Matrix metalloproteinase-3 (stromelysin-1). Identification as the cartilage acid metalloprotease and effect of pH on catalytic properties and calcium affinity.

Abstract

Human pro-MMP-3 (pro-matrix metalloproteinase-3) was purified from three sources: articular cartilage and conditioned media from synovial fibroblasts and Chinese hamster ovary cells expressing recombinant pro-MMP-3. All three preparations reacted with two monoclonal antibodies specific for human fibroblast pro-MMP-3. Each preparation of active MMP-3 possessed properties identical to those previously reported for the cartilage acid metalloproteinase (MMP-6; Azzo and Woessner, J. F., Jr. (1986) J. Biol. Chem. 261, 5434-5441): an acid pH optimum of 5.3-5.5 for digestion of cartilage aggrecan; digestion of oxidized insulin B-chain at Ala14-Leu15 and Tyr16-Leu17 in a ratio of 3:1; and heat stability at neutral pH. Further characterization of MMP-3 establishes that the acid pH optimum for cartilage aggrecan is not due to substrate denaturation since the same optimum is found by viscosity assay, by SDS-polyacrylamide gel electrophoresis assay of G1 domain, and by digestion of aggrecan in fresh cartilage fragments in vitro. Fibronectin was also digested optimally at pH 5.5 and NH2-terminal sequence analysis revealed no pH change in a major proteolytic site of cleavage at the Pro689-Leu690 bond. The specificity constant kcat/Km is maximal at pH 5.5 as determined in a quenched fluorescence peptide assay. This is due to an increase in kcat at pH 5.5 without any substantial effect on Km. The affinity of MMP-3 for calcium is decreased about 10-fold at pH 5.3 compared to neutral pH. Finally, the neutral cartilage metalloproteinase is identified as 72-kDa pro-MMP-2 based on M(r), specificity of insulin B-chain cleavage, and reactivity with a specific polyclonal antibody to human MMP-2.