Aggrecanase molecules

This application claims the benefit of U.S. Provisional Application No. 60/242,317 filed Oct. 20, 2000.

The present invention relates to the discovery of nucleotide sequences encoding novel aggrecanase molecules, the aggrecanase proteins and processes for producing them. The invention further relates to the development of inhibitors of, as well as antibodies to the aggrecanase enzymes. These inhibitors and antibodies may be useful for the treatment of various aggrecanase-associated conditions including osteoarthritis.

Aggrecan is a major extracellular component of articular cartilage. It is a proteoglycan responsible for providing cartilage with its mechanical properties of compressibility and elasticity. The loss of aggrecan has been implicated in the degradation of articular cartilage in arthritic diseases. Osteoarthritis is a debilitating disease which affects at least 30 million Americans (MacLean et al. J Rheumatol 25:2213-8. (1998)). Osteoarthritis can severely reduce quality of life due to degradation of articular cartilage and the resulting chronic pain. An early and important characteristic of the osteoarthritic process is loss of aggrecan from the extracellular matrix (Brandt, K D. and Mankin H J. Pathogenesis of Osteoarthritis , Textbook of Rheumatology, W B Saunders Company, Philadelphia, Pa. pgs. 1355-1373. (1993)). The large, sugar-containing portion of aggrecan is thereby lost from the extra-cellular matrix, resulting in deficiencies in the biomechanical characteristics of the cartilage.

A proteolytic activity termed “aggrecanase” is thought to be responsible for the cleavage of aggrecan thereby having a role in cartilage degradation associated with osteoarthritis and inflammatory joint disease. Work has been conducted to identify the enzyme responsible for the degradation of aggrecan in human osteoarthritic cartilage. Two enzymatic cleavage sites have been identified within the interglobular domain of aggrecan. One (Asn 34 1-Phe 342 ) is observed to be cleaved by several known metalloproteases (Flannery, C R et al. J Biol Chem 267:1008-14. 1992; Fosang, A J et al. Biochemical J . 304:347-351. (1994)). The aggrecan fragment found in human synovial fluid, and generated by IL-1 induced cartilage aggrecan cleavage is at the Glu 373 -Ala3 74 bond (Sandy, J D, et al. J Clin Invest 69:1512-1516. (1992); Lohmander L S, et al. Arthritis Rheum 36: 1214-1222. (1993); Sandy J D et al. J Biol Chem . 266: 8683-8685. (1991)), indicating that none of the known enzymes are responsible for aggrecan cleavage in vivo.

Recently, identification of two enzymes, aggrecanase-1(ADAMTS 4) and aggrecanase-2(ADAMTS-11) within the “Disintegrin-like and Metalloprotease with Thrombospondin type 1 motif” (ADAM-TS) family have been identified which are synthesized by IL-1 stimulated cartilage and cleave aggrecan at the appropriate site (Tortorella M D, et al. Science 284:1664-6. (1999); Abbaszade, I, et al. J Biol Chem 274: 23443-23450. (1999)). It is possible that these enzymes could be synthesized by osteoarthritic human articular cartilage. It is also contemplated that there are other, related enzymes in the ADAM-TS family which are capable of cleaving aggrecan at the Glu 373 -Ala3 74 bond and could contribute to aggrecan cleavage in osteoarthritis.

The present invention is directed to the identification of aggrecanase protein molecules capable of cleaving aggrecanase, the nucleotide sequences which encode the aggrecanase enzymes, and processes for the production of aggrecanases. These enzymes are contemplated to be characterized as having proteolytic aggrecanase activity. The invention further includes compositions comprising these enzymes as well as antibodies to these enzymes. In addition, the invention includes methods for developing inhibitors of aggrecanase which block the enzyme's proteolytic activity. These inhibitors and antibodies may be used in various assays and therapies for treatment of conditions characterized by the degradation of articular cartilage.

The nucleotide sequence of the aggrecanase molecule of the present invention is set forth in SEQ ID NO: 3. In another embodiment, the nucleotide sequence of the aggrecanase molecule of the present invention is set forth SEQ ID NO: 1 from nucleotide #1 to #3766. In another embodiment the nucleotide sequence of the invention comprises nucleotide #1086(TCG) to #3396(CGC) of SEQ ID NO: 1. The invention further includes equivalent degenerative codon sequences of the sequences set forth in SEQ ID NO: 1, as well as fragments thereof which exhibit aggrecanase activity.

The amino acid sequence of an isolated aggrecanase molecule of the invention comprises the sequence set forth in SEQ ID NO: 4. The amino acid sequence of an isolated aggrecanase molecule comprises the sequence set forth in SEQ ID NO: 2. The invention further includes fragments of the amino acid sequence which encode molecules exhibiting aggrecanase activity.

The human aggrecanase protein or a fragment thereof may be produced by culturing a cell transformed with a DNA sequence of SEQ ID NO: 3 or SEQ ID NO: 1 comprising nucleotide #1 to #3766 of SEQ ID NO: 1 or comprising nucleotide #1086 to #3396 of SEQ ID NO: 1 and recovering and purifying from the culture medium a protein characterized by the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 2, respectively, substantially free from other proteinaceous materials with which it is co-produced. For production in mammalian cells, the DNA sequence further comprises a DNA sequence encoding a suitable propeptide 5′ to and linked in frame to the nucleotide sequence encoding the aggrecanase enzyme.

The invention includes methods for obtaining additional aggrecanase molecules, the DNA sequence obtained by this method and the protein encoded thereby. The method for isolation of the full length sequence involves utilizing the aggrecanase sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 1 from nucleotide #1086 to #3396 to design probes for screening using standard procedures known to those skilled in the art.

It is expected that other species have DNA sequences homologous to human aggrecanase enzyme. The invention, therefore, includes methods for obtaining the DNA sequences encoding other aggrecanase molecules, the DNA sequences obtained by those methods, and the protein encoded by those DNA sequences. This method entails utilizing the nucleotide sequence of the invention or portions thereof to design probes to screen libraries for the corresponding gene from other species or coding sequences or fragments thereof from using standard techniques. Thus, the present invention may include DNA sequences from other species, which are homologous to the human aggrecanase protein and can be obtained using the human sequence. The present invention may also include functional fragments of the aggrecanase protein, and DNA sequences encoding such functional fragments, as well as functional fragments of other related proteins. The ability of such a fragment to function is determinable by assay of the protein in the biological assays described for the assay of the aggrecanase protein.

