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Reactive Site-modified Tissue Inhibitor of Metalloproteinases-2 Inhibits the Cell-mediated Activation of Progelatinase A

1999; Elsevier BV; Volume: 274; Issue: 15 Linguagem: Inglês

10.1074/jbc.274.15.10497

1083-351X

Shouichi Higashi, Kaoru Miyazaki,

Enzyme Production and Characterization

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is supposed to play a regulatory role in the cell-mediated activation of progelatinase A. To investigate the mechanism of the regulation, we prepared and characterized a chemically modified TIMP-2, and examined its effects on the activation of progelatinase A. We found that treatment of TIMP-2 with cyanate ion led to loss of inhibitory activity toward matrilysin or gelatinase A. Structural and functional analyses of the modified TIMP-2 showed that carbamylation of the α-amino group of the NH2-terminal Cys1 of TIMP-2 led to complete loss of the inhibitory activity. When the reactive-site modified TIMP-2 was added to culture medium of concanavalin A-stimulated HT1080 cells, the conversion of endogenous progelatinase A to the intermediate form was partially inhibited, whereas that of the intermediate form to the mature one was strongly inhibited. The reactive site-modified TIMP-2 also prevented an accumulation of active gelatinase A on the cell surface. We speculate that occupation of the hemopexin-like domain of gelatinase A by the reactive site-modified TIMP-2 makes it unable for gelatinase A to be retained on the cell surface, thus preventing the autocatalytic conversion of the intermediate form of gelatinase A to its mature form. Tissue inhibitor of metalloproteinases-2 (TIMP-2) is supposed to play a regulatory role in the cell-mediated activation of progelatinase A. To investigate the mechanism of the regulation, we prepared and characterized a chemically modified TIMP-2, and examined its effects on the activation of progelatinase A. We found that treatment of TIMP-2 with cyanate ion led to loss of inhibitory activity toward matrilysin or gelatinase A. Structural and functional analyses of the modified TIMP-2 showed that carbamylation of the α-amino group of the NH2-terminal Cys1 of TIMP-2 led to complete loss of the inhibitory activity. When the reactive-site modified TIMP-2 was added to culture medium of concanavalin A-stimulated HT1080 cells, the conversion of endogenous progelatinase A to the intermediate form was partially inhibited, whereas that of the intermediate form to the mature one was strongly inhibited. The reactive site-modified TIMP-2 also prevented an accumulation of active gelatinase A on the cell surface. We speculate that occupation of the hemopexin-like domain of gelatinase A by the reactive site-modified TIMP-2 makes it unable for gelatinase A to be retained on the cell surface, thus preventing the autocatalytic conversion of the intermediate form of gelatinase A to its mature form. matrix metalloproteinase(s) tissue inhibitor of metalloproteinases membrane-type MMP p-aminophenyl mercuric acetate Tris-buffered saline conditioned medium Matrix metalloproteinases (MMPs)1 are zinc-dependent endopeptidases that degrade components of extracellular matrix and play an essential role in tissue remodeling under physiological and pathological conditions such as morphogenesis, angiogenesis, tissue repair, and tumor invasion (1Docherty A.J.P. O'Connell J. Crabbe T. Angal S. Murphy G. Trends Biotechnol. 1992; 10: 200-207Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 2Matrisian L.M. Bioessays. 1992; 14: 455-463Crossref PubMed Scopus (1324) Google Scholar, 3Stetler-Stevenson W.G. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1515) Google Scholar). Most MMPs are secreted as a zymogen and are activated by serine proteases or some activated MMPs. The activities of activated MMPs are regulated by a family of specific inhibitors known as tissue inhibitor of metalloproteinases (TIMPs). Among the MMP family, gelatinase A (MMP-2) and gelatinase B (MMP-9) are critical in the invasion of tumor cells across basement membranes because of their strong activity against type IV collagen, a major component of basement membranes (4Liotta L.A. Cancer Res. 1986; 46: 1-7Crossref PubMed Scopus (1) Google Scholar, 5Collier I.E. Wilhelm S.M. Eisen A.Z. Marmer B.L. Grant G.A. Seltzer J.L. Kronberger A. He C. Bauer E.A. Goldberg G.I. J. Biol. Chem. 1988; 263: 6579-6587Abstract Full Text PDF PubMed Google Scholar, 6Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar). Unlike other zymogen of MMPs, progelatinase A is not activated by serine proteases or soluble MMPs and had been reported to be activated by a MMP-like activity on the surface of cancer and fibroblastic cells (7Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar, 8Brown P.D. Levy A.T. Margulies I.M. