Assays And Peptide Substrate For Determining Aggrecan Degrading Metallo Protease Activity

Abstract

This invention is directed to assays to determine the presence or absence of proteins that exhibit aggrecanase or ADMP activity. This invention also relates to peptides that acts as a substrates for ADMPs, their use in various assays to determine the presence or absence of ADMP activity, and their use as inhibitors of ADMP activity.

Description

[0001] This application is a divisional application of U.S. application Ser. No. 09/975,813, filed on Oct. 12, 2001, which is a divisional application of U.S. application Ser. No. 09/122,127, filed on Jul. 25, 1997 (now U.S. Pat. No. 6,326,162), which claims benefit to three U.S. Provisional Applications: U.S. Provisional Application No. 60/053850 filed on Jul. 25, 1997, U.S. Provisional Application No. 60/055836 filed on Aug. 15, 1997; and U.S. Provisional Application No. 60/062,169, filed on Oct. 16, 1997. Each of the priority applications is incorporated by reference in their entireties.

FIELD OF INVENTION

[0002] This invention is directed to various assays for determining aggrecanase or aggrecan degrading metallo protease (ADMP) activity. This invention also relates to a peptide substrate for ADMPs; the use of the peptide substrate in various assays to determine the presence or absence of ADMP activity; and the use of the substrate as an inhibitor of ADMP activity.

BACKGROUND OF THE INVENTION

[0003] Aggrecan is the major proteoglycan of cartilage and provides this tissue with its mechanical properties of compressibility and elasticity. In arthritic conditions one of the earliest changes observed in cartilage morphology is the depletion of aggrecan, which appears to be due to an increased rate of degradation. Mankin et al., (1970) J. Bone Joint Surg. 52A, 424-434.

[0004] The aggrecan molecule comprises two N-terminal globular domains: G1 and G2. The G1 and G2 domains are separated by an approximately 150 residue interglobular domain (IGD), followed by a long central glycosaminoglycan (GAG) attachment region and a C-terminal globular domain, G3. Hardingham et al., (1992) in Articular Cartilage and Osteoarthritis: Aggrecan, The Chondroitin Sulfate/Keratan Sulfate Proteoglycan from Cartilage (Kuettner et al.) pp. 5-20, Raven Press, New York and Paulson et al., (1987) Biochem. J. 245, 763-772. These aggrecan molecules interact through the G1 domain with hyaluronic acid and a link protein, to form large molecular weight aggregates which are trapped within the cartilage matrix. Hardingham et al., (1972) Biochim. Biophys. Acta 279, 401-405; Heinegard et al., (1974) J. Biol. Chem. 249, 4250-4256; and Hardingham, T. E. (1979) Biochem. J. 177, 237-247. Loss of aggrecan from cartilage in arthritic conditions involves proteolytic cleavage of the aggrecan core protein within the IGD, producing an N-terminal G-1 fragment bound to hyaluronic acid and the link protein within the matrix. A large C-terminal GAG-containing aggrecan fragment is released and diffuses out of the cartilage matrix. Loss of the C-terminal fragment results in cartilage having deficient mechanical properties. The deficiency arises because the GAGs, which are present on the C-terminal portion of the aggrecan core protein, comprise components of aggrecan that impart beneficial mechanical properties to the molecule through their high negative charge and water binding capacity.

[0005] IGD has two major proteolytic cleavage sites: one between amino acid residues Asn.sup.341-Phe.sup.342 and the other between residues Glu.sup.373-Ala.sup.374 (human sequence enumeration). G1 fragments formed as a result of cleavage at the Asn.sup.341-Phe.sup.342 site and at the Glu.sup.373-Ala.sup.374 site have been identified within articular cartilage. However, the "ARGSVIL", is the only N-terminal sequence identified on GAG-containing aggrecan C-terminal aggrecan fragments in synovial fluids of patients with osteoarthritis, inflammatory joint disease, and in the media from cartilage explant and chondrocyte cultures stimulated with interleukin-1 or retinoic acid. This finding suggests that the fragments were formed by cleavage between amino acid residues Glu.sup.373-Ala..sup.374 These observations suggest that cleavage at this site may be responsible for cartilage degradation. See Flannery et al., (1992) J. Biol. Chem. 267, 1008-1014; Sandy et al., (1992) J. Clin. Invest. 69, 1512-1516; Lohmander et al., (1993) Arthritis Rheum. 36, 1214-1222; Sandy et al., (1991) J. Biol. Chem. 266, 8198; Sandy et al., (1991) J. Biol. Chem. 266, 8683-8685; Leulakis et al., (1992) Biochem. J. 264, 589-593; Ilic et al., (1992) Arch. Biochem. Biophys. 294, 115-122; and Lark et al., (1995) J. Biol. Chem. 270, 2550-2556.

[0006] Although many matrix metalloproteases (e.g., MMP-1, -2, -3, -7, -8,-9 and 13) have been shown to cleave aggrecan at the Asn.sup.341-Phe.sup.342 site in vitro, digestion of aggrecan with a number of purified proteases demonstrates that there is no cleavage at the Glu.sup.373-Ala.sup.374 site. Fosang et al., (1992) J. Biol. Chem. 267, 19470-19474; Flannery et al., (1992) J. Biol. Chem. 267, 1008-1014; Fosang et al., (1993) Biochem. J. 295, 273-276; Fosang et al., (1996) FEBS Lett. 380, 17-20; Flannery et al., (1993) Orthop. Trans., 17, 677; and Fosang et al., (1994) Biochem. J. 305, 347-351. Cleavage at this site has been attributed to a novel, proteolytic activity termed "aggrecanase" activity.

[0007] In addition to the Glu.sup.373-Ala.sup.374 bond within the IGD of aggrecan, four potential aggrecanase-sensitive sites have been identified within the C-terminus of the aggrecan core protein outside of the IGD. Loulakis et al., (1992) Biochem. J. 264, 589-593. and Sandy et al., (1995) Acta Orhtop Scand (Suppl 266) 66, 26-32. Although cleavage at these sites outside of the IGD would not be expected to release the major portion of the aggrecan molecule from the matrix, the cleavage sites may nonetheless be involved in processing of aggrecan within the matrix.