The aggrecanase proteins of the present invention may be produced by culturing a cell transformed with the DNA sequence of SEQ ID NO: 3 or SEQ ID NO: 1 comprising nucleotide #1 to #3766 of SEQ ID NO: 1 or comprising nucleotide #1086 to #3396 of SEQ ID NO: 1 and recovering and purifying aggrecanase protein from the culture medium. In one embodiment the protein comprises amino acid sequence of SEQ ID NO: 4 or amino acid #1 to #770 of SEQ ID NO: 2. The purified expressed protein is substantially free from other proteinaceous materials with which it is co-produced, as well as from other contaminants. The recovered purified protein is contemplated to exhibit proteolytic aggrecanase activity cleaving aggrecan. Thus, the proteins of the invention may be further characterized by the ability to demonstrate aggrecan proteolytic activity in an asssay which determines the presence of an aggrecan-degrading molecule. These assays or the development thereof is within the knowledge of one skilled in the art. Such assays may involve contacting an aggrecan substrate with the aggrecanase molecule and monitoring the production of aggrecan fragments (see for example, Hughes et al., Biochem J 305: 799-804(1995); Mercuri et al., J. Bio Chem . 274:32387-32395 (1999)).

In another embodiment, the invention includes methods for developing inhibitors of aggrecanase and the inhibitors produced thereby. These inhibitors prevent cleavage of aggrecan. The method may entail the determination of binding sites based on the three dimensional structure of aggrecanase and aggrecan and developing a molecule reactive with the binding site. Candidate molecules are assayed for inhibitory activity. Additional standard methods for developing inhibitors of the aggrecanase molecule are known to those skilled in the art. Assays for the inhibitors involve contacting a mixture of aggrecan and the inhibitor with an aggrecanase molecule followed by measurement of the aggrecanase inhibition, for instance by detection and measurement of aggrecan fragments produced by cleavage at an aggrecanase susceptible site.

Another aspect of the invention therefore provides pharmaceutical compositions containing a therapeutically effective amount of aggrecanase inhibitors, in a pharmaceutically acceptable vehicle. Aggrecanase-mediated degradation of aggrecan in cartilage has been implicated in osteoarthritis and other inflammatory diseases. Therefore, these compositions of the invention may be used in the treatment of diseases characterized by the degradation of aggrecan and/or an upregulation of aggrecanase. The compositions may be used in the treatment of these conditions or in the prevention thereof.

The invention includes methods for treating patients suffering from conditions characterized by a degradation of aggrecan or preventing such conditions. These methods, according to the invention, entail administering to a patient needing such treatment, an effective amount of a composition comprising an aggrecanase inhibitor which inhibits the proteolytic activity of aggrecanase enzymes.

Still a further aspect of the invention are DNA sequences coding for expression of an aggrecanase protein. Such sequences include the sequence of nucleotides in a 5′ to 3′ direction illustrated in SEQ ID NO: 1 comprising nucleotide #1 to #3766 or comprising nucleotide #1086 to #3396 of SEQ ID NO: 1 or SEQ ID NO: 3 and DNA sequences which, but for the degeneracy of the genetic code, are identical to the DNA sequence of SEQ ID NO: 1 and SEQ ID NO: 3, and encode an aggrecanase protein. Further included in the present invention are DNA sequences which hybridize under stringent conditions with the DNA sequence of SEQ ID NO: 1 and SEQ ID NO: 3 and encode a protein having the ability to cleave aggrecan. Preferred DNA sequences include those which hybridize under stringent conditions (see, T. Maniatis et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to 389. It is generally preferred that such DNA sequences encode a polypeptide which is at least about 80% homologous, and more preferably at least about 90% homologous, to the sequence of set forth in SEQ ID NO: 3 or SEQ ID NO: 1 comprising nucleotide #1 to #3766 or comprising nucleotide #1086 to #3396 of SEQ ID NO: 1. Finally, allelic or other variations of the sequences of SEQ ID NO: 1 or SEQ ID NO: 3, whether such nucleotide changes result in changes in the peptide sequence or not, but where the peptide sequence still has aggrecanase activity, are also included in the present invention. The present invention also includes fragments of the DNA sequence shown in SEQ ID NO: 1 which encode a polypeptide which retains the activity of aggrecanase.

The DNA sequences of the present invention are useful, for example, as probes for the detection of mRNA encoding aggrecanase in a given cell population. Thus, the present invention includes methods of detecting or diagnosing genetic disorders involving the aggrecanase, or disorders involving cellular, organ or tissue disorders in which aggrecanase is irregularly transcribed or expressed. The DNA sequences may also be useful for preparing vectors for gene therapy applications as described below.

A further aspect of the invention includes vectors comprising a DNA sequence as described above in operative association with an expression control sequence therefor. These vectors may be employed in a novel process for producing an aggrecanase protein of the invention in which a cell line transformed with a DNA sequence encoding an aggrecanase protein in operative association with an expression control sequence therefor, is cultured in a suitable culture medium and an aggrecanase protein is recovered and purified therefrom. This process may employ a number of known cells both prokaryotic and eukaryotic as host cells for expression of the polypeptide. The vectors may be used in gene therapy applications. In such use, the vectors may be transfected into the cells of a patient ex vivo, and the cells may be reintroduced into a patient. Alternatively, the vectors may be introduced into a patient in vivo through targeted transfection.

Still a further aspect of the invention are aggrecanase proteins or polypeptides. Such polypeptides are characterized by having an amino acid sequence including the sequence illustrated in SEQ ID NO: 2 or 4, variants of the amino acid sequence of SEQ ID NO: 2 or 4, including naturally occurring allelic variants, and other variants in which the protein retains the ability to cleave aggrecan characteristic of aggrecanase molecules. Preferred polypeptides include a polypeptide which is at least about 80% homologous, and more preferably at least about 90% homologous, to the amino acid sequence shown in SEQ ID NO: 2 or 4. Finally, allelic or other variations of the sequences of SEQ ID NO: 2 or 4, whether such amino acid changes are induced by mutagenesis, chemical alteration, or by alteration of DNA sequence used to produce the polypeptide, where the peptide sequence still has aggrecanase activity, are also included in the present invention. The present invention also includes fragments of the amino acid sequence of SEQ ID NO: 2 or 4 which retain the activity of aggrecanase protein.

The purified proteins of the present inventions may be used to generate antibodies, either monoclonal or polyclonal, to aggrecanase and/or other aggrecanase-related proteins, using methods that are known in the art of antibody production. Thus, the present invention also includes antibodies to aggrecanase or other related proteins. The antibodies may be useful for detection and/or purification of aggrecanase or related proteins, or for inhibiting or preventing the effects of aggrecanase. The aggrecanase of the invention or portions thereof may be utilized to prepare antibodies that specifically bind to aggrecanase.

The human aggrecanase of the present invention comprises the nucleotide sequence set in SEQ ID NO: 3. In another embodiment, the human aggrecanase of the present invention comprises nucleotides #1 to #3766 or nucleotides #1086 to #3396 of SEQ ID NO: 1. The human aggrecanase protein sequence comprises the amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, the human aggrecanase protein sequence comprises amino acids #1 to #770 set forth in SEQ ID NO: 2. Further sequences of the aggrecanase of the present invention may be obtained using the sequences of SEQ ID NO: 3 or SEQ ID NO: 1 comprising nucleotides #1086 to #3396 to design probes for screening for the full sequence using standard techniques.