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191PubMed Google Scholar, 9Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 10Azzam H.S. Thompson E.W. Cancer Res. 1992; 52: 4540-4544PubMed Google Scholar). Sato et al. (11Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar) recently identified a novel membrane-type MMP, named MT-MMP as an activator of progelatinase A on the cell surface. The cell-mediated activation of progelatinase A includes two steps of processing: MT-MMP-catalyzed cleavage of progelatinase A at a peptide bond between Asn37 and Leu38, first converts the zymogen into an intermediate form, and then autocatalytic cleavage of a Asn80-Tyr81 bond converts the intermediate form into a mature one (12Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). Several studies suggest that both steps are greatly accelerated by binding of (pro)gelatinase A onto the cell surface, and therefore, the receptor of (pro)gelatinase A on the cell surface is important for the activation. Carboxyl-terminal hemopexin-like domain of gelatinase A is reported to be essential for the interaction with the cell surface receptor (12Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar, 13Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). The NH2-terminal reactive site of TIMP-2 binds to the active site of MT-MMP to form a protease-inhibitor complex, whereas the COOH-terminal region of TIMP-2 has an affinity to the hemopexin-like domain of gelatinase A. Therefore, it is hypothesized that a complex formed between MT-MMP and TIMP-2 acts as a receptor of progelatinase A. This hypothesis appears to be supported by a finding that overexpression of MT-MMP results in an accumulation of gelatinase A on the cell surface (11Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). Another candidate for the gelatinase A receptor is integrin αvβ3, which forms a sodium dodecyl sulfate stable complex with gelatinase A also by binding to the hemopexin-like domain (14Brooks P.C. Strömblad S. Sanders L.C. von Schalscha T.L. Aimes R.T. Stetler-Stevenson W.G. Quigley J.P. Cheresh D.A. Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1420) Google Scholar, 15Brooks P.C. Silletti S. von Schalscha T.L. Friedlander M. Cheresh D.A. Cell. 1998; 92: 391-400Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). TIMP-2 is a bifunctional regulator of the cell-mediated activation of progelatinase A. Strongin et al. (13Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar) demonstrated that a small amount of TIMP-2 facilitates the activation of progelatinase A by the MT-MMP-containing cell membrane, whereas excess TIMP-2 strongly inhibits the activation. This could be explained that the binding of TIMP-2 to MT-MMP provides a receptor for progelatinase A and also leads to an inhibition of catalytic activity of MT-MMP. However, the detailed mechanism remains to be clarified. Recently, we examined expression levels of gelatinase A, TIMP-2, and three MT-MMPs in human cancer cell lines and found that activation of progelatinase A has a strong inverse correlation only with the level of TIMP-2 secreted into culture medium (16Shofuda K. Moriyama K. Nishihashi A. Higashi S. Mizushima H. Yasumitsu H. Miki K. Sato H. Seiki M. Miyazaki K. J. Biochem. (Tokyo). 1998; 124: 462-470Crossref PubMed Scopus (82) Google Scholar), suggesting that TIMP-2 is a key regulator of the activation of progelatinase A. In this study, we prepared a chemically modified TIMP-2 of which the reactive site is destroyed, and the modified inhibitor was examined for its effect on the cell-mediated activation of progelatinase A. Mechanisms related to the TIMP-2 regulation of the activation of progelatinase A are discussed.DISCUSSIONTo explore the reactive site of TIMP-2 involved in the interaction with the active site of MMPs, we treated TIMP-2 with cyanate ions under controlled conditions, and identified an amino group essential for the inhibitory activity of TIMP-2. We also examined effects of the reactive site-modified TIMP-2 on the cell-mediated activation of progelatinase A. We found that carbamylation of the α-amino group of the NH2-terminal Cys1 of TIMP-2 led to complete losses of its inhibitory activity and binding ability to matrilysin. The crystal structure of the complex formed between TIMP-1 and stromelysin suggests that the unprotonated α-amino group and carbonyl oxygen of the NH2-terminal Cys1 of TIMP-1 coordinate the catalytic zinc atom of stromelysin, thus being involved in the inhibitory action (19Gomis-Rüth F.X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). Quite recently, the crystal structure of the complex formed between TIMP-2 and catalytic domain of MT1-MMP was also determined (21Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). According to their data, the α-amino group and carbonyl oxygen of the NH2-terminal Cys1 of TIMP-2 similarly interact with the catalytic zinc of the protease, suggesting that chelation of the catalytic zinc atom by the NH2-terminal Cys1 of TIMPs is a common mechanism for the inhibition of MMPs activity. Carbamylation of Cys1 of TIMP-2 must lead to a reduction of basicity of the Nα nitrogen of the α-amino group, which probably makes it unable for the Nα nitrogen to coordinate the catalytic zinc atom of MMPs, thereby abolishing the inhibitory activity of TIMP-2. There is an alternative explanation that the carbamylated α-amino group of Cys1 may not be able to interact with the catalytic zinc atom due to steric hindrance. The crystal structures of the two MMP·TIMP complexes also indicate that TIMPs have wide range contacts with the corresponding MMPs. However, the