ctacaattct 660

cagggcaaaa tgagaaaaat cgagttccca gagccgctac atcaagaacg gaaagagtat 720

ggcctggagg cgttattcct tatgttatag gaggaaactt cactggcagc cagagagcca 780

tgttcaagca ggccatgagg cactgggaaa agcacacatg tgtgactttc atagaaagaa 840

gtgatgaaga gagttacatt gtattcacct ataggccttg tggatgctgc tcctatgtag 900

gtcggcgagg aagtggacct caggcaatct ctatcggcaa gaactgtgat aaatttggga 960

ttgttgttca tgaattgggt catgtgatag gcttttggca tgaacacaca agaccagatc 1020

gagataacca cgtaactatc ataagagaaa acatccagcc aggtcaagag tacaattttc 1080

tgaagatgga gcctggagaa gcaaactcac ttggagaaag atatgatttc gacagtatca 1140

tgcactatgc caggaacacc ttctcaaggg ggatgtttct ggataccatt ctcccctccc 1200

gtgatgataa tggcatacgt cctgcaattg gtcagcgaac ccgtctaagc aaaggagata 1260

tcgcacaggc aagaaagctg tatagatgtc cagcatgtgg agaaactcta caagaatcca 1320

atggcaacct ttcctctcca ggatttccca atggctaccc ttcttacaca cactgcatct 1380

ggagagtttc tgtgacccca ggggagaaga ttgttttaaa ttttacaacg atggatctat 1440

acaagagtag tttgtgctgg tatgactata ttgaagtaag agacgggtac tggagaaaat 1500

cacctctcct tggtagattc tgtggggaca aattgcctga agttcttact tctacagaca 1560

gcagaatgtg gattgagttt cgtagcagca gtaattgggt aggaaaaggc tttgcagctg 1620

tctatgaagc gatctgtgga ggtgagatac gtaaaaatga aggacagatt cagtctccca 1680

attatcctga tgactatcgc ccgatgaagg aatgtgtgtg gaaaataaca gtgtctgaga 1740

gctaccacgt cgggctgacc tttcagtcct ttgagattga aagacatgac aattgtgctt 1800

atgactacct ggaagttaga gatggaacca gtgaaaatag ccctttgata gggcgtttct 1860

gtggttatga caaacctgaa gacataagat ctacctccaa tactttgtgg atgaagtttg 1920

tttctgacgg aactgtgaac aaagcagggt ttgctgctaa cttttttaaa gaggaagatg 1980

agtgtgccaa acctgaccgt ggaggctgtg agcagcgatg tctgaacact ctgggcagtt 2040

accagtgtgc ctgtgagcct ggctatgagc tgggcccaga cagaaggagc tgtgaagctg 2100

cttgtggtgg acttcttacc aaacttaacg gcaccataac cacccctggc tggcccaagg 2160

agtaccctcc taataagaac tgtgtgtggc aagtggttgc accaacccag tacagaattt 2220

ctgtgaagtt tgagtttttt gaattggaag gcaatgaggt ttgcaaatat gattatgtgg 2280

agatctggag tggtctttcc tctgagtcta aactgcatgg caaattctgt ggcgctgaag 2340

tgcctgaagt gatcacatcc cagttcaaca atatgagaat tgaattcaaa tctgacaata 2400

ctgtatccaa gaagggcttc aaagcacatt ttttctcaga caaagatgaa tgctctaagg 2460

ataatggtgg atgtcagcac gaatgtgtca acacgatggg gagctacatg tgtcaatgcc 2520

gtaatggatt tgtgctacat gacaataaac atgattgcaa ggaagctgag tgtgaacaga 2580

agatccacag tccaagtggc ctcatcacca gtcccaactg gccagacaag tacccaagca 2640

ggaaagaatg cacttgggaa atcagcgcca ctcctggcca ccgaatcaaa ttagccttta 2700

gtgaatttga gattgagcag catcaagaat gtgcttatga ccacttagaa gtatttgatg 2760