The aggrecanase proteins of the present invention, include polypeptides comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and having the ability to cleave aggrecan.

The aggrecanase proteins recovered from the culture medium are purified by isolating them from other proteinaceous materials from which they are co-produced and from other contaminants present. The isolated and purified proteins may be characterized by the ability to cleave aggrecan substrate. The aggrecanase proteins provided herein also include factors encoded by the sequences similar to those of SEQ ID NO: 3 or the sequence of SEQ ID NO: 1 comprising nucleotide #1 to #3766 or comprising nucleotide #1086 to #3396 of SEQ ID NO: 1, but into which modifications or deletions are naturally provided (e.g. allelic variations in the nucleotide sequence which may result in amino acid changes in the polypeptide) or deliberately engineered. For example, synthetic polypeptides may wholly or partially duplicate continuous sequences of the amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 4. These sequences, by virtue of sharing primary, secondary, or tertiary structural and conformational characteristics with aggrecanase molecules may possess biological properties in common therewith. It is know, for example that numerous conservative amino acid substitutions are possible without significantly modifying the structure and conformation of a protein, thus maintaining the biological properties as well. For example, it is recognized that conservative amino acid substitutions may be made among amino acids with basic side chains, such as lysine (Lys or K), arginine (Arg or R) and histidine (His or H); amino acids with acidic side chains, such as aspartic acid (Asp or D) and glutamic acid (Glu or E); amino acids with uncharged polar side chains, such as asparagine (Asn or N), glutamine (Gln or Q), serine (Ser or S), threonine (Thr or T), and tyrosine (Tyr or Y); and amino acids with nonpolar side chains, such as alanine (Ala or A), glycine (Gly or G), valine (Val or V), leucine (leu or L), isoleucine (Ile or I), proline (Pro or P), phenylalanine (Phe or F), methionine (Met or M), tryptophan (Trp or W) and cysteine (Cys or C). Thus, these modifications and deletions of the native aggrecanase may be employed as biologically active substitutes for naturally-occurring aggrecanase and in the development of inhibitors other polypeptides in therapeutic processes. It can be readily determined whether a given variant of aggrecanase maintains the biological activity of aggrecanase by subjecting both aggrecanase and the variant of aggrecanase, as well as inhibitors thereof, to the assays described in the examples.

Other specific mutations of the sequences of aggrecanase proteins described herein involve modifications of glycosylation sites. These modifications may involve O-linked or N-linked glycosylation sites. For instance, the absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at asparagine-linked glycosylation recognition sites. The asparagine-linked glycosylation recognition sites comprise tripeptide sequences which are specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or asparagine-X-serine, where X is usually any amino acid. A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence. Additionally, bacterial expression of aggrecanase-related protein will also result in production of a non-glycosylated protein, even if the glycosylation sites are left unmodified.

The present invention also encompasses the novel DNA sequences, free of association with DNA sequences encoding other proteinaceous materials, and coding for expression of aggrecanase proteins. These DNA sequences include those depicted in SEQ ID NO: 1 or 3 in a 5′ to 3′ direction and those sequences which hybridize thereto under stringent hybridization washing conditions (for example, 0.1×SSC, 0.1% SDS at 65° C.; see, T. Maniatis et al., Molecular Cloning ( A Laboratory Manual ), Cold Spring Harbor Laboratory (1982), pages 387 to 389) and encode a protein having aggrecanase proteolytic activity. These DNA sequences also include those which comprise the DNA sequence of SEQ ID NO: 1 and those which hybridize thereto under stringent hybridization conditions and encode a protein which maintain the other activities disclosed for aggrecanase.

Similarly, DNA sequences which code for aggrecanase proteins coded for by the sequences of SEQ ID NO: 1 comprising nucleotide #1 to #3766 or comprising nucleotide #1086 to #3396 of SEQ ID NO: 1 or the sequence of SEQ ID NO: 3 or aggrecanase proteins which comprise the amino acid sequence of SEQ ID NO: 2 or 4, but which differ in codon sequence due to the degeneracies of the genetic code or allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) also encode the novel factors described herein. Variations in the DNA sequences of SEQ ID NO: 3 or SEQ ID NO: 1 comprising nucleotide #1 to #3766 or comprising nucleotide #1086 to #3396 of SEQ ID NO: 1 which are caused by point mutations or by induced modifications (including insertion, deletion, and substitution) to enhance the activity, half-life or production of the polypeptides encoded are also encompassed in the invention.

Another aspect of the present invention provides a novel method for producing aggrecanase proteins. The method of the present invention involves culturing a suitable cell line, which has been transformed with a DNA sequence encoding a aggrecanase protein of the invention, under the control of known regulatory sequences. The transformed host cells are cultured and the aggrecanase proteins recovered and purified from the culture medium. The purified proteins are substantially free from other proteins with which they are co-produced as well as from other contaminants.

Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO). The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature , 293:620-625 (1981), or alternatively, Kaufman et al., Mol. Cell. Biol ., 5(7): 1750-1759 (1985) or Howley et al., U.S. Pat. No. 4,419,446. Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey COS-1 cell line. The mammalian cell CV-1 may also be suitable.

Bacterial cells may also be suitable hosts. For example, the various strains of E. coli (e.g., HB101, MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis , Pseudomonas, other bacilli and the like may also be employed in this method. For expression of the protein in bacterial cells, DNA encoding the propeptide of Aggrecanase is generally not necessary.

Many strains of yeast cells known to those skilled in the art may also be available as host cells for expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al., Genetic Engineering , 8:277-298 (Plenum Press 1986) and references cited therein.

Another aspect of the present invention provides vectors for use in the method of expression of these novel aggrecanase polypeptides. Preferably the vectors contain the full novel DNA sequences described above which encode the novel factors of the invention. Additionally, the vectors contain appropriate expression control sequences permitting expression of the aggrecanase protein sequences. Alternatively, vectors incorporating modified sequences as described above are also embodiments of the present invention. Additionally, the sequence of SEQ ID NO: 3 or SEQ ID NO: 1 or other sequences encoding aggrecanase proteins could be manipulated to express composite aggrecanase molecules. Thus, the present invention includes chimeric DNA molecules encoding an aggrecanase protein comprising a fragment from SEQ ID NO: 3 or SEQ ID NO: 1 comprising nucleotide #1 to #3766 or comprising nucleotide #1086 to #3396 of SEQ ID NO: 1 linked in correct reading frame to a DNA sequence encoding another aggrecanase polypeptide.