[0008] It follows from the foregoing considerations that a sensitive and specific assay for detection of aggrecanase activity by ADMPs would be beneficial to identify inhibitors of members of the ADMP family, which could serve as potential therapeutic agents for treating aggrecanase-related disorders.

SUMMARY OF THE INVENTION

[0009] A preferred embodiment of the invention provides an assay that facilitates determination of ADMP activity.

[0010] A preferred embodiment of the invention provides an assay using purified native aggrecan or recombinant aggrecan as the substrate and monitoring product generation via an enzyme-linked immunosorbent assay (ELISA) using neoepitope antibodies to detect the newly formed N- or C-terminal aggrecan epitopes formed by specific cleavage at an ADMP-sensitive site in the aggrecan core protein.

[0011] A preferred embodiment of the invention provides peptides that have been found to act as substrates for the family of ADMPs. One peptide, based on the human aggrecan sequence around the Glu.sup.373-Ala.sup.374 ADMP-sensitive site has the sequence: TABLE-US-00001 (SEQ ID NO:1) QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL

[0012] A second peptide, based on the bovine aggrecan sequence around the Glu.sup.373-Ala.sup.374 ADMP-sensitive site has the sequence: TABLE-US-00002 (SEQ ID NO:2) QTVTWPDVELPLPRNITEGE-ARGSVILTAKPDFEVSPTAPE

[0013] Both peptides are capable of being cleaved at this specific recognition site by members of the family of ADMP proteins. A third peptide, based on the human aggrecan sequence around the Ala.sup.1714-Gly.sup.1715 ADMP-sensitive site has the sequence: TABLE-US-00003 (SEQ ID NO:3) ITFVDTSLVEVTPTTFKEEE-GLGSVELSGLPSGELGVSGT

and is capable of being cleaved at this specific recognition site by the family of ADMP proteins.

[0014] A preferred embodiment of the invention provides assay formats and methods of utilizing these peptide substrates for the detection and quantification of ADMP activity.

[0015] Another preferred embodiment of the invention provides a modified version of the peptide substrates and a method for their use as an inhibitor of ADMP activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Preferred embodiments of the invention have been chosen for purposes of illustration and description, but are not intended in any way to restrict the scope of the invention. The preferred embodiments of certain aspects of the invention are shown in the accompanying drawings described below.

[0017] FIG. 1. Shows the activity of a biotinylated form of the 41-mer peptide substrate (41-PS) against ADMP enzymatic activity using the microplate assay format and the inhibition of that activity by a hydroxymate inhibitor compound.

[0018] FIG. 2. Shows the activity of a biotinylated form of the 41-mer peptide substrate (41-PS) against ADMP enzymatic activity using the HPLC assay format.

[0019] FIG. 3. Shows the activity of the biotinylated form of the 41-mer peptide substrate (41-PS) against ADMP enzymatic activity using the microplate assay format and the inhibition of that activity by the 30-mer inhibitor peptide (30-IP), QTVTWP-DMELPLPRNITEGQARGSVILTVK-Biotin, the sequence of which is based upon the sequence of the 41-PS.

DETAILED DESCRIPTION OF THE INVENTION

[0020] A family of ADMP proteins cleave the aggrecan core protein at the Glu.sup.373-Ala.sup.374 peptide bond and exhibit enzymatic activity referred to as "aggrecanase" activity. Flannery et al., (1992) J. Biol. Chem. 267, 1008-1014. The presence of ADMP enzymatic activity can be determined by monitoring the production of aggrecan fragments generated exclusively by cleavage at the Glu.sup.373-Ala.sup.374 peptide bond within the aggrecan core protein. These aggrecan fragments are detected using neoepitope antibodies to the N- or C-terminal epitopes produced by specific cleavage at this ADMP-sensitive site. The neoepitope antibodies that may be used include, but are not limited to, BC-3 monoclonal antibodies. See (Hughes, C. E., et al., Biochem. J. 306:799-804, 1995; U.S. Provisional Patent Application Ser. No. 60/006,684 and U.S. patent application Ser. No. 08/743,439.

[0021] ADMP activity may be detected by monitoring the production of fragments formed by cleavage at alternative ADMP-sensitive sites using neoepitope antibodies to the C-or N-terminal epitopes generated by ADMP-specific cleavage at these sites. Alternative sites in the aggrecan core protein encompass, but are not limited to, the E.sup.1545-G.sup.1546, E.sup.1714-G.sup.1715, E.sup.1819-A.sup.1820, or E.sup.1919-L.sup.1920 bond (numbering based on the human aggrecan core protein sequence).

[0022] A preferred assay format involves using purified native aggrecan or recombinant aggrecan as the substrate with product detection via ELISA, herein referred to as the "Problot assay", using neoepitope antibodies to the C- or N-terminal epitopes generated upon specific cleavage at ADMP-sensitive sites within the aggrecan core protein. Alternative sites in the aggrecan core protein encompass, but are not limited to, the E.sup.1545-G.sup.1546, E.sup.1714-G.sup.1715, E.sup.1819-A.sup.1820, or E.sup.1919-L.sup.1920 bond (numbering based on the human aggrecan core protein sequence). These human aggrecan ADMP-sensitive cleavage sites are conserved in aggrecan from various animal species although the absolute numbering based on the sequence of the aggrecan core protein may vary from species to species. Conserved amino acid sequences in various species around conserved ADMP-sensitive sites are shown below. TABLE-US-00004 Human NITEGE.sup.373 .sup.374ARGSVILT Bovine NITEGE ARGSVILT Rat NITEGE ARGNVILT Mouse NVTEGE ALGSVILT Pig NITEGE ARGTVILT Sheep NITEGE ARGNVILT Chicken NVTEEE ARGSI Horse NITEGE ARGNVILT Human ASTASELE.sup.1545 .sup.1546GRGTIGIS Bovine ATTAGELE GRGTIDIS Mouse ATTSSELE GRGTIGIS Rat ATTASELE GRGTISVS Human PTTFKEEE.sup.1714 .sup.1715GLGSVELS Bovine PTTFKEEE GLGSVELS Rat PTTFREEE GLGSVELS Mouse PTTFREEE GLGSVELS Human TQAPTAQE.sup.1819 .sup.1820AGEGPSGI Bovine TQAPTAQE AGEGPSGI Rat TLAPTAQE AGEGPSSI Mouse TQAPTAQE AGEGPSGI Chicken TQTSVAQE VGEGPSGM Human TEPTISQE.sup.1919 .sup.1920LGQRPPVT Bovine TEPTVSQE LGQRPPVT Rat TEPTVSQE LGHGPSMT Mouse TEPTVSQE LGHGPSMT Chicken TRPTVSQE LGGETAVT Dog TEPTVSQE LAQRPPVT

[0023] Thus, aggrecan from various animal species, including but not limited to, bovine, dog, pig, rat, mouse, sheep, horse and chicken may also be used as a substrate for detecting ADMP activity.