gagaaacaga aaagtcaccg attcttggac gactatgtgg caacaagata ccagatcccc 2820

ttgtggctac tggaaataaa atgtttgttc ggtttgtttc tgatgcatct gttcaaagaa 2880

aaggctttca agccacacat tctacagagt gtggcggacg attgaaagca gaatcaaaac 2940

caagagatct gtactcacat gctcagtttg gtgataacaa ctacccagga caggttgact 3000

gtgaatggct attagtatca gaacggggct ctcgacttga attatccttc cagacatttg 3060

aagtggagga agaagcggac tgtggctatg actatgtgga gctctttgat ggtcttgatt 3120

caacagctgt ggggcttggt cgattctgtg gatccgggcc accagaagag atttattcaa 3180

ttggagattc agttttaatt catttccaca ctgatgacac aatcaacaag aagggatttc 3240

atataagata caaaagcata agatatccag ataccacaca taccaaaaaa taacaccaaa 3300

acctctgtca gaacacaaag gaatgtgcat aatggagaga agacatattt tttttaaaac 3360

tgaagatatt ggcacaaatg ttttatacaa agagtttgaa caaaaaatcc ctgtaagacc 3420

agaattatct ttgtactaaa agagaagttt ccagcaaaac cctcatcagc attacaagga 3480

tatttgaact ccatgcttga tggtattaat aaagctggtg aaagggcatc atatacttca 3540

aggaagactc tacaagcttt tgttcacagc ttgaaataga tgcctcacaa ttcagacagt 3600

ttaattcagg aactgtgacc ctgaagtgtt ctttttgaca atttgtcaag atttagggac 3660

ataaaatgat cttgcaggtc gtaaactgga aaacagtatt ttggttgtct taggataatt 3720

gctgactttg tatcttggat acagtgtaaa ccagatccat ataaggtgaa tgtgaaatgg 3780

gagtcttctg agggtgattt gtactttcca tgtgtatgtg tgtgtctggt gtttggaaac 3840

tgggatattt cagcttcatt atttccactt gcaggccagc ttaacctctg aaacacaaat 3900

gatcttgaga ccactttagt gtacttacat ttagatgagt ttgaaatctc aatggtgtct 3960

aattattgca gttaaattct agacatcagt tctttaagtc tcagaaaacg cccagtgaat 4020

tggtaaactt agttcttttt tttggaagtg ctgccttttc acaccaaatc caagaagcct 4080

gtgatgtctt atgaacctta tgagaaaact ccgaagaggt gtgagcagga ttcttctgaa 4140

tgactgtctg gatggttcat tactcaagtt actgctgctg ctattgtctt tcctttgttg 4200

tcgatctgtt attgttgtat tattattgtt gatgttgtca tggttaatct attttttaaa 4260

attgaaatga agcagaagta ggccttgtga gaactgaaag gtctctttca tttttctctt 4320

cctgggattc attttttcaa aacacaatgc tggaaaaaaa agatttgttt ctgaaagact 4380

tcttatggtg ctattccata aacttttttt caaacaagtt tttgaccttt gagccaaccc 4440

acccgtagac tacgaatgtc tccctatggc tggtagcatt tgaagactaa agacttgtca 4500

aatatatcaa gagtatatca ttgcaagggc agcacttgtc ctgtggaaca actacttata 4560

atgccttaga attcctgcac atgatcaaac agatcctcct aaaacacacc ttttgaaatg 4620

ttgaacataa tagtgtatgt taattaacag ctctatgaag aaaatccatt tccatgactg 4680

aagcattgga tataaatatg gtgtcctgct ttttttgtag aaaatgtaat ttgaggatga 4740

attttctgct ttaaaggcat gtgtgttttt aaaattaatg aatgtagatg tgtgattgtc 4800

tgagtgagtg aaactacaag aggtaaaaaa taatgggtgg ttgaaaagtt aaaatgtatg 4860

