Analysis of tissue inhibitor of metalloproteinases-2 effect on pro-matrix metalloproteinase-2 activation by membrane-type 1 matrix metalloproteinase using baculovirus/insect-cell expression system

Membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14) is known to activate pro-matrix metalloproteinase-2 (pro-MMP-2; progelatinase A) on the cell surface. To analyse the tissue inhibitor of metalloproteinases-2 (TIMP-2) effect on activation of pro-MMP-2 by MT1-MMP, we have expressed the full-size MT1-MMP (fMT1-MMP) and a transmembrane (TM)-domain-deleted soluble MT1-MMP (sMT1-MMP) in the baculovirus/Sf9 (Spodoptera frugiperda 9) insect-cell system, where neither endogenous gelatinolytic MMPs nor TIMP-2 are expressed. Both fMT1-MMP and sMT1-MMP expressed in the expression system were found not to contain the pro-domain and were able to activate the TIMP-2-free pro-MMP-2. Both in the insect cells and in vitro, activation of pro-MMP-2 by fMT1-MMP was enhanced at low concentrations of TIMP-2 and inhibited by its higher concentrations. The maximal enhancing effect was detected at 0.05 molar fraction of TIMP-2/fMT1-MMP. In contrast, activation of pro-MMP-2 by sMT1-MMP was dose-dependently inhibited by TIMP-2. These results demonstrate that the TM domain of MT1-MMP is not required for the ability to activate pro-MMP-2, but is required for the enhancing effect of TIMP-2 on pro-MMP-2 activation by recruiting pro-MMP-2 to the MT1-MMP-TIMP-2 complex as a cell-surface pro-MMP-2 receptor. Moreover, our data strongly suggest that the pro-domain of MT1-MMP is not required for the TIMP-2-mediated enhancing effect on pro-MMP-2 activation. In addition, the pro-MMP-2 in the MT1-MMP-TIMP-2-pro-MMP-2 ternary complex was not activated without external activator, but readily by addition of sMT1-MMP. This result demonstrates that MT1-MMP free of TIMP-2 would be the enzyme responsible for activation of the pro-MMP-2 in the ternary complex under physiological conditions.