[0024] The direct ELISA assay employs 96-well filtration plates containing polyvinyl-denedifluoride (PVDF) cationically charged membranes. These membranes are semi-selective in binding the highly negatively-charged aggrecan, which allows for binding of detectable levels of neoepitope antibody-reactive aggrecan fragments from solutions containing high levels of other proteins.

[0025] Utilizing neoepitope antibodies allows detection of fragments formed specifically by ADMP-mediated cleavage even in the presence of other proteolytic activities which may be present in crude preparations. Thus, the Problot assay can be used to monitor ADMP activity in culture medium containing other proteases, as well as to monitor the activity of the purified ADMP enzyme. Therefore, this assay has particular use in following ADMP activity during purification from tissue or media samples, as well as for use in enzymatic assays to evaluate inhibitors of the ADMP enzyme. The Problot assay can also be used to detect ADMP-generated aggrecan fragments in culture media from tissue or cell cultures stimulated to induce ADMP-mediated degradation. This assay may also be useful for detecting ADMP-generated aggrecan fragments in cartilage, synovial fluid, serum, urine or other biological samples from patients with ADMP-associated diseases.

[0026] Peptide substrates are commonly employed in a variety of assays to determine the presence of enzymes that catalyze the hydrolysis of proteins. One skilled in the art would rely on the use of peptide substrates that are relatively short in length, generally consisting of approximately six to ten amino acids in length. These peptide substrates typically encompass amino acid sequences that bracket the known hydrolysis site of the natural protein substrates. These peptide substrates, including those for matrix metalloproteases, serine proteases, aspartyl proteases, and aminopeptidases, are readily available for use in a variety of enzymatic assays.

[0027] Further provided are peptides that have been found to act as substrates for ADMPs. It is commonly known that short peptide sequences which contain the proper substrate cleavage site are quite acceptable substrates for many proteases. Copeland, R. A., Enzymes: A Practical Introduction to Structure, Mechanism and Data Analysis, VCH/Wiley, N.Y., 1996. However, no such peptide, even those containing as many as twenty amino acids, has been determined that will act as a suitable substrate for ADMPs. The peptides of the instant invention are unique in that it was unexpectedly found that these longer, forty amino acid sequence acted as very good substrates for ADMPs. One such peptide provided by the invention, of the sequence: TABLE-US-00005 (SEQ ID NO:1) QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL

[0028] comprises a 40 amino acid segment of the human aggrecan protein that contains the ITEGE.sup.373-.sup.374ARGS cleavage site present in aggrecan, and is capable of being cleaved at this specific recognition site by the ADMPs. Since the human aggrecan ADMP-sensitive cleavage sites are conserved in aggrecan from various animal species, peptides based on the amino acid sequence around the ADMP-sensitive cleavage sites from other species can also serve as substrates for ADMPs. A peptide substrate, similar to SEQ ID NO: 1, based on a 41 amino acid segment of the bovine aggrecan protein, of the sequence TABLE-US-00006 (SEQ ID NO:2) QTVTWPDVELPLPRNITEGE-ARGSVILTAKPDFEVSPTAPE

containing the E.sup.373-A.sup.374 cleavage site is also capable of being cleaved at this specific recognition site by the ADMPS.

[0029] Cleavage products are easily detected by using neoepitope antibodies to the N-terminal or C-terminal fragments produced by specific cleavage at the E.sup.373-A.sup.374 bond. Such antibodies include the monoclonal antibody BC-3. Hughes, C. E., et al., Biochem. J. 306:799-804, 1995. The BC-3 antibody recognizes the N-terminal epitope comprising the sequence "ARGS" which is the amino terminal portion of one of the product peptides resulting from the ADMP activity of the enzyme.

[0030] One skilled in the art could readily design peptides of similar size encompassing the alternative ADMP-sensitive cleavage sites in the aggrecan core protein, encompassing, but not limited to, regions of the molecule containing the E.sup.1545-G.sup.1546, E.sup.1714-G.sup.1715, E.sup.1819-A.sup.1820, or E.sup.1919-L.sup.1920 bond (numbering based on the human aggrecan core protein sequence). One such peptide provided by the invention, of the sequence: TABLE-US-00007 (SEQ ID NO:3) ITFVDTSLVEVTPTTFKEEE-GLGSVELSGLPSGELGVSGT

comprises a 40 amino acid segment of the human aggrecan protein that contains the KEEE.sup.174-.sup.1715GLGS cleavage site present in the natural protein substrate, aggrecan, and is capable of being cleaved at this specific recognition site by the ADMPs.

[0031] A preferred form of the peptide substrate is a peptide that is biotinylated at the carboxy terminus or amino terminus. Several streptavidin coated supports may be used. These include, but are not limited to microplates, metallic and non-metallic beads, and membranes.

[0032] Another preferred assay format involves the direct analysis, by high-performance liquid chromatography (HPLC), of the cleavage fragments from the substrate that are generated by ADMP activity.

[0033] Another preferred embodiment of the invention is a substrate that inhibits ADMP activity. It was discovered that esterification of the P1 glutamic acid residue (GLU.sup.373) of the substrate peptide of SEQ ID NO: 1, or its replacement by glutamine abolish catalytic hydrolysis. Unexpectedly, the peptide containing the GLU to GLN substitution at amino acid position 373 (the P1-glutamine containing peptide) was shown to be a competitive inhibitor of the enzyme. Thus, a carboxylate residue at position P1 of the substrate appears to be involved in turnover by ADMPs, but exerts less influence over initial substrate binding to the enzyme. This feature can be readily exploited by one trained in the art to design specific peptide and non-peptide inhibitors of this enzyme.

DEFINITIONS

[0034] As used herein, the following terms and expressions have the indicated meanings.

