The carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin.
The complete carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin was elucidated through chemical and enzymatic methods including gas chromatography-mass spectrometry (GC-MS) and lectin affinity chromatography. Pronase digestion of thiostatin yielded a major glycopeptide fraction with asparagine the most abundant amino acid present. Based on one mole of aspartic acid, the following molar ratios obtained for the four major amino acids: aspartic acid (1.0), threonine (0.53), glycine (0.48) and serine (0.30). Neutral sugar analysis yielded a 3:2 molar ratio for mannose to galactose based on an assigned value to mannose of 3. On this basis, the fraction also contained 3 residues of sialic acid and, on average, 0 to 1 residue of fucose. GC-MS of partially methylated alditol acetates from the glycopeptide fraction identified the presence of biantennary and triantennary structure. Analyses of the neutral sugar and amino-acid composition, together with methylation data, support a biantennary N-linked structure for this major glycopeptide fraction and a triantennary N-linked structure as a lesser component. Sequencing of the desialyated 14C-labelled glycopeptide fraction by sequential exoglycosidase digestion and lectin affinity chromatography uncovered the following saccharide order: terminal galactose, N-acetylglucosamine and pentasaccharide inner core. This sequence is consistent with the N-linked glycan structures demonstrated by methylation and compositional analyses.
Rusiniak
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ME
Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo 14260.
Bedi
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GS
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D006020:Glycopeptides / Q000302:isolation & purification; D006026:Glycoside Hydrolases; D007704:Kininogens / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D011402:Pronase; D051381:Rats; D013402:Sugar Alcohols / Q000032:analysis
D000596:Amino Acids; D002241:Carbohydrates; D006020:Glycopeptides; D007704:Kininogens; D009844:Oligosaccharides; D013402:Sugar Alcohols; D001216:Asparagine; D006026:Glycoside Hydrolases; D011402:Pronase