Multiple sites of the propeptide region of human stromelysin-1 are required for maintaining a latent form of the enzyme.

Latency of matrix metalloproteinase 3 (MMP-3) is regulated by the interaction of a free cysteine residue (Cys-75) in the conserved amino acid sequence Pro-Arg-Cys-Gly-Val-Pro-Asp located in the COOH-terminal portion of the propeptide with a chelated zinc atom in the active site of the catalytic domain. Proteolytic activation of full-length human pro-MMP-3 involves the removal of approximately 35 amino acids from the NH2-terminal portion of the propeptide, forming a 53-kDa unstable intermediate that undergoes intermolecular autocatalysis to form the 45-kDa mature active enzyme. In this study, we have evaluated the contribution of the NH2-terminal 35 amino acids to the maintenance of latency. Full-length human pro-MMP-3 was expressed in Escherichia coli and refolded to form latent pro-MMP-3 capable of activation by chymotrypsin or aminophenylmercuric acetate. Renaturation of pro-MMP-3 expressed in bacteria with 20 or more amino acids removed from the NH2-terminal region of the propeptide yielded only an active enzyme. COS-7 cells transiently transfected with pro-MMP-3 expression vectors containing the single amino acid substitutions Y20A, L21A, and C75S also secreted active forms of the enzyme. These data suggest that simultaneous interactions of the NH2- and COOH-terminal regions of the propeptide are required for maintenance of the latent form of the enzyme.