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
References
Jurlander, B., Thim, L., Klausen, N.K., Persson, E., Kjalke, M., Rexen, P., Jorgensen, T.B., Ostergaard, P.B., Erhardtsen, E., Bjorn, S.E.: Recombinant activated factor VII (rFVIIa): characterization, manufacturing, and clinical development. Semin. Thromb. Hemost 27, 373–384 (2001)
Hagen, F.S., Gray, C.L., O’Hara, P., Grant, F.J., Saari, G.C., Woodbury, R.G., Hart, C.E., Insley, M., Kisiel, W., Kurachi, K.: Characterization of a cDNA coding for human factor VII. Proc. Natl. Acad. Sci. U. S. A 83, 2412–2416 (1986)
Hansson, K., Stenflo, J.: Post-translational modifications in proteins involved in blood coagulation. J. Thromb. Haemost 3, 2633–2648 (2005)
Bolt, G., Kristensen, C., Steenstrup, T.D.: Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII. Glycobiology 15, 541–547 (2005)
Iino, M., Foster, D.C., Kisiel, W.: Functional consequences of mutations in Ser-52 and Ser-60 in human blood coagulation factor VII. Arch. Biochem. Biophys 352, 182–192 (1998)
Klausen, N.K., Bayne, S., Palm, L.: Analysis of the site-specific asparagine-linked glycosylation of recombinant human coagulation factor VIIa by glycosidase digestions, liquid chromatography, and mass spectrometry. Mol. Biotechnol 9, 195–204 (1998)
Thim, L., Bjoern, S., Christensen, M., Nicolaisen, E.M., Lund-Hansen, T., Pedersen, A.H., Hedner, U.: Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry 27, 7785–7793 (1988)
Bjoern, S., Foster, D.C., Thim, L., Wiberg, F.C., Christensen, M., Komiyama, Y., Pedersen, A.H., Kisiel, W.: Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine. J. Biol. Chem 266, 11051–11057 (1991)
Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Takao, T., Shimonishi, Y., Iwanaga, S.: Identification of a disaccharide (Xyl-Glc) and a trisaccharide (Xyl2-Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor-like domain of human factors VII and IX and protein Z and bovine protein Z. J. Biol. Chem 264, 20320–20325 (1989)
Harvey, D.J.: Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Mass Spectrom. Rev 18, 349–450 (1999)
Morelle, W., Michalski, J.C.: The mass spectrometric analysis of glycoproteins and their glycan structures. Curr. Anal. Chem 1, 29–57 (2005)
Medzihradszky, K.F.: Characterization of protein N-glycosylation. Methods Enzymol 405, 116–138 (2005)
Peter-Katalinic, J.: Methods in enzymology: O-glycosylation of proteins. Methods Enzymol 405, 139–171 (2005)
Nemeth, J.F., Hochgesang Jr., G.P., Marnett, L.J., Caprioli, R.M.: Characterization of the glycosylation sites in cyclooxygenase-2 using mass spectrometry. Biochemistry 40, 3109–3116 (2001)
Harazono, A., Kawasaki, N., Itoh, S., Hashii, N., Ishii-Watabe, A., Kawanishi, T., Hayakawa, T.: Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry. Anal. Biochem 348, 259–268 (2006)
Zaia, J., Boynton, R., Heinegard, D., Barry, F.: Posttranslational modifications to human bone sialoprotein determined by mass spectrometry. Biochemistry 40, 12983–12991 (2001)
Woosley, B., Xie, M., Wells, L., Orlando, R., Garrison, D., King, D., Bergmann, C.: Comprehensive glycan analysis of recombinant Aspergillus niger endo-polygalacturonase C. Anal. Biochem 354, 43–53 (2006)
Lyubarskaya, Y., Houde, D., Woodard, J., Murphy, D., Mhatre, R.: Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity. Anal. Biochem 348, 24–39 (2006)
Nemeth-Cawley, J.F., Rouse, J.C.: Identification and sequencing analysis of intact proteins via collision-induced dissociation and quadrupole time-of-flight mass spectrometry. J. Mass Spectrom 37, 270–282 (2002)
Schirm, M., Schoenhofen, I.C., Logan, S.M., Waldron, K.C., Thibault, P.: Identification of unusual bacterial glycosylation by tandem mass spectrometry analyses of intact proteins. Anal. Chem 77, 7774–7782 (2005)
Chabbat, J., Hampikian-Lenin, S., Toully, V., Gaillandre, A., Pejaudier, L., Steinbuch, M.: A human factor VIIa concentrate and its effects in the hemophilic A dog. Thromb. Res 54, 603–612 (1989)
