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Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.

A method was developed for generating soluble, active "aggrecanase" in conditioned media from interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine nasal cartilage conditioned media as a source of the aggrecanase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleavage products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage between amino acid residues Glu373 and Ala374). Using this assay we have characterized cartilage aggrecanase with respect to assay kinetics, pH and salt optima, heat sensitivity, and stability upon storage. Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, suggesting that aggrecanase is a metalloproteinase. Sensitivity to known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the notion that aggrecanase is a metalloproteinase potentially related to the ADAM family or MMP family of proteases previously implicated in the catabolism of the extracellular matrix.

Cleavage of aggrecan core protein at the Glu373-Ala374 site by the unidentified enzyme, "aggrecanase," is thought to play an important role in cartilage degradation. To examine aggrecan cleavage by MMP-8 at this aggrecanase site, we evaluated the release of fragments with the N terminus ARGSVIL from freeze-thawed bovine nasal cartilage using the monoclonal antibody BC-3. Recombinant human MMP-8 catalytic domain cleaved native aggrecan in a concentration-related manner between 0.2 and 2 microg/ml, with complete release of glycosaminoglycan at 2 microg/ml or greater. Cleavage at the aggrecanase site was observed only at MMP-8 concentrations resulting in complete release of glycosaminoglycan from the cartilage, suggesting that preferential cleavage occurs at a different site. Time course studies indicated that only following depletion of substrate containing the preferred clip site did MMP-8 rapidly cleave at the aggrecanase site. Finally, MMP-8 resulted in a different pattern of BC-3-reactive fragments from that produced by endogenous aggrecanase in live cartilage, and SA751(N-(1(R)-carboxyethyl) -alpha-(S)-(4-phenyl-3-butynyl)glycyl-L-O-methyltyrosine, N-methylamide), a potent inhibitor of MMP-8 (Ki = 2 nM) which was effective in blocking cleavage by MMP-8 at the aggrecanase site with an IC50 in the nanomolar range, did not prevent aggrecan degradation or specific cleavage at this site by endogenously generated aggrecanase at concentrations up to 100 microM. Taken together these data suggest that MMP-8 does not represent cartilage aggrecanase.