[0035] The term "aggrecan degrading metallo protease" ("ADMP") activity as referred to herein, refers to the enzymatic activity of a family of polypeptides which specifically cleave the protein aggrecan within the interglobular domain at the Glu.sup.373-Ala.sup.374 peptide bond, but do not readily cleave at the Asn341-Phe342 bond which is preferentially cleaved by matrix metalloproteases.

[0036] The term "amino acid" as used herein means an organic compound containing both a basic amino group and an acidic carboxyl group.

[0037] The term "amino acid residue" as used herein means that portion of an amino acid (as defined herein) that is present in a peptide.

[0038] The term "peptide" as used herein means a compound that consists of two or more amino acids (as defined herein) that are linked by means of a peptide bond. The term "peptide" also includes compounds containing both peptide and non-peptide components, such as pseudopeptide or peptide mimetic residues or other non-amino acid components. Such a compound containing both peptide and non-peptide components may also be referred to as a "peptide analog".

[0039] The term "peptide bond" means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid.

[0040] The term "substrate" refers to a molecule that is bound by the active site and acted upon by the enzyme.

[0041] The term "solid-phase peptide synthesis" refers to the direct chemical synthesis of peptides utilizing an insoluble polymeric support as the anchor for the growing peptide, which is built up one amino acid at a time using a standard set of reactions in a repeating cycle. Merrifield, R. B., Science 232, 341-347 1986.

[0042] As used herein, the term "TMB" refers to 3,3',5,5'-tetramethylbenzidine.

[0043] The term "neoepitope antibody" refers to an antibody which specifically recognizes a new N-terminal amino acid sequence or new C-terminal amino acid sequence generated by proteolytic cleavage but does not recognize these same sequences of amino acids when they are present within the intact protein.

[0044] As used herein, the cleavage site "E.sup.373-.sup.374A" refers to the ITEGE.sup.373-.sup.374 ARGS bond of human aggrecan as well as to the homologous aggrecanase-sensitive cleavage site in aggrecan from various animal species, the cleavage site "E.sup.1545-.sup.1546G" refers to the SELE.sup.1545-.sup.1546GRGT bond of human aggrecan as well as to the homologous aggrecanase-sensitive cleavage site in aggrecan from various animal species, the cleavage site "E.sup.1714-.sup.1715G" refers to the KEEE.sup.1714-.sup.1715GLGS bond of human aggrecan as well as to the homologous aggrecanase-sensitive cleavage site in aggrecan from various animal species, the cleavage site "E.sup.1819-.sup.1820A" refers to the TAQE.sup.1819-.sup.1820AGEG bond of human aggrecan as well as to the homologous aggrecanase-sensitive cleavage site in aggrecan from various animal species, the cleavage site "E.sup.1919-.sup.1920L" refers to the ISQE.sup.1919-.sup.1920LGQR bond of human aggrecan as well as to the homologous aggrecanase-sensitive cleavage site in aggrecan from various animal species.

[0045] The term "aggrecan" as used herein refers to the aggregating proteoglycan, aggrecan, of cartilage from human or various animal species, as the native aggrecan isolated from tissue, as recombinant full-length aggrecan or as a recombinant protein representing a portion of the aggrecan molecule.

[0046] As used herein the term "ADMP-susceptible cleavage site" refers to the E.sup.373-.sup.374A bond, the E.sup.1545-.sup.1546G bond, the E.sup.1545-.sup.1546G bond, the E.sup.1819-.sup.1820A bond, and the E.sup.1919-.sup.1920L bond of aggrecan from human and various animal species, and to a peptide bond of a protein containing an amino acid sequence which has a glutamine in the P1 position and shows at least 65% homology with the P1, P2, P3, P1', P2' and P3' amino acids of one or more of the ADMP-sensitive sites in the aggrecan molecule.

[0047] The term "sissel bonds" refers to the peptide bond of a polypeptide that is to be cleaved by a protease. The term "P1" as used herein refers to the amino acid residue on the N-terminal side of the sissel bond. The term "P2" as used herein refers to the amino acid residue adjacent to P1 on the N-terminal side of the sissel bond. The term "P3" as used herein refers to the amino acid residue adjacent to P2 on the N-terminal side of the sissel bond. The term "P 1'" as used herein refers to the amino acid residue on the C-terminal side of the sissel bond. The term "P2'" as used herein refers to the amino acid residue adjacent to P1' on the C-terminal side of the sissel bond. The term "P3'" as used herein refers to the amino acid residue adjacent to P2' on the C-terminal side of the sissel bond.

[0048] The term "BC-3 antibody" refers to a monoclonal antibody that reacts specifically with the newly-formed amino-terminal sequence ARGS on fragments produced by proteolytic cleavage at the Glu.sup.373-Ala.sup.374 aggrecan cleavage site, but does not recognize this same sequence of amino acids when they are present within the intact interglobular domain of the protein. Hughes, C. E., et al., Biochem. J. 306:799-804, 1995.

[0049] The term "SEQ ID NO:1" refers to the peptide sequence: TABLE-US-00008 QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL.

[0050] The term "SEQ ID NO:2" refers to the peptide sequence: TABLE-US-00009 QTVTWPDVELPLPRNITEGE-ARGSVILTAKPDFEVSPTAPE.

[0051] The term "SEQ ID NO:3" refers to the peptide sequence: TABLE-US-00010 ITFVDTSLVEVTPTTFKEEE-GL-GSVELSGLPSGELGVSGT.

[0052] The term "41-PS" and "SEQ ID NO:4" refer to the peptide sequence: TABLE-US-00011 QTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPL- (BIOTINYL)K.

[0053] The term "SEQ ID NO:5" refers to the peptide sequence: TABLE-US-00012 ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K.

[0054] The term "SEQ ID NO:6" refers to the peptide sequence: K(BIOTINYL)- TABLE-US-00013 QTVTWPDMELPLPRNITEGE.

[0055] The term "30-IP" and "SEQ ID NO:7" refer to the peptide sequence TABLE-US-00014 QTVTWPDMELPLPRNITEGQARGSVILTV--(BIOTINYL)K.

[0056] The invention can be further understood by the following examples. These examples provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention recited in the appended claims. In the following examples all methods described are conventional unless otherwise specified.

