Thrombospondin Type 1 Repeats Interact with Matrix Metalloproteinase 2

doi:10.1074/jbc.M003834200

Thrombospondin Type 1 Repeats Interact with Matrix Metalloproteinase 2

REGULATION OF METALLOPROTEINASE ACTIVITY*

Kiflai Bein and
Michael Simons‡

From the Angiogenesis Research Center, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

Abstract

Thrombospondins are thought to function as inhibitors of angiogenesis. However, the mechanism(s) of this activity is not well understood. In this study, we have used the yeast two-hybrid system to identify proteins that interact with the thrombospondins 1 (TSP1) and 2 (TSP2) properdin-like type 1 repeats (TSR). One of the proteins identified that interacted with both TSR was matrix metalloproteinase 2 (MMP2). The isolated MMP2 cDNA clone encoded amino acid residues 237–633, which include the fibronectin-like gelatin binding region flanking the catalytic center and the carboxyl hemopexin-like region. Further testing of this clone demonstrated that the TSR interacted with the NH₂-terminal region of the MMP2 that contains the catalytic domain. The protein interaction observed in yeast was further demonstrated by immunoprecipitation and Western blotting using purified intact TSP1, TSP2, MMP2, and MMP9. Although MMP2 interacted with TSP1 and TSP2 via its gelatin-binding domain or a closely mapping site, neither TSP1 nor TSP2 was degraded by MMP2 in vitro. Tissue culture and in vitro assays demonstrated that the presence of purified TSR and intact TSP1 resulted in inhibition of MMP activity. The ability of TSP1 to inhibit MMP3-dependent activation of pro-MMP9 and thrombin-induced activation of pro-MMP2 suggests that the TSPs may inhibit MMP activity by preventing activation of the MMP2 and MMP9 zymogens.

Footnotes

↵* 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.
↵‡ To whom correspondence should be addressed: Cardiovascular Division, RW453, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617 667 5364; Fax: 617 975 5201; E-mail: msimons@bidmc.harvard.edu.
Published, JBC Papers in Press, July 18, 2000, DOI 10.1074/jbc.M003834200
↵2 P. Bornstein, personal communication.
↵3 J. Lawler, personal communication.
Abbreviations:

TSP

thrombospondin

TSR

TSP1 and -2 properdin-like type 1repeat(s)

BAEC

bovine aortic endothelial cells

PAGE

polyacrylamide gel electrophoresis

GST

glutathioneS-transferase

APMA

p-aminophenylmercuric acetate

X-gal

5-bromo-4-chloro-3-indolyl β-d-galactopyranoside

hTSP1

human platelet TSP1

hMMP2

human MMP2

PBS

phosphate-buffered saline
- Received May 3, 2000.
- Revision received July 18, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.