RECIPROCATED MATRIX METALLOPROTEINASE ACTIVATION - A PROCESS PERFORMED BY INTERSTITIAL COLLAGENASE AND PROGELATINASE-A

Gelatinase A, a member of the matrix metalloproteinase (MMP) family, i s secreted possessing an 80 amino acid N-terminal propeptide that must be removed in order to generate the active enzyme. Purified progelati nase A was activated to 38% of maximum by a 6 h incubation at 37 degre es C with equimolar concentrations of trypsin-activated interstitial c ollagenase (another MMP). The increase in activity was accompanied by cleavage of the M(r) 72 000 progelatinase A to the M(r) 66 000 active enzyme that has Y-81 as its N-terminus. At low concentrations, progela tinase A was processed via an inactive intermediate, suggesting that i ts activation is a biphasic process. This was confirmed by the action of collagenase on proE(375-->)A (a mutant of progelatinase A that cann ot become active) because, in this instance, only an M(r) 68 000 speci es with L(38) as the N-terminus was produced. The remaining propeptide amino acids to Y-81 could be readily removed by added active gelatina se A, indicating that collagenase works by generating an intermediate that is susceptible to autolytic activation. Although relatively slow, the rate of activation could be increased approximately 10-fold by th e addition of 100 mu g/mL heparin. This binds to the C-terminal domain of collagenase and progelatinase A and presumably acts as a template that positions the reactants close to one another. Collagenase activat ed by trypsin retains 8 or 14 amino acids of its propeptide. The activ ated gelatinase A was able to remove these by cleaving the Q(80)-F-81 peptide bond, an event that has been shown to significantly increase t he activity of collagenase against fibrillar collagen [Suzuki, K., Eng hild, J. J., Morodomi, T., Salvesen, G., and Nagase, H. (1990) Biochem istry 29, 10261-10270]. The fact that the complete degradation of nati ve collagen requires the activities of both a collagenase and a gelati nase provides a functional basis for this reciprocated mechanism of ac tivation.