AU2002245758A1 - Novel aggrecanase molecules - Google Patents

Description

TITLE OF THE INVENTION

NOVEL AGGRECANASE MOLECULES

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.

BACKGROUND OF THE INVENTION

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, KD. and Mankin HJ. Pathogenesis of Osteoarthritis. in Textbook of Rheumatology, WB 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 (Asn³ l-Phe³⁴²) is observed to be cleaved by several known metalloproteases [Flannery, CR et al. J Biol Chem 267:1008-14. 1992; Fosang, AJ 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 Glu³⁷³-Ala3⁷⁴ bond [Sandy, JD, et al. J Clin Invest 69:1512-1516. (1992); Lohmander LS, et al.

Arthritis Rheum 36: 1214-1222. (1993): Sandy JD 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( AD AMTS 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 MD, 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 Glu³⁷³-Ala3⁷⁴ bond and could contribute to aggrecan cleavage in osteoarthritis. SUMMARY OF THE INVENTION

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 dete minable 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.

DETAILED DESCRIPTION OF THE INVENTION

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 (Gin 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 (He 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, 0.1X 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. Patent 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 CN-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, DΝA 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.

EXAMPLES

EXAMPLE 1 Isolation of DNA

Potential novel aggrecanase family members were identified using a database

screening approach. Aggrecanase-1 [Science284: 1664-1666 (1999)] has at least six domains: signal, propeptide, catalytic domain, disintegrin, tsp and c-terminal. The catalytic domain contains a zinc binding signature region, TAAHELGHVKF and a "MET turn" which are responsible for protease activity. Substitutions within the zinc binding region in the number of the positions still allow protease activity, but the histidine (H) and glutamic acid (E) residues must be present. The thrombospondin domain of Aggrecanase-1 is also a critical domain for substrate recognition and cleavage. It is these two domains that determine our classification of a novel aggrecanase family member.

The protein sequence of the Aggrecanase-1 DNA sequence was used to query against the GeneBank ESTs focusing on human ESTs using TBLASTN. The resulting sequences were the starting point in the effort to identify full length sequence for potential family members. The nucleotide sequence of the aggrecanase of the present invention is comprised of one EST (AA588434) that contains homology over the catalytic domain and zinc binding motif of Aggrecanase-1 (ADAMTS4).

This human aggrecanase sequence was isolated from a dT-primed cDNA library constructed in the plasmid vector pED6-dpc2. cDNA was made from human testes RNA purchased from Clontech. The probe to isolate the aggrecanase of the present invention was generated from the sequence obtained from the database search. The sequence of the probe was as follows: 5'-GGTCAAATCGCGTCAGTGTAAATACGGG-3' . The DNA probe was radioactively labeled with ²P and used to screen the human testes dT-primed cDNA library, under high stringency hybridization/washing conditions, to identify clones containing sequences of the human candidate #8.