The vectors may be employed in the method of transforming cell lines and contain selected regulatory sequences in operative association with the DNA coding sequences of the invention which are capable of directing the replication and expression thereof in selected host cells. Regulatory sequences for such vectors are known to those skilled in the art and may be selected depending upon the host cells. Such selection is routine and does not form part of the present invention.

Various conditions such as osteoarthritis are known to be characterized by degradation of aggrecan. Therefore, an aggrecanase protein of the present invention which cleaves aggrecan may be useful for the development of inhibitors of aggrecanase. The invention therefore provides compositions comprising an aggrecanase inhibitor. The inhibitors may be developed using the aggrecanase in screening assays involving a mixture of aggrecan substrate with the inhibitor followed by exposure to aggrecan. The compositions may be used in the treatment of osteoarthritis and other conditions exhibiting degradation of aggrecan.

The invention further includes antibodies which can be used to detect aggrecanase and also may be used to inhibit the proteolytic activity of aggrecanase.

The therapeutic methods of the invention includes administering the aggrecanase inhibitor compositions topically, systemically, or locally as an implant or device. The dosage regimen will be determined by the attending physician considering various factors which modify the action of the aggrecanase protein, the site of pathology, the severity of disease, the patient's age, sex, and diet, the severity of any inflammation, time of administration and other clinical factors. Generally, systemic or injectable administration will be initiated at a dose which is minimally effective, and the dose will be increased over a preselected time course until a positive effect is observed. Subsequently, incremental increases in dosage will be made limiting such incremental increases to such levels that produce a corresponding increase in effect, while taking into account any adverse affects that may appear. The addition of other known factors, to the final composition, may also effect the dosage.

Progress can be monitored by periodic assessment of disease progression. The progress can be monitored, for example, by x-rays, MRI or other imaging modalities, synovial fluid analysis, and/or clinical examination.

The following examples illustrate practice of the present invention in isolating and characterizing human aggrecanase and other aggrecanase-related proteins, obtaining the human proteins and expressing the proteins via recombinant techniques.

