STEPWISE ACTIVATION MECHANISMS OF THE PRECURSOR OF MATRIX METALLOPROTEINASE-3 (STROMELYSIN) BY PROTEINASES AND (4-AMINOPHENYL)MERCURIC ACETATE

The mechanisms of activation of the precursor of human matrix metalloproteinase 3 (proMMP-3/prostromelysin) by proteinases and (4-aminophenyl)mercuric acetate (APMA) were investigated by kinetic and sequence analyses. Incubation of proMMP-3 with neutrophil elastase, plasma kallikrein, plasmin, or chymotrypsin at 37 °C resulted in the formation of MMP-3 of M(r) = 45000 by cleaving of the His82-Phe83 bond. Since this bond is unlikely to be cleaved by these proteinases it was postulated that an initial attack of an activator proteinase on proMMP-3 creates an intermediate form, which is then processed to a more stable form of M(r) = 45000. To test this hypothesis proMMP-3 was incubated with these serine proteinases under conditions that minimize the action of MMP-3. This led to the accumulation of major intermediates of M(r) = 53000 and two minor forms of M(r) = 49000 and 47000. The 53000 M(r) intermediate generated by human neutrophil elastase resulted from cleavage of the Val35-Arg36 bond, whereas plasma kallikrein cleaved the Arg36-Arg37 and Lys38-Asp39 bonds and chymotrypsin the Phe34-Val35 bond, all of which are located near the middle of the propeptide. Conversion of these intermediates to the fully active 45000 M(r) form of MMP-3 resulted from a bimolecular reaction of the intermediates. A similar short-lived intermediate of M(r) = 46000 generated by APMA was a result of the intramolecular cleavage of the Glu68-Val69 bond, and it was then converted to a stable MMP-3 of M(r) = 45000 by a intermolecular reaction of MMP-3. However, MMP-3 failed to activate proMMP-3. These results indicate the removal of the NH2-terminal 34-38 residues by proteinases or 68 residues by APMA is the crucial step for activation of proMMP-3. This initial processing of the propeptide allows the His82-Phe83 bond, which is hindered from proteolysis in native pro-MMP-3, to be correctly oriented for cleavage by activated intermediates, thereby producing stable 45000 M(r) MMP-3. This stepwise activation process allows proMMP-3 to be activated by various proteinases with distinct substrate specificities.