EXAMPLE 1

Microplate Assay Format for Detection of ADMP Activity

[0057] The substrate and product peptides were prepared in the following manner. A 41 amino acid form (41-PS) SEQ ID NO:4 of the peptide substrate SEQ ID NO:1 was prepared by solid phase peptide synthesis. The peptide was prepared commercially (Quality Controlled Biochemicals, Inc. Hopkinton, Mass.) as a biotin conjugate by adding an additional lysine residue at the carboxy terminus of the peptide SEQ ID NO: 1. Biotin was then covalently attached through the lysine .epsilon.-amino side chain. 41-PS sequence: TABLE-US-00015 (SEQ ID NO:4) QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL- (BIOTINYL)K

[0058] A 21 amino acid peptide representing the product of ADMP-mediated cleavage of the 41 -PS containing the ARGS N-terminus was prepared in a similar manner and had the following sequence: TABLE-US-00016 (SEQ ID NO:5) ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K

[0059] The substrate and product peptide microplates were prepared in the following manner. A 0.1 mM stock of 41-PS was made by dissolving it in distilled water. From this a working solution of 7.times.10.sup.8 M 41-PS was prepared in 1.times.PBS, 0.05% Tween 20. Aliquots of 100 .mu.L of this solution were added to the microplate wells of a streptavidin coated microtiter strip plate (DUPONT, NEN Products, Catalog# NEF-711). The solutions were allowed to sit at 25.degree. C. overnight in order for the biotin moiety of the peptides to bind to the streptavidin which was coated on the microplate. The plate was washed three times with 200 .mu.L of 1.times.PBS, 0.05% Tween 20, after which the plate was inverted, blotted dry, sealed and stored at 4.degree. C.

[0060] The assay was performed in the following manner. Microplate strips (eight wells each) were rinsed once with 100 .mu.L of 1.times.Assay Buffer (Assay Buffer consists of: 50 mM Tris, pH 7.5, 10 mM CaCl.sub.2, and 100 mM NaCl) and blotted dry. Reactions were prepared in duplicate in a final volume of 100 .mu.L, containing: 50 .mu.L of 2.times.Assay Buffer (100 mM Tris, pH 7.5, 20 mM CaCl.sub.2, and 200 mM NaCl), 25 .mu.L of a hydroxymate inhibitor compound (final concentrations consisting of 5.0, 1.0, 0.75, 0.5, 0.25, 0.125, 0.05, 0.001, and 0.0 .mu.M), and 25 .mu.L of soluble ADMP (0.05 .eta.M). The microplate strips were incubated for 3 hours at 37.degree. C. The microplate wells were then washed six times with 200 .mu.L of 1.times.PBS, 0.05% Tween 20 using a Denley Well Wash 4 micro plate washer.

[0061] A BC-3 antibody solution was prepared by adding 4 .mu.L of BC-3 antibody (0.405 mg/mL in PBS) to 2 mL of antibody dilution buffer (DB), which consisted of: 0.1 g BSA (Boehringer Mannheim Catalog 238-031), 10 mL 1.times.PBS, and 10 .mu.L Tween 20. 100 .mu.L of this solution was added to each well. The microplate strips were incubated for 1 hour at 25.degree. C. The microplate wells were washed six times with 200 .mu.L of 1.times.PBS, 0.05% Tween 20.

[0062] The secondary (detection) antibody solution was prepared by adding 4 .mu.L of Goat anti-Mouse-HRP antibody conjugate (Pierce Cat #31430) (0.8 mg/mL in PBS) to 2 mL of antibody dilution buffer (DB). 100 .mu.L of this solution was added to each well. The microplate strips were incubated for 1 hour at 25.degree. C. The microplate wells were washed six times with 200 .mu.L of 1.times.PBS, 0.05% Tween 20. TMB Substrate (100 .mu.L per well, DAKO Cat S1600) was added and the microplate strips incubated at 25.degree. C. for 15 minutes. The reaction was quenched with 100 .mu.L of 1N HCl. The optical density was read at 450 nm using a Molecular Devices Spectromax 250 microplate reader. By coating the plate with the product peptide, one can create a standard curve which can be used to convert optical density values to units of activity.

[0063] ADMP activity can easily be followed by this method and inhibition of ADMP activity can be monitored. The IC.sub.50 for the inhibition of ADMP by the hydroxamate inhibitor tested was 0.413 .mu.M.

EXAMPLE 2

HPLC Assay for Detection of ADMP Activity

[0064] The High Performance Liquid Chromatography (HPLC) instrument used for the assay was from Hewlett-Packard, model number HP1090, equipped with a HP ChemStation. A 250.times.4.6 mm Vydac C.sub.18 column with 10.mu. particle size was obtained from The Separations Group, Tesperia, Calif.; HEPES from Research Organics, Cleveland, Ohio.; Brij-35 from Technicon Corp., Tarrington, N.Y.; Other chemicals from Sigma, St. Louis, Mo.; the lyophilized peptide substrate 41-PS, QTVTWPDMELPLRNITEGEARGSVILT-VKPIFEVSPSPL-(BIOTINYL)K (SEQ ID NO:4), which includes the ADMP E373-A374 cleavage site, was obtained from Quality Controlled Biochemicals, Inc. Hopkinton, Mass. A 21 amino acid product peptide (SEQ ID NO:5) with the sequence, ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K, used as a standard for quantitation of product formation, was obtained from the same vendor.

[0065] The HPLC assay is performed in the following manner. The reaction buffer contains 50 mM HEPES, 10 mM CaCl.sub.2, 100 mM NaCl and 0.05% Brij-35, pH 7.5. 30 .mu.M 41-PS was incubated with 1 unit ADMP activity (1 unit=the amount of ADMP activity resulting in 1 pMole of 21-mer peptide product produced per hr at 37.degree..) at 37.degree. C. for 2.5 hours and then the reaction was quenched with 50 mM EDTA. A 10 .mu.L portion of the reaction mixture was injected onto a reverse-phase HPLC C.sub.18 column. The peptides were eluted with a mobile phase of 0.1% trifluoroacetic acid and a 25-45% acetonitrile gradient in 20 minutes. UV absorbance was measured at 220 nm and peak integration was performed on a Hewlett-Packard HP ChemStation. The 21-mer product peptide was used as a standard for quantitation of product formation. The 41-PS and 21-mer product are well separated with retention times of 14.2 and 10.5 minutes, respectively. A standard curve was prepared using the 21-mer peptide to allow quantitation of product formation. Effect of incubation time was evaluated and found to be linear over the timecourse of the assay (FIG. 2).

