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Human Tissue Inhibitor of Metalloproteinases 3 Interacts with Both the N- and C-terminal Domains of Gelatinases A and B

1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês

10.1074/jbc.274.16.10846

ISSN

1083-351X

Autores

Georgina S. Butler, Suneel Apte, Frances Willenbrock, Gillian Murphy,

Tópico(s)

Peptidase Inhibition and Analysis

Resumo

We compared the association constants of tissue inhibitor of metalloproteinases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A makes an important contribution to the interaction with TIMP-3. The exchange of portions of the gelatinase A hemopexin domain with that of stromelysin revealed that residues 568–631 of gelatinase A were required for rapid association with TIMP-3. The N-terminal domain of gelatinase B alone also showed slower association with TIMP-3, again implying significant C-domain interactions. The isolation of complexes between TIMP-3 and progelatinases A and B on gelatin-agarose demonstrated that TIMP-3 binds to both proenzymes. We analyzed the effect of various polyanions on the inhibitory activity of TIMP-3 in our soluble assay. The association rate was increased by dextran sulfate, heparin, and heparan sulfate, but not by dermatan sulfate or hyaluronic acid. Because TIMP-3 is sequestered in the extracellular matrix, the presence of certain heparan sulfate proteoglycans could enhance its inhibitory capacity. We compared the association constants of tissue inhibitor of metalloproteinases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A makes an important contribution to the interaction with TIMP-3. The exchange of portions of the gelatinase A hemopexin domain with that of stromelysin revealed that residues 568–631 of gelatinase A were required for rapid association with TIMP-3. The N-terminal domain of gelatinase B alone also showed slower association with TIMP-3, again implying significant C-domain interactions. The isolation of complexes between TIMP-3 and progelatinases A and B on gelatin-agarose demonstrated that TIMP-3 binds to both proenzymes. We analyzed the effect of various polyanions on the inhibitory activity of TIMP-3 in our soluble assay. The association rate was increased by dextran sulfate, heparin, and heparan sulfate, but not by dermatan sulfate or hyaluronic acid. Because TIMP-3 is sequestered in the extracellular matrix, the presence of certain heparan sulfate proteoglycans could enhance its inhibitory capacity. The tissue inhibitors of metalloproteinases (TIMPs) 1The abbreviations used are: TIMP, tissue inhibitor of metalloproteinases; MMP, matrix metalloproteinase; (Δ1–414)gelatinase A, C-terminal domain of gelatinase A; (Δ418–631)gelatinase A, N-terminal domain of gelatinase A; proE375A-gelatinase A, catalytically inactive mutant of gelatinase A; (Δ426–688)gelatinase B, N-terminal domain of gelatinase B; N-G.C-SGG, gelatinase A mutant with residues 418–474 replaced with residues 248–305 of stromelysin-1; N-G.C-SGS, as N-G.C-SGG but with the additional replacement of residues 568–631 of gelatinase A with residues 400–460 of stromelysin-1; N-GL.C-SL, gelatinase A residues 1–417 fused with residues 248–460 of stromelysin-1; TCABN, 50 mm Tris, pH 7.5, 150 mm NaCl, 10 mmCaCl2, 0.025% Brij 35, and 0.02% azide; MT, membrane type.1The abbreviations used are: TIMP, tissue inhibitor of metalloproteinases; MMP, matrix metalloproteinase; (Δ1–414)gelatinase A, C-terminal domain of gelatinase A; (Δ418–631)gelatinase A, N-terminal domain of gelatinase A; proE375A-gelatinase A, catalytically inactive mutant of gelatinase A; (Δ426–688)gelatinase B, N-terminal domain of gelatinase B; N-G.C-SGG, gelatinase A mutant with residues 418–474 replaced with residues 248–305 of stromelysin-1; N-G.C-SGS, as N-G.C-SGG but with the additional replacement of residues 568–631 of gelatinase A with residues 400–460 of stromelysin-1; N-GL.C-SL, gelatinase A residues 1–417 fused with residues 248–460 of stromelysin-1; TCABN, 50 mm Tris, pH 7.5, 150 mm NaCl, 10 mmCaCl2, 0.025% Brij 35, and 0.02% azide; MT, membrane type. are specific protein inhibitors of the matrix metalloproteinases (MMPs), a group of zinc-dependent enzymes that include collagenases, gelatinases, and stromelysins. Four forms of human TIMP have been cloned: TIMP-1 (1Docherty A.J.P. Lyons A. Smith B.J. Wright E.M. Stephens P.E. Harris T.J.R. Murphy G. Reynolds J.J. Nature. 