tgccaagttc tactagaatt ccatttgaaa tagcaccttc cttaggtttc atggacaaat 4920

aatgggaact tctaattttg atcaatccca ttaaaaaaag gctctttcct ttagagaaac 4980

tctattttga tgtcaatata gattactgta tgaagtagct ttgtgtctgt tacctgtcca 5040

tgagcataca acattgaata caattgggtg tattctttca gttttacaca attaaagtat 5100

acacacagat gtaaaaaaaa aaaaaaaaaa aaaaaaaaac tcgag 5145

2

1013

2

Met Gly Leu Gly Thr Leu Ser Pro Arg Met Leu Val Trp Leu Val Ala

1 5 10 15

Ser Gly Ile Val Phe Tyr Gly Glu Leu Trp Val Cys Ala Gly Leu Asp

20 25 30

Tyr Asp Tyr Thr Phe Asp Gly Asn Glu Glu Asp Lys Thr Glu Thr Ile

35 40 45

Asp Tyr Lys Asp Pro Cys Lys Ala Ala Val Phe Trp Gly Asp Ile Ala

50 55 60

Leu Asp Asp Glu Asp Leu Asn Ile Phe Gln Ile Asp Arg Thr Ile Asp

65 70 75 80

Leu Thr Gln Asn Pro Phe Gly Asn Leu Gly His Thr Thr Gly Gly Leu

85 90 95

Gly Asp His Ala Met Ser Lys Lys Arg Gly Ala Leu Tyr Gln Leu Ile

100 105 110

Asp Arg Ile Arg Arg Ile Gly Phe Gly Leu Glu Gln Asn Asn Thr Val

115 120 125

Lys Gly Lys Val Pro Leu Gln Phe Ser Gly Gln Asn Glu Lys Asn Arg

130 135 140

Val Pro Arg Ala Ala Thr Ser Arg Thr Glu Arg Val Trp Pro Gly Gly

145 150 155 160

Val Ile Pro Tyr Val Ile Gly Gly Asn Phe Thr Gly Ser Gln Arg Ala

165 170 175

Met Phe Lys Gln Ala Met Arg His Trp Glu Lys His Thr Cys Val Thr

180 185 190

Phe Ile Glu Arg Ser Asp Glu Glu Ser Tyr Ile Val Phe Thr Tyr Arg

195 200 205

Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg Gly Ser Gly Pro Gln

210 215 220

Ala Ile Ser Ile Gly Lys Asn Cys Asp Lys Phe Gly Ile Val Val His

225 230 235 240

Glu Leu Gly His Val Ile Gly Phe Trp His Glu His Thr Arg Pro Asp

245 250 255

Arg Asp Asn His Val Thr Ile Ile Arg Glu Asn Ile Gln Pro Gly Gln

260 265 270

Glu Tyr Asn Phe Leu Lys Met Glu Pro Gly Glu Ala Asn Ser Leu Gly

275 280 285

Glu Arg Tyr Asp Phe Asp Ser Ile Met His Tyr Ala Arg Asn Thr Phe

290 295 300

Ser Arg Gly Met Phe Leu Asp Thr Ile Leu Pro Ser Arg Asp Asp Asn

305 310 315 320

Gly Ile Arg Pro Ala Ile Gly Gln Arg Thr Arg Leu Ser Lys Gly Asp

325 330 335

Ile Ala Gln Ala Arg Lys Leu Tyr Arg Cys Pro Ala Cys Gly Glu Thr

340 345 350

Leu Gln Glu Ser Asn Gly Asn Leu Ser Ser Pro Gly Phe Pro Asn Gly

355 360 365

Tyr Pro Ser Tyr Thr His Cys Ile Trp Arg Val Ser Val Thr Pro Gly

370 375 380

Glu Lys Ile Val Leu Asn Phe Thr Thr Met Asp Leu Tyr Lys Ser Ser

385 390 395 400

Leu Cys Trp Tyr Asp Tyr Ile Glu Val Arg Asp Gly Tyr Trp Arg Lys

405 410 415

Ser Pro Leu Leu Gly Arg Phe Cys Gly Asp Lys Leu Pro Glu Val Leu

420 425 430

Thr Ser Thr Asp Ser Arg Met Trp Ile Glu Phe Arg Ser Ser Ser Asn

