Membrane Type 4 Matrix Metalloproteinase (MMP17) Has Tumor Necrosis Factor-α Convertase Activity but Does Not Activate Pro-MMP2

Abstract

Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein. We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated. Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2. The pro-catalytic domain was expressed in Escherichia coli as insoluble inclusions and active enzyme recovered after refolding. Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3. The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin. mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor ␣ (TNF␣) cleavage site, a glutathione S-transferase-pro-TNF␣ fusion protein, and was found to shed pro-TNF␣ when co-transfected in COS-7 cells. MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNF␣-converting activity suggests a role in inflammation.Matrix metalloproteinases (MMPs) 1 are a large family of structurally related zinc-dependent endopeptidases that are essential in tissue remodeling under physiological and pathological conditions. These include morphogenesis, angiogenesis, tissue repair, arthritis, tumor invasion, and migration of inflammatory leukocytes (1-5) A subset of the MMP family, the membrane type MMPs (MT-MMPs) have more recently been identified (6, 7). To date six such enzymes have been described, and MT1-, -2-, and -3-MMP have been shown to have a wide range of activities against extracellular matrix proteins (8 -13). The activity of MT-MMPs has been implicated in cell invasion of the ECM (4). Possibly more importantly, they are also activators of pro-MMP2 and pro-MMP13 which has been correlated with the invasiveness of tumor and other cells (7,9,14,15). Like other MMPs, the activity of MT-MMPs can be modulated by the tissue inhibitors of MMPs or TIMPs; however, TIMP1 was found to be a poor inhibitor of MT1-, -2-, and -5-MMP (9, 10, 16). TIMP2 and MT1-MMP can also associate to form a pro-MMP2 receptor at the cell surface, leaving the C terminus of TIMP2 free to bind pro-MMP2. This allows efficient activation of pro-MMP2 by adjacent TIMP2-free MT1-MMP and may be a common mechanism for pro-MMP2 activation by .The fourth member of the MT-MMP family, MT4-MMP, has the least degree of sequence identity to the other family members (7, 18). The human cDNA (hMT4-MMP) originally isolated from a human breast carcinoma cell line was found to lack the signal sequence and part of the pro-domain. Northern blot analysis of the tissue mRNA distribution showed hMT4-MMP to be localized in brain, colon, ovary...

The Shedding of Betaglycan Is Regulated by Pervanadate and Mediated by Membrane Type Matrix Metalloprotease-1

“…This result, along with that obtained with mutant cells defective in TACE activity (M1 cells), confirms that TACE is not involved in the pervanadate-stimulated release of betaglycan and argue in favor of members of MT-MMPs. To date six members of the subfamily of MT-MMP have been described (37)(38)(39)(47)(48)(49). It has been shown that among the six MTMMPs, only MT4-MMP is not inhibited by TIMP-2 (37), and MT6-MMP is inhibited by TIMP-2 as well as TIMP-1 (56), ruling out MT4-and MT6-MMP as the pervanadate-stimulated sBG-90 sheddase.…”

“…The effect of the TIMPs has been used previously to characterize the identity of the metalloprotease(s) involved in a given shedding event. For example, TACE is inhibited by TIMP-3, but not by TIMP-2 or TIMP-1 (55), and the majority of the MT-MMPs are inhibited by TIMP-2, but not by TIMP-1 or TIMP-3 (37,38,(47)(48)(49)56). Our data demonstrate that the pervanadate-activated proteolytic activity that results in the production of the sBG-90 fragment in L6E9 cells is inhibited by TIMP-2 but not by TIMP-1.…”

“…In contrast, other metalloproteases putatively involved in ectodomain shedding, such as certain MT-MMPs, are inhibited by TIMP-2 (37). To date, six MT-MMPs have been described (37)(38)(39)(47)(48)(49). To analyze the putative role of MTMMPs on the shedding of sBG-90, we analyzed the effect of the overexpression of these metalloproteases on the production of sBG-90 by L6E9 cells.…”

Delineating the Molecular Basis of the Inactivity of Tissue Inhibitor of Metalloproteinase-2 against Tumor Necrosis Factor-α-converting Enzyme

“…There are four variants of mammalian TIMPs (TIMP-1 to -4), and each TIMP has its own profile of MP selectivity. For instance, whereas the majority of the soluble MMPs are well inhibited by all TIMP-1 to -4, membrane type MMPs and MMP-19 are less sensitive to TIMP-1 (4,5). The ADAMs, on the other hand, are generally more sensitive to TIMP-3 than TIMP-1, -2, or -4 (6 -10).…”

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

“…Apparently, MT2-MMP is able to perform this activation in a TIMP-2-independent manner. 24 The proMMP-2 activation ability has been associated with a characteristic loop insertion (the MT-specific loop) 25 between strands sII and sIII of the catalytic domain, 26 and, accordingly, the inefficiency in proMMP-2 activation of the classical MMPs and MT-MMPs 4 27 and 6 28,29 with the lack of this loop. The more severe phenotype exhibited by MT1-MMP knock-out mice compared with MMP-2 deficient mice 30,31 indicates, however, that the MT-MMPs 1, 2, 3 and 5 play a much broader role than simply to activate proMMP-2.…”