1985; 318: 66-69Crossref PubMed Scopus (579) Google Scholar), TIMP-2 (2Boone T.C. Johnson M.J. DeClerck Y.A. Langley K.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2800-2804Crossref PubMed Scopus (179) Google Scholar), TIMP-3 (3Apte S.S. Mattei M.-G. Olsen B.R. Genomics. 1994; 19: 86-90Crossref PubMed Scopus (195) Google Scholar, 4Silbiger S.M. Jacobsen V.L. Cupples R.L. Koski R.A. Gene (Amst.). 1994; 141: 293-297Crossref PubMed Scopus (65) Google Scholar, 5Uria J.A. Ferrando A.A. Velasco G. Freije J.M.P. Lopez-Otin C. Cancer Res. 1994; 54: 2091-2094PubMed Google Scholar, 6Wilde C.G. Hawkins P.R. Coleman R.T. Levine W.B. Delegeane A.M. Okamoto P.M. Ito L.Y. Scott R.W. Seilhamer J.J. DNA Cell Biol. 1994; 13: 711-718Crossref PubMed Scopus (40) Google Scholar), and, more recently, TIMP-4 (7Greene J. Wang M. Lin Y.E. Raymond L.A. Rosen C. Shi Y.E. J. Biol. Chem. 1996; 271: 30375-30380Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). TIMP-1 and TIMP-2 are secreted by many cell types in culture and are found in body fluids and tissue extracts. TIMP-3 is unique in that it appears to be a component of the extracellular matrix (8Blenis J. Hawkes S.P. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 770-774Crossref PubMed Scopus (31) Google Scholar, 9Staskus P.W. Masiarz F.R. Pallanck L.J. Hawkes S.P. J. Biol. Chem. 1991; 266: 449-454Abstract Full Text PDF PubMed Google Scholar, 10Kishnani N.S. Staskus P.W. Yang T.T. Masiarz F.R. Hawkes S.P. Matrix Biol. 1994; 14: 479-488Crossref Scopus (36) Google Scholar) and occurs in relatively small amounts, possibly being expressed during specific cellular events (11Wick M. Bürger C. Brüsselbach S. Lucibello F.C. Müller R. J. Biol. Chem. 1994; 269: 18953-18960Abstract Full Text PDF PubMed Google Scholar). The TIMPs have comparable abilities to inhibit the active forms of the MMPs when assessed using macromolecular substrates (12Ward R.V. Hembry R.M. Reynolds J.J. Murphy G. Biochem. J. 1991; 278: 179-187Crossref PubMed Scopus (162) Google Scholar, 13Apte S.S. Olsen B.R. Murphy G. J. Biol. Chem. 1995; 270: 14313-14318Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) and have been shown to make tight binding noncovalent complexes with active MMPs with a 1:1 stoichiometry (14Cawston T.E. Galloway W.A. Mercer E. Murphy G. Reynolds J.J. Biochem. J. 1981; 195: 159-165Crossref PubMed Scopus (262) Google Scholar, 15Welgus H.G. Campbell E.J. Bar-Shavit Z. Senior R.M. Teitelbaum S.L. J. Clin. Invest. 1985; 76: 219-224Crossref PubMed Scopus (149) Google Scholar, 16Okada Y. Nagase H. Harris Jr., E.D. J. Biol. Chem. 1986; 261: 14245-14255Abstract Full Text PDF PubMed Google Scholar, 17Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (238) Google Scholar). The inhibitors have related primary and secondary structures, consisting of an N-terminal subdomain of three disulfide bonded loops and a smaller C-terminal region also containing three loops (18Williamson R.A. Marston F.A.O. Angal S. Koklitis P. Panico M. Morris H.R. Carne A.F. Smith B.J. Harris T.J.R. Freedman R.B. Biochem. J. 1990; 268: 267-274Crossref PubMed Scopus (152) Google Scholar, 19Murphy G. Houbrechts A. Cockett M.I. Williamson R.A. O'Shea M. Docherty A.J.P. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (281) Google Scholar, 20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar). The N-terminal domain of TIMP-1 and TIMP-2 can act as a functional inhibitor (19Murphy G. Houbrechts A. Cockett M.I. Williamson R.A. O'Shea M. Docherty A.J.P. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (281) Google Scholar, 21DeClerck Y.A. Yean T.D. Lee Y. Tomich J.M. Langley K. Biochem. J. 1993; 289: 65-69Crossref PubMed Scopus (57) Google Scholar, 22Gomis-Rüth F.X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkow G.B. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (503) Google Scholar), interacting with the catalytic domain of the enzymes such that competition with low molecular weight substrate analogue inhibitors can be observed (23Leliévre Y. Bouboutou R. Boiziau J. Faucher D. Achard D. Cartwright T. Matrix. 1990; 10: 292-299Crossref PubMed Scopus (24) Google Scholar). 