435 440 445

Trp Val Gly Lys Gly Phe Ala Ala Val Tyr Glu Ala Ile Cys Gly Gly

450 455 460

Glu Ile Arg Lys Asn Glu Gly Gln Ile Gln Ser Pro Asn Tyr Pro Asp

465 470 475 480

Asp Tyr Arg Pro Met Lys Glu Cys Val Trp Lys Ile Thr Val Ser Glu

485 490 495

Ser Tyr His Val Gly Leu Thr Phe Gln Ser Phe Glu Ile Glu Arg His

500 505 510

Asp Asn Cys Ala Tyr Asp Tyr Leu Glu Val Arg Asp Gly Thr Ser Glu

515 520 525

Asn Ser Pro Leu Ile Gly Arg Phe Cys Gly Tyr Asp Lys Pro Glu Asp

530 535 540

Ile Arg Ser Thr Ser Asn Thr Leu Trp Met Lys Phe Val Ser Asp Gly

545 550 555 560

Thr Val Asn Lys Ala Gly Phe Ala Ala Asn Phe Phe Lys Glu Glu Asp

565 570 575

Glu Cys Ala Lys Pro Asp Arg Gly Gly Cys Glu Gln Arg Cys Leu Asn

580 585 590

Thr Leu Gly Ser Tyr Gln Cys Ala Cys Glu Pro Gly Tyr Glu Leu Gly

595 600 605

Pro Asp Arg Arg Ser Cys Glu Ala Ala Cys Gly Gly Leu Leu Thr Lys

610 615 620

Leu Asn Gly Thr Ile Thr Thr Pro Gly Trp Pro Lys Glu Tyr Pro Pro

625 630 635 640

Asn Lys Asn Cys Val Trp Gln Val Val Ala Pro Thr Gln Tyr Arg Ile

645 650 655

Ser Val Lys Phe Glu Phe Phe Glu Leu Glu Gly Asn Glu Val Cys Lys

660 665 670

Tyr Asp Tyr Val Glu Ile Trp Ser Gly Leu Ser Ser Glu Ser Lys Leu

675 680 685

His Gly Lys Phe Cys Gly Ala Glu Val Pro Glu Val Ile Thr Ser Gln

690 695 700

Phe Asn Asn Met Arg Ile Glu Phe Lys Ser Asp Asn Thr Val Ser Lys

705 710 715 720

Lys Gly Phe Lys Ala His Phe Phe Ser Asp Lys Asp Glu Cys Ser Lys

725 730 735

Asp Asn Gly Gly Cys Gln His Glu Cys Val Asn Thr Met Gly Ser Tyr

740 745 750

Met Cys Gln Cys Arg Asn Gly Phe Val Leu His Asp Asn Lys His Asp

755 760 765

Cys Lys Glu Ala Glu Cys Glu Gln Lys Ile His Ser Pro Ser Gly Leu

770 775 780

Ile Thr Ser Pro Asn Trp Pro Asp Lys Tyr Pro Ser Arg Lys Glu Cys

785 790 795 800

Thr Trp Glu Ile Ser Ala Thr Pro Gly His Arg Ile Lys Leu Ala Phe

805 810 815

Ser Glu Phe Glu Ile Glu Gln His Gln Glu Cys Ala Tyr Asp His Leu

820 825 830

Glu Val Phe Asp Gly Glu Thr Glu Lys Ser Pro Ile Leu Gly Arg Leu

835 840 845

Cys Gly Asn Lys Ile Pro Asp Pro Leu Val Ala Thr Gly Asn Lys Met

850 855 860

Phe Val Arg Phe Val Ser Asp Ala Ser Val Gln Arg Lys Gly Phe Gln

865 870 875 880

Ala Thr His Ser Thr Glu Cys Gly Gly Arg Leu Lys Ala Glu Ser Lys

885 890 895

Pro Arg Asp Leu Tyr Ser His Ala Gln Phe Gly Asp Asn Asn Tyr Pro

900 905 910

Gly Gln Val Asp Cys Glu Trp Leu Leu Val Ser Glu Arg Gly Ser Arg

915 920 925

Leu Glu Leu Ser Phe Gln Thr Phe Glu Val Glu Glu Glu Ala Asp Cys

930 935 940

Gly Tyr Asp Tyr Val Glu Leu Phe Asp Gly Leu Asp Ser Thr Ala Val

945 950 955 960

