Title : Mass spectrometric characterization of
N- and O-glycans of plasma-derived
coagulation factor VII
Abstract :
- Factor VII ( FVII ) is a vitamin K-dependent glycoprotein which, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX
- FVII is secreted as single peptide chain of 406 residues
- Plasma-derived FVII undergoes many post-translational modifications such as gamma-carboxylation, N- and O-glycosylation, beta-hydroxylation
- Despite glycosylation of recombinant FVIIa has been fully characterized, nothing is reported on the N- and O-glycans of plasma-derived FVII (pd- FVII ) and on their structural heterogeneity at each glycosylation site
- N- and O-glycosylation sites and site specific heterogeneity of pd- FVII were studied by various complementary qualitative and quantitative techniques
- A MALDI-MS analysis of the native protein indicated that FVII is a 50.1 kDa glycoprotein modified on two sites by diantennary, disialylated non-fucosylated (A2S2) glycans
- LC-ESIMS/MS analysis revealed that both light chain and heavy chain were N-glycosylated mainly by A2S2 but also by triantennary sialylated glycans
- Nevertheless, lower amounts of triantennary structures were found on Asn(322) compared to Asn(145)
- Moreover, the triantennary glycans were shown to be fucosylated
- In parallel, quantitative analysis of the isolated glycans by capillary electrophoresis indicated that the diantennary structures represented about 50% of the total glycan content
- Glycan sequencing using different glycanases led to the identification of triantennary difucosylated structures
- Last, MS and MS/MS analysis revealed that FVII is O-glycosylated on the light chain at position Ser(60) and Ser(52) which are modified by oligosaccharide structures such as fucose and Glc(Xyl)(0-1-2), respectively
- These latter three O-glycans coexist in equal amounts in plasma-derived FVII