THE SELF-ASSOCIATION AND FIBRONECTIN-BINDING SITES OF FIBULIN-1 MAP TO CALCIUM-BINDING EPIDERMAL GROWTH FACTOR-LIKE DOMAINS

Fibulin-1 is a modular glycoprotein with amino-terminal anaphylatoxin- like modules followed by nine epidermal growth factor (EGF)-like modul es and, depending on alternative splicing, four possible carboxyl term ini. Fibulin-1 has been shown to self-associate as well as to bind cal cium, fibronectin (FN), laminin, nidogen, and fibrinogen. To map ligan d-binding sites within fibulin-1, polypeptides corresponding to variou s regions of fibulin-1 were expressed recombinantly and evaluated for their capacity to bind calcium, FN, or fibulin-1. A calcium-binding si te(s) was mapped to EGF-like modules 5-9. A fibulin-1 self-association site was localized to EGF-like modules 5 and 6 (amino acid residues 3 56-440), as was a binding site for FN. The self-association interactio n mediated by this pair of modules involved calcium since divalent cat ion chelators reduced the binding affinity of the interaction. By cont rast, FN binding to EGF-like modules 5 and 6 was unaffected by the pre sence of divalent cation chelators. It can be concluded that EGF-like modules 5 and 6 bind calcium and mediate homotypic interaction between EGF-like modules 5 and 6 present in different fibulin-1 molecules and heterotypic interaction between EGF-like modules 5 and 6 and type III repeats 13 and 14 in FN. While additional binding sites for calcium o r FN were not detected, another fibulin-1 self-association site was fo und within amino acid residues 30-173. However, unlike the self-associ ation site in EGF-like modules 5 and 6, which was functional in the na tive protein, the amino-terminal site was cryptic and revealed only af ter the protein was denatured.