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 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.
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus 43210.
M L
ML
Da Silva
T
T
Tamura
T
T
McBroom
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KG
Rice
D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005340:Fibrinogen / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D013552:Swine; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; C025469:asialooligosaccharides; C016735:sialooligosaccharides; C088322:tert-butoxycarbonyl-tyrosine; D014443:Tyrosine; D005340:Fibrinogen; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase