Analysis of the site-specific asparagine-linked glycosylation of recombinant human coagulation factor VIIa by glycosidase digestions, liquid chromatography, and mass spectrometry.
The two asparagine-linked glycosylation sites of recombinant coagulation factor VIIa have been characterized by glycosidase digestions, size-exclusion chromatography (SEC), and mass spectrometry (MS). Nine structures were characterized as core fucosylated bi-and triantennary structures with 0-3 sialic-acid residues which were alpha 2-3 linked to galactose exclusively. Three of the structures had one or two galactose residues substituted by N-acetylgalactosamine. Significant differences were found between the oligosaccharide profiles for the two glycosylation sites in rFVIIa. At Asn322, the degree of sialylation was lower and higher amounts of structures containing N-acetylgalactosamine were found compared to Asn145.
Department of Protein Chemistry Ge, Novo Nordisk A/S, Gentofte, Denmark. nkk@novo.dk
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Klausen
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Bayne
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Palm
D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D001665:Binding Sites; D002460:Cell Line; D002853:Chromatography, Liquid; D006224:Cricetinae; D015942:Factor VIIa / Q000737:chemistry* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D009844:Oligosaccharides / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D062367:ortho-Aminobenzoates
D009844:Oligosaccharides; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; D001216:Asparagine; D006026:Glycoside Hydrolases; D015942:Factor VIIa; C000219:anthranilamide