Activation of Progelatinase A and Progelatinase A/TIMP-2 Complex by Membrane Type 2-Matrix Metalloproteinase

Abstract

C-terminal truncated membrane-type 2 matrix metalloproteinase (MT2-MMP1-269), comprising prodomain and catalytic domain, was expressed as a soluble protein in Escherichia coli. Unlike the corresponding form of MT1-MMP, which can be isolated as a 31 kDa protein, MT2-MMP1-269 proved to be comparatively instable, and already the freshly isolated preparation displayed several proteins in SDS-PAGE representing MT2-MMP1-269 (33 kDa) and four N-truncated forms with N-termini methionine32 (30 kDa), isoleucine37 (30 kDa), leucine84 (24 kDa), and leucine93 (22 kDa), the catalytic domain. After thawing of frozen preparations the 33 and the 30 kDa proforms were no longer detectable in SDS-PAGE, and only the 24 and 22 kDa forms remained. The catalytic domain of MT2-MMP activated progelatinase A as well as the progelatinase A/TMP-2 complex by cleaving the 72 kDa progelatinase A to yield 67 kDa gelatinase A, which is then transformed into 62 kDa gelatinase A. The 62 kDa form is about twice as active as the 67 kDa form towards the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. No significant difference in activity was found between free and complexed gelatinase A forms. the activation of the progelatinase A/TIMP-2 complex proceeds in two steps: At first MT2-MMP is inhibited by the progelatinase A/TIMP-2/MT2-MMP, complex, whereby a ternary complex, progelatinase A/TIMP-2/ MT-2MMP is generated. This ternary complex is then activated by excess MT2-MMP. Our results suggest a mechanism for spatially regulated extracellular gelatinase A activity mediated by activation with membrane-type MMPs; Free gelatinase A is released into the extracellular space, while gelatinase A/TIMP-2 bound to MT-MMP remains anchored on the cell surface.

The Propeptide Domain of Membrane Type 1-Matrix Metalloproteinase Acts as an Intramolecular Chaperone when Expressed in transwith the Mature Sequence in COS-1 Cells

“…Because purified MT-MMPs cannot readily be evaluated in their membranebound state, most experiments designed to study the function of MT-MMPs have employed secreted (soluble) mutant MT1-MMP recombinants lacking the transmembrane domain (15)(16)(17)(18). Based on these studies, it has been concluded that cleavage of the N-terminal domain of membrane-bound MT-MMPs by furin, presumably in the trans-Golgi network, is required for function of the enzyme in activating progelatinase A and degrading other substrates (15,16,18,19).…”

Crystal Structure of the Catalytic Domain of MMP-16/MT3-MMP: Characterization of MT-MMP Specific Features

“…11 MT1-MMP seems to be the major activator of proMMP-2, mainly via presentation of proMMP-2 by a MT1-MMP-TIMP-2 "receptor". [15][16][17][18] However, the catalytic domains or the fulllength molecules of the other MT-MMPs [19][20][21][22][23] have also been shown in vitro or expressed in mammalian cells, respectively, to activate proMMP-2. Apparently, MT2-MMP is able to perform this activation in a TIMP-2-independent manner.…”

Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

“…MT1-MMP appears to be the major activator of MMP-2 as shown using MT1-MMP null mice, however, some activation of MMP-2 in these mice was observed (30). Indeed other members of the MT-MMP sub-family have been shown to activate MMP-2, using either catalytic domains in vitro (MT-MMPs 2, 4, and 5) (13,(31)(32)(33) or the full-length molecule expressed in mammalian cells (MT-MMPs 3, 5, and 6) (6,34,35). However, the requirement for TIMP-2 in MMP-2 activation by MT-MMPs 2-6 has not been demonstrated, nor has the potential role of TIMP-4 in MMP-2 activation by these MT-MMPs been addressed.…”

“…Expression of MT2-MMP in glioblastomas correlated with MMP-2 activation and degree of malignancy (40), and expression of MT2-MMP in COS cells was found to confer invasiveness (41). Although the soluble MT2-MMP catalytic domain produced in Escherichia coli was shown to activate MMP-2 in the absence of TIMP-2 in vitro (31,32), this was thought to occur due to an unphysiologically high concentration of the MT2-MMP. Other reports are unclear regarding the potential for the cellular activation of MMP-2 by full-length MT2-MMP, and the requirement for TIMP-2 has not specifically been addressed.…”