Gly Leu Gly Arg Phe Cys Gly Ser Gly Pro Pro Glu Glu Ile Tyr Ser

965 970 975

Ile Gly Asp Ser Val Leu Ile His Phe His Thr Asp Asp Thr Ile Asn

980 985 990

Lys Lys Gly Phe His Ile Arg Tyr Lys Ser Ile Arg Tyr Pro Asp Thr

995 1000 1005

Thr His Thr Lys Lys

1010

3

3690

3

gaattcggca cgagctcgtg ccgctcgtgc cgcgggtact ggagaaaatc acctctcctt 60

gattctgtgg ggacaaattg cctgaagttc ttacttctac agacagcaga atgtggattg 120

agtttcgtag cagcagtaat tgggtaggaa aaggctttgc agctgtctat gaagcgatct 180

gtggaggtga gatacgtaaa aatgaaggac agattcagtc tcccaattat cctgatgact 240

atcgcccgat gaaagaatgt gtgtggaaaa taacagtgtc tgagagctac cacgtcgggc 300

tgacctttca gtcctttgag attgaaagac atgacaattg tgcttatgac tacctggaag 360

ttagagatgg aaccagtgaa aatagccctt tgatagggcg tttctgtggt tatgacaaac 420

ctgaagacat aagatctacc tccaatactt tgtggatgaa gtttgtttct gacggaactg 480

tgaacaaagc agggtttgct gctaactttt ttaaagagga agatgagtgt gccaaacctg 540

accgtggagg ctgtgagcag cgatgtctga acactctggg cagttaccag tgtgcctgtg 600

agcctggcta tgagctgggc ccagacagaa ggagctgtga agctgcttgt ggtggacttc 660

ttaccaaact taacggcacc ataaccaccc ctggctggcc caaggagtac cctcctaata 720

agaactgtgt gtggcaagtg gttgcaccaa cccagtacag aatttctgtg aagtttgagt 780

tttttgaatt ggaaggcaat gaagtttgca aatatgatta tgtggagatc tggagtggtc 840

tttcctctga gtctaaactg catggcaaat tctgtggcgc tgaagtgcct gaagtgatca 900

catcccagtt caacaatatg agaattgaat tcaaatctga caatactgta tccaagaagg 960

gcttcaaagc acattttttc tcagacaaag atgaatgctc taaggataat ggtggatgtc 1020

agcacgaatg tgtcaacacg atggggagct acatgtgtca atgccgtaat ggatttgtgc 1080

tacatgacaa taaacatgat tgcaaggaag ctgagtgtga acagaagatc cacagtccaa 1140

gtggcctcat caccagtccc aactggccag acaagtaccc aagcaggaaa gaatgcactt 1200

gggaaatcag cgccactcct ggccaccgaa tcaaattagc ctttagtgaa tttgagattg 1260

agcagcatcg ggaatgtgct tatgaccact tagaagtatt tgatggagaa acagaaaagt 1320

caccgattct tggacgacta tgtggcaaca agataccaga tccccttgtg gctactggaa 1380

ataaaatgtt tgttcggttt gtttctgatg catctgttca aagaaaaggc tttcaagcca 1440

cacattctac agagtgtggc ggacgattga aagcagaatc aaaaccaaga gatctgtact 1500

cacatgctca gtttggtgat aacaactacc caggacaggt tgactgtgaa tggctattag 1560

tatcagaacg gggctctcga cttgaattat ccttccagac atttgaagtg gaggaagaag 1620

cagactgtgg ctatgactat gtggagctct ttgatggtct tgattcaaca gctgtggggc 1680

ttggtcgatt ctgtggatcc gggccaccag aagagattta ttcaattgga gattcagttt 1740

taattcattt ccacactgat gacacaatca acaagaaggg atttcatata agatacaaaa 1800

gcataagata tccagatacc acacatacca aaaaataaca ccaaaacctc tgtcagaaca 1860

caaaggaatg tgcataatgg agagaagaca tatttttttt aaaactgaag atattggcac 1920