EXAMPLE 3

Inhibition of ADMP Activity by a Peptide Inhibitor

[0066] A peptide inhibitor was prepared based upon the sequence of the 40 amino acid peptide SEQ ID NO: 1, but designed such that it contained a Glu to Gln substitution at the P1 of the Glu373-Ala374 bond. This peptide (30-IP) of the sequence: TABLE-US-00017 (SEQ ID NO:7) QTVTWPDMELPLPRNITEGQARGSVILTVK-Biotin

[0067] was prepared with the n-terminus acetylated and the c-terminal residue present as the amide (Quality Controlled Biochemicals, Inc., Hopkinton, Mass.). This peptide was employed in a microplate assay as described in Example 1 wherein the 30-IP inhibitor was substituted for the hydroxamate inhibitor used in that example. The 30-IP inhibitor was employed at final concentrations of 0.01, 0.1, 1.0, 3.0, 5.0, 10.0, 30.0 and 100.0 .mu.M.

[0068] ADMP activity was inhibited as shown in FIG. 3. The IC.sub.50 for the inhibition of ADMP activity by 30-IP was 11 .mu.M.

EXAMPLE 4

Problot Assay

[0069] This example describes a method for analyzing ADMP enzymatic activity and inhibitors of this activity by monitoring cleavage at the E373-A374 bond using the BC-3 antibody to detect fragments with the new N-terminus, ARGS. Samples containing ADMP activity (10 units/ml) were incubated with 500 nM bovine aggrecan monomer in a final volume of 200 .mu.L in 0.05 M Tris, pH 7.6, containing 0.1 M NaCl and 10 mM CaCl.sub.2. Reactions were incubated for 4 hr at 37.degree. C., quenched with 20 mM EDTA, and analyzed for aggrecan fragments with the new N-terminus, ARGS, generated by specific ADMP-mediated cleavage using the Problot assay.

[0070] The Immobolin PVDF membrane plate (#MAIPN4550; Millipore Corp., Bedford, Mass.) was prewet with 50 .mu.L per well 70% ethanol, incubated for 30 seconds at room temperature then flushed two times each with 200 .mu.L of purified H.sub.2O. The plate was then coated with aggrecan equivalent to 36 .mu.g of glycosaminoglycan (GAG) as detected by the dimethyl methylene blue dye assay (Farndale R. W., et al., (1982) Conn. Tiss. Res. 9, 247-248) in 150 .mu.L of 50 mM carbonate-bicarbonate buffer, pH 9.6, overnight at 4.degree. C. with gentle agitation. The coating solution was then filtered off of the plate using a vacuum manifold and membranes were washed once with 200 .mu.L of Buffer A (Buffer A comprises 20 mM Tris, 500 mM NaCl, pH 7.5) allowing 20 seconds of contact with the membrane. Membranes were then blocked with 150 .mu.L of 5% BSA/TBS solution for 1 hour at room temperature with gentle agitation. The blocking solution was filtered off of the plate and the membranes washed one time with 200 .mu.L of 1.times.TBS buffer, allowing 20 seconds of contact with membrane per wash.

[0071] Removal of the glycosaminoglycan (GAG) side chains from aggrecan is necessary for the BC-3 antibody to recognize the epitope on the core protein. Therefore, to remove GAGs from the bound aggrecan, samples were treated with deglycosylation enzymes as follows: 0.01 units chondroitinase ABC (#EC4.2.2.4; Seikaguku Co., Kogyo, Japan) per 10 .mu.g GAG in 150 .mu.L of Buffer B (Buffer B comprises 50 mM sodium acetate, 100 mM NaCl, pH 6.5) was added to each well and incubated at 37.degree. C. for 1 hour. Following incubation the enzyme solution was filtered out of the plate and 0.01 units chondroitinase ABC per 10. .mu.g GAG, 0.01 units keratanase I (#EC3.2.1.103; Co., Kogyo, Japan) per 10 .mu.g GAG, and 0.0005 units keratanase II (Seikaguku Co., Kogyo, Japan) per 10 .mu.g GAG in 150 .mu.L Buffer B were added and allowed to incubate an additional 2 hours at 37.degree. C. Enzyme solution was filtered out and membranes rinsed one time with 200 .mu.L of Buffer A.

[0072] 150 .mu.L of BC-3 antibody was added at a 1:500 dilution in 1% BSA in Buffer A and incubated for 1 hour at room temperature with gentle agitation. BC-3 antibody was removed and membranes washed three times each with 200 .mu.L Buffer A allowing membrane contact for 20 seconds per wash. Next 150 .mu.L of goat anti-mouse IgG AP conjugate (#S3721; Promega, Madison, Wis.) was added at a 1:2500 dilution in 1% BSA/TBS buffer and allowed to incubate for 1 hour at room temperature with gentle agitation. The secondary antibody was prefiltered prior to use, using 0.22 .mu.M syringe filters to remove aggregates which cause high background. Following the incubation, secondary antibody was removed and wells were washed three times each with 200 .mu.L Buffer A allowing contact for 20 seconds per wash. Then 100 .mu.L of p-NPP AP substrate solution (#50-80-00; Kirkegaard & Perry Lab., Gaithersburg, Md.) was added to each well and incubated in the dark at room temperature for 30 minutes. The solution was then filtered into a corresponding ELISA plate. The filter plate was washed with 100 .mu.L of 500 mM EDTA and the wash was combined with the corresponding samples in the ELISA plate. Absorbance of the samples was read at 405 nm (Thermomax plate reader).

[0073] By coating the plate with the target peptide, ARGS, linked to BSA representing the ADMP-generated product, one can create a standard curve which can be used to convert optical density values to units of activity. A unit of ADMP activity is defined as that resulting in product produced equivalent to 1 .mu.g BSA peptide per hour at 37.degree. C.