present study showed that the modified TIMP-2 bearing a single carbamylated α-amino group had essentially no affinity with matrilysin. This discrepancy might be explained by sequential interactions: the primary interaction between the Cys1 of TIMPs and the catalytic zinc atom of MMPs may trigger a rearrangement of residues to make secondary interactions. Further study will be required to clarify this mechanism. Previously, it has been reported that chemical modification of TIMP-1 with diethyl pyrocarbonate abolishes the inhibitory activity. The modified residues are His95, His144, and His164 of TIMP-1, and the modification of His95 is proposed to be responsible for the loss of activity (22Williamson R.A. Smith B.J. Angal S. Freedman R.B. Biochim. Biophys. Acta. 1993; 1203: 147-154Crossref PubMed Scopus (12) Google Scholar). However, mutational study has revealed that replacement of His95 to glutamine does not affect the inhibitory activity of TIMP-1 (22Williamson R.A. Smith B.J. Angal S. Freedman R.B. Biochim. Biophys. Acta. 1993; 1203: 147-154Crossref PubMed Scopus (12) Google Scholar). Furthermore, the H95Q mutant is still sensitive to diethyl pyrocarbonate treatment. So far, there is no explanation for the effect of diethyl pyrocarbonate on the TIMP-1 activity. It is possible, however, to speculate that the α-amino group of Cys1 of TIMP-1 had been modified during treatment with diethyl pyrocarbonate, because the α-amino group, as well as the imidazole group, are reactive with diethyl pyrocarbonate.As the carbamylated TIMP-2 in the matrilysin-unbound fraction had an ability to bind to progelatinase A, it is likely that a site of TIMP-2 essential for the interaction with the hemopexin-like domain of (pro)gelatinase A is not affected by the carbamylation. We found that the reactive site-modified TIMP-2 could prevent an accumulation of the active form of gelatinase A on the surface of concanavalin A-stimulated HT1080 cells. It is hypothesized that a complex formed between MT-MMP and TIMP-2 acts as a cell surface receptor of (pro)gelatinase A (12Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar,13Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). Accordingly, the disappearance of the cell-associated gelatinase A could be explained by speculation that the competitive binding of the reactive site-modified TIMP-2 to the hemopexin-like domain of gelatinase A makes it unable for gelatinase A to be retained on the cell surface, because TIMP-2 cannot interact with MT-MMP. We also found that the reactive site-modified TIMP-2 partially inhibited the conversion of progelatinase A to the intermediate form and strongly inhibited the conversion of the intermediate form to the mature one. As the conversion of progelatinase A to the intermediate form is thought to be facilitated by cell association of progelatinase A (20Kinoshita T. Sato H. Okada A. Ohuchi E. Imai K. Okada Y. Seiki M. J. Biol. Chem. 1998; 273: 16098-16103Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), the partial inhibition of the processing of progelatinase A is likely to be caused by the prevention of cell association of the zymogen by the reactive site-modified TIMP-2 (Fig.7 A). We also speculate that the conversion of the intermediate form of gelatinase A to the mature one depends upon the cell associated activity of gelatinase A, and therefore, deprivation of the cell-associated active form of gelatinase A by the reactive site-modified TIMP-2 causes an inhibition of production of the mature form. In the presence of high concentrations of reactive site-modified TIMP-2, the disappearance of the mature form of gelatinase A in the CM was indeed in parallel with the diminution of the cell-associated active gelatinase A (Fig. 6). Recent studies (15Brooks P.C. Silletti S. von Schalscha T.L. Friedlander M. Cheresh D.A. Cell. 1998; 92: 391-400Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar,23Kinoshita T. Sato H. Takino T. Itoh M. Akizawa T. Seiki M. Cancer Res. 1996; 56: 2535-2538PubMed Google Scholar, 24Pei D.Q. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 25Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17213Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 26Lichte A. Kolkenbrock H. Tschesche H. FEBS Lett. 1996; 397: 277-282Crossref PubMed Scopus (61) Google Scholar) suggest that transmembrane domainless variants of MT-MMP convert progelatinase A to the intermediate form but hardly to the mature one. It is also reported that cell-mediated processing of mutant progelatinase A of which the active site residue is replaced does not produce the mature form of the mutant (27Atkinson S.J. Crabbe T. Cowell S. Ward R.V. Butler M.J. Sato H. Seiki M. Reynolds J.J. Murphy G. J. Biol. Chem. 1995; 270: 30479-30485Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 28Sato H. Takino T. Kinoshita T. Imai K. Okada Y. Stetler-Stevenson W.G. Seiki M. FEBS Lett. 1996; 385: 238-240Crossref PubMed Scopus (174) Google Scholar). These studies suggest the importance of cell associated activity of gelatinase A for the conversion of the intermediate form of gelatinase A to its mature form. Considering the importance of formation of the ternary complex consisting of MT-MMP, TIMP-2, and (pro)gelatinase A, the inhibition of the cell-mediated activation of progelatinase A by TIMP-2 could be explained in two alternative ways. One explanation is that excess TIMP-2 occupies both the active site of MT-MMP and the TIMP-2-binding site in hemopexin-like domain of (pro)gelatinase A, thus preventing the formation of the ternary complex (Fig. 7 B). The other explanation is that TIMP-2 inhibits the catalytic activity of MT-MMP, thus inhibiting the proteolytic processing of progelatinase A. We found that native TIMP-2, as well as reactive site-modified TIMP-2, could prevent accumulation of active gelatinase A on the cell surface, without increasing the cell-associated progelatinase A. These data suggest that prevention of the formation of ternary complex contributes to the TIMP-2 inhibition of the cell-mediated activation of progelatinase A. Native TIMP-2, but not the reactive site-modified TIMP-2, inhibited production of the intermediate form of gelatinase A. Therefore, it is also likely that inhibition of the catalytic activity of MT-MMP by TIMP-2 contributes to inhibition of the processing of progelatinase A. As disappearance of the mature and the intermediate forms of gelatinase A in the CM and diminution of the cell-associated active gelatinase A were observed at similar concentrations of unmodified TIMP-2 (Fig. 6), prevention of formation of the ternary complex and inhibition of MT-MMP activity may occur simultaneously, at a critical concentration of TIMP-2 (Fig. 7 B). It is likely that both the mechanisms make TIMP-2 a potent regulator of the cell-mediated activation of progelatinase A. As described here, reactive site-modified TIMP-2 could inhibit the activation of progelatinase A without inhibiting the catalytic activity of MT-MMP. The reactive site-modified TIMP-2 might be a useful tool to distinguish the functions of MT-MMP and cell-associated gelatinase A. We are now using this modified TIMP-2 to explore the role of MT-MMP and/or cell-associated gelatinase A in the processing of cell-surface proteins. Matrix metalloproteinases (MMPs)1 are zinc-dependent endopeptidases that degrade components of extracellular matrix and play an essential role in tissue remodeling under physiological and pathological conditions such as morphogenesis, angiogenesis, tissue repair, and tumor invasion (1Docherty A.J.P. O'Connell J. Crabbe T. Angal S. Murphy G. Trends Biotechnol. 1992; 10: 200-207Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 2Matrisian L.M. Bioessays. 1992; 14: 455-463Crossref PubMed Scopus (1324) Google Scholar, 3Stetler-Stevenson W.G. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1515) Google Scholar). Most MMPs are secreted as a zymogen and are activated by serine proteases or some activated MMPs. The activities of activated MMPs are regulated by a family of specific inhibitors known as tissue inhibitor of metalloproteinases (TIMPs). Among the MMP family, gelatinase A (MMP-2) and gelatinase B (MMP-9) are critical in the invasion of tumor cells across basement membranes because of their strong activity against type IV collagen, a major component of basement membranes (4Liotta L.A. Cancer Res. 1986; 46: 1-7Crossref PubMed Scopus (1) Google Scholar, 5Collier I.E. Wilhelm S.M. Eisen A.Z. Marmer B.L. Grant G.A. Seltzer J.L. Kronberger A. He C. Bauer E.A. Goldberg G.I. J. Biol. Chem. 1988; 263: 6579-6587Abstract Full Text PDF PubMed Google Scholar, 6Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar). Unlike other zymogen of MMPs, progelatinase A is not activated by serine proteases or soluble MMPs and had been reported to be activated by a MMP-like activity on the surface of cancer and fibroblastic cells (7Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar, 8Brown P.D. Levy A.T. Margulies I.M. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191PubMed Google Scholar, 9Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 10Azzam H.S. Thompson E.W. Cancer Res. 1992; 52: 4540-4544PubMed Google Scholar). Sato et al. (11Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar) recently identified a novel membrane-type MMP, named MT-MMP as an activator of progelatinase A on the cell surface. The cell-mediated activation of progelatinase A includes two steps of processing: MT-MMP-catalyzed cleavage of progelatinase A at a peptide bond between Asn37 and Leu38, first converts the zymogen into an intermediate form, and then autocatalytic cleavage of a Asn80-Tyr81 bond converts the intermediate form into a mature one (12Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). Several studies suggest that both steps are greatly accelerated by binding of (pro)gelatinase A onto the cell surface, and therefore, the receptor of (pro)gelatinase A on the cell surface is important for the activation. Carboxyl-terminal hemopexin-like domain of gelatinase A is reported to be essential for the interaction with the cell surface receptor (12Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar, 13Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). The NH2-terminal reactive site of TIMP-2 binds to the active site of MT-MMP to form a protease-inhibitor complex, whereas the COOH-terminal region of TIMP-2 has an affinity to the hemopexin-like domain of gelatinase A. Therefore, it is hypothesized that a complex formed between MT-MMP and TIMP-2 acts as a receptor of progelatinase A. This hypothesis appears to be supported by a finding that overexpression of MT-MMP results in an accumulation of gelatinase A on the cell surface (11Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). Another candidate for the gelatinase A receptor is integrin αvβ3, which forms a sodium dodecyl sulfate stable complex with gelatinase A also by binding to the hemopexin-like domain (14Brooks P.C. Strömblad S. Sanders L.C. von Schalscha T.L. Aimes R.T. Stetler-Stevenson W.G. Quigley J.P. Cheresh D.A. Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1420) Google Scholar, 15Brooks P.C. Silletti S. von Schalscha T.L. Friedlander M. Cheresh D.A. Cell. 1998; 92: 391-400Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). TIMP-2 is a bifunctional regulator of the cell-mediated activation of progelatinase A. Strongin et al. (13Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar) demonstrated that a small amount of TIMP-2 facilitates the activation of progelatinase A by the MT-MMP-containing cell membrane, whereas excess TIMP-2 strongly inhibits the activation. This could be explained that the binding of TIMP-2 to MT-MMP provides a receptor for progelatinase A and also leads to an inhibition of catalytic activity of MT-MMP. However, the detailed mechanism remains to be clarified. Recently, we examined expression levels of gelatinase A, TIMP-2, and three MT-MMPs in human cancer cell lines and found that activation of progelatinase A has a strong inverse correlation only with the level of TIMP-2 secreted into culture medium (16Shofuda K. Moriyama K. Nishihashi A. Higashi S. Mizushima H. Yasumitsu H. Miki K. Sato H. Seiki M. Miyazaki K. J. Biochem. (Tokyo). 1998; 124: 462-470Crossref PubMed Scopus (82) Google Scholar), suggesting that TIMP-2 is a key regulator of the activation of progelatinase A. In this study, we prepared a chemically modified TIMP-2 of which the reactive site is destroyed, and the modified inhibitor was examined for its effect on the cell-mediated activation of progelatinase A. Mechanisms related to the TIMP-2 regulation of the activation of progelatinase A are discussed. DISCUSSIONTo explore the reactive site of TIMP-2 involved in the interaction with the active site of MMPs, we treated TIMP-2 with cyanate ions under controlled conditions, and identified an amino group essential for the inhibitory activity of TIMP-2. We also examined effects of the reactive site-modified TIMP-2 on the cell-mediated activation of progelatinase A. We found that carbamylation of the α-amino group of the NH2-terminal Cys1 of TIMP-2 led to complete losses of its inhibitory activity and binding ability to matrilysin. The crystal structure of the complex formed between TIMP-1 and stromelysin suggests that the unprotonated α-amino group and carbonyl oxygen of the NH2-terminal Cys1 of TIMP-1 coordinate the catalytic zinc atom of stromelysin, thus being involved in the inhibitory action (19Gomis-Rüth F.X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). Quite recently, the crystal structure of the complex formed between TIMP-2 and catalytic domain of MT1-MMP was also determined (21Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). According to their data, the α-amino group and carbonyl oxygen of the NH2-terminal Cys1 of TIMP-2 similarly interact with the catalytic zinc of the protease, suggesting that chelation of the catalytic zinc atom by the NH2-terminal Cys1 of TIMPs is a common mechanism for the inhibition of MMPs activity. Carbamylation of Cys1 of TIMP-2 must lead to a reduction of basicity of the Nα nitrogen of the α-amino group, which probably makes it unable for the Nα nitrogen to coordinate the catalytic zinc atom of MMPs, thereby abolishing the inhibitory activity of TIMP-2. There is an alternative explanation that the carbamylated α-amino group of Cys1 may not be able to interact with the catalytic zinc atom due to steric hindrance. The crystal structures of the two MMP·TIMP complexes also indicate that TIMPs have wide range contacts with the corresponding MMPs. However, the present study showed that the modified TIMP-2 bearing a single carbamylated α-amino group had essentially no affinity with matrilysin. This discrepancy might be explained by sequential interactions: the primary interaction between the Cys1 of TIMPs and the catalytic zinc atom of MMPs may trigger a rearrangement of residues to make secondary interactions. Further study will be required to clarify this mechanism. Previously, it has been reported that chemical modification of TIMP-1 with diethyl pyrocarbonate abolishes the inhibitory activity. The modified residues are His95, His144, and His164 of TIMP-1, and the modification of His95 is proposed to be responsible for the loss of activity (22Williamson R.A. Smith B.J. Angal S. Freedman R.B. Biochim. Biophys. Acta. 1993; 1203: 147-154Crossref PubMed Scopus (12) Google Scholar). However, mutational study has revealed that replacement of His95 to glutamine does not affect the inhibitory activity of TIMP-1 (22Williamson R.A. Smith B.J. Angal S. Freedman R.B. Biochim. Biophys. Acta. 