aaatgtttta tacaaagagt ttgaacaaaa aatccctgta agaccagaat tatctttgta 1980

ctaaaagaga agtttccagc aaaaccctca tcagcattac aaggatattt gaactccatg 2040

cttgatggta ttaataaagc tggtgaaagg gcatcatata cttcaaggaa gactctacaa 2100

gcttttgttc acagcttgaa atagatgcct cacaattcag acagtttaat tcaggaactg 2160

tgaccctgaa gtgttctttt tgacaatttg tcaagattta gggacataaa atgatcttgc 2220

aggtcgtaaa ctggaaaaca gtattttggt tgtcttagga taattgctga ctttgtatct 2280

tggatacagt gtaaaccaga tccatataag gtgaatgtga aatgggagtc ttctgagggt 2340

gatttgtact ttccatgtgt atgtgtgtgt ctggtgtttg gaaactggga tatttcagct 2400

tcattatttc cacttgcagg ccagcttaac ctctgaaaca caaatgatct tgagaccact 2460

ttagtgtact tacatttaga tgagtttgaa atctcaatgg tgtctaatta ttgcagttaa 2520

attctagaca tcagttcttt aagtctcaga aaacgcccag tgaattggta aacttagttc 2580

ttttttttgg aagtgctgcc ttttcacacc aaatccaaga agcctgtgat gtcttatgaa 2640

ccttatgaga aaactccgaa gaggtgtgag caggattctt ctgaatgact gtctggatgg 2700

ttcattactc aagttactgc tgctgctatt gtctttcctt tgttgtcgat ctgttattgt 2760

tgtattatta ttgttgatgt tgtcatggtt aatctatttt ttaaaattga aatgaagcag 2820

aagtaggcct tgtgagaact gaaaggtctc tttcattttt ctcttcctgg gattcatttt 2880

ttcaaaacac aatgctggaa aaaaaagatt tgtttctgaa agacttctta tggtgctatt 2940

ccataaactt tttttcaaac aagtttttga cctttgagcc aacccacccg tagactacga 3000

atgtctccct atggctggta gcatttgaag actaaagact tgtcaaatat atcaagagta 3060

tatcattgca agggcagcac ttgtcctgtg gaacaactac ttataatgcc ttagaattcc 3120

tgcacatgat caaacagatc ctcctaaaac acaccttttg aaatgttgaa cataatagtg 3180

tatgttaatt aacagctcta tgaagaaaat ccatttccat gactgaagca ttggatataa 3240

atatggtgtc ctgctttttt tgtagaaaat gtaatttgag gatgaatttt ctgctttaaa 3300

ggcatgtgtg tttttaaaat taatgaatgt agatgtgtga ttgtctgagt gagtgaaact 3360

acaagaggta aaaaataatg ggtggttgaa aagttaaaat gtatgtgcca agttctacta 3420

gaattccatt tgaaatagca ccttccttag gtttcatgga caaataatgg gaacttctaa 3480

ttttgatcaa tcccattaaa aaaaggctct ttcctttaga gaaactctat tttgatgtca 3540

atatagatta ctgtatgaag tagctttgtg tctgttacct gtccatgagc atacaacatt 3600

gaatacaatt gggtgtattc tttcagtttt acacaattaa agtatacaca cagatgtaaa 3660

aaaaaaaaaa aaaaaaaaaa aaaactcgag 3690

4

591

4

Phe Cys Gly Asp Lys Leu Pro Glu Val Leu Thr Ser Thr Asp Ser Arg

1 5 10 15

Met Trp Ile Glu Phe Arg Ser Ser Ser Asn Trp Val Gly Lys Gly Phe

20 25 30

Ala Ala Val Tyr Glu Ala Ile Cys Gly Gly Glu Ile Arg Lys Asn Glu

35 40 45

Gly Gln Ile Gln Ser Pro Asn Tyr Pro Asp Asp Tyr Arg Pro Met Lys

50 55 60

Glu Cys Val Trp Lys Ile Thr Val Ser Glu Ser Tyr His Val Gly Leu