Unknown Organism

Description of Unknown Organism Nucleotide

sequence of the aggrecanase molecule

1

gcggccgctg aattctaggg aggccccggg cgcggcgcag gctccaaaga agaagaaacc 60

aaggcccaga gagggaggcc caggtgcagg gagcaggcga gggaaggatc cgtacagggg 120

cccaacacta ctccaccaac cgaagccccc aaaaggagcc cggtgatgct gcgaaggctg 180

tgaacagggg aggcggcact gtgggggctg ccggcagccg gggctgggga gagacatgtg 240

gacacgtggc ctctatggct cccgcctgcc agatcctccg ctgggccctc gccctggggc 300

tgggcctcat gttcgaggtc acgcacgcct tccggtctca agatgagttc ctgtccagtc 360

tggagagcta tgagatcgcc ttccccaccc gcgtggacca caacggggca ctgctggcct 420

tctcgccacc tcctccccgg aggcagcgcc gcggcacggg ggccacagcc gagtcccgcc 480

tcttctacaa agtggcctcg cccagcaccc acttcctgct gaacctgacc cgcagctccc 540

gtctactggc agggcacgtc tccgtggagt actggacacg ggagggcctg gcctggcaga 600

gggcggcccg gccccactgc ctctacgctg gtcacctgca gggccaggcc agcagctccc 660

atgtggccat cagcacctgt ggaggcctgc acggcctgat cgtggcagac gaggaagagt 720

acctgattga gcccctgcac ggtgggccca agggttctcg gagcccggag gaaagtggac 780

cacatgtggt gtacaagcgt tcctctctgc gtcaccccca cctggacaca gcctgtggag 840

tgagagatga gaaaccgtgg aaagggcggc catggtggct gcggaccttg aagccaccgc 900

ctgccaggcc cctggggaat gaaacagagc gtggccagcc aggcctgaag cgatcggtca 960

gccgagagcg ctacgtggag accctggtgg tggctgacaa gatgatggtg gcctatcacg 1020

ggcgccggga tgtggagcag tatgtcctgg ccatcatgaa cattgttgcc aaacttttcc 1080

aggactcgag tctgggaagc accgttaaca tcctcgtaac tcgcctcatc ctgctcacgg 1140

aggaccagcc cactctggag atcacccacc atgccgggaa gtccctggac agcttctgta 1200

agtggcagaa atccatcgtg aaccacagcg gccatggcaa tgccattcca gagaacggtg 1260

tggctaacca tgacacagca gtgctcatca cacgctatga catctgcatc tacaagaaca 1320

aaccctgcgg cacactaggc ctggccccgg tgggcggaat gtgtgagcgc gagagaagct 1380

gcagcgtcaa tgaggacatt ggcctggcca cagcgttcac cattgcccac gagatcgggc 1440

acacattcgg catgaaccat gacggcgtgg gaaacagctg tggggcccgt ggtcaggacc 1500

cagccaagct catggctgcc cacattacca tgaagaccaa cccattcgtg tggtcatcct 1560

gcagccgtga ctacatcacc agctttctag actcagggcc tggggctctg cctgaacaac 1620

cggcccccca gacaggactt tgtgtacccg acagtggcac cgggccaagc ctacgatgca 1680

gatgagcaat gccgctttca gcatggagtc aaatcgcgtc agtgtaaata cgggaggtct 1740

gcagcgagct gtggtgtctg agcaagagca accggtgcat caccaacagc atcccggccg 1800

ccgagggcac gctgtgccag acgcacacca tcgacaaggg gtggtgctac aaacgggtct 1860

gtgtcccctt tgggtcgcgc ccagagggtg tggacggagc ctgggggccg tggactccat 1920

ggggcgactg cagccggacc tgtggcggcg gcgtgtcctc ttctagccgt cactgcgaca 1980

gccccaggcc aaccatcggg ggcaagtact gtctgggtga gagaaggcgg caccgctcct 2040

gcaacacgga tgactgtccc cctggctccc aggacttcag agaagtgcag tgttctgaat 2100

ttgacagcat ccctttccgt gggaaattct acaagtggaa aacgtaccgg ggagggggcg 2160

tgaaggcctg ctcgctcacg tgcctagcgg aaggcttcaa cttctacacg gagagggcgg 2220

cagccgtggt ggacgggaca ccctgccgtc cagacacggt ggacatttgc gtcagtggcg 2280

aatgcaagca cgtgggctgc gaccgagtcc tgggctccga cctgcgggag gacaagtgcc 2340

gagtgtgtgg cggtgacggc agtgcctgcg agaccatcga gggcgtcttc agcccagcct 2400

cacctggggc cgggtacgag gatgtcgtct ggattcccaa aggctccgtc cacatcttca 2460

tccaggatct gaacctctct ctcagtcact tggccctgaa gggagaccag gagtccctgc 2520

tgctggaggg gctgcccggg accccccagc cccaccgtct gcctctagct gggaccacct 2580

ttcaactgcg acaggggcca gaccaggtcc agagcctcga agccctggga ccgattaatg 2640

catctctcat cgtcatggtg ctggcccgga ccgagctgcc tgccctccgc taccgcttca 2700

atgcccccat cgcccgtgac tcgctgcccc cctactcctg gcactatgcg ccctggacca 2760

agtgctcggc ccagtgtgca ggcggtagcc aggtgcaggc ggtggagtgc cgcaaccagc 2820

tggacagctc cgcggtcgcc ccccactact gcagtgccca cagcaagctg cccaaaaggc 2880

agcgcgcctg caacacggag ccttgccctc cagactgggt tgtagggaac tggtcgctct 2940

gcagccgcag ctgcgatgca ggcgtgcgca gccgctcggt cgtgtgccag cgccgcgtct 3000

ctgccgcgga ggagaaggcg ctggacgaca gcgcatgccc gcagccgcgc ccacctgtac 3060

tggaggcctg ccacggcccc acttgccctc cggagtgggc ggccctcgac tggtctgagt 3120

gcacccccag ctgcgggccg ggcctccgcc accgcgtggt cctttgcaag agcgcagacc 3180

accgcgccac gctgcccccg gcgcactgct cacccgccgc caagccaccg gccaccatgc 3240

gctgcaactt gcgccgctgc cccccggccc gctgggtggc tggcgagtgg ggtgagtgct 3300

ctgcacagtg cggcgtcggg cagcggcagc gctcggtgcg ctgcaccagc cacacgggcc 3360

aggcgtcgca cgagtgcacg gaggccctgc ggccgcccac cacgcagcaa tgtgaggcca 3420

agtgcgacag cccaaccccc gggggcggcc ctgaagagtg caaggatgtg aacaaggtcg 3480

cctactgccc cctggtgctc aaatttcagt tctgcagccg agcctacttc cgccagatgt 3540

gctgcaaaac ctgccagggc cactaggggg cgcgcggcac ccggagccac agctggcggg 3600

gtctccgccg ccagtcctgc agcgggccgg ccagaggggg ccccgggggg gcgggaactg 3660

ggagggaagg gtgagacgga gccggaagtt atttattggg aacccctgca gggccctggc 3720

tggggggatg gagaggggct ggctatccac ctgcccgggc ggccgc 3766

2

770

Unknown Organism

Description of Unknown Organism Amino acid

sequence of the aggrecanase molecule

2

Ser Ser Leu Gly Ser Thr Val Asn Ile Leu Val Thr Arg Leu Ile Leu

1 5 10 15

Leu Thr Glu Asp Gln Pro Thr Leu Glu Ile Thr His His Ala Gly Lys

20 25 30

Ser Leu Asp Ser Phe Cys Lys Trp Gln Lys Ser Ile Val Asn His Ser

35 40 45

Gly His Gly Asn Ala Ile Pro Glu Asn Gly Val Ala Asn His Asp Thr

50 55 60

Ala Val Leu Ile Thr Arg Tyr Asp Ile Cys Ile Tyr Lys Asn Lys Pro

65 70 75 80

Cys Gly Thr Leu Gly Leu Ala Pro Val Gly Gly Met Cys Glu Arg Glu

85 90 95