[0074] To evaluate inhibition of ADMP activity, compounds are prepared as 10 mM stocks in dimethyl sulfoxide (DMSO), water or other solvents and diluted to appropriate concentrations in water. Drug (50 .mu.L) was added to 50 .mu.L of 2 mg/mL aggrecan substrate and 50 .mu.l of ADMP (40 units/ml) and brought to a final volume of 200 .mu.L by addition of 50 .mu.l of 0.2 M Tris, pH 7.6, containing 0.4 M NaCl and 40 mM CaCl.sub.2. The reaction mixture was incubated for 4 hr at 37.degree. C., quenched with 20 mM EDTA and analyzed for ADMP-generated products. A sample containing enzyme and substrate without drug was included as a positive control and enzyme prequenched with EDTA served as a measure of background.

[0075] IC.sub.50 values for inhibitors of ADMP enzymatic activity determined using the Problot assay with the BC-3 antibody for analysis of product generation by cleavage at the E373-A374 bond correlated (r.sup.2=0.99) with those determined using a BC-3 Western blot analysis to detect product formation.

Sequence CWU 1

1

61 1 40 PRT Homo Sapiens 1 Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu Pro Arg Asn Ile 1 5 10 15 Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr Val Lys Pro Ile 20 25 30 Phe Glu Val Ser Pro Ser Pro Leu 35 40 2 41 PRT Bovine 2 Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Leu Pro Arg Asn Ile 1 5 10 15 Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr Ala Lys Pro Asp 20 25 30 Phe Glu Val Ser Pro Thr Ala Pro Glu 35 40 3 40 PRT Homo Sapiens 3 Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val Thr Pro Thr Thr Phe 1 5 10 15 Lys Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu Pro Ser 20 25 30 Gly Glu Leu Gly Val Ser Gly Thr 35 40 4 41 PRT Artificial synthetic MISC_FEATURE (41)..(41) biotinylated lysine MISC_FEATURE (41)..(41) biotinylated lysine residue 4 Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu Pro Arg Asn Ile 1 5 10 15 Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr Val Lys Pro Ile 20 25 30 Phe Glu Val Ser Pro Ser Pro Leu Lys 35 40 5 21 PRT Artificial synthetic MISC_FEATURE (21)..(21) biotinylated lysine residue 5 Ala Arg Gly Ser Val Ile Leu Thr Val Lys Pro Ile Phe Glu Val Ser 1 5 10 15 Pro Ser Pro Leu Lys 20 6 21 PRT Artificial synthetic MISC_FEATURE (1)..(1) biotinylated lysine residue 6 Lys Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu Pro Arg Asn 1 5 10 15 Ile Thr Glu Gly Glu 20 7 30 PRT Artificial synthetic 7 Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu Pro Arg Asn Ile 1 5 10 15 Thr Glu Gly Gln Ala Arg Gly Ser Val Ile Leu Thr Val Lys 20 25 30 8 6 PRT Homo sapiens 8 Asn Ile Thr Glu Gly Glu 1 5 9 8 PRT Homo sapiens 9 Ala Arg Gly Ser Val Ile Leu Thr 1 5 10 6 PRT Bovine 10 Asn Ile Thr Glu Gly Glu 1 5 11 8 PRT Bovine 11 Ala Arg Gly Ser Val Ile Leu Thr 1 5 12 6 PRT Rat 12 Asn Ile Thr Glu Gly Glu 1 5 13 8 PRT Rat 13 Ala Arg Gly Asn Val Ile Leu Thr 1 5 14 6 PRT Mouse 14 Asn Val Thr Glu Gly Glu 1 5 15 8 PRT Mouse 15 Ala Leu Gly Ser Val Ile Leu Thr 1 5 16 6 PRT Pig 16 Asn Ile Thr Glu Gly Glu 1 5 17 8 PRT Pig 17 Ala Arg Gly Thr Val Ile Leu Thr 1 5 18 6 PRT Sheep 18 Asn Ile Thr Glu Gly Glu 1 5 19 8 PRT Sheep 19 Ala Arg Gly Asn Val Ile Leu Thr 1 5 20 6 PRT Chicken 20 Asn Val Thr Glu Glu Glu 1 5 21 5 PRT Chicken 21 Ala Arg Gly Ser Ile 1 5 22 6 PRT Horse 22 Asn Ile Thr Glu Gly Glu 1 5 23 8 PRT Horse 23 Ala Arg Gly Asn Val Ile Leu Thr 1 5 24 8 PRT Homo Sapiens 24 Ala Ser Thr Ala Ser Glu Leu Glu 1 5 25 8 PRT Homo Sapiens 25 Gly Arg Gly Thr Ile Gly Ile Ser 1 5 26 8 PRT Bovine 26 Ala Thr Thr Ala Gly Glu Leu Glu 1 5 27 8 PRT Bovine 27 Gly Arg Gly Thr Ile Asp Ile Ser 1 5 28 8 PRT Mouse 28 Ala Thr Thr Ser Ser Glu Leu Glu 1 5 29 8 PRT Mouse 29 Gly Arg Gly Thr Ile Gly Ile Ser 1 5 30 8 PRT Rat 30 Ala Thr Thr Ala Ser Glu Leu Glu 1 5 31 8 PRT Rat 31 Gly Arg Gly Thr Ile Ser Val Ser 1 5 32 8 PRT Homo Sapiens 32 Pro Thr Thr Phe Lys Glu Glu Glu 1 5 33 8 PRT Homo Sapiens 33 Gly Leu Gly Ser Val Glu Leu Ser 1 5 34 8 PRT Bovine 34 Pro Thr Thr Phe Lys Glu Glu Glu 1 5 35 8 PRT Bovine 35 Gly Leu Gly Ser Val Glu Leu Ser 1 5 36 8 PRT Rat 36 Pro Thr Thr Phe Arg Glu Glu Glu 1 5 37 8 PRT Rat 37 Gly Leu Gly Ser Val Glu Leu Ser 1 5 38 8 PRT Mouse 38 Pro Thr Thr Phe Arg Glu Glu Glu 1 5 39 8 PRT Mouse 39 Gly Leu Gly Ser Val Glu Leu Ser 1 5 40 8 PRT Homo Sapiens 40 Thr Gln Ala Pro Thr Ala Gln Glu 1 5 41 8 PRT Homo Sapiens 41 Ala Gly Glu Gly Pro Ser Gly Ile 1 5 42 8 PRT Bovine 42 Thr Gln Ala Pro Thr Ala Gln Glu 1 5 43 8 PRT Bovine 43 Ala Gly Glu Gly Pro Ser Gly Ile 1 5 44 8 PRT Rat 44 Thr Leu Ala Pro Thr Ala Gln Glu 1 5 45 8 PRT Rat 45 Ala Gly Glu Gly Pro Ser Ser Ile 1 5 46 8 PRT Mouse 46 Thr Gln Ala Pro Thr Ala Gln Glu 1 5 47 8 PRT Mouse 47 Ala Gly Glu Gly Pro Ser Gly Ile 1 5 48 8 PRT Chicken 48 Thr Gln Thr Ser Val Ala Gln Glu 1 5 49 8 PRT Chicken 49 Val Gly Glu Gly Pro Ser Gly Met 1 5 50 8 PRT Homo Sapiens 50 Thr Glu Pro Thr Ile Ser Gln Glu 1 5 51 8 PRT Homo Sapiens 51 Leu Gly Gln Arg Pro Pro Val Thr 1 5 52 8 PRT Bovine 52 Thr Glu Pro Thr Val Ser Gln Glu 1 5 53 8 PRT Bovine 53 Leu Gly Gln Arg Pro Pro Val Thr 1 5 54 8 PRT Rat 54 Thr Glu Pro Thr Val Ser Gln Glu 1 5 55 8 PRT Rat 55 Leu Gly His Gly Pro Ser Met Thr 1 5 56 8 PRT Mouse 56 Thr Glu Pro Thr Val Ser Gln Glu 1 5 57 8 PRT Mouse 57 Leu Gly His Gly Pro Ser Met Thr 1 5 58 8 PRT Chicken 58 Thr Arg Pro Thr Val Ser Gln Glu 1 5 59 8 PRT Chicken 59 Leu Gly Gly Glu Thr Ala Val Thr 1 5 60 8 PRT Dog 60 Thr Glu Pro Thr Val Ser Gln Glu 1 5 61 8 PRT Dog 61 Leu Gly Gly Glu Thr Ala Val Thr 1 5