Fifty thousand library transformants were plated at a density of approximately 5000 transformants per plate on 10 plates. Nitrocellulose replicas of the transformed colonies were hybridized to the ³²P labeled DNA probe in standard hybridization buffer (IX Blotto[25X Blotto = %5 nonfat dried milk,0.02% azide in dH2O] + 1% NP-40 + 6X SSC +0.05% Pyrophosphate) under high stringency conditions (65°C for 2 hours shaking). After 2 hours hybridization, the radioactively labeled DNA probe containing hybridization solution was removed and the filters were washed under high stringency conditions (3X SSC, 0.05% Pyrophosphate for 5 minutes at RT; followed by 2.2X SSC, 0.05% Pyrophosphate for 15 minutes at RT; followed by 2.2X SSC, 0.05% Pyrophosphate for 1-2 minutes at 65 °C). The filters were wrapped in Saran wrap and exposed to X-ray film for overnight. The autoradiographs were developed and positively hybridizing transformants of various signal intensities were identified. These positive clones were picked; grown for 12 hours in selective medium(L-broth plus lOOμg/ml ampicillin) and plated at low density (approximately 100 colonies per plate). Nitrocellulose replicas of the colonies were hybridized to the ³²P labeled probe in standard hybridization buffer ((IX Blotto[25X Blotto = %5 nonfat dried milk, 0.02% azide in dH2O] + 1% NP-40 + 6X SSC +0.05% Pyrophosphate) under high stringency conditions (65°C for 2 hours). After 2 hours hybridization, the radioactively labeled DNA probe containing hybridization solution was removed and the filters were washed under high stringency conditions (3X SSC, 0.05% Pyrophosphate for 5 minutes at RT; followed by 2.2X SSC, 0.05% Pyrophosphate for 15 minutes at RT; followed by 2.2X SSC, 0.05% Pyrophosphate for 1-2 minutes at 65°C). The filters were wrapped in Saran wrap and exposed to X-ray film for overnight. The autoradiographs were developed and positively hybridizing transformants were identified. Bacterial stocks of purified hybridization positive clones were made and plasmid DNA was isolated. The sequence of the cDNA insert was determined and is set forth in SEQ ID NO. 1 from nucleotide # 1086 (TCG) through # 3396 (CGC). This sequence has been deposited in the American Type Culture Collection 10801 University Blvd. Manassas, VA 20110-2209 USA as PTA -2284. The cDNA insert contained the sequences of the DNA probe used in the hybridization. The 5'(prime) and 3' (prime) sequences of this isolated sequence was then extracted using the RACE protocol. The fully determined sequence is set forth in SEQ ID NO: 1 from nucleotide # 1 to # 3766 . The human candidate #8 sequence obtained aligns with several ESTs in the public database. Candididate #8 shows homology with ADAMTS 7 and 6. The aggrecanase of the present invention contains the zinc binding signature region, a "MET turn", and tsp type -1 motif, however is missing the signal and propeptide regions and c-terminal spacer regions. It is with these criteria that candidate #8 is considered a novel Aggrecanase family member.

The aggrecanase sequence of the invention can be used to design probes for further screening for full length clones containing the isolated sequence.

The 5P (signal and propeptide) and 3P (C-terminal spacer regions) ends of the

full-length version of EST8 were determined by RACE PCR using the Clontech Marathon

cDNA Amplification Kit. The testes and stomach Marathon cDNA sources were used as

substrates for the RACE reactions. 5P RACE primers used in the reactions were; GSP1 -

AGTCTAGAAAGCTGGTGATGTAGTCACGGC and GSP2 -

TAGATGCATATGTCATAGCGTGTGATGAGCACTGC (contains a Nsil site). The

Advantage-2 PCR Kit from Clontech was used to set up nested RACE reactions following

instructions in the user manual for the Marathon cDNA Amplification Kit; the amount the

GSP primers used was 0.2 pmol/ul of each PCR primer/ul of reaction mix.

GSP1 primer was used for the first round of PCR and GSP2 primer was used for the

nested reaction. Products from the nested RACE reactions were digested with Nsil (on

the GSP2 primer) and Notl (on the AP2 primer provided in the Clontech kit and used in

the nested RACE PCR) and ligated into the CS2+ vector cut with Nsil and Notl. Ligated

products were transformed into ElectroMAX DH10B cells from Life Technologies. Cloned RACE products were plated at low density (approximately 300 colonies per plate). Nitrocellulose replicas of the transformed colonies were hybridized to a ³²P labeled DNA probe in standard hybridization buffer (IX Blotto [25X Blotto =5% nonfat dried milk, 0.02% azide], 1% NP-40, 6X SSC, 0.05% pyrophosphate) under high stringency conditions (65°C for 2 hours shaking). Sequence at the 5P end of candidate 8- 1 was used as a DNA probe: CTCGAGTCTGGGAAGCACCGTTAACATCC.

After 2 hours, the hybridization solution (hybridization buffer containing lxlO⁶ cpm ³² P labeled DNA probe) was removed and the filters were washed under high stringency conditions (3X SSC, 0.05% pyrophosphate for 5 minutes at RT standing; followed by 2.2X SSC, 0.05% pyrophosphate for 15 minutes shaking at RT; followed by 2.2X SSC, 0.05% pyrophosphate for 1-2 minutes shaking at 65 °C). The filters were covered with Saran Wrap and exposed to X-ray film overnight. The autoradiographs were developed and positively hybridizing transformants of various signal intensities were identified. These positive clones were picked and then grown for 12 hours in selective medium (L-broth plus lOOug/ml ampicillin). Plasmid DNA was prepared and sent for DNA sequence analysis. A second round of hybridizations was performed using a probe that was made to sequence more 5P than candidate 8-1. The DNA probe sequence was deduced from the 5P RACE products. The second probe sequence was as follows:

