Cellular Mechanisms for Human Procollagenase-3 (MMP-13) Activation

doi:10.1074/jbc.271.29.17124

Cellular Mechanisms for Human Procollagenase-3 (MMP-13) Activation

EVIDENCE THAT MT1-MMP (MMP-14) AND GELATINASE A (MMP-2) ARE ABLE TO GENERATE ACTIVE ENZYME*

^‡ Department of Cell and Molecular Biology, Strangeways Research Laboratory, Worts' Causeway, Cambridge, CB1 4RN, United Kingdom,
^¶ In ViTek GmbH, Robert-Roessle-Strasse 10, D-13125 Berlin-Buch, Germany,
^∥ Universidad de Oviedo, Departamento de Biologia Funcional, 33006 Oviedo, Spain, and
” Celltech Ltd., 216 Bath Road, Slough SL1 4EN, United Kingdom

^§To whom correspondence should be addressed. Tel.: 44-1223-243231; Fax: 44-1223-411609.

Abstract

Gelatinase A and membrane-type metalloproteinase (MT1-MMP) were able to process human procollagenase-3 (M_r 60,000) to the fully active enzyme (Tyr⁸⁵ N terminus; M_r 48,000). MT1-MMP activated procollagenase-3 via a M_r 56,000 intermediate (Ile³⁶ N terminus) to 48,000 which was the result of the cleavage of the Glu⁸⁴-Tyr⁸⁵ peptide bond. We have established that the activation rate of procollagenase-3 by MT1-MMP was enhanced in the presence of progelatinase A, thereby demonstrating a unique new activation cascade consisting of three members of the matrix metalloproteinase family.

In addition, procollagenase-3 can be activated by plasmin, which cleaved the Lys³⁸-Glu³⁹ and Arg⁷⁶-Cys⁷⁷ peptide bonds in the propeptide domain. Autoproteolysis then resulted in the release of the rest of the propeptide domain generating Tyr⁸⁵ N-terminal active collagenase-3. However, plasmin cleaved the C-terminal domain of collagenase-3 which results in the loss of its collagenolytic activity.

Concanavalin A-stimulated fibroblasts expressing MT1-MMP and fibroblast-derived plasma membranes were able to process human procollagenase-3 via a M_r 56,000 intermediate form to the final M_r 48,000 active enzyme which, by analogy with progelatinase A activation, may represent a model system for in vivo activation. Inhibition experiments using tissue inhibitor of metalloproteinases, plasminogen activator inhibitor-2, or aprotinin demonstrated that activation in the cellular model system was due to MT1-MMP/gelatinase A and excluded the participation of serine proteinases such as plasmin during procollagenase-3 activation. We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by crude plasma membrane preparations from concanavalin A-stimulated fibroblasts, thus confirming our results using purified progelatinase A and MT1-MMP. This new activation cascade may be significant in human breast cancer pathology, where all three enzymes have been implicated as playing important roles.

Footnotes

↵* This work was supported in part by the Arthritis and Rheumatism Council and the Medical Research Council, United Kingdom, and by the Wellcome Trust. 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 metalloproteinase

MT1-MMP

membrane type matrix metalloproteinase (MMP-14)

CT1399

N4-hydroxy-N1-(1-(S-morpholinosulfonylaminoethylaminocarbonyl)-2-cyclohexyl-ethyl)-2-(R)-(4-chlorophenylpropyl)succinamide)

PAI-2

plasminogen activator inhibitor 2

ConA

concanavalin A

McaPLGLDpaAR

(7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-[3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl)Ala-ArgNH₂

DMEM

Dulbecco's modified Eagle's medium

PAGE

polyacrylamide gel electrophoresis

HPLC

high performance liquid chromatography.
↵² V. Knäuper and G. Murphy, unpublished results.
↵³ G. Murphy, H. Will, and V. Knäuper, unpublished results.
↵⁴ Will, H., Atkinson, S. J., Butler, G. S., Smith, B., and Murphy, G. (1996) J. Biol. Chem. 271, 17119-17123.
↵⁵ S. Atkinson, personal communication.
↵⁶ S. Atkinson, M. Butler, and G. Murphy, unpublished results.
- Received January 24, 1996.
- Revision received March 27, 1996.