Claims

1. A method for the determination of the presence of aggrecan-degrading metalloprotease activity comprising: (a) binding an ADMP substrate peptide to a streptavidin-coated microtiter plate; (b) rinsing the microtiter plate with assay buffer; (c) incubating the microtiter plate with an ADMP-containing sample; (d) rinsing the microtiter plate; (e) incubating the microtiter plate with a neoepitope antibody solution; (f) rinsing the microtiter plate; (g) incubating microtiter plates with secondary-detection antibody solution; (h) incubating the microtiter plate with an appropriate substrate solution; (i) quenching the reaction; and (j) reading the optical density.

2. The method of claim 1, wherein said ADMP peptide substrate comprises a covalently-linked linking-moiety.

3. A method for the determination of ADMP activity by quantifying the appearance of a product peptide comprising: (a) incubating an ADMP substrate peptide with assay buffer and an ADMP-containing sample; (b) quenching the reaction; (c) injecting a portion of the reaction mixture onto a reverse-phase HPLC column; (d) eluting the peptide with an organic solvent; (e) reading the absorbance; and (f) determining the quantity based on a standard curve.

4. A method for assaying compounds for activity against an ADMP comprising: (a) providing an ADMP and an ADMP substrate; (b) contacting said ADMP with a candidate inhibitory compound in the presence of said ADMP; and (c) measuring the inhibition of the ADMP activity.

5. An assay for detecting ADMP activity which comprises: (a) incubating a sample containing soluble ADMPs or aggrecanase activity with an aggrecan substrate; and (b) monitoring production of aggrecan fragments produced by specific cleavage at an ADMP-susceptible site using a neoepitope antibody to the new N-terminus or the new C-terminus generated by specific ADMP-mediated cleavage by the Problot assay comprising: (1) incubating a polyvinyl-denedifluoride (PVDF) cationically charged membrane, secured in a welled filtration plate, with a sample containing ADMP-degraded aggrecan; (2) washing any unbound aggrecan from the filtration plate; (3) coupling any unreacted cationic sites on the PVDF membrane with a solution of bovine serum albumin (BSA); (4) washing any unbound BSA from the filtration plate; (5) removing glycosaminoglycan side chains from the bound aggrecan with deglycosylation enzymes, wash membrane; (6) incubating PVDF membrane with a neoepitope antibody to fragments generated by cleavage at an ADMP-sensitive site, wash membrane; (7) incubating PVDF membrane with secondary detection antibody, wash membrane; (8) incubating PVDF membrane with detection substrate; and (9) draining solution into welled plate, obtain absorbance readings on individual samples; compare values to those obtained for standard curve.

6. An assay according to claim 5 wherein the sample is derived from cartilage or chondrocytes.

7. An assay according to claim 5 wherein the aggrecan substrate is native aggrecan isolated from human or animal tissue.

8. An assay according to 5 wherein the aggrecan substrate is a recombinant aggrecan molecule or recombinant portion of the aggrecan molecule containing an aggrecanase-sensitive cleavage site.

9. An assay according to claim 5 wherein the recombinant portion of the aggrecan molecule contains the E.sup.374-.sup.374A bond.

10. An assay according to claim 5 wherein the recombinant aggrecan fragment contains the EL.sup.1545-.sup.546G bond.

11. An assay according to claim 5 wherein the portion of the aggrecan molecule contains the E.sup.1714-.sup.1715G bond.

12. An assay according to claim 5 wherein the recombinant portion of the aggrecan molecule contains the E.sup.1819-.sup.1820A bond.

13. An assay according to claim 5 wherein the recombinant portion of the aggrecan molecule contains the E.sup.1919-.sup.1920L bond.

14. A method according to claim 5 wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E.sup.373 -A.sup.374 bond.

15. The method of claim 5 wherein the neoepitope antibody is a BC-3 monoclonal antibody.

16. The method of claim 5 wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E.sup.1545-G.sup.1546 bond.

17. The method of claim 5 wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E.sup.1714-G.sup.1715 bond.

18. The method of claim 5 wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E.sup.1819-A.sup.1820 bond.

19. The method of claim 5 wherein the neoepitope antibody recognizes the new N-terminus or new C-terminus generated by cleavage at the E.sup.19 9-L.sup.1920 bond.