Different Collagenase Gene Products Have Different Roles in Degradation of Type I Collagen

doi:10.1074/jbc.271.45.28509

Different Collagenase Gene Products Have Different Roles in Degradation of Type I Collagen*

From the ^‡ Department of Medicine, Harvard Medical School and the Arthritis Unit, Massachusetts General Hospital, Boston, Massachusetts 02114,
the ^¶ Connective Tissue Group, University of Louvain and International Institute of Cellular and Molecular Pathology, Avenue Hippocrate, 75, B-1200 Bruxelles, Belgium,
the ** Division of Hematology, Department of Medicine, Albany Medical College, Albany, New York 12208, and
the ^§§ Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142

^§ To whom correspondence should be addressed:
Dept. of Medicine, Harvard Medical School and the Arthritis Unit, Massachusetts General Hospital, Fruit St., Boston, MA 02114
. Tel. 617-726-2870; Fax: 617-726-2872; E-mail: kranes{at}A1.mgh.harvard.edu.

Abstract

Vertebrate collagenases, matrix metalloproteinases (MMPs), cleave type I collagen at a single helical locus. We show here that rodent interstitial collagenases (MMP-13), but not human fibroblast collagenase (MMP-1), cleave type I collagen at an additional aminotelopeptide locus. Collagenase cDNAs and chimeric constructs in pET-3d, juxtaposing MMP-13 sequences amino-terminal to the active site in the catalytic domain and MMP-1 sequences carboxyl-terminal and vice versa, were expressed in Escherichia coli. Assays utilized collagen from wild type (+/+) mice or mice that carry a targeted mutation (r/r) that encodes substitutions in α1(I) chains that prevent collagenase cleavage at the helical locus. MMP-13 and chimeric molecules that contained the MMP-13 sequences amino-terminal to the active site cleaved (+/+) collagen at the helical locus and cleaved cross-linked (r/r) collagen in the aminotelopeptide (β components converted to α chains). Human MMP-1 and chimeric MMP-1/MMP-13 with MMP-1 sequences amino-terminal to the active site cleaved collagen at the helical locus but not in the aminotelopeptide. All activities were inhibited by TIMP-1, 1,10-phenanthroline, and EDTA. Sequences in the distal two-thirds of the catalytic domain determine the aminotelopeptide-degrading capacity of MMP-13.

Footnotes

↵^¶ Supported in part as Research Fellows of the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture.
↵^‡‡ Supported in part as an Established Investigator of the Arthritis Foundation.
↵* This work was supported in part by National Institutes of Health Research Grants AR-03564 and TR-AR-07258 (to S. M. K.), HD-05291 (to J. J. J.), and HL-41484 (to R. J.) and by Grant 3.4522.91F of the Belgian Fonds de la Recherche Scientifique Médicale and a Grant from the Belgian Programme on Interuniversity Poles of Attraction, Prime Minister's Office, Federal Services of Scientific, Technical and Cultural Affairs (to Y. E.). 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 metalloproteinases

PCR

polymerase chain reaction

TIMP

tissue inhibitor of metalloproteinases

bp

base pair(s)

APMA

p-aminophenylmercuric acetate

PAGE

polyacrylamide gel electrophoresis

CLH

recombinant human fibroblast (MMP-1) collagenase

CLM

mouse interstitial collagenase.
↵² V. Lemaître, P. Henriet, and Y. Eeckhout, manuscript in preparation.
↵³ J. P. Witter, S. M. Krane, M. H. Byrne, C. López-Otín, and M. B. Goldring, unpublished observations.
- Received May 25, 1996.
- Revision received August 19, 1996.