Fenaille, F., Le Mignon, M., Groseil, C., Siret, L., Bihoreau, N.: Combined use of 2,4,6-trihydroxyacetophenone as matrix and enzymatic deglycosylation in organic-aqueous solvent systems for the simultaneous characterization of complex glycoproteins and N-glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom 21, 812–816 (2007)
Fenaille, F., Le Mignon, M., Groseil, C., Ramon, C., Riande, S., Siret, L., Bihoreau, N.: Site-specific N-glycan characterization of human complement factor H. Glycobiology 17, 932–944 (2007)
Colangelo, J., Orlando, R.: On-target exoglycosidase digestions/MALDI-MS for determining the primary structures of carbohydrate chains. Anal. Chem 71, 1479–1482 (1999)
Harvey, S.B., Stone, M.D., Martinez, M.B., Nelsestuen, G.L.: Mutagenesis of the gamma-carboxyglutamic acid domain of human factor VII to generate maximum enhancement of the membrane contact site. J. Biol. Chem 278, 8363–8369 (2003)
Kolarich, D., Weber, A., Turecek, P.L., Schwarz, H.P., Altmann, F.: Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms. Proteomics 6, 3369–3380 (2006)
Rudd, P.M., Dwek, R.A.: Glycosylation: heterogeneity and the 3D structure of proteins. Crit Rev. Biochem. Mol. Biol 32, 1–100 (1997)
Kornfelt, T., Persson, E., Palm, L.: Oxidation of methionine residues in coagulation factor VIIa. Arch. Biochem. Biophys 363, 43–54 (1999)
Nicolaisen, E.M., Thim, L., Jacobsen, J.K., Nielsen, P.F., Mollerup, I., Jorgensen, T., Hedner, U.: FVIIa derivatives obtained by autolytic and controlled cathepsin G mediated cleavage. FEBS Lett 317, 245–249 (1993)
Johnson Jr., R.W., Ahmed, T.F., Miesbauer, L.J., Edalji, R., Smith, R., Harlan, J., Dorwin, S., Walter, K., Holzman, T.: Protein fragmentation via liquid chromatography-quadrupole time-of-flight mass spectrometry: the use of limited sequence information in structural characterization. Anal. Biochem 341, 22–32 (2005)
Carr, S.A., Huddleston, M.J., Bean, M.F.: Selective identification and differentiation of N- and O-linked oligosaccharides in glycoproteins by liquid chromatography-mass spectrometry. Protein Sci 2, 183–196 (1993)
Huddleston, M.J., Bean, M.F., Carr, S.A.: Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests. Anal. Chem 65, 877–884 (1993)
Breci, L.A., Tabb, D.L., Yates III, J.R., Wysocki, V.H.: Cleavage N-terminal to proline: analysis of a database of peptide tandem mass spectra. Anal. Chem 75, 1963–1971 (2003)
Paizs, B., Suhai, S.: Fragmentation pathways of protonated peptides. Mass Spectrom. Rev 24, 508–548 (2005)
Krokhin, O., Ens, W., Standing, K.G., Wilkins, J., Perreault, H.: Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides. Rapid Commun. Mass Spectrom 18, 2020–2030 (2004)
Wada, Y., Azadi, P., Costello, C.E., Dell, A., Dwek, R.A., Geyer, H., Geyer, R., Kakehi, K., Karlsson, N.G., Kato, K., Kawasaki, N., Khoo, K.H., Kim, S., Kondo, A., Lattova, E., Mechref, Y., Miyoshi, E., Nakamura, K., Narimatsu, H., Novotny, M.V., Packer, N.H., Perreault, H., Peter-Katalinic, J., Pohlentz, G., Reinhold, V.N., Rudd, P.M., Suzuki, A., Taniguchi, N.: Comparison of the methods for profiling glycoprotein glycans-HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study. Glycobiology 17, 411–422 (2007)
Guile, G.R., Harvey, D.J., O’Donnell, N., Powell, A.K., Hunter, A.P., Zamze, S., Fernandes, D.L., Dwek, R.A., Wing, D.R.: Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland. Eur. J. Biochem 258, 623–656 (1998)
Macek, B., Hofsteenge, J., Peter-Katalinic, J.: Direct determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom 15, 771–777 (2001)
Acknowledgement
The authors would like to thank Michel Nogré and Alain Lejars (LFB) for the purification of FVII.
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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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DOI: https://doi.org/10.1007/s10719-008-9143-7