1993; 1203: 147-154Crossref PubMed Scopus (12) Google Scholar). Furthermore, the H95Q mutant is still sensitive to diethyl pyrocarbonate treatment. So far, there is no explanation for the effect of diethyl pyrocarbonate on the TIMP-1 activity. It is possible, however, to speculate that the α-amino group of Cys1 of TIMP-1 had been modified during treatment with diethyl pyrocarbonate, because the α-amino group, as well as the imidazole group, are reactive with diethyl pyrocarbonate.As the carbamylated TIMP-2 in the matrilysin-unbound fraction had an ability to bind to progelatinase A, it is likely that a site of TIMP-2 essential for the interaction with the hemopexin-like domain of (pro)gelatinase A is not affected by the carbamylation. We found that the reactive site-modified TIMP-2 could prevent an accumulation of the active form of gelatinase A on the surface of concanavalin A-stimulated HT1080 cells. It is hypothesized that a complex formed between MT-MMP and TIMP-2 acts as a cell surface receptor of (pro)gelatinase A (12Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar,13Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). Accordingly, the disappearance of the cell-associated gelatinase A could be explained by speculation that the competitive binding of the reactive site-modified TIMP-2 to the hemopexin-like domain of gelatinase A makes it unable for gelatinase A to be retained on the cell surface, because TIMP-2 cannot interact with MT-MMP. We also found that the reactive site-modified TIMP-2 partially inhibited the conversion of progelatinase A to the intermediate form and strongly inhibited the conversion of the intermediate form to the mature one. As the conversion of progelatinase A to the intermediate form is thought to be facilitated by cell association of progelatinase A (20Kinoshita T. Sato H. Okada A. Ohuchi E. Imai K. Okada Y. Seiki M. J. Biol. Chem. 1998; 273: 16098-16103Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), the partial inhibition of the processing of progelatinase A is likely to be caused by the prevention of cell association of the zymogen by the reactive site-modified TIMP-2 (Fig.7 A). We also speculate that the conversion of the intermediate form of gelatinase A to the mature one depends upon the cell associated activity of gelatinase A, and therefore, deprivation of the cell-associated active form of gelatinase A by the reactive site-modified TIMP-2 causes an inhibition of production of the mature form. In the presence of high concentrations of reactive site-modified TIMP-2, the disappearance of the mature form of gelatinase A in the CM was indeed in parallel with the diminution of the cell-associated active gelatinase A (Fig. 6). Recent studies (15Brooks P.C. Silletti S. von Schalscha T.L. Friedlander M. Cheresh D.A. Cell. 1998; 92: 391-400Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar,23Kinoshita T. Sato H. Takino T. Itoh M. Akizawa T. Seiki M. Cancer Res. 1996; 56: 2535-2538PubMed Google Scholar, 24Pei D.Q. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 25Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17213Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 26Lichte A. Kolkenbrock H. Tschesche H. FEBS Lett. 1996; 397: 277-282Crossref PubMed Scopus (61) Google Scholar) suggest that transmembrane domainless variants of MT-MMP convert progelatinase A to the intermediate form but hardly to the mature one. It is also reported that cell-mediated processing of mutant progelatinase A of which the active site residue is replaced does not produce the mature form of the mutant (27Atkinson S.J. Crabbe T. Cowell S. Ward R.V. Butler M.J. Sato H. Seiki M. Reynolds J.J. Murphy G. J. Biol. Chem. 1995; 270: 30479-30485Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 28Sato H. Takino T. Kinoshita T. Imai K. Okada Y. Stetler-Stevenson W.G. Seiki M. FEBS Lett. 1996; 385: 238-240Crossref PubMed Scopus (174) Google Scholar). These studies suggest the importance of cell associated activity of gelatinase A for the conversion of the intermediate form of gelatinase A to its mature form. Considering the importance of formation of the ternary complex consisting of MT-MMP, TIMP-2, and (pro)gelatinase A, the inhibition of the cell-mediated activation of progelatinase A by TIMP-2 could be explained in two alternative ways. One explanation is that excess TIMP-2 occupies both the active site of MT-MMP and the TIMP-2-binding site in hemopexin-like domain of (pro)gelatinase A, thus preventing the formation of the ternary complex (Fig. 7 B). The other explanation is that TIMP-2 inhibits the catalytic activity of MT-MMP, thus inhibiting the proteolytic processing of progelatinase A. We found that native TIMP-2, as well as reactive site-modified TIMP-2, could prevent accumulation of active gelatinase A on the cell surface, without increasing the cell-associated progelatinase A. These data suggest that prevention of the formation of ternary complex contributes to the TIMP-2 inhibition of the cell-mediated activation of progelatinase A. Native TIMP-2, but not the reactive site-modified TIMP-2, inhibited production of the intermediate form of gelatinase A. Therefore, it is also likely that inhibition of the catalytic