Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII.
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.
Fenaille
François
F
Laboratoire Français du Fractionnement et des Biotechnologies, 3 avenue des Tropiques, BP 305, Les Ulis, 91958, Courtaboeuf cedex, France. francois.fenaille@cea.fr
Groseil
Catherine
C
Ramon
Christine
C
Riandé
Sandrine
S
Siret
Laurent
L
Chtourou
Sami
S
Bihoreau
Nicolas
N
D000478:Alkylation; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D019075:Electrophoresis, Capillary; D005167:Factor VII / Q000032:analysis / Q000737:chemistry*; D005453:Fluorescence; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000737:chemistry*; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
D006020:Glycopeptides; D011134:Polysaccharides; D021122:Protein Subunits; D005167:Factor VII; D014357:Trypsin