Transmembrane-deletion Mutants of the Membrane-type Matrix Metalloproteinase-1 Process Progelatinase A and Express Intrinsic Matrix-degrading Activity

doi:10.1074/jbc.271.15.9135

Transmembrane-deletion Mutants of the Membrane-type Matrix Metalloproteinase-1 Process Progelatinase A and Express Intrinsic Matrix-degrading Activity (*)

From the Division of Hematology/Oncology, Department of Internal Medicine, the University of Michigan Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 48109

^¶ To whom correspondence should be addressed:
Division of Hematology/Oncology, 5220 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109-0640.
Tel.: 313-764-0030; Fax: 313-764-0101.

Abstract

Membrane-type matrix metalloproteinase-1 (MT-MMP-1) has been proposed to play a critical role in regulating the expression of tissue-invasive phenotypes in normal and neoplastic cells by directly or indirectly mediating the activation of progelatinase A. To begin characterizing MT-MMP-1 structure-function relationships, transmembrane-deletion mutants were constructed, and the processing of the zymogens as well as the enzymic activity of the mature proteinases was analyzed. We now demonstrate that pro-MT-MMP-1 mutants are efficiently processed to active proteinases following post-translational endoproteolysis immediately downstream of an Arg-Arg-Lys-Arg basic motif by a proprotein convertase-dependent pathway. The secreted form of active MT-MMP-1 not only displays an N terminus identical with that described for the processed wild-type enzyme at Tyr (Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grants, G. A., and Goldberg, G. I.(1995) J. Biol. Chem. 270, 5331-5338), but also directly mediated progelatinase A activation via a two-step proteolytic cascade indistinguishable from that observed with intact cells. Furthermore, although the only function previously ascribed to MT-MMP-1 is its ability to act as a progelatinase A activator, purified transmembrane deletion mutants also expressed proteolytic activities against a wide range of extracellular matrix molecules. Given recent reports that MT-MMP-1 ectodomains may undergo intercellular transfer in vivo (Okada, A., Bellocq, J.-P., Rouyer, N., Chenard, M.-P., Rio, M.-C., Chambon, P., and Basset, P.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2730-2734), our data suggest that soluble forms of the proteinase confer recipient cells with the ability to not only process progelatinase A, but also directly degrade extracellular matrix components.

Footnotes

↵^§ Supported in part by an Oncology research training grant from NHLBI, National Institutes of Health.
↵* This study was supported in part by Grant AI23876 from the National Institutes of Health, the Susan G. Komen Breast Cancer Foundation, and United States Army Medical Research Command Grant DAMD17-94-J-4322. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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

αPI

α-proteinase inhibitor

αPI

Pittsburgh mutant of α-proteinase inhibitor

ECM

extracellular matrix

MT-MMP-1

-2, −3, membrane-type matrix metalloproteinase-1, −2, and −3

ΔMT-MMP-1

transmembrane-deleted MT-MMP-1

MT-MMP-1

MT-MMP-1

MT-MMP-1

MT-MMP-1

TM

transmembrane

MDCK

Madin-Darby canine kidney cells

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction.
↵²All MT-MMPs also contain a homologous 8-amino acid insert within their catalytic domains whose function remains undefined(4, 5, 6).
↵³D. Pei and S. J. Weiss, unpublished observation.
↵⁴Although stromelysin-3 and MT-MMP-1 both contain proprotein convertase recognition motifs, comparisons of their genomic organization and chromosomal localization indicate that the two metalloproteinases are not closely related and belong to separate branches of the phylogenetic tree (D. Pei and S. J. Weiss, unpublished observation).
↵⁵However, a portion of the MT-MMP-1 zymogen does undergo processing since the mature enzyme has been isolated from HT-1080 plasma membranes (10) and is required for progelatinase activation.
- Received January 25, 1996.