Membrane Type 1 Matrix Metalloproteinase Digests Interstitial Collagens and Other Extracellular Matrix Macromolecules

doi:10.1074/jbc.272.4.2446

Membrane Type 1 Matrix Metalloproteinase Digests Interstitial Collagens and Other Extracellular Matrix Macromolecules*

From the Departments of ^‡ Molecular Immunology and Pathology, and
** Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920,
^§ Fuji Chemical Industries, Ltd., Takaoka, Toyama 933, and
^| Department of Chemistry, Fukui Medical School, Fukui 910-11, Japan

^‡‡ To whom correspondence should be addressed:
Dept. of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920, Japan.
Tel.: 81-762-34-4507; Fax: 81-762-34-4508; E-mail: yasokada{at}kenroku.ipc.kanazawa-u.ac.jp

Abstract

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (ΔMT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly⁷⁷⁵-Ile⁷⁷⁶ bond for α1(I) chains and the Gly⁷⁷⁵-Leu⁷⁷⁶ and Gly⁷⁸¹-Ile⁷⁸² bonds for α2(I) chains. ΔMT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of ΔMT1 and MMP-1 indicate that ΔMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of ΔMT1 is 8-fold higher than that of MMP-1. ΔMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as α₁-proteinase inhibitor and α₂-macroglobulin. The activity of ΔMT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.

Footnotes

↵^¶ Recipient of research fellowships from the Japan Society for the Promotion of Science for Young Scientists.
↵* This work was supported by Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan (to Y. O.). 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

proMMP

corresponding zymogen form

MT-MMP

membrane-type MMP

ΔMT1

MT1-MMP lacking transmembrane domain

APMA

p-aminophenylmercuric acetate

PAGE

polyacrylamide gel electrophoresis

TIMP-2

tissue inhibitor of metalloproteinases 2

ECM

extracellular matrix

CHO

Chinese hamster ovary

Mca

(7-methoxycoumarin-4-yl)acetyl

Dpa

N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl.
↵² E. Ohuchi K. Imai, and Y. Okada, unpublished data.
↵³ H. Sato and M. Seiki, manuscript in preparation.
↵⁴ K. Imai, S. Ohta, and Y. Okada, unpublished data.
- Received June 24, 1996.
- Revision received September 20, 1996.