GAAGGCGATCTCATAGCTCTCCAGACT. Cloned RACE products were again plated and the same hybridization protocol was followed, except using the more 5P probe. The initiator Met was deduced from a consensus sequence derived from the 5P RACE products generated from both the testes and the stomach cDNAs. 3P RACE primers used were; GSP1 - GCTCTAGACTGGTCTGAGTGCACCCCCAGCT and GSP2 -

GTCCTTTGCAAGAGCGCAGACCAC. The Advantage GC-2 PCR Kit from Clontech

was used to set up nested RACE reactions. Reactions were set up following the

instructions in the user manual for the Marathon cDNA Amplification Kit; with the

exception that the amount of GC melt used was 5ul per 50ul reaction; the amount the GSP

primers used was 0.2 pmol/ul of each PCR primer/ul of reaction mix. GSP1 primer was

used for the first round of PCR and GSP2 primer was used for the nested reaction.

Products from the nested RACE reactions were ligated into the pT-Adv vector using the

AdvanTAge PCR Cloning Kit, per manufacturer's instructions. Ligated products were

transformed into ElectroMAX DH10B cells from Life Technologies. Cloned RACE

products were plated at low density (approximately 300 colonies per plate).

Nitrocellulose replicas of the transformed colonies were hybridized to a ³²P labeled DNA

probe in standard hybridization buffer (IX Blotto [25X Blotto =5% nonfat dried milk,

0.02% azide], 1% NP-40, 6X SSC, 0.05% pyrophosphate) under high stringency

conditions (65^°C for 2 hours shaking). Sequence at the 3P end of candidate 8-1 was used

as a DNA probe: GCACTGTGCAGAGCACTCACCCCA. After 2 hours, the

hybridization solution (hybridization buffer containing 1x10^s cpm ³²P labeled DNA

probe) was removed and the filters were washed under high stringency conditions (3X

SSC, 0.05% pyrophosphate for 5 minutes at RT standing; followed by 2.2X SSC, 0.05%

pyrophosphate for 15 minutes shaking at RT; followed by 2.2X SSC, 0.05%

pyrophosphate for 1-2 minutes shaking at 65°C). The filters were covered with Saran

Wrap and exposed to X-ray film overnight. The autoradiographs were developed and positively hybridizing transformants of various signal intensities were identified. The

positive clones were picked and then grown for 12 hours in selective medium (L-broth

plus lOOug/ml ampicillin). Plasmid DNA was prepared and sent for DNA sequence

analysis. The stop codon was deduced from a consensus sequence derived from the 3P

RACE products generated from both the testes and the stomach cDNAs.

With the exception of the region from base pair 1332 to 1517(for this description

base pair #1 is A of the initiator Met (ATG), the full-length sequence of EST8 was

confirmed. A search of the public databases revealed a partial sequence for EST8 ,

termed ADAMTS10. We used the sequence from this partial clone to construct the

contiguous region of our EST8 (base pair 1332 to 1517) with synthetic oligonucleotides.

The full-length sequence for EST8(SEQ ID NO:3) was the consensus sequence

derived from the hybridization positive candidate 8-1, the publicly available sequences

representing EST8, and the PCR products from the Clontech testes and stomach cDNAs.

The final EST8 expression construct was assembled from 4 EST8 specific fragments.

The 5P portion of EST8, from base pair 1 - 1342, was PCR amplified from a pool of

stomach and testes cDNAs and will be termed fragment 1. The following primers were

used; 5P PCR primer -

AAATGGGCGAATTCCCACCATGGCTCCCGCCTGCCAGATCCTCCG (contains an

8 base pair linker (AAATGGGC) an EcoRl cloning site (GAATTC) and a Kozak

sequence (CCACC) upstream of the initiator Met) and 3P PCR primer -

CCGAGTCTAGAAAGCTGGTGATGTAG (contains an Xbal site (TCTAGA)). This PCR product was digested with EcoRl and Xbal using standard digestion conditions. The next portion of the gene, fragment 2, was constructed using synthetic

oligonucleotides. The synthetic fragment stretched from an Xbal site to a BsrFl site

representing base pair 1333 to 1517 of EST8. The synthetic oligonucleotides consisted of

the following sequence: the top strand consisted of - CTAGACTCGGGCCTGGGGCTCTGCCTGAACAACCGACCCCCCAGACAGGACT