Arg Ser Cys Ser Val Asn Glu Asp Ile Gly Leu Ala Thr Ala Phe Thr

100 105 110

Ile Ala His Glu Ile Gly His Thr Phe Gly Met Asn His Asp Gly Val

115 120 125

Gly Asn Ser Cys Gly Ala Arg Gly Gln Asp Pro Ala Lys Leu Met Ala

130 135 140

Ala His Ile Thr Met Lys Thr Asn Pro Phe Val Trp Ser Ser Cys Ser

145 150 155 160

Arg Asp Tyr Ile Thr Ser Phe Leu Asp Ser Gly Pro Gly Ala Leu Pro

165 170 175

Glu Gln Pro Ala Pro Gln Thr Gly Leu Cys Val Pro Asp Ser Gly Thr

180 185 190

Gly Pro Ser Leu Arg Cys Arg Xaa Ala Met Pro Leu Ser Ala Trp Ser

195 200 205

Gln Ile Ala Ser Val Xaa Ile Arg Glu Val Cys Ser Glu Leu Trp Cys

210 215 220

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

225 230 235 240

Gly Thr Leu Cys Gln Thr His Thr Ile Asp Lys Gly Trp Cys Tyr Lys

245 250 255

Arg Val Cys Val Pro Phe Gly Ser Arg Pro Glu Gly Val Asp Gly Ala

260 265 270

Trp Gly Pro Trp Thr Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly

275 280 285

Gly Val Ser Ser Ser Ser Arg His Cys Asp Ser Pro Arg Pro Thr Ile

290 295 300

Gly Gly Lys Tyr Cys Leu Gly Glu Arg Arg Arg His Arg Ser Cys Asn

305 310 315 320

Thr Asp Asp Cys Pro Pro Gly Ser Gln Asp Phe Arg Glu Val Gln Cys

325 330 335

Ser Glu Phe Asp Ser Ile Pro Phe Arg Gly Lys Phe Tyr Lys Trp Lys

340 345 350

Thr Tyr Arg Gly Gly Gly Val Lys Ala Cys Ser Leu Thr Cys Leu Ala

355 360 365

Glu Gly Phe Asn Phe Tyr Thr Glu Arg Ala Ala Ala Val Val Asp Gly

370 375 380

Thr Pro Cys Arg Pro Asp Thr Val Asp Ile Cys Val Ser Gly Glu Cys

385 390 395 400

Lys His Val Gly Cys Asp Arg Val Leu Gly Ser Asp Leu Arg Glu Asp

405 410 415

Lys Cys Arg Val Cys Gly Gly Asp Gly Ser Ala Cys Glu Thr Ile Glu

420 425 430

Gly Val Phe Ser Pro Ala Ser Pro Gly Ala Gly Tyr Glu Asp Val Val

435 440 445

Trp Ile Pro Lys Gly Ser Val His Ile Phe Ile Gln Asp Leu Asn Leu

450 455 460

Ser Leu Ser His Leu Ala Leu Lys Gly Asp Gln Glu Ser Leu Leu Leu

465 470 475 480

Glu Gly Leu Pro Gly Thr Pro Gln Pro His Arg Leu Pro Leu Ala Gly

485 490 495

Thr Thr Phe Gln Leu Arg Gln Gly Pro Asp Gln Val Gln Ser Leu Glu

500 505 510

Ala Leu Gly Pro Ile Asn Ala Ser Leu Ile Val Met Val Leu Ala Arg

515 520 525

Thr Glu Leu Pro Ala Leu Arg Tyr Arg Phe Asn Ala Pro Ile Ala Arg

530 535 540

Asp Ser Leu Pro Pro Tyr Ser Trp His Tyr Ala Pro Trp Thr Lys Cys

545 550 555 560

Ser Ala Gln Cys Ala Gly Gly Ser Gln Val Gln Ala Val Glu Cys Arg

565 570 575

Asn Gln Leu Asp Ser Ser Ala Val Ala Pro His Tyr Cys Ser Ala His

580 585 590

Ser Lys Leu Pro Lys Arg Gln Arg Ala Cys Asn Thr Glu Pro Cys Pro

595 600 605

Pro Asp Trp Val Val Gly Asn Trp Ser Leu Cys Ser Arg Ser Cys Asp

610 615 620

Ala Gly Val Arg Ser Arg Ser Val Val Cys Gln Arg Arg Val Ser Ala

625 630 635 640

Ala Glu Glu Lys Ala Leu Asp Asp Ser Ala Cys Pro Gln Pro Arg Pro

645 650 655

Pro Val Leu Glu Ala Cys His Gly Pro Thr Cys Pro Pro Glu Trp Ala

660 665 670

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

675 680 685

His Arg Val Val Leu Cys Lys Ser Ala Asp His Arg Ala Thr Leu Pro

690 695 700

Pro Ala His Cys Ser Pro Ala Ala Lys Pro Pro Ala Thr Met Arg Cys

705 710 715 720

Asn Leu Arg Arg Cys Pro Pro Ala Arg Trp Val Ala Gly Glu Trp Gly

725 730 735

Glu Cys Ser Ala Gln Cys Gly Val Gly Gln Arg Gln Arg Ser Val Arg

740 745 750

Cys Thr Ser His Thr Gly Gln Ala Ser His Glu Cys Thr Glu Ala Leu

755 760 765

Arg Pro

770

3

3377

Unknown Organism

Description of Unknown Organism Nucleotide

sequence of the aggrecanase molecule

3

gaattcccac catggctccc gcctgccaga tcctccgctg ggccctcgcc ctggggctgg 60

gcctcatgtt cgaggtcacg cacgccttcc ggtctcaaga tgagttcctg tccagtctgg 120

agagctatga gatcgccttc cccacccgcg tggaccacaa cggggcactg ctggccttct 180

cgccacctcc tccccggagg cagcgccgcg gcacgggggc cacagccgag tcccgcctct 240

tctacaaagt ggcctcgccc agcacccact tcctgctgaa cctgacccgc agctcccgtc 300

tactggcagg gcacgtctcc gtggagtact ggacacggga gggcctggcc tggcagagag 360

cggcccggcc ccactgcctc tacgctggtc acctgcaggg ccaggccagc agctcccatg 420

tggccatcag cacctgtgga ggcctgcacg gcctgatcgt ggcagacgag gaagagtacc 480

tgattgagcc cctgcacggt gggcccaagg gttctcggag cccggaggaa agtggaccac 540

atgtggtgta caagcgttcc tctctgcgtc acccccacct ggacacagcc tgtggagtga 600

gagatgagaa accgtggaaa gggcggccat ggtggctgcg gaccttgaag ccaccgcctg 660

ccaggcccct ggggaatgaa acagagcgtg gccagccagg cctgaagcga tcggtcagcc 720

gagagcgcta cgtggagacc ctggtggtgg ctgacaagat gatggtggcc tatcacgggc 780

gccgggatgt ggagcagtat gtcctggcca tcatgaacat tgttgccaaa cttttccagg 840

actcgagtct gggaagcacc gttaacatcc tcgtaactcg cctcatcctg ctcacggagg 900

accagcccac tctggagatc acccaccatg ccgggaagtc cctggacagc ttctgtaagt 960

ggcagaaatc catcgtgaac cacagcggtc atggcaatgc cattccagag aacggtgtgg 1020

ctaaccatga cacagcagtg ctcatcacac gctatgacat ctgcatctac aagaacaaac 1080

cctgcggcac actaggcctg gccccggtgg gcggaatgtg tgagcgcgag agaagctgca 1140

gcgtcaatga ggacattggc ctggccacag cgttcaccat tgcccacgag atcgggcaca 1200

cattcggcat gaaccatgac ggcgtgggaa acagctgtgg ggcccgtggt caggacccag 1260

ccaagctcat ggctgcccac attaccatga agaccaaccc gttcgtgtgg tcatcctgca 1320

gccgtgacta catcaccagc tttctagact cgggcctggg gctctgcctg aacaaccgac 1380

cccccagaca ggactttgtg tacccgacag tggcaccggg ccaagcctac gatgcagatg 1440

agcaatgccg ctttcagcat ggagtcaaat cgcgtcagtg taaatacggg gaggtctgca 1500