activity of MT-MMP by TIMP-2 contributes to inhibition of the processing of progelatinase A. As disappearance of the mature and the intermediate forms of gelatinase A in the CM and diminution of the cell-associated active gelatinase A were observed at similar concentrations of unmodified TIMP-2 (Fig. 6), prevention of formation of the ternary complex and inhibition of MT-MMP activity may occur simultaneously, at a critical concentration of TIMP-2 (Fig. 7 B). It is likely that both the mechanisms make TIMP-2 a potent regulator of the cell-mediated activation of progelatinase A. As described here, reactive site-modified TIMP-2 could inhibit the activation of progelatinase A without inhibiting the catalytic activity of MT-MMP. The reactive site-modified TIMP-2 might be a useful tool to distinguish the functions of MT-MMP and cell-associated gelatinase A. We are now using this modified TIMP-2 to explore the role of MT-MMP and/or cell-associated gelatinase A in the processing of cell-surface proteins. To explore the reactive site of TIMP-2 involved in the interaction with the active site of MMPs, we treated TIMP-2 with cyanate ions under controlled conditions, and identified an amino group essential for the inhibitory activity of TIMP-2. We also examined effects of the reactive site-modified TIMP-2 on the cell-mediated activation of progelatinase A. We found that carbamylation of the α-amino group of the NH2-terminal Cys1 of TIMP-2 led to complete losses of its inhibitory activity and binding ability to matrilysin. The crystal structure of the complex formed between TIMP-1 and stromelysin suggests that the unprotonated α-amino group and carbonyl oxygen of the NH2-terminal Cys1 of TIMP-1 coordinate the catalytic zinc atom of stromelysin, thus being involved in the inhibitory action (19Gomis-Rüth F.X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). Quite recently, the crystal structure of the complex formed between TIMP-2 and catalytic domain of MT1-MMP was also determined (21Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). According to their data, the α-amino group and carbonyl oxygen of the NH2-terminal Cys1 of TIMP-2 similarly interact with the catalytic zinc of the protease, suggesting that chelation of the catalytic zinc atom by the NH2-terminal Cys1 of TIMPs is a common mechanism for the inhibition of MMPs activity. Carbamylation of Cys1 of TIMP-2 must lead to a reduction of basicity of the Nα nitrogen of the α-amino group, which probably makes it unable for the Nα nitrogen to coordinate the catalytic zinc atom of MMPs, thereby abolishing the inhibitory activity of TIMP-2. There is an alternative explanation that the carbamylated α-amino group of Cys1 may not be able to interact with the catalytic zinc atom due to steric hindrance. The crystal structures of the two MMP·TIMP complexes also indicate that TIMPs have wide range contacts with the corresponding MMPs. However, the present study showed that the modified TIMP-2 bearing a single carbamylated α-amino group had essentially no affinity with matrilysin. This discrepancy might be explained by sequential interactions: the primary interaction between the Cys1 of TIMPs and the catalytic zinc atom of MMPs may trigger a rearrangement of residues to make secondary interactions. Further study will be required to clarify this mechanism. Previously, it has been reported that chemical modification of TIMP-1 with diethyl pyrocarbonate abolishes the inhibitory activity. The modified residues are His95, His144, and His164 of TIMP-1, and the modification of His95 is proposed to be responsible for the loss of activity (22Williamson R.A. Smith B.J. Angal S. Freedman R.B. Biochim. Biophys. Acta. 1993; 1203: 147-154Crossref PubMed Scopus (12) Google Scholar). However, mutational study has revealed that replacement of His95 to glutamine does not affect the inhibitory activity of TIMP-1 (22Williamson R.A. Smith B.J. Angal S. Freedman R.B. Biochim. Biophys. Acta. 1993; 1203: 147-154Crossref PubMed Scopus (12) Google Scholar). Furthermore, the H95Q mutant is still sensitive to diethyl pyrocarbonate treatment. So far, there is no explanation for the effect of diethyl pyrocarbonate on the TIMP-1 activity. It is possible, however, to speculate that the α-amino group of Cys1 of TIMP-1 had been modified during treatment with diethyl pyrocarbonate, because the α-amino group, as well as the imidazole group, are reactive with diethyl pyrocarbonate. As the carbamylated TIMP-2 in the matrilysin-unbound fraction had an ability to bind to progelatinase A, it is likely that a site of TIMP-2 essential for the interaction with the hemopexin-like domain of (pro)gelatinase A is not affected by the carbamylation. We found that the reactive site-modified TIMP-2 could prevent an accumulation of the active form of gelatinase A on the surface of concanavalin A-stimulated HT1080 cells. It is hypothesized that a complex formed between MT-MMP and TIMP-2 acts as a cell surface receptor of (pro)gelatinase A (12Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar,13Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). Accordingly, the disappearance of the cell-associated gelatinase A could be explained by speculation that the competitive