We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

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 inflammationassociated 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.Aggrecan is the major proteoglycan of cartilage and is responsible for its compressibility and stiffness. Aggrecan contains two N-terminal globular domains, G 1 and G 2 , separated by a proteolyticaly sensitive interglobular domain, followed by a glycosaminoglycan attachment region and a C-terminal globular domain (G 3 ). The G 1 domain of aggrecan interacts with hyaluronic acid and link protein to form large aggregates containing multiple aggrecan monomers that are trapped within the cartilage matrix. Cleavage of aggrecan has been shown to occur at Asn 341 -Phe 342 and Glu 373 -Ala 374 within the interglobular domain, with the cleaved aggrecan being free to exit the matrix since it lacks the G 1 domain, which is responsible for formation of the high molecular weight complexes. Results from several studies suggest that cleavage at the Glu 373 -Ala 374 site is responsible for the increased aggrecan degradation observed in inflammatory joint disease. Products resulting from cleavage at the Glu 373 -Ala 374 site have been shown to accumulate in cartilage explants and chondrocyte cultures treated with interleukin-1 and retinoic acid (1-5) and in the synovial fluid of patients with osteoarthritis and inflammatory joint disease (6, 7). While several characterized matrix metalloproteases 1 have been shown to cleave at the Asn 341 -Phe 342 site (8 -14), they are not responsible for the observed cleavage at Glu 373 -Ala 374 . A novel proteolytic activity, termed "aggrecanase," has been hypothesized to be respo...

In cell cultures, the key residues associated with HIV-1 resistance to cyclic urea-based HIV-1 protease (PR) inhibitors are Val82 and Ile84 of HIV-1 PR. To gain an understanding of how these two residues modulate inhibitor binding, we have measured the Ki values of three recombinant mutant proteases, I84V, V82F, and V82F/I84V, for DMP323 and DMP450, and determined the three-dimensional structures of their complexes to 2.1-1.9 A resolution with R factors of 18.7-19.6%. The Ki values of these mutants increased by 25-, 0.5-, and 1000-fold compared to the wild-type values of 0.8 and 0.4 nM for DMP323 and DMP450, respectively. The wild-type and mutant complexes overall are very similar (rms deviations of 0.2-0.3 A) except for differences in the patterns of their van der Waals (vdw) interactions, which appear to modulate the Ki values of the mutants. The loss of the CD1 atom of Ile84, in the I84V mutant complexes, creates a hole in the S1 subsite, reducing the number of vdw contacts and increasing the Ki values. The V82F mutant binds DMP323 more tightly than wild type because the side chain of Phe82 forms additional vdw and edge-to-face interactions with the P1 group of DMP323. The Ki values of the single mutants are not additive because the side chain of Phe82 rotates out of the S1 subsite in the double mutant (the chi 1 angles of Phe82 and -182 in the V82F and V82F/I84V mutants differ by 90 and 185 degrees, respectively), further reducing the vdw interactions. Finally, compensatory shifts in the I84V and V82F/ I84V complexes pick up a small number of new contacts, but too few to offset the initial loss of interactions caused by the mutations. Therefore, our data suggest that variants persist in the presence of DMP323 and DMP450 because of a decrease in vdw interactions between the mutant proteases and inhibitors.