2J. O'Connell and G. Murphy, unpublished data. 2J. O'Connell and G. Murphy, unpublished data. Using peptide substrate assays, it has been possible to demonstrate that TIMP-MMP complexes interact with K i values of 10−9 to 10−12m (24Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (241) Google Scholar). Comparative studies of the association rates of TIMP-1 and TIMP-2 with different members of the MMP family in our laboratory have shown exceptionally strong C-terminal domain interactions between TIMP-1 and gelatinase B and between TIMP-2 and gelatinase A, suggesting that complexes between the respective pro forms of these enzymes, the active sites of which are inaccessible, and inhibitors can also occur (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar, 25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar, 26O'Connell J.P. Willenbrock F. Docherty A.J.P. Eaton D. Murphy G. J. Biol. Chem. 1994; 269: 14967-14973Abstract Full Text PDF PubMed Google Scholar). This supports other biochemical studies of these complexes (27Collier I.E. Willhelm 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, 28Howard E.W. Banda M.J. J. Biol. Chem. 1991; 266: 17972-17977Abstract Full Text PDF PubMed Google Scholar, 29Goldberg G.I. Strongin A. Collier I.E. Genrich L.T. Marmer B.L. J. Biol. Chem. 1992; 267: 4583-4591Abstract Full Text PDF PubMed Google Scholar, 30Kleiner D.E. Unsworth E.J. Krutzsch H.C. Stetler-Stevenson W.G. Biochemistry. 1992; 31: 1665-1672Crossref PubMed Scopus (54) Google Scholar). In this study, we have assessed the ability of TIMP-3 to associate with active MMPs using a kinetic method, and we have compared this with TIMP-1 and TIMP-2. We have also investigated the contribution of the C-terminal domains of both gelatinase A and gelatinase B to the interaction with TIMP-3, because this has important implications for the regulation of proenzyme activation. We have tested the effect of heparin and other polyanions on TIMP-3 activity in our soluble kinetic assay to determine whether interaction with similar components of the extracellular matrix could affect the capacity of TIMP-3 to inhibit MMPs. All chemicals and reagents were purchased from Sigma, ICN Flow, or Pierce unless stated otherwise. Quenched fluorescent peptides (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl)-Ala-Arg-NH2(Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) and (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Ala-Norval-(3-(2, 4-dinitrophenyl)-l-2,3-diaminopropionyl)-Ala-Arg-NH2(Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2) were made by Dr C. G. Knight (Biochemistry Department, University of Cambridge, Cambridge, United Kingdom). The following polyanions were purchased from Sigma: heparin (porcine intestinal mucosa, H3149); de-N-sulfated heparin (porcine intestinal mucosa, D4776); heparan sulfate (bovine kidney, H7640; bovine intestinal mucosa, H7641); hyaluronic acid (human umbilical cord, H1504); dermatan sulfate (chondroitin sulfate B; bovine mucosa, C0320); dextran sulfate (averageM r 10,000; D6924). TIMP-1, TIMP-2, and TIMP-3 were expressed from NS0 myeloma cells and purified as described previously (13Apte S.S. Olsen B.R. Murphy G. J. Biol. Chem. 1995; 270: 14313-14318Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 19Murphy G. Houbrechts A. Cockett M.I. Williamson R.A. O'Shea M. Docherty A.J.P. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (281) Google Scholar, 20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar). Progelatinase A, (Δ1–414)gelatinase A, pro(Δ418–631)gelatinase A, and the catalytically inactive mutant proE375A-gelatinase A were prepared as described previously (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar,31Ward R.V. Atkinson S.J. Reynolds J.J. Murphy G. Biochem. J. 1994; 304: 263-269Crossref PubMed Scopus (87) Google Scholar, 32Murphy G. Willenbrock F. Ward R.V. Cockett M.I. Eaton D. Docherty A.J.P. Biochem. J. 1992; 283: 637-641Crossref PubMed Scopus (244) Google Scholar, 33Crabbe T. Zucker S. Cockett M.I. Willenbrock F. Tickle S. O'Connell J.P. Scothern J.M. Murphy G. Docherty A.J.P. Biochemistry. 1994; 33: 6684-6690Crossref PubMed Scopus (61) Google Scholar). Progelatinase B and pro(Δ426–688)gelatinase B were prepared as described in Ref. 26O'Connell J.P. Willenbrock F. Docherty A.J.P. Eaton D. Murphy G. J. Biol. Chem. 1994; 269: 14967-14973Abstract Full Text PDF PubMed Google Scholar. Stromelysin-1 and matrilysin were prepared as recombinant pro forms as described previously (34Koklitis P.A. Murphy G. Sutton C. Angal S. Biochem. J. 1991; 276: 217-221Crossref PubMed Google Scholar, 35Crabbe T. Willenbrock F. Eaton D. Hynds P. Carne A.F. Murphy G. Docherty A.J.P. Biochemistry. 