CLEAVAGE OF NATIVE CARTILAGE AGGRECAN BY NEUTROPHIL COLLAGENASE (MMP-8) IS DISTINCT FROM ENDOGENOUS CLEAVAGE BY AGGRECANASE

Cleavage of aggrecan core protein at the Glu(373)-Ala(374) 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 t he 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 mu g/ml, with complete release of glycosaminoglycan at 2 mu g/m l or greater, Cleavage at the aggrecanase site was observed only at MM P-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 depl etion of substrate containing the preferred clip site did MMP-8 rapidl y cleave at the aggrecanase site, Finally, MMP-8 resulted in a differe nt pattern of BC-3-reactive fragments from that produced by endogenous aggrecanase in live cartilage, and SA751 (S)-(4-phenyl-3-butynyl)glyc yl-L-O-methyltyrosine, N-methylamide), a potent inhibitor of MMP-8 (K- i = 2 nM) which was effective in blocking cleavage by MMP-8 at the agg recanase site with an IC50 in the nanomolar range, did not prevent agg recan degradation or specific cleavage at this site by endogenously ge nerated aggrecanase at concentrations up to 100 mu M. Taken together t hese data suggest that MMP-8 does not represent cartilage aggrecanase.