Partial glycosylation of Asn(2181) in human factor V as a cause of molecular and functional heterogeneity. Modulation of glycosylation efficiency by mutagenesis of the consensus sequence for N-linked glycosylation

Coagulation factor V (FV) circulates in two forms, FV1 and FV2, having slig htly different molecular masses and phospholipid-binding properties. The ai m was to determine whether this heterogeneity is due to the degree of glyco sylation of Asn(2181). FVa1 and FVa2 were isolated and digested with endogl ycosidase PNGase F. As judged by Western blotting, the FVa2 light chain con tained two N-linked carbohydrates, whereas FVa1 contained three. Wild-type FV and three mutants, Asn(2181)Gln, Ser(2183)Thr, and Ser(2183)Ala, were ex pressed in COS1 cells, activated by thrombin, and analyzed by Western blott ing. Wild-type FVa contained the 71 kDa-74 kDa doublet, whereas the Asn(218 1)Gln and Ser(2183)Ala mutants contained only the 71 kDa light chain. In co ntrast, the Ser(2183)Thr mutant gave a 74 kDa light chain. This demonstrate d that the third position in the Asn-X-Ser/Thr consensus affects glycosylat ion efficiency, Thr being associated with a higher degree of glycosylation than Ser. The Ser(2183)Thr mutant FVa was functionally indistinguishable fr om plasma-purified FVa1, whereas Asn(2181)Gln and Ser(2183)Ala mutants beha ved like FVa2. Thus, the carbohydrate at Asn2181 impaired the interaction b etween FVa and the phospholipid membrane, an interpretation consistent with a structural analysis of a three-dimensional model of the C2 domain and th e position of a proposed phospholipid-binding site. In conclusion, we show that the FV1-FV2 heterogeneity is caused by differential glycosylation of A sn(2181) related to the presence of a Ser rather than a Thr at the third po sition in the consensus sequence of glycosylation.