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 modules and, depending on alternative splicing, four possible carboxyl termini. Fibulin-1 has been shown to self-associate as well as to bind calcium, fibronectin (FN), laminin, nidogen, and fibrinogen. To map ligand-binding sites within fibulin-1, polypeptides corresponding to various regions of fibulin-1 were expressed recombinantly and evaluated for their capacity to bind calcium, FN, or fibulin-1. A calcium-binding site(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 356-440), as was a binding site for FN. The self-association interaction mediated by this pair of modules involved calcium since divalent cation chelators reduced the binding affinity of the interaction. By contrast, FN binding to EGF-like modules 5 and 6 was unaffected by the presence 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 or FN were not detected, another fibulin-1 self-association site was found within amino acid residues 30-173. However, unlike the self-association site in EGF-like modules 5 and 6, which was functional in the native protein, the amino-terminal site was cryptic and revealed only after the protein was denatured.
Tran
H
H
Biochemistry Department, American Red Cross, Rockville, Maryland 20855, USA.
VanDusen
W J
WJ
Argraves
W S
WS
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002135:Calcium-Binding Proteins; D005353:Fibronectins; D011994:Recombinant Proteins; C060820:fibulin; D004815:Epidermal Growth Factor; D002118:Calcium