The Hemopexin-like Domain (C Domain) of Human Gelatinase A (Matrix Metalloproteinase-2) Requires Ca2+ for Fibronectin and Heparin Binding

doi:10.1074/jbc.272.11.7473

The Hemopexin-like Domain (C Domain) of Human Gelatinase A (Matrix Metalloproteinase-2) Requires Ca²⁺ for Fibronectin and Heparin Binding

BINDING PROPERTIES OF RECOMBINANT GELATINASE A C DOMAIN TO EXTRACELLULAR MATRIX AND BASEMENT MEMBRANE COMPONENTS*

From the Faculty of Dentistry and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, 2199 Wesbrook Mall, Vancouver, British Columbia V6T 1Z3, Canada

^§ Recipient of a Medical Research Council of Canada Clinician Scientist Award. To whom correspondence should be addressed. Tel.: 604-822-2958; Fax: 604-822-8279; E-mail: overall{at}unixg.ubc.ca

↵^‡ Present address: Dept. of Biochemistry, University of Washington, Seattle, WA.

Abstract

The binding properties of the COOH-terminal hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2, 72-kDa gelatinase) were investigated to determine whether the C domain has binding affinity for extracellular matrix and basement membrane components. Recombinant C domain (rC domain) (Gly⁴¹⁷-Cys⁶³¹) was expressed in Escherichia coli, and the purified protein, identified using two antipeptide antibodies, was determined by electrospray mass spectrometry to have a mass of 25,925 Da, within 0.1 Da of that predicted. As assessed by microwell substrate binding assays and by column affinity chromatography, the matrix proteins laminin, denatured type I collagen, elastin, SPARC (secreted protein that is acidic and rich in cysteine), tenascin, and Matrigel™ were not bound by the rC domain. Unlike the hemopexin-like domains of collagenase and stromelysin, the rC domain also did not bind native type I collagen. Nor were native or denatured types II, IV, V, and X collagen, or the NC1 domain of type VII collagen bound. However, binding to heparin and fibronectin (K_d, 1.1 × 10⁻⁶ M) could be disrupted by 0.58-0.76 and 0.3 M NaCl, respectively. Using nonoverlapping chymotrypsin-generated fragments of fibronectin, binding sites for the rC domain were found on both the 40-kDa heparin binding and the 120-kDa cell binding fibronectin domains (K_d values, ∼4-6 × 10⁻⁷ M). The Ca²⁺ ion, but not the potential structural Zn²⁺ ion, were found to be essential for maintaining the binding properties of the protein. The apo-form of the rC domain did not bind heparin, and both ethylenediaminetetraacetic acid and the specific Ca²⁺ ion chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid, but not the Zn²⁺ ion chelator 1,10-phenanthroline, eluted the holo form of the rC domain from both heparin-Sepharose and fibronectin. Inductive coupled plasma mass spectrometry also did not detect a Zn²⁺ ion in the rC domain. In contrast, reduction with 65 mM dithiothreitol did not interfere with heparin binding, further emphasizing the crucial structural role played by the Ca²⁺ ion. Together, these data demonstrate for the first time that the hemopexin-like domain of gelatinase A has a binding site for fibronectin and heparin, and that Ca²⁺ ions are important in maintaining the structure and function of the domain.

Footnotes

↵* This work was supported in part by Grant 006388 from the National Cancer Institute of Canada and initially by a grant from the Medical Research Council of Canada. 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

gelatinase A

also termed 72-kDa gelatinase, 72-kDa type IV collagenase

MMP-2

gelatinase B

also termed 92-kDa gelatinase, 92-kDa type IV collagenase

MMP-9

C domain

gelatinase A COOH-terminal domain

BAPTA

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

ConA

concanavalin A

DTT

dithiothreitol

ECL

enhanced chemiluminesence

FPLC

fast protein liquid chromatography

PAGE

polyacrylamide gel electrophoresis

rCBD

recombinant collagen binding domain of gelatinase A, consisting of fibronectin type II-like modules 1-3

rC domain

recombinant COOH-terminal hemopexin-like domain

SPARC

secreted protein that is acidic and rich in cysteine

TIMP

tissue inhibitor of matrix metalloproteinases

MT

membrane type.
↵² R. S. Abbey, B. Steffensen, and C. M. Overall, manuscript in preparation.
↵³ B. Steffensen and C. M. Overall, manuscript in preparation.
↵⁴ A. Iamaroon, U. M. Wallon, C. M. Overall, and V. M. Diewert, resubmitted for publication.
↵⁵ C. M. Overall, A. King, M. Wallon, T. Lau, A. Ong, and D. Sam, unpublished data.
- Received July 17, 1996.
- Revision received December 20, 1996.