binding of the reactive site-modified TIMP-2 to the hemopexin-like domain of gelatinase A makes it unable for gelatinase A to be retained on the cell surface, because TIMP-2 cannot interact with MT-MMP. We also found that the reactive site-modified TIMP-2 partially inhibited the conversion of progelatinase A to the intermediate form and strongly inhibited the conversion of the intermediate form to the mature one. As the conversion of progelatinase A to the intermediate form is thought to be facilitated by cell association of progelatinase A (20Kinoshita T. Sato H. Okada A. Ohuchi E. Imai K. Okada Y. Seiki M. J. Biol. Chem. 1998; 273: 16098-16103Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), the partial inhibition of the processing of progelatinase A is likely to be caused by the prevention of cell association of the zymogen by the reactive site-modified TIMP-2 (Fig.7 A). We also speculate that the conversion of the intermediate form of gelatinase A to the mature one depends upon the cell associated activity of gelatinase A, and therefore, deprivation of the cell-associated active form of gelatinase A by the reactive site-modified TIMP-2 causes an inhibition of production of the mature form. In the presence of high concentrations of reactive site-modified TIMP-2, the disappearance of the mature form of gelatinase A in the CM was indeed in parallel with the diminution of the cell-associated active gelatinase A (Fig. 6). Recent studies (15Brooks P.C. Silletti S. von Schalscha T.L. Friedlander M. Cheresh D.A. Cell. 1998; 92: 391-400Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar,23Kinoshita T. Sato H. Takino T. Itoh M. Akizawa T. Seiki M. Cancer Res. 1996; 56: 2535-2538PubMed Google Scholar, 24Pei D.Q. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 25Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17213Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 26Lichte A. Kolkenbrock H. Tschesche H. FEBS Lett. 1996; 397: 277-282Crossref PubMed Scopus (61) Google Scholar) suggest that transmembrane domainless variants of MT-MMP convert progelatinase A to the intermediate form but hardly to the mature one. It is also reported that cell-mediated processing of mutant progelatinase A of which the active site residue is replaced does not produce the mature form of the mutant (27Atkinson S.J. Crabbe T. Cowell S. Ward R.V. Butler M.J. Sato H. Seiki M. Reynolds J.J. Murphy G. J. Biol. Chem. 1995; 270: 30479-30485Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 28Sato H. Takino T. Kinoshita T. Imai K. Okada Y. Stetler-Stevenson W.G. Seiki M. FEBS Lett. 1996; 385: 238-240Crossref PubMed Scopus (174) Google Scholar). These studies suggest the importance of cell associated activity of gelatinase A for the conversion of the intermediate form of gelatinase A to its mature form. Considering the importance of formation of the ternary complex consisting of MT-MMP, TIMP-2, and (pro)gelatinase A, the inhibition of the cell-mediated activation of progelatinase A by TIMP-2 could be explained in two alternative ways. One explanation is that excess TIMP-2 occupies both the active site of MT-MMP and the TIMP-2-binding site in hemopexin-like domain of (pro)gelatinase A, thus preventing the formation of the ternary complex (Fig. 7 B). The other explanation is that TIMP-2 inhibits the catalytic activity of MT-MMP, thus inhibiting the proteolytic processing of progelatinase A. We found that native TIMP-2, as well as reactive site-modified TIMP-2, could prevent accumulation of active gelatinase A on the cell surface, without increasing the cell-associated progelatinase A. These data suggest that prevention of the formation of ternary complex contributes to the TIMP-2 inhibition of the cell-mediated activation of progelatinase A. Native TIMP-2, but not the reactive site-modified TIMP-2, inhibited production of the intermediate form of gelatinase A. Therefore, it is also likely that inhibition of the catalytic activity of MT-MMP by TIMP-2 contributes to inhibition of the processing of progelatinase A. As disappearance of the mature and the intermediate forms of gelatinase A in the CM and diminution of the cell-associated active gelatinase A were observed at similar concentrations of unmodified TIMP-2 (Fig. 6), prevention of formation of the ternary complex and inhibition of MT-MMP activity may occur simultaneously, at a critical concentration of TIMP-2 (Fig. 7 B). It is likely that both the mechanisms make TIMP-2 a potent regulator of the cell-mediated activation of progelatinase A. As described here, reactive site-modified TIMP-2 could inhibit the activation of progelatinase A without inhibiting the catalytic activity of MT-MMP. The reactive site-modified TIMP-2 might be a useful tool to distinguish the functions of MT-MMP and cell-associated gelatinase A. We are now using this modified TIMP-2 to explore the role of MT-MMP and/or cell-associated gelatinase A in the processing of cell-surface proteins. We thank M. Isaji and K. Hoshida (Biosciences Research Laboratory, Mochida Pharmaceutical Co., Ltd., Tokyo) for amino acid sequence analysis and mass spectrometric analysis. We are grateful to Drs. N. Koshikawa and S. Miyata for help with purification of TIMP-2.