The Effect of O-Fucosylation on the First EGF-like Domain from Human Blood Coagulation Factor VII

Abstract

The first epidermal growth factor-like domain (EGF-1) from blood coagulation factor VII (FVII) contains two unusual O-linked glycosylation sites at Ser-52 and Ser-60. We report here a detailed study of the effect of O-fucosylation 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 the nonglycosylated FVII EGF-1 was achieved by using O-fucosyltransferase purified from Chinese hamster ovary cells. Distance and dihedral constraints derived from NMR data were used to determine the solution structures of both nonglycosylated and fucosylated FVII EGF-1 in the presence of CaCl2. The overall structure of fucosylated FVII EGF-1 is very similar to the nonfucosylated form even for the residues near the fucosylation site. The Ca2+ dissociation constants (Kd) 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 EGF-1 domain binds to the extracellular part of TF with a low affinity (Kd approximately 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 FVII 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 interaction of full-length FVIIa with TF by influencing its Ca2+-binding affinity.

neurotic, a novel maternal neurogenic gene, encodes anO-fucosyltransferase that is essential for Notch-Delta interactions

“…The simplest interpretation of these phenomena would be that association of Delta to its receptor involves lectin-like protein-carbohydrate interaction. This hypothesis is consistent with the result that O-fucosylation induces little conformational change in EGF repeat of factor VII (Kao et al, 1999). The first protein shown to be O-fucosylated was urinary type plasminogen activator (uPA) (Kentzer et al, 1990), followed by tissue-type plasminogen activators and several clotting factors.…”

Mammalian Notch1 Is Modified with Two Unusual Forms ofO-Linked Glycosylation Found on Epidermal Growth Factor-like Modules

“…They then compared the structure of fucosylated and unfucosylated factor VII EGF modules. Although the addition of fucose did not alter the tertiary structure of the module, the presence or the sugar resulted in a large secondary feature resembling a knob extending from the module (40). Spatially, it poses as a significant structure on one face of the EGF module, which could help or hinder protein complexes.…”

“…Kao et al (40) examined the effect of a single fucose residue on the EGF module of factor VII using nuclear magnetic resonance spec- Fig. 2A.…”

Role of unusual O-glycans in intercellular signaling

“…This included decreases in the rate of deuterium exchange for amide protons remote from the site of fucosylation (Mer et al, 1996). Similarly, the presence of an O -fucose on a factor VII EGF repeat showed no significant effect on backbone structure, but showed NMR chemical shift effects in regions distant from the site of glycosylation (Kao et al, 1999). Interestingly, the O -fucose glycan produced a modest increase in the affinity of the amino-terminal calcium binding domain for calcium (Kao et al, 1999).…”