The Soluble Catalytic Domain of Membrane Type 1 Matrix Metalloproteinase Cleaves the Propeptide of Progelatinase A and Initiates Autoproteolytic Activation

doi:10.1074/jbc.271.29.17119

The Soluble Catalytic Domain of Membrane Type 1 Matrix Metalloproteinase Cleaves the Propeptide of Progelatinase A and Initiates Autoproteolytic Activation

REGULATION BY TIMP-2 AND TIMP-3*

^‡ In Vitek GmbH, Robert-Roessle-Strasse 10, D-13125 Berlin-Buch, Federal Republic of Germany, the
^§Department of Cell & Molecular Biology, Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN, and
^¶ Celltech Research, 216 Bath Road, Slough SL1 4EN, United Kingdom

^∥To whom correspondence should be addressed:
Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN. United Kingdom.
Tel: 44 1223 243231; Fax: 44 1223 411609; E-mail gm{at}mole.bio.cam.ac.uk

Abstract

It has been proposed that the cell-mediated activation of progelatinase A requires binding of the C-terminal domain of the proenzyme to a membrane-associated complex of the membrane type matrix metalloproteinase MT1-MMP and TIMP-2. Subsequent sequential proteolysis of the propeptide by MT1-MMP and gelatinase A is thought to generate the active form of gelatinase A. We have prepared the proform of the catalytic domain of the MT1-MMP and demonstrated that this may be activated in vitro by trypsin proteolysis to yield a functional proteinase capable of cleaving typical metalloproteinase peptide substrates, gelatin and casein. The active catalytic domain of MT1-MMP was also shown to activate progelatinase A to a fully active form. Using the inactive mutant pro-E375A gelatinase A, we dissected the propeptide processing events that occur. MT1-MMP cleaves the propeptide at the sequence Asn³⁷-Leu³⁸ only. Further cleavage of the mutant enzyme propeptide at Asn⁸⁰-Tyr⁸¹, equivalent to that of the active wild type gelatinase A, could only be effected by addition of gelatinase A to the system. TIMP-1 was essentially unable to prevent MT1-MMP processing of wild type or E375A progelatinase A, whereas TIMP-2 and TIMP-3 were good inhibitors of these events. Analysis of the rate of binding of TIMPs to the catalytic domain of MT1-MMP using kinetic methods showed that TIMP-1 is an extremely poor inhibitor of MT1-MMP. In comparison, TIMP-2 and TIMP-3 are excellent inhibitors, binding more rapidly to the catalytic domain of MT1-MMP than to the catalytic domain of gelatinase A. These data demonstrate the basic mechanism of MT1-MMP action on progelatinase A and the reason for the lack of inhibition by TIMP-1 previously demonstrated in cell-based activation studies.

Footnotes

↵* This work was supported by the Arthritis and Rheumatism Council, the Wellcome Trust, the Medical Research Council, U. K., and the Deutsche Forschungsgemeinschaft, Germany. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MMP

matrix metalloproteinase

TIMP

tissue inhibitor of matrix metalloproteinase

MT1-MMP

membrane type 1 matrix metalloproteinase, MMP14

N-MT1-MMP

Δ269-559 MT1-MMP, equivalent to the catalytic domain

Mca-PLGL-Dpa-AR

(7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-[3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl)]-Ala-Arg-NH₂

SBTI

soybean trypsin inhibitor

Mca

methoxycoumarin, APMA, 4-aminophenylmercuric acetate

PAGE

polyacrylamide gel electrophoresis.
↵² S. J. Atkinson, R. M. Hembry, and G. Murphy, unpublished observations.
- Received March 14, 1996.