The effect of O-fucosylation on the first EGF-like domain from human bloodcoagulation factor VII

The first epidermal growth factor-like domain (EGF-1) from blood coagulatio n factor VII (FVII) contains two unusual O-linked glycosylation sites at Se r-52 and Ser-60, We report here a detailed study of the effect of O-fucosyl ation at Ser-60 on the structure of FVII EGF-1, its Ca2+-binding affinity, and its interaction with tissue factor (TF). The in vitro fucosylation of t he nonglycosylated FVII EGF-1 was achieved by using O-fucosyltransferase pu rified from Chinese hamster ovary cells. Distance and dihedral constraints derived from NMR data were used to determine the solution structures of bot h nonglycosylated and fucosylated FVII EGF-1 in the presence of CaCl2, The overall structure of fucosylated FVII EGF-1 is very similar to the nonfucos ylated form even for the residues near the fucosylation site. The Ca2+ diss ociation constants (K-d) for the nonfucosylated and fucosylated FVII EGF-1 were found to be 16.4 +/- 1.8 and 8.6 +/- 1.4 mM, respectively. The FVII EG F-1 domain binds to the extracellular part of TF with a low affinity (K-d a pproximate to 0.6 mM), and the addition of fucose appears to have no effect on this affinity. These results indicate that the FVII EGF-1 alone cannot form a tight complex with TF and suggest that the high binding affinity of FVIIa for TF requires cooperative interaction among the four domains in FVI I with TF, Although the fucose has no significant effect on the interaction between TF and the individual FVII EGF-1 domain, it may affect the interac tion of full-length FVIIa with TF by influencing its Ca2+- binding affinity .