1992; 31: 8500-8507Crossref PubMed Scopus (91) Google Scholar). N-GL.C-SL was prepared as described previously (25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar). The preparation of gelatinase A mutants N-G.C-SGG and N-G.C-SGS was as described previously (36Butler G.S. Butler M.J. Atkinson S.J. Will H. Tamura T. Schade van Westrum S. Crabbe T. Clements J. d'Ortho M.P. Murphy G. J. Biol. Chem. 1998; 273: 871-880Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). Gelatinase A and gelatinase A C-terminal domain mutants were activated at 100 μg/ml with 2 mm 4-aminophenylmercuric acetate for 1 h at 25 °C. Stromelysin-1 was activated with trypsin using the standard method (37Koklitis P.A. Murphy G. Sutton C. Angal S. Biochem. J. 1991; 276: 217-221Crossref PubMed Scopus (67) Google Scholar). Gelatinase B and (Δ426–688)gelatinase B were activated at 2 μm with 0.1 μm active stromelysin-1 at 37 °C for 2 h. Matrilysin was activated at 22 μg/ml with 1 mm 4-aminophenylmercuric acetate at 37 °C for 1 h. Active enzymes were active site titrated against a standard preparation of TIMP-1 (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar). TIMP-2 and TIMP-3 were active site titrated with stromelysin-1 that had been titrated against the standard TIMP-1. Assays were performed at 25 °C for gelatinase A and gelatinase B or at 37 °C for stromelysin-1 and matrilysin in a buffer containing 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm CaCl2, and 0.05% Brij 35 (fluorometry buffer). Hydrolysis of 1 μmsubstrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 for the gelatinases and matrilysin or Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2 for stromelysin-1 was followed using a Perkin Elmer LS 50B fluorescence spectrometer (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar, 25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar,38Knight C.G. Willenbrock F. Murphy G. FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (669) Google Scholar). Inhibition of the matrix metalloproteinases by TIMPs was analyzed under pseudo-first-order conditions using suitable ratios of enzymes:inhibitors as described previously (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar, 25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar). Association rate constants (k on) were estimated from the progress curves using published equations (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar, 25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar) and the Enzfitter (Biosoft) or Grafit (Erithacus Software) program. The effect of ionic strength was analyzed by increasing the concentration of NaCl in the standard buffer from 0.1 m to 0.25 m and 0.5m. For competition assays, various concentrations of (Δ1–414)gelatinase A or proE375A-gelatinase A were added to the cuvette with the gelatinase A before the addition of TIMP-2 or TIMP-3. Because the K i values for the TIMP:gelatinase A interaction are unknown, the K i and theK d for the TIMP:competitor interaction are expressed as relative values using an arbitrary value of 1 for theK i. The relationship between the two dissociation constants is given in Equation 1: Kd=(Ff·EI)(FI·Ef)·KiEquation 1 in which EI and E f are the TIMP:gelatinase A complex and free gelatinase A, respectively, whereasFI and F f are the TIMP:competitor complex and free competitor, respectively. Equation 1 can be rewritten as: Kd=(Ft−FI)(Et−Ef)(It−[Et−Ef]−If)·Ef·KiEquation 2 in which F t, E t, and I t are total reagent concentrations. In our assays, F t≫FI, andI f is negligible, so Equation 2 can be simplified to Equation 3, from which the relative K dcan be readily calculated. Kd=Ft·(Et−Ef)(It−Et+Ef)·Ef·KiEquation 3 The effect of various polyanions on the rate of association was carried out using a constant amount of enzyme and inhibitor (concentrations similar to those used to calculate thek on values listed in Table I) with increasing concentrations of each test polyanion in the fluorometry buffer.Table IComparison of the rate constants for the interaction of TIMPs with matrix metalloproteinasesk onTIMP-1TIMP-2TIMP-3(× 10−6) M−1·s−1Gelatinase A3.42014(Δ418–631)Gelatinase A0.010.110.01Gelatinase B11.10.300.25(Δ426–688)Gelatinase B0.0020.160.02Stromelysin-10.600.490.17Matrilysin0.300.120.11Association