65 70 75 80

Thr Phe Gln Ser Phe Glu Ile Glu Arg His Asp Asn Cys Ala Tyr Asp

85 90 95

Tyr Leu Glu Val Arg Asp Gly Thr Ser Glu Asn Ser Pro Leu Ile Gly

100 105 110

Arg Phe Cys Gly Tyr Asp Lys Pro Glu Asp Ile Arg Ser Thr Ser Asn

115 120 125

Thr Leu Trp Met Lys Phe Val Ser Asp Gly Thr Val Asn Lys Ala Gly

130 135 140

Phe Ala Ala Asn Phe Phe Lys Glu Glu Asp Glu Cys Ala Lys Pro Asp

145 150 155 160

Arg Gly Gly Cys Glu Gln Arg Cys Leu Asn Thr Leu Gly Ser Tyr Gln

165 170 175

Cys Ala Cys Glu Pro Gly Tyr Glu Leu Gly Pro Asp Arg Arg Ser Cys

180 185 190

Glu Ala Ala Cys Gly Gly Leu Leu Thr Lys Leu Asn Gly Thr Ile Thr

195 200 205

Thr Pro Gly Trp Pro Lys Glu Tyr Pro Pro Asn Lys Asn Cys Val Trp

210 215 220

Gln Val Val Ala Pro Thr Gln Tyr Arg Ile Ser Val Lys Phe Glu Phe

225 230 235 240

Phe Glu Leu Glu Gly Asn Glu Val Cys Lys Tyr Asp Tyr Val Glu Ile

245 250 255

Trp Ser Gly Leu Ser Ser Glu Ser Lys Leu His Gly Lys Phe Cys Gly

260 265 270

Ala Glu Val Pro Glu Val Ile Thr Ser Gln Phe Asn Asn Met Arg Ile

275 280 285

Glu Phe Lys Ser Asp Asn Thr Val Ser Lys Lys Gly Phe Lys Ala His

290 295 300

Phe Phe Ser Asp Lys Asp Glu Cys Ser Lys Asp Asn Gly Gly Cys Gln

305 310 315 320

His Glu Cys Val Asn Thr Met Gly Ser Tyr Met Cys Gln Cys Arg Asn

325 330 335

Gly Phe Val Leu His Asp Asn Lys His Asp Cys Lys Glu Ala Glu Cys

340 345 350

Glu Gln Lys Ile His Ser Pro Ser Gly Leu Ile Thr Ser Pro Asn Trp

355 360 365

Pro Asp Lys Tyr Pro Ser Arg Lys Glu Cys Thr Trp Glu Ile Ser Ala

370 375 380

Thr Pro Gly His Arg Ile Lys Leu Ala Phe Ser Glu Phe Glu Ile Glu

385 390 395 400

Gln His Arg Glu Cys Ala Tyr Asp His Leu Glu Val Phe Asp Gly Glu

405 410 415

Thr Glu Lys Ser Pro Ile Leu Gly Arg Leu Cys Gly Asn Lys Ile Pro

420 425 430

Asp Pro Leu Val Ala Thr Gly Asn Lys Met Phe Val Arg Phe Val Ser

435 440 445

Asp Ala Ser Val Gln Arg Lys Gly Phe Gln Ala Thr His Ser Thr Glu

450 455 460

Cys Gly Gly Arg Leu Lys Ala Glu Ser Lys Pro Arg Asp Leu Tyr Ser

465 470 475 480

His Ala Gln Phe Gly Asp Asn Asn Tyr Pro Gly Gln Val Asp Cys Glu

485 490 495

Trp Leu Leu Val Ser Glu Arg Gly Ser Arg Leu Glu Leu Ser Phe Gln

500 505 510

Thr Phe Glu Val Glu Glu Glu Ala Asp Cys Gly Tyr Asp Tyr Val Glu

515 520 525

Leu Phe Asp Gly Leu Asp Ser Thr Ala Val Gly Leu Gly Arg Phe Cys

530 535 540

Gly Ser Gly Pro Pro Glu Glu Ile Tyr Ser Ile Gly Asp Ser Val Leu

545 550 555 560

Ile His Phe His Thr Asp Asp Thr Ile Asn Lys Lys Gly Phe His Ile

565 570 575

Arg Tyr Lys Ser Ile Arg Tyr Pro Asp Thr Thr His Thr Lys Lys

580 585 590