TTGTGTACCCGACAGTGGCACCGGGCCAAGCCTACGATGCAGATGAGCAATG

CCGCTTTCAGCATGGAGTCAAATCGCGTCAGTGTAAATACGGGGAGGTCTGC

AGCGAGCTGTGGTGTCTGAGCAAGAGCAA; the bottom strand consisted of -

CCGGTTGCTCTTGCTCAGACACCACAGCTCGCTGCAGACCTCCCCGTATTTAC

ACTGACGCGATTTGACTCCATGCTGAAAGCGGCATTGCTCATCTGCATCGTAG

GCTTGGCCCGGTGCCACTGTCGGGTACACAAAGTCCTGTCTGGGGGGTCGGTT

GTTCAGGCAGAGCCCCAGGCCCGAGT. The next portion of EST8, fragment 3, was

a BsrFl to Sphl fragment digested from candidate 8-1. This represented from base pair

1518 to 2783 of the full-length version of EST8. The 3P portion of EST8, termed

fragment 4 (base pair 2663 to 3314), was PCR amplified. The following primers were

used; 5P - GGGTTGTAGGGAACTGGTCGCTCTG (located within fragment 3,

upstream of the Sphl site) and 3P -

AAATGGGCCTCGAGCCCTAGTGGCCCTGGCAGGTTTTGC (contains an 8 base pair linker (AAATGGGC) and a Xhol site (CTCGAG) downstream of the stop codon

(TAG)). This PCR product was digested with Sphl and Xhol using standard digestion

conditions. A full-length version of EST8 was constructed by ligating these 4 described

fragments, 5P fragment 1 (EcoRl/Xbal), internal fragment 2 (Xbal/BsrFl), internal fragment 3 (BsrFl/Sphl), and 3P fragment 4 (Sphl/Xhol) into the Cos expression vector

pED6-dpc2 (digested with EcoRl and Xhol). The final construct had a mutation in the

Xhol cloning site, which was destroyed in the ligation. This did not effect the EST8

coding sequence and was left in the construct.

EXAMPLE 2

Expression of Aggrecanase

In order to produce murine, human or other mammalian aggrecanase-related

proteins, the DNA encoding it is transferred into an appropriate expression vector and

introduced into mammalian cells or other preferred eukaryotic or prokaryotic hosts

including insect host cell culture systems by conventional genetic engineering techniques.

Expression system for biologically active recombinant human aggrecanase is

contemplated to be stably transformed mammalian cells, insect, yeast or bacterial cells.

One skilled in the art can construct mammalian expression vectors by employing

the sequence of SEQ ID NO: 3 or the sequence of SEQ ID NO: 1 comprising nucleotide

# 1 to # 3766 or comprising nuclotide # 1086 to #3396 of SEQ ID NO lor other DNA

sequences encoding aggrecanase-related proteins or other modified sequences and known

vectors, such as pCD [Okayama et al, Mol. Cell Biol.. 2:161-170 (1982)], pJL3, pJL4 [Gough et al., EMBO I, 4:645-653 (1985)] and pMT2 CXM.

The mammalian expression vector pMT2 CXM is a derivative of p91023(b)

(Wong et al, Science 228:810-815, 1985) differing from the latter in that it contains the

ampicillin resistance gene in place of the tetracycline resistance gene and further contains a Xhol site for insertion of cDNA clones. The functional elements of pMT2 CXM have

been described (Kaufman, R J., 1985, Proc. Natl. Acad. Sci. USA 82:689-693) and

include the adenovirus VA genes, the SN40 origin of replication including the 72 bp

enhancer, the adenovirus major late promoter including a 5' splice site and the majority of

the adenovirus tripartite leader sequence present on adenovirus late mRΝAs, a 3' splice

acceptor site, a DHFR insert, the SN40 early polyadenylation site (SV40), and pBR322

sequences needed for propagation in K coli.

Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which has

been deposited with the American Type Culture Collection (ATCC), Rockville, MD

(USA) under accession number ATCC 67122. EcoRI digestion excises the cDΝA insert

present in pMT2-NWF, yielding pMT2 in linear form which can be ligated and used to

transform E. coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2 DΝA can be

prepared by conventional methods. pMT2 CXM is then constructed using loopout/in

mutagenesis [Morinaga, et al.. Biotechnology 84: 636 (1984). This removes bases 1075

to 1145 relative to the Hind -HI site near the SN40 origin of replication and enhancer

sequences of pMT2. In addition it inserts the following sequence:

5' PO-CATGGGCAGCTCGAG-3'

at nucleotide 1145. This sequence contains the recognition site for the restriction

endonuclease Xho I. A derivative of pMT2CXM, termed pMT23, contains recognition

sites for the restriction endonucleases Pstl, Eco RI, Sail and Xhol. Plasmid pMT2 CXM

and pMT23 DΝA may be prepared by conventional methods. pEMC2βl derived from pMT21 may also be suitable in practice of the invention.

pMT21 is derived from pMT2 which is derived from pMT2-NWF. As described above

EcoRI digestion excises the cDΝA insert present in pMT-NWF, yielding pMT2 in linear

form which can be ligated and used to transform R Coli HB 101 or DH-5 to ampicillin

resistance. Plasmid pMT2 DΝA can be prepared by conventional methods.

pMT21 is derived from pMT2 through the following two modifications. First, 76

bp of the 5' untranslated region of the DHFR cDΝA including a stretch of 19 G residues

from G/C tailing for cDΝA cloning is deleted. In this process, a Xhol site is inserted to

obtain the following sequence immediately upstream from DHFR: 5' -

CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3'

Pstl Eco RI Xhol

Second, a unique Clal site is introduced by digestion with EcoRN and Xbal, treatment

with Klenow fragment of DΝA polymerase I, and ligation to a Clal linker (CATCGATG).

This deletes a 250 bp segment from the adenovirus associated RΝA (NAI) region but

does not interfere with NAI RΝA gene expression or function. pMT21 is digested with

EcoRI and Xhol, and used to derive the vector pEMC2Bl.

A portion of the EMCN leader is obtained from pMT2-ECATl [S.K. Jung, et al, L

Nirol 63:1651-1660 (1989)] by digestion with Eco RI and Pstl, resulting in a 2752 bp

fragment. This fragment is digested with Taql yielding an Eco RI-Taql fragment of 508

bp which is purified by electrophoresis on low melting agarose gel. A 68 bp adapter and its complementary strand are synthesized with a 5' Taql protruding end and a 3' Xhol

protruding end which has the following sequence:

5'-CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT Taql

GAAAAACACGATTGC-3' Xhol

This sequence matches the EMC virus leader sequence from nucleotide 763 to 827. It

also changes the ATG at position 10 within the EMC virus leader to an ATT and is

followed by a Xhol site. A three way ligation of the pMT21 Eco RI-16hoI fragment, the

EMC virus EcoRI-Taql fragment, and the 68 bp

oligonucleotide adapter TaqI-16hoI adapter resulting in the vector pEMC2βl.

This vector contains the SN40 origin of replication and enhancer, the adenovirus

major late promoter, a cDΝA copy of the majority of the adenovirus tripartite leader

sequence, a small hybrid intervening sequence, an SN40 polyadenylation signal and the

adenovirus NA I gene, DHFR and β-lactamase markers and an EMC sequence, in

appropriate relationships to direct the high level expression of the desired cDΝA in

mammalian cells.

The construction of vectors may involve modification of the aggrecanase-related

DΝA sequences. For instance, aggrecanase cDΝA can be modified by removing the non-

coding nucleotides on the 5' and 3' ends of the coding region. The deleted non-coding nucleotides may or may not be replaced by other sequences known to be beneficial for

expression. These vectors are transformed into appropriate host cells for expression of

aggrecanase-related proteins. Additionally, the sequence of SEQ ID NO: 3 or SEQ ID

NO: 1 comprising nucleotide # 1 to # 3766 or comprising nuclotide # 1086 to #3396 of

SEQ ID NO lor other sequences encoding aggrecanase-related proteins can be

manipulated to express a mature aggrecanase-related protein by deleting aggrecanase

encoding propeptide sequences and replacing them with sequences encoding the complete

propeptides of other aggrecanase proteins.

