Partial glycosylation of Asn2181 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 slightly different molecular masses and phospholipid-binding properties. The aim was to determine whether this heterogeneity is due to the degree of glycosylation of Asn(2181). FVa1 and FVa2 were isolated and digested with endoglycosidase PNGase F. As judged by Western blotting, the FVa2 light chain contained 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 expressed in COS1 cells, activated by thrombin, and analyzed by Western blotting. Wild-type FVa contained the 71 kDa-74 kDa doublet, whereas the Asn(2181)Gln and Ser(2183)Ala mutants contained only the 71 kDa light chain. In contrast, the Ser(2183)Thr mutant gave a 74 kDa light chain. This demonstrated that the third position in the Asn-X-Ser/Thr consensus affects glycosylation efficiency, Thr being associated with a higher degree of glycosylation than Ser. The Ser(2183)Thr mutant FVa was functionally indistinguishable from plasma-purified FVa1, whereas Asn(2181)Gln and Ser(2183)Ala mutants behaved like FVa2. Thus, the carbohydrate at Asn(2181) impaired the interaction between FVa and the phospholipid membrane, an interpretation consistent with a structural analysis of a three-dimensional model of the C2 domain and the position of a proposed phospholipid-binding site. In conclusion, we show that the FV1-FV2 heterogeneity is caused by differential glycosylation of Asn(2181) related to the presence of a Ser rather than a Thr at the third position in the consensus sequence of glycosylation.
Nicolaes
G A
GA
Wallenberg Laboratory, Department of Clinical Chemistry, Malmö University Hospital, Lund University, Sweden. Gerry.Nicolaes@klkemi.mas.lu.se
Villoutreix
B O
BO
Dahlbäck
B
B
D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002852:Chromatography, Ion Exchange; D016384:Consensus Sequence / Q000235:genetics*; D005165:Factor V / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015943:Factor Va / Q000737:chemistry / Q000235:genetics; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011517:Prothrombin Time; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012694:Serine / Q000235:genetics; D013912:Threonine / Q000235:genetics; D013917:Thrombin / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D015943:Factor Va; D001216:Asparagine; D005165:Factor V; D006026:Glycoside Hydrolases; D013917:Thrombin