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Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

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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 γ-carboxylation, N- and O-glycosylation, β-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 Asn322 compared to Asn145. 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 Ser60 and Ser52 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.

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Abbreviations

A2S1:

diantennary monosialylated glycan

A2S2:

diantennary disialylated glycan

A2S2F:

diantennary disialylated fucosylated glycan

A3S3:

triantennary trisialylated glycan

A3S3F:

triantennary trisialylated fucosylated glycan

DHB:

2,5-dihydroxybenzoic acid

Endo H:

endoglycosidase H

Fuc:

fucose

FVII:

factor VII

FVIIa:

activated factor VII

Glc:

glucose

GN:

N-acetylglucosamine

H:

hexose

HPCE-LIF:

high performance capillary electrophoresis-laser induced fluorescence

HC:

heavy chain of activated factor VII

HCCA:

α-cyano-4-hydroxycinnamic acid

LC:

light chain of activated factor VII

LC–ESIMS/MS:

liquid chromatography coupled to electrospray ionization tandem mass spectrometry

MALDI-TOFMS:

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

PNGase F:

peptide-N-glycosidase F

PTMs:

post-translational modifications

Q-TOF:

quadrupole time-of-flight mass spectrometer

TFA:

trifluoroacetic acid

THAP:

2,4,6-trihydroxyacetophenone

Xyl:

xylose

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Acknowledgement

The authors would like to thank Michel Nogré and Alain Lejars (LFB) for the purification of FVII.

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Correspondence to Nicolas Bihoreau.

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Fenaille, F., Groseil, C., Ramon, C. et al. Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII. Glycoconj J 25, 827–842 (2008). https://doi.org/10.1007/s10719-008-9143-7

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