Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin)

Abstract

The mechanism of activation of tissue procollagenase by matrix metalloproteinase 3 (MMP-3)/stromelysin was investigated by kinetic and sequence analyses. MMP-3 slowly activated procollagenase by cleavage of the Gln80-Phe81 bond to generate a fully active collagenase of Mr = 41 000. The specific collagenolytic activity of this species was 27000 units/mg (1 unit = 1 .mu.g of collagen digested in 1 min at 37.degree.C). Treatment of procollagenase with plasmin or plasma kallikrein gave intermediates of Mr = 46 000. These intermediates underwent rapid autolytic activation, via cleaving the Thr64 - Leu65 bond, to give a collagenase species of Mr = 43 000 that exhibited only about 15% of the maximal specific activity. Similarly, (4-aminophenyl)mercuric acetate (APMA) activated procollagenase by intramolecular cleavage of the Val67-Met68 bond to generate a collagenase species of Mr = 43, 000 , but with only about 25% of the maximal specific activity. Subsequent incubation of the 43000-Mr species with MMP-3 resulted in rapid, full activation and generated the 41 000-Mr collagenase by cleaving the Gln80-Phe81 bond. In the case of the proteinase-generated 43000-Mr species, the action of MMP-3 was approximately 24 000 times faster than that on the native procollagenase. This indicates that the removal of a portion of the propeptide of procollagenase induces conformational changes around the Gln80-Phe81 bond, rendering it readily susceptible to MMP-3 activation. Prolonged treatment of procollagenase with APMA in the absence of MMP-3 also generated a 41 000-Mr collagenase, but this species had only 40% of the full activity and contained Val82 and Leu83 as NH2 termini. Thus, cleavage of the Gln80-Phe81 bond by MMP-3 is crucial for the expression of full collagenase activity. These results suggest that the activation of procollagenase by MMP-3 is regulated by two pathways: one with direct, slow activation by MMP-3 and the other with rapid activation in conjuction with tissue and/plasma proteinases. The latter event may explain an accelerated degradation of collagens under certain physiological and pathological conditions.