gcgagctgtg gtgtctgagc aagagcaacc ggtgcatcac caacagcatc ccggccgccg 1560

agggcacgct gtgccagacg cacaccatcg acaaggggtg gtgctacaaa cgggtctgtg 1620

tcccctttgg gtcgcgccca gagggtgtgg acggagcctg ggggccgtgg actccatggg 1680

gcgactgcag ccggacctgt ggcggcggcg tgtcctcttc tagccgtcac tgcgacagcc 1740

ccaggccaac catcgggggc aagtactgtc tgggtgagag aaggcggcac cgctcctgca 1800

acacggatga ctgtccccct ggctcccagg acttcagaga agtgcagtgt tctgaatttg 1860

acagcatccc tttccgtggg aaattctaca agtggaaaac gtaccgggga gggggcgtga 1920

aggcctgctc gctcacgtgc ctagcggaag gcttcaactt ctacacggag agggcggcag 1980

ccgtggtgga cgggacaccc tgccgtccag acacggtgga catttgcgtc agtggcgaat 2040

gcaagcacgt gggctgcgac cgagtcctgg gctccgacct gcgggaggac aagtgccgag 2100

tgtgtggcgg tgacggcagt gcctgcgaga ccatcgaggg cgtcttcagc ccagcctcac 2160

ctggggccgg gtacgaggat gtcgtctgga ttcccaaagg ctccgtccac atcttcatcc 2220

aggatctgaa cctctctctc agtcacttgg ccctgaaggg agaccaggag tccctgctgc 2280

tggaggggct gcccgggacc ccccagcccc accgtctgcc tctagctggg accacctttc 2340

aactgcgaca ggggccagac caggtccaga gcctcgaagc cctgggaccg attaatgcat 2400

ctctcatcgt catggtgctg gcccggaccg agctgcctgc cctccgctac cgcttcaatg 2460

cccccatcgc ccgtgactcg ctgcccccct actcctggca ctatgcgccc tggaccaagt 2520

gctcggccca gtgtgcaggc ggtagccagg tgcaggcggt ggagtgccgc aaccagctgg 2580

acagctccgc ggtcgccccc cactactgca gtgcccacag caagctgccc aaaaggcagc 2640

gcgcctgcaa cacggagcct tgccctccag actgggttgt agggaactgg tcgctctgca 2700

gccgcagctg cgatgcaggc gtgcgcagcc gctcggtcgt gtgccagcgc cgcgtctctg 2760

ccgcggagga gaaggcgctg gacgacagcg catgcccgca gccgcgccca cctgtactgg 2820

aggcctgcca cggccccact tgccctccgg agtgggcggc cctcgactgg tctgagtgca 2880

cccccagttg cgggccgggc ctccgccacc gcgtggtcct ttgcaagagc gcagaccacc 2940

gcgccacgct gcccccggcg cactgctcac ccgccgccaa gccaccggcc accatgcgct 3000

gcaacttgcg ccgctgcccc ccggcccgct gggtggctgg cgagtggggt gagtgctctg 3060

cacagtgcgg cgtcgggcag cggcagcgct cggtgcgctg caccagccac acgggccagg 3120

cgtcgcacga gtgcacggag gccctgcggc cgcccaccac gcagcagtgt gaggccaagt 3180

gcgacagccc aacccccggg gacggccctg aagagtgcaa ggatgtgaac aaggtcgcct 3240

actgccccct ggtgctcaaa tttcagttct gcagccgagc ctacttccgc cagatgtgct 3300

gcaaaacctg ccagggccac tagggtcgag gcccatttaa gccgaattct gcagatatcc 3360

atcacactgg cggccgc 3377

4

1104

Unknown Organism

Description of Unknown Organism Amino acid

sequence of the aggrecanase molecule

4

Met Ala Pro Ala Cys Gln Ile Leu Arg Trp Ala Leu Ala Leu Gly Leu

1 5 10 15

Gly Leu Met Phe Glu Val Thr His Ala Phe Arg Ser Gln Asp Glu Phe

20 25 30

Leu Ser Ser Leu Glu Ser Tyr Glu Ile Ala Phe Pro Thr Arg Val Asp

35 40 45

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

50 55 60

Arg Arg Gly Thr Gly Ala Thr Ala Glu Ser Arg Leu Phe Tyr Lys Val

65 70 75 80

Ala Ser Pro Ser Thr His Phe Leu Leu Asn Leu Thr Arg Ser Ser Arg

85 90 95

Leu Leu Ala Gly His Val Ser Val Glu Tyr Trp Thr Arg Glu Gly Leu

100 105 110

Ala Trp Gln Arg Ala Ala Arg Pro His Cys Leu Tyr Ala Gly His Leu

115 120 125

Gln Gly Gln Ala Ser Ser Ser His Val Ala Ile Ser Thr Cys Gly Gly

130 135 140

Leu His Gly Leu Ile Val Ala Asp Glu Glu Glu Tyr Leu Ile Glu Pro

145 150 155 160

Leu His Gly Gly Pro Lys Gly Ser Arg Ser Pro Glu Glu Ser Gly Pro

165 170 175

His Val Val Tyr Lys Arg Ser Ser Leu Arg His Pro His Leu Asp Thr

180 185 190

Ala Cys Gly Val Arg Asp Glu Lys Pro Trp Lys Gly Arg Pro Trp Trp

195 200 205

Leu Arg Thr Leu Lys Pro Pro Pro Ala Arg Pro Leu Gly Asn Glu Thr

210 215 220

Glu Arg Gly Gln Pro Gly Leu Lys Arg Ser Val Ser Arg Glu Arg Tyr

225 230 235 240

Val Glu Thr Leu Val Val Ala Asp Lys Met Met Val Ala Tyr His Gly

245 250 255

Arg Arg Asp Val Glu Gln Tyr Val Leu Ala Ile Met Asn Ile Val Ala

260 265 270

Lys Leu Phe Gln Asp Ser Ser Leu Gly Ser Thr Val Asn Ile Leu Val

275 280 285

Thr Arg Leu Ile Leu Leu Thr Glu Asp Gln Pro Thr Leu Glu Ile Thr

290 295 300

His His Ala Gly Lys Ser Leu Asp Ser Phe Cys Lys Trp Gln Lys Ser

305 310 315 320

Ile Val Asn His Ser Gly His Gly Asn Ala Ile Pro Glu Asn Gly Val

325 330 335

Ala Asn His Asp Thr Ala Val Leu Ile Thr Arg Tyr Asp Ile Cys Ile

340 345 350

Tyr Lys Asn Lys Pro Cys Gly Thr Leu Gly Leu Ala Pro Val Gly Gly

355 360 365

Met Cys Glu Arg Glu Arg Ser Cys Ser Val Asn Glu Asp Ile Gly Leu

370 375 380

Ala Thr Ala Phe Thr Ile Ala His Glu Ile Gly His Thr Phe Gly Met

385 390 395 400

Asn His Asp Gly Val Gly Asn Ser Cys Gly Ala Arg Gly Gln Asp Pro

405 410 415

Ala Lys Leu Met Ala Ala His Ile Thr Met Lys Thr Asn Pro Phe Val

420 425 430

Trp Ser Ser Cys Ser Arg Asp Tyr Ile Thr Ser Phe Leu Asp Ser Gly

435 440 445

Leu Gly Leu Cys Leu Asn Asn Arg Pro Pro Arg Gln Asp Phe Val Tyr

450 455 460

Pro Thr Val Ala Pro Gly Gln Ala Tyr Asp Ala Asp Glu Gln Cys Arg

465 470 475 480

Phe Gln His Gly Val Lys Ser Arg Gln Cys Lys Tyr Gly Glu Val Cys

485 490 495

Ser Glu Leu Trp Cys Leu Ser Lys Ser Asn Arg Cys Ile Thr Asn Ser

500 505 510

Ile Pro Ala Ala Glu Gly Thr Leu Cys Gln Thr His Thr Ile Asp Lys

515 520 525

Gly Trp Cys Tyr Lys Arg Val Cys Val Pro Phe Gly Ser Arg Pro Glu

530 535 540

Gly Val Asp Gly Ala Trp Gly Pro Trp Thr Pro Trp Gly Asp Cys Ser

545 550 555 560

Arg Thr Cys Gly Gly Gly Val Ser Ser Ser Ser Arg His Cys Asp Ser

565 570 575

