Human 72-kilodalton type IV collagenase forms a complex with a tissue inhibitor of metalloproteases designated TIMP-2.

Abstract

Simian virus 40 (SV40)-transformed human lung fibroblasts secrete both 72-kDa type IV collagenase and a closely related 92-kDa type IV collagenase that was not detected in the parental cell line. The 92-kDa type IV procollagenase purified from these cells exists in a noncovalent complex with the tissue inhibitor of metalloproteases, TIMP. Here we report that the 72-kDa type IV procollagenase purified from HRAS-transformed human bronchial epithelial cells, SV40-transformed lung fibroblasts, and normal skin fibroblasts exists in a stable but noncovalent stoichiometric complex with a 24-kDa inhibitor referred to here as "TIMP-2." TIMP-2 is closely related to TIMP, as demonstrated by comparison of the partial amino acid sequence of this protein to that of TIMP, although it does not cross-react with TIMP-specific antibody. The TIMP-2 inhibitor interacts with the 72-kDa type IV collagenase in preference to the 92-kDa type IV collagenase that forms a complex exclusively with TIMP. The 72-kDa type IV collagenase-TIMP-2 complex can be activated with organomercurials to yield a catalytically competent enzyme. Activation occurs concomitantly with autoproteolytic cleavage of the amino terminus of the protein and does not require dissociation of the complex. Both activity and activation of the complex can be completely inhibited by further addition of stoichiometric quantities of purified TIMP-2 or recombinant TIMP.Tissue inhibitor of metalloproteases (TIMP) is a ubiquitous (1) glycoprotein (Mr 30,000) that originally was purified from serum-free medium conditioned by normal human skin fibroblasts (2). Inhibition of interstitial collagenase is stoichiometric, with a 1:1 molar ratio of inhibitor to enzyme being required for complete inhibition of enzyme activity. Interstitial procollagenase can neither interact with TIMP nor bind to collagen; both require extracellular activation of the collagenase zymogen (3, 4). Active enzyme binds with nearly equal affinity to both monomeric collagen and aggregated fibrils. The K, of enzyme-inhibitor binding was determined to be less than 10-9 M (4).The gene encoding TIMP has been cloned as a collagenase inhibitor (5, 6) and as a factor with erythroid-potentiating activity (7) that stimulates colony formation by relatively mature erythroid precursors (CFU-E). The TIMP gene has been localized to the X chromosome (8, 9). The secreted protein consists of 184 amino acid residues and contains six disulfide bonds and two glycosylation sites containing Nlinked oligosaccharides. The sequence of the inhibitor has no substantial homology to previously sequenced protease inhibitors with the exception of the metalloprotease inhibitor isolated from bovine scapular cartilage (10) and from normal murine fibroblasts (11, 12). The inhibitor extracted from cartilage has a molecular mass of 27,400 with an NH2-terminal sequence of 45 residues that shows 65% homology to TIMP.Recent observations suggest that expression of TIMP may be involved in suppression of tumorigenicity of immortal murine 3T3 cells...

Specific, High Affinity Binding of Tissue Inhibitor of Metalloproteinases-4 (TIMP-4) to the COOH-terminal Hemopexin-like Domain of Human Gelatinase A

“…This demonstrates that both inhibitors cannot bind simultaneously, suggesting the presence of a common or overlapping binding sites on the rC domain. Latent gelatinase A from ROS 17/2.8 cell conditioned culture medium is present mainly as an enzyme-TIMP-2 complex (22,33). The reduced binding of this enzyme to exogenous TIMP-4 and TIMP-2 therefore further indicates competition for a common or overlapping binding sites.…”

“…However, the association of TIMPs with the proforms of the MMPs is more specific, and so far only two such interactions have been described: that of TIMP-2 with the proform of gelatinase A (22,(33)(34)(35) and that of TIMP-1 with the proform of gelatinase B (13,45). In this report, we present evidence for the first time of a third TIMP-progelatinase interaction, that is, of the newly cloned TIMP-4 with progelatinase A.…”

“…Gelatinase A is unique among the MMPs in that the latent enzyme binds TIMP-2 to form a tightly bound 1:1 molar stoichiometric complex (22,(33)(34)(35), whereas only the active forms of the other MMPs can bind this inhibitor. Binding of TIMP-2 to progelatinase A occurs via the C domains of the enzyme (17,18,34,36) and inhibitor (37,38); and with the other MMPs, an additional binding site for the N domain of TIMP-2 is also present in the catalytic domain of active gelatinase A (18,22,33,34). A further binding site for the N domain of TIMP-2 is also found on the C domain of gelatinase A (39), although the biological significance of this interaction is not yet clear.…”

“…A new fourth member of the TIMP family (TIMP-4) has very recently been identified (31,32); the predicted sequence of this protein shares a 37% homology with TIMP-1 but a 51% identity with TIMP-2 and TIMP-3 (31). Gelatinase A is unique among the MMPs in that the latent enzyme binds TIMP-2 to form a tightly bound 1:1 molar stoichiometric complex (22,(33)(34)(35), whereas only the active forms of the other MMPs can bind this inhibitor. Binding of TIMP-2 to progelatinase A occurs via the C domains of the enzyme (17,18,34,36) and inhibitor (37,38); and with the other MMPs, an additional binding site for the N domain of TIMP-2 is also present in the catalytic domain of active gelatinase A (18,22,33,34).…”

Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins

“…22 In contrast, TIMP-2 binds selectively to pro-MMP-2, an interaction that can serve to present the enzyme for activation by membrane type MMP-1 (MT1-MMP). [22][23][24][25] Higher concentrations of TIMP-2, but not TIMP-1, inhibit MT1-MMP. 26,27 In a recent study of adenovirus-mediated TIMP gene transfer into isolated SMC, 19 TIMP-2, but not TIMP-1, was shown to inhibit proliferation, an effect not mediated by inhibition of MMPs.…”