rate constants (k on) were estimated from the inhibition progress curves using equations described previously (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar, 25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar). The data for TIMP-1 were taken from our previous work [25, 26]. Open table in a new tab Association rate constants (k on) were estimated from the inhibition progress curves using equations described previously (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar, 25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar). The data for TIMP-1 were taken from our previous work [25, 26]. TIMP-3 was incubated in the presence or absence of progelatinases in TCABN for 1–2 h at 25 °C. Complexes with progelatinases were isolated on gelatin-Sepharose that had been blocked with 0.2 mg/ml bovine serum albumin in TCABN. The column was washed with TCABN, and bound material was eluted with TCABN containing 15% dimethyl sulfoxide. Eluates were analyzed by rabbit collagenase diffuse collagen fibril assays (39Murphy G. Cawston T.E. Galloway W.A. Barnes M.J. Bunning R.A.D. Mercer E. Reynolds J.J. Burgeson R.E. Biochem. J. 1981; 199: 807-811Crossref PubMed Scopus (82) Google Scholar) and reverse zymography (40Atkinson 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). Approximately 1 μg of each TIMP was applied to heparin-agarose (blocked with 0.2 mg/ml bovine serum albumin) in TCABN buffer. Columns were washed with TCABN, and proteins were eluted stepwise with the same buffer containing 0.5m NaCl and then 2 m NaCl. Bound and unbound fractions were analyzed for TIMP content by SDS-polyacrylamide gel electrophoresis and silver staining and by rabbit collagenase diffuse collagen fibril assay (39Murphy G. Cawston T.E. Galloway W.A. Barnes M.J. Bunning R.A.D. Mercer E. Reynolds J.J. Burgeson R.E. Biochem. J. 1981; 199: 807-811Crossref PubMed Scopus (82) Google Scholar). 5 μg of TIMP-3 or TIMP-1 were incubated for 4 h at 37 °C in the presence or absence of 1250 units of PNGase F (New England Biolabs). TIMPs were diluted in fluorometry buffer and used in assays as above. We analyzed the inhibition of active gelatinase A, gelatinase B, stromelysin-1, and matrilysin by TIMP-3 using continuous fluorometric assays with the appropriate fluorescent peptide substrate (see "Experimental Procedures"). As discussed previously for TIMP-1 and TIMP-2 (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar, 25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar), we were unable to obtain accurate values ofK i (<200 pm). Our measurements were therefore limited to the association rate constants (k on) at low reagent concentrations, over a range where the observed rate was linear with TIMP concentration. In Table I, the data are compared withk on values for TIMP-2 that were re-assayed at the same time and k on values for TIMP-1 derived from our previous work (25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar, 26O'Connell J.P. Willenbrock F. Docherty A.J.P. Eaton D. Murphy G. J. Biol. Chem. 1994; 269: 14967-14973Abstract Full Text PDF PubMed Google Scholar). All three TIMPs bound relatively slowly to stromelysin-1 and matrilysin. In general, we found that TIMP-3 was more like TIMP-2 than TIMP-1, showing rapid binding to gelatinase A and slower association with gelatinase B. The contribution of the C-terminal domains of gelatinase A and gelatinase B to TIMP-3 binding was assessed by measuring the association rate of the isolated catalytic domains, (Δ418–631)gelatinase A and (Δ426–688)gelatinase B. Whereas TIMP-2 binding was only affected by the loss of the gelatinase A C-terminal domain, TIMP-3 association was slower in the absence of the C-terminal domains of both gelatinase A and gelatinase B (1400-fold and 12.5-fold, respectively). The effect of ionic strength on the rate of association of gelatinase A and TIMP-3 was analyzed at increasing NaCl concentrations. Similar to TIMP-2 (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar), there was a marked decrease in k onfrom 16.0 × 106m−1·s−1 in 0.1 mNaCl to 9.6 × 106m−1·s−1 (0.25 mNaCl) and 7.3 × 106m−1·s−1 (0.5 mNaCl), suggesting that ionic interactions are involved in the association of gelatinase A and TIMP-3. The contribution of the C-terminal domain of gelatinase A to TIMP-3 binding was assessed by measuring the effect of adding increasing amounts of (Δ1–414)gelatinase A (the isolated C-terminal domain) or proE375A-gelatinase A (an inactive form of progelatinase A) to the inhibition assay and observing the effect on the association rate for active full-length gelatinase A. The effect on inhibition by TIMP-2 was also measured for comparison. The increase in the final steady-state velocity and the decreased rate of inhibition observed with increasing concentrations of (Δ1–414)gelatinase A and proE375A-gelatinase A were deduced to be due to an effective decrease in TIMP-3 concentration by binding to the C-terminal domain, as was seen for TIMP-2 (20Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (215) Google Scholar). The data were analyzed as described under "Experimental Procedures" to obtain an estimate for K d, the dissociation constant, relative to the K i for the appropriate TIMP:gelatinase A interaction (Table II). The interaction of TIMP-3 with (1–414)gelatinase A was significant but was around 16-fold weaker than the interaction of TIMP-2. The interaction between TIMP-3 and proE375A-gelatinase A was about five times weaker than that for TIMP-2. In both cases, the interaction of the TIMPs with proE375A-gelatinase A was stronger than that with the isolated C-terminal domain, which suggests that additional sites of interaction exist in the proenzyme-TIMP complex.Table IIEstimation of the dissociation constant for binding of (Δ1–414)gelatinase A or proE375A-gelatinase A to TIMP-3 and TIMP-2TIMPCompetitorRelativeK d ± S.E.TIMP-3(Δ1–414)Gelatinase A1366 ± 425 (n = 10)TIMP-2(Δ1–414)Gelatinase A88 ± 15 (n = 10)TIMP-3proE375A-gelatinase A231 ± 66 (n = 10)TIMP-2proE375A-gelatinase A50 ± 8 (n = 8)Inhibition assays were carried out at 25 °C with increasing amounts of either (Δ1–414)gelatinase A or proE375A-gelatinase A. The dissociation constant (K d) was estimated as described under "Experimental Procedures" and is given as a relative value compared to that of the appropriate TIMP:gelatinase A interaction. n is the number of assays carried out. Open table in a new tab Inhibition assays were carried out at 25 °C with increasing amounts of either (Δ1–414)gelatinase A or proE375A-gelatinase A. The dissociation constant (K d) was estimated as described under "Experimental Procedures" and is given as a relative value compared to that of the appropriate TIMP:gelatinase A interaction. n is the number of assays carried out. To further characterize the region of gelatinase A responsible for the C-terminal domain interaction, we used two C-terminal domain mutants: regions of the C-terminal domain of gelatinase A were exchanged for the corresponding regions of the C-terminal domain of stromelysin-1, which does not interact significantly with the TIMPs (25Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar). As was the case for TIMP-2 (36Butler G.S. Butler M.J. Atkinson S.J. Will H. Tamura T. Schade van Westrum S. Crabbe T. Clements J. d'Ortho M.P. Murphy G. J. Biol. Chem. 1998; 273: 871-880Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar), replacement of residues 418–474 in N-G.C-SGG did not affect the rate of association with TIMP-3 (k on= 17.0 × 106m−1·s−1, compared with 16.5 × 106m−1·s−1 for gelatinase A). However, the additional substitution of residues 568–631 in N-G.C-SGS reduced the rate of association of TIMP-3 with gelatinase A by a factor of 100 to 0.1 × 106m−1·s−1, suggesting that residues 568–631 of gelatinase A are crucial for the interaction with TIMP-3. Because the kinetic data suggested that TIMP-3 has significant interactions with the hemopexin domains of gelatinase A and gelatinase B, we assessed the ability of TIMP-3 to bind to various pro form constructs of gelatinases A and B, in which normal catalytic domain interactions are precluded due to the presence of the propeptide domain (Table III). A small amount of TIMP-3 alone bound to the gelatin-Sepharose matrix. Enhanced retention of TIMP-3 was observed after preincubation with progelatinase A or progelatinase B, suggesting that TIMP-3 shows significant binding to both proenzymes. TIMP-3 was recovered in the unbound fraction after incubation with pro(Δ418–631)gelatinase A or pro(Δ426–688)gelatinase B. TIMP-3 bound to gelatin-Sepharose after preincubation with proN-G.C-SGG but did not bind if proN-G.C-SGS or proN-GL.C-SL were used. TIMP-2 was retaine

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