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

580 585 590

His Arg Ser Cys Asn Thr Asp Asp Cys Pro Pro Gly Ser Gln Asp Phe

595 600 605

Arg Glu Val Gln Cys Ser Glu Phe Asp Ser Ile Pro Phe Arg Gly Lys

610 615 620

Phe Tyr Lys Trp Lys Thr Tyr Arg Gly Gly Gly Val Lys Ala Cys Ser

625 630 635 640

Leu Thr Cys Leu Ala Glu Gly Phe Asn Phe Tyr Thr Glu Arg Ala Ala

645 650 655

Ala Val Val Asp Gly Thr Pro Cys Arg Pro Asp Thr Val Asp Ile Cys

660 665 670

Val Ser Gly Glu Cys Lys His Val Gly Cys Asp Arg Val Leu Gly Ser

675 680 685

Asp Leu Arg Glu Asp Lys Cys Arg Val Cys Gly Gly Asp Gly Ser Ala

690 695 700

Cys Glu Thr Ile Glu Gly Val Phe Ser Pro Ala Ser Pro Gly Ala Gly

705 710 715 720

Tyr Glu Asp Val Val Trp Ile Pro Lys Gly Ser Val His Ile Phe Ile

725 730 735

Gln Asp Leu Asn Leu Ser Leu Ser His Leu Ala Leu Lys Gly Asp Gln

740 745 750

Glu Ser Leu Leu Leu Glu Gly Leu Pro Gly Thr Pro Gln Pro His Arg

755 760 765

Leu Pro Leu Ala Gly Thr Thr Phe Gln Leu Arg Gln Gly Pro Asp Gln

770 775 780

Val Gln Ser Leu Glu Ala Leu Gly Pro Ile Asn Ala Ser Leu Ile Val

785 790 795 800

Met Val Leu Ala Arg Thr Glu Leu Pro Ala Leu Arg Tyr Arg Phe Asn

805 810 815

Ala Pro Ile Ala Arg Asp Ser Leu Pro Pro Tyr Ser Trp His Tyr Ala

820 825 830

Pro Trp Thr Lys Cys Ser Ala Gln Cys Ala Gly Gly Ser Gln Val Gln

835 840 845

Ala Val Glu Cys Arg Asn Gln Leu Asp Ser Ser Ala Val Ala Pro His

850 855 860

Tyr Cys Ser Ala His Ser Lys Leu Pro Lys Arg Gln Arg Ala Cys Asn

865 870 875 880

Thr Glu Pro Cys Pro Pro Asp Trp Val Val Gly Asn Trp Ser Leu Cys

885 890 895

Ser Arg Ser Cys Asp Ala Gly Val Arg Ser Arg Ser Val Val Cys Gln

900 905 910

Arg Arg Val Ser Ala Ala Glu Glu Lys Ala Leu Asp Asp Ser Ala Cys

915 920 925

Pro Gln Pro Arg Pro Pro Val Leu Glu Ala Cys His Gly Pro Thr Cys

930 935 940

Pro Pro Glu Trp Ala Ala Leu Asp Trp Ser Glu Cys Thr Pro Ser Cys

945 950 955 960

Gly Pro Gly Leu Arg His Arg Val Val Leu Cys Lys Ser Ala Asp His

965 970 975

Arg Ala Thr Leu Pro Pro Ala His Cys Ser Pro Ala Ala Lys Pro Pro

980 985 990

Ala Thr Met Arg Cys Asn Leu Arg Arg Cys Pro Pro Ala Arg Trp Val

995 1000 1005

Ala Gly Glu Trp Gly Glu Cys Ser Ala Gln Cys Gly Val Gly Gln Arg

1010 1015 1020

Gln Arg Ser Val Arg Cys Thr Ser His Thr Gly Gln Ala Ser His Glu

1025 1030 1035 1040

Cys Thr Glu Ala Leu Arg Pro Pro Thr Thr Gln Gln Cys Glu Ala Lys

1045 1050 1055

Cys Asp Ser Pro Thr Pro Gly Asp Gly Pro Glu Glu Cys Lys Asp Val

1060 1065 1070

Asn Lys Val Ala Tyr Cys Pro Leu Val Leu Lys Phe Gln Phe Cys Ser

1075 1080 1085

Arg Ala Tyr Phe Arg Gln Met Cys Cys Lys Thr Cys Gln Gly His Xaa

1090 1095 1100

5

11

Artificial Sequence

Description of Artificial Sequence Synthetic

zinc binding signature sequence

5

Thr Ala Ala His Glu Leu Gly His Val Lys Phe

1 5 10

6

28

Artificial Sequence

Description of Artificial Sequence Probe

6

ggtcaaatcg cgtcagtgta aatacggg 28

7

30

Artificial Sequence

Description of Artificial Sequence Primer

7

agtctagaaa gctggtgatg tagtcacggc 30

8

35

Artificial Sequence

Description of Artificial Sequence Primer

8

tagatgcata tgtcatagcg tgtgatgagc actgc 35

9

29

Artificial Sequence

Description of Artificial Sequence Probe

9

ctcgagtctg ggaagcaccg ttaacatcc 29

10

27

Artificial Sequence

Description of Artificial Sequence Probe

10

gaaggcgatc tcatagctct ccagact 27

11

31

Artificial Sequence

Description of Artificial Sequence Primer

11

gctctagact ggtctgagtg cacccccagc t 31

12

24

Artificial Sequence

Description of Artificial Sequence Primer

12

gtcctttgca agagcgcaga ccac 24

13

24

Artificial Sequence

Description of Artificial Sequence Probe

13

gcactgtgca gagcactcac ccca 24

14

45

Artificial Sequence

Description of Artificial Sequence Primer

14

aaatgggcga attcccacca tggctcccgc ctgccagatc ctccg 45

15

26

Artificial Sequence

Description of Artificial Sequence Primer

15

ccgagtctag aaagctggtg atgtag 26

16

185

Artificial Sequence

Description of Artificial Sequence Synthetic

oligonucleotide

16

ctagactcgg gcctggggct ctgcctgaac aaccgacccc ccagacagga ctttgtgtac 60

ccgacagtgg caccgggcca agcctacgat gcagatgagc aatgccgctt tcagcatgga 120

gtcaaatcgc gtcagtgtaa atacggggag gtctgcagcg agctgtggtg tctgagcaag 180

agcaa 185

17

185

Artificial Sequence

Description of Artificial Sequence Synthetic

oligonucleotide

17

ccggttgctc ttgctcagac accacagctc gctgcagacc tccccgtatt tacactgacg 60

cgatttgact ccatgctgaa agcggcattg ctcatctgca tcgtaggctt ggcccggtgc 120

cactgtcggg tacacaaagt cctgtctggg gggtcggttg ttcaggcaga gccccaggcc 180

cgagt 185

18

25

Artificial Sequence

Description of Artificial Sequence Primer

18

gggttgtagg gaactggtcg ctctg 25

19

39

Artificial Sequence

Description of Artificial Sequence Primer

19

aaatgggcct cgagccctag tggccctggc aggttttgc 39

20

15

Artificial Sequence

Description of Artificial Sequence Synthetic

oligonucleotide

20

catgggcagc tcgag 15

21

34

Artificial Sequence

Description of Artificial Sequence Synthetic

oligonucleotide

21

ctgcaggcga gcctgaattc ctcgagccat catg 34

22

68

Artificial Sequence

Description of Artificial Sequence Synthetic

oligonucleotide

22

cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 60

acgattgc 68