One skilled in the art can manipulate the sequences of SEQ ID NO: 3 or SEQ ID

NO: 1 by eliminating or replacing the mammalian regulatory sequences flanking the

coding sequence with bacterial sequences to create bacterial vectors for intracellular or

extracellular expression by bacterial cells. For example, the coding sequences could be

further manipulated (e.g. ligated to other known linkers or modified by deleting non-

coding sequences therefrom or altering nucleotides therein by other known techniques).

The modified aggrecanase-related coding sequence could then be inserted into a known

bacterial vector using procedures such as described in T. Taniguchi et al., Proc. Natl

Acad. Sci. USA. 77:5230-5233 (1980). This exemplary bacterial vector could then be

transformed into bacterial host cells and a aggrecanase-related protein expressed thereby.

For a strategy for producing extracellular expression of aggrecanase-related proteins in

bacterial cells, see, e.g. European patent application EPA 177,343.

Similar manipulations can be performed for the construction of an insect vector [See, e.g. procedures described in published European patent application 155,476] for expression in insect cells. A yeast vector could also be constructed employing yeast

regulatory sequences for intracellular or extracellular expression of the factors of the

present invention by yeast cells. [See, e.g., procedures described in published PCT

application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a aggrecanase-related protein of the

invention in mammalian, bacterial, yeast or insect host cell systems may involve the

construction of cells containing multiple copies of the heterologous Aggrecanase-related

gene. The heterologous gene is linked to an amplifiable marker, e.g. the dihydrofolate

reductase (DHFR) gene for which cells containing increased gene copies can be selected

for propagation in increasing concentrations of methotrexate (MTX) according to the

procedures of Kaufman and Sharp, J. Mol. Biol.. 159:601-629 (1982). This approach can

be employed with a number of different cell types.

For example, a plasmid containing a DNA sequence for an aggrecanase-related

protein of the invention in operative association with other plasmid sequences enabling

expression thereof and the DHFR expression plasmid pAdA26SN(A)3 [Kaufman and

Sharp, Mol. Cell. Biol.. 2:1304 (1982)] can be co-introduced into DHFR-deficient CHO

cells, DUKX-BII, by various methods including calcium phosphate coprecipitation and

transfection, electroporation or protoplast fusion. DHFR expressing transformants are

selected for growth in alpha media with dialyzed fetal calf serum, and subsequently

selected for amplification by growth in increasing concentrations of MTX (e.g. sequential

steps in 0.02, 0.2, 1.0 and 5uM MTX) as described in Kaufman et al., Mol Cell Biol..

5:1750 (1983). Transformants are cloned, and biologically active aggrecanase expression is monitored by the assays described above. Aggrecanase protein expression should

increase with increasing levels of MTX resistance. Aggrecanase polypeptides are

characterized using standard techniques known in the art such as pulse labeling with

[35S] methionine or cysteine and polyacrylamide gel electrophoresis. Similar procedures

can be followed to produce other related aggrecanase-related proteins.

In one example the aggrecanase gene of the present invention set forth in SEQ ID

NO:3 is cloned into the expression vector pED6 [Kaufman et al., Nucleic Acid Res.

19:44885-4490(1991)]. COS and CHO DUKX Bll cells are transiently transfected with

the aggrecanase sequence of the invention (+/- co-transfection of PACE on a separate

pED6 plaasmid) by lipofection(LF2000, Invitrogen). Duplicate tranfections are

performed for each gene of interest: (a) one for harvesting conditioned media for activity

assay and (b) one for 35-S-methionine/cysteine metabolic labeling.

On day one media is changed to DME(COS) or alpha(CHO) media + 1% heat-

inactivated fetal calf serum+/- lOOμg/ml heparin on wells(a) to be harvested for activity

assay. After 48h (day4), conditioned media is harvested for activity assay.

