THE HEMOPEXIN-LIKE DOMAIN (C-DOMAIN) OF HUMAN GELATINASE-A (MATRIX METALLOPROTEINASE-2) REQUIRES CA2-BINDING - BINDING-PROPERTIES OF RECOMBINANT GELATINASE A C-DOMAIN TO EXTRACELLULAR-MATRIX AND BASEMENT-MEMBRANE COMPONENTS( FOR FIBRONECTIN AND HEPARIN)

The binding properties of the COOH-terminal hemopexin-like domain (C d omain) of human gelatinase A (matrix metalloproteinase-2, 72-kDa gelat inase) were investigated to determine whether the C domain has binding affinity for extracellular matrix and basement membrane components. R ecombinant C domain (rC domain) (Gly(417)-Cys(631)) was expressed in E scherichia coli, and the purified protein, identified using two antipe ptide 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 chromato graphy, the matrix proteins laminin, denatured type I collagen, elasti n, SPARC (secreted protein that is acidic and rich in cysteine), tenas cin, and MatrigelTM were not bound by the rC domain. Unlike the hemope xin-like domains of collagenase and stromelysin, the rC domain also di d 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 boun d. However, binding to heparin and fibronectin (K-d, 1.1 x 10(-6) M) c ould be disrupted by 0.58-0.76 and 0.3 M NaCl, respectively. Using non overlapping chymotrypsin-generated fragments of fibronectin, binding s ites for the rC domain were found on both the 40-kDa heparin binding a nd the 120-kDa cell binding fibronectin domains (K-d values, similar t o 4-6 x 10(-7) M). The Ca2+ ion, but not the potential structural Zn2 ion, were found to be essential for maintaining the binding propertie s of the protein. The ape-form of the rC domain did not bind heparin, and both ethylenediaminetetraacetic acid and the specific Ca2+ ion che lator 1,2-bis(2-amino-phenoxy) ethane=N,N,N',N'-tetraacetic acid, but not the Zn2+ ion chelator 1,10-phenanthroline, eluted the hole 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 mhn dithiothreitol did not interfere with heparin binding, further emphasizing the crucial struct ural role played by the Ca2+ ion. Together, these data demonstrate for the first time that the hemopexin-like domain of gelatinase A has a b inding site for fibronectin and heparin, and that Ca2+ ions are import ant in maintaining the structure and function of the domain.