The spectrum of N-linked oligosaccharide structures detected by enzymic microsequencing on a recombinant soluble CD4 glycoprotein from Chinese hamster ovary cells.
Structures of the N-linked oligosaccharides of a recombinant soluble form of human CD4 glycoprotein (sCD4) have been investigated by enzymic microsequencing. The glycoprotein has two N-glycosylation sites, Asn271 and Asn300, at both of which evidence for the presence of complex type biantennary sialo-oligosaccharides has been obtained previously by mass spectrometric analyses [Carr, S.A., Hemling, M.E., Folena-Wasserman, G., Sweet, R.W., Anumula, K., Barr, J.R., Huddleston, M.J. & Taylor, P. (1989) J. Biol. Chem. 264, 21,286-21,295]. Among oligosaccharides released from sCD4 by hydrazinolysis and labelled with NaB3H4, neutral (12.8%) and acidic (87.2%) oligosaccharides were detected by paper electrophoresis. The latter were rendered neutral following sialidase treatment indicating that acidity was due exclusively to the presence of sialic acid residues. By enzymic microsequencing of the sialidase-treated oligosaccharides (fractionated on affinity columns of Ricinis communis agglutinin 120 and concanavalin A) in conjunction with methylation data from the earlier study, 14 sequences were identified. These accounted for over 80% of the sialidase-treated oligosaccharides of sCD4 as follows: [formula: see text] where +/- indicates residues present on only a proportion of chains. The spectrum of oligosaccharide structures released from each glycosylation site was assessed as being similar to that of total oligosaccharides on the basis of their chromatographic profiles on the lectin columns and on Bio-Gel P-4.
Yuen
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CT
Glycoconjugates Section, Medical Research Council Clinical Research Centre, Harrow, England.
Carr
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SA
Feizi
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D000818:Animals; D015704:CD4 Antigens / Q000235:genetics; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D004590:Electrophoresis, Paper; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009439:Neuraminidase; D009844:Oligosaccharides / Q000302:isolation & purification*; D010053:Ovary; D011994:Recombinant Proteins; D014162:Transfection
D015704:CD4 Antigens; D009844:Oligosaccharides; D011994:Recombinant Proteins; D009439:Neuraminidase