On day 3, the duplicate wells(b) were changed to MEM (methiooine-

free/cyysteine free) media + 1% heat-inactivated fetal calif serum +100μg/ml heparin +

lOOμCi/ml 35S-methioine/cysteine (Redivue Pro mix, Amersham). Following 6h

incubation at 37°C, conditioned media was harvested and run on SDS-PAGE gels under reducing conditions. Proteins are visualized by autoradiography. EXAMPLE 3

Biological Activity of Expressed Aggrecanase

To measure the biological activity of the expressed aggrecanase-related proteins

obtained in Example 2 above, the proteins are recovered from the cell culture and purified

by isolating the aggrecanase-related proteins from other proteinaceous materials with

which they are co-produced as well as from other contaminants. The purified protein may

be assayed in accordance with assays described above. Purification is carried out using

standard techniques known to those skilled in the art.

Protein analysis is conducted using standard techniques such as SDS-PAGE

acrylamide [Laemmli, Nature 227:680 (1970)] stained with silver [Oakley, et al. Anal.

Biochem. 105:361 (1980)] and by immunoblot [Towbin, et al. Proc. Natl. Acad. Sci. USA 76:4350 (1979)].

The foregoing descriptions detail presently preferred embodiments of the present

invention. Numerous modifications and variations in practice thereof are expected to

occur to those skilled in the art upon consideration of these descriptions. Those

modifications and variations are believed to be encompassed within the claims appended hereto.

Claims (20)

What is claimed is:

1. An isolated DNA molecule comprising a DNA sequence set forth in SEQ ID NO.

2. An isolated DNA molecule comprising a DNA sequence set forth in SEQ ID NO.

1 from nucleotide #1086 to # 3396.

3. An isolated DNA molecule comprising a DNA sequence selected from the group

consisting of

a) the sequence of SEQ ID NO. 3, b) the sequence of SEQ ID NO: 1 comprising nuclotide # 1086 to #3396

naturally occurring human allelic sequences and equivalent degenerative

codon sequences of a) and b).

4. A vector comprising a DNA molecule of claim 1 in operative association with an

expression control sequence therefor.

5. A vector comprising a DNA molecule of claim 2 in operative association with an

expression control sequence therefor.

6. A host cell transformed with the DNA sequence of claim 1.

7. A host cell transformed with a DNA sequence of claim 2.

8. A method for producing a purified human aggrecanase protein, said method comprising the steps of:

(a) culturing a host cell transformed with a DNA molecule according to claim 1; and

(b) recovering and purifying said aggrecanase protein from the culture medium.

9. A method for producing a purified human aggrecanase protein, said method comprising the steps of:

(a) culturing a host cell transformed with a DNA molecule according to claim 2; and

(b) recovering and purifying said aggrecanase protein from the culture medium.

10. A purified aggrecanase polypeptide comprising the amino acid sequence of SEQ

ID NO:4.

11. A purified aggrecanase polypeptide comprising the amino acid sequence set forth in SEQ ID NO 2.

12. A purified aggrecanase polypeptide produced by the steps of (a) culturing a cell transformed with a DNA molecule according to claim 1;

and

(b) recovering and purifying from said culture medium a polypeptide

comprising the amino acid sequence set forth in SEQ ID NO.4.

13. A purified aggrecanase polypeptide produced by the steps of

(a) culturing a cell transformed with a DNA molecule according to claim 2; and

(b) recovering and purifying from said culture medium a polypeptide

comprising the amino acid sequence set forth in SEQ ID NO. 2.

14. An antibody that binds to a purified aggrecanase protein of claim 10.

15. A method for developing inhibitors of aggrecanase comprising the use of

aggrecanase protein set forth in SEQ ID NO. 4 or a fragment thereof.

16. A method for developing inhibitors of aggrecanase comprising the use of

aggrecanase protein set forth in SEQ ID NO. 2 or a fragment thereof.

17. The method of claim 15 or 16 wherein said method comprises three dimensional structural analysis.

18. The method of claim 15 or 16 wherein said method comprises computer aided

drug design.

19. A composition for inhibiting the proteolytic activity of aggrecanase comprising a

peptide molecule which binds to the aggrecanase inhibiting the proteolytic

degradation of aggrecan.

20. A method for inhibiting the cleavage of aggrecan in a mammal comprising

administering to said mammal an effective amount of a compound that inhibits

aggrecanase activity.