The Role of the C-terminal Domain of Human Collagenase-3 (MMP-13) in the Activation of Procollagenase-3, Substrate Specificity, and Tissue Inhibitor of Metalloproteinase Interaction

doi:10.1074/jbc.272.12.7608

The Role of the C-terminal Domain of Human Collagenase-3 (MMP-13) in the Activation of Procollagenase-3, Substrate Specificity, and Tissue Inhibitor of Metalloproteinase Interaction*

From the ^‡ Department of Cell and Molecular Biology, Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN, United Kingdom
^¶ Celltech Limited, 216 Bath Road, Slough SL1 4EN, United Kingdom, the
^∥ Departamento de Bioquimica y Biologia Molecular, Universidad de Oviedo, 33006 Oviedo, Spain, and the
** Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genoa, Italy

^§ To whom correspondence should be addressed. Tel.: 44-1223-243231; Fax: 44-1223-411609; E-mail: vk{at}srl.mrc-lmb.cam.ac.uk

Abstract

Recombinant human procollagenase-3 and a C-terminal truncated form (Δ_249-451 procollagenase-3) have been stably expressed in myeloma cells and purified. The truncated proenzyme could be processed by aminophenylmercuric acetate via a short-lived intermediate form (N-terminal Leu⁵⁸) to the final active form (N-terminal Tyr⁸⁵). The kinetics of activation were not affected by removal of the hemopexin-like C-terminal domain. The specific activities of both collagenase-3 and Δ_249-451 collagenase-3 were found to be similar using two quenched fluorescent substrates, but Δ_249-451 collagenase-3 failed to cleave native triple helical collagens (types I and II) into characteristic one- and three-quarter fragments. It was noted, however, that the β1,2(I) chains of type I collagen were susceptible to Δ_249-451 collagenase-3, which indicates that the catalytic domain displays telopeptidase activity, thereby generating α1,2(I) chains that are slightly shorter than those in native type I collagen. It can be concluded that the C-terminal domain is only essential for the triple helicase activity of collagenase-3. Binding of procollagenase-3 and active collagenase-3 to type I collagen is mediated by the C-terminal domain. Both collagenase-3 and Δ_249-451 collagenase-3 hydrolyzed the large tenascin C isoform, fibronectin, recombinant fibronectin fragments, and type IV, IX, X, and XIV collagens; thus, these events were independent from C-terminal domain interactions. In contrast, the minor cartilage type XI collagen was resistant to cleavage. Kinetic analysis of the mechanism of inhibition of wild-type and Δ_249-451 collagenase-3 by wild-type and mutant tissue inhibitors of metalloproteinase (TIMPs) revealed that the association rates for complex formation were influenced by both N- and C-terminal domain interactions. The C-terminal domain of wild-type collagenase-3 promoted increased association rates with the full-length inhibitors TIMP-1 and TIMP-3 and the hybrid N.TIMP-2/C.TIMP-1 by a factor of up to 33. In contrast, the association rates for complex formation with TIMP-2 and N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain interactions.

Footnotes

↵* This work was supported by the Arthritis and Rheumatism Council, the Wellcome Trust, the Medical Research Council (United Kingdom), and the European Union Biomed 2 Program. 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:

MMPs

matrix metalloproteinases

APMA

aminophenylmercuric acetate

MT

membrane-type

TIMP

tissue inhibitor of metalloproteinase

N.TIMP-1/C.TIMP-2

chimeric TIMP constructed from N-terminal TIMP-1 and C-terminal TIMP-2

N.TIMP-2/C.TIMP-1

chimeric TIMP constructed from N-terminal TIMP-2 and C-terminal TIMP-1

Mca-PLGL-Dpa-AR-NH₂

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

PAGE

polyacrylamide gel electrophoresis

TN-C

tenascin C

FN

fibronectin

HPLC

high pressure liquid chromatography.
↵² J.-J. Wu, D. R. Eyre, V. Knäuper, and G. Murphy, unpublished results.
↵³ V. Knäuper and G. Murphy, unpublished results.
- Received August 5, 1996.
- Revision received December 16, 1996.