Tyrosine derivatization and preparative purification of the sialyl and asialy-N-linked oligosaccharides from porcine fibrinogen

The N-linked oligosaccharides from porcine fibrinogen were purified following their release from glycopeptides using N-glycosidase F. In separate experiments, both sialyl and asialyl oligosaccharides were prepared from 5 g of fibrinogen. The reducing oligosaccharides were reacted with ammonium bicar...

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Published inArchives of biochemistry and biophysics Vol. 312; no. 1; pp. 151 - 157
Main Authors Da Silva, M L, Tamura, T, McBroom, T, Rice, K G
Format Journal Article
LanguageEnglish
Published United States 01.07.1994
Subjects
Animals
Carbohydrate Sequence
Chromatography, High Pressure Liquid
Fibrinogen - chemistry
Glycopeptides - chemistry
Glycoproteins - chemistry
Magnetic Resonance Spectroscopy
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Molecular Sequence Data
Oligosaccharides - chemistry
Swine
Tyrosine - analogs & derivatives
Tyrosine - chemistry
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Summary:The N-linked oligosaccharides from porcine fibrinogen were purified following their release from glycopeptides using N-glycosidase F. In separate experiments, both sialyl and asialyl oligosaccharides were prepared from 5 g of fibrinogen. The reducing oligosaccharides were reacted with ammonium bicarbonate to form/oligosaccharide-glycosylamines and then derivatized with tert-butoxycarbonyl-L-tyrosine N-hydroxysuccinimidyl ester. Tyrosinamide--oligosaccharides were purified first by gel filtration chromatography and then by reverse-phase HPLC and the products were characterized by proton NMR and fast atom bombardment-MS. Porcine fibrinogen was found to have predominantly a single asialyl biantennary oligosaccharide containing a fucose linked alpha 1-6 to GlcNAc 1. The oligosaccharide possesses two sialylation patterns with a major form (70%) having a single N-acetyl neuraminic acid (NeuAc) residue linked alpha 2-6 to galactose on only one antenna and a minor form (30%) possessing two NeuAc residues linked alpha 2-6 to both terminal galactose residues. In addition to developing an isolation procedure and establishing the structures of porcine fibrinogen oligosaccharides, this study improves on the tyrosine derivatization technique as a general approach to isolate structurally diverse N-linked oligosaccharides from glycoproteins.
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ISSN:0003-9861