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STUDIES ON THE STRUCTURE OF CARBOHYDRATE CHAINS OF GLYCOPROTEINS
Vliegenthart, J. F. G. Hard, K. de Waard, P. Kamerling, J. P.
Vliegenthart, J. F. G.
Hard, K.
de Waard, P.
Kamerling, J. P.
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Issue Date
1991
Submitted date
2024-02-20
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Abstract
To obtain detailed information on the primary structures ofthe carbohydrate chains of glycoproteins,it is still necessary to degradethe glycoprotein to partial structures each representing only one glycosylation position. The partial structures suitable for analysis, can be oligosaccharides, oligosaccharide-alditols or glycopeptides. In our approachfor the analysis of N,O-glycoproteins we cleavefirst the N-linked chains with the aid of PNGaseF. Theextentofthis reaction has to be checked carefully, in order to makesure that in case the reaction doesnot lead to complete removal of the carbohydrate chains, the chainsthat are still linked to the protein do not have a specific structure. Forthis purpose several methodscan be applied. The pool of N-glycans is separated from the O-glycoprotein, whichis then isolated and subjected to alkaline borohydride treatment. The pools correspondingto the N- and O-linked chains, respectively, are fractionated to homogeneous compounds asfar as possible. Analysis of the compoundsis carried out by 500-MHz !H-NMR spectroscopy. The occurrence of non-carbohydrate substituentslike alkyl, acyl, sulfate and phosphate groups may giverise to serious complications. Often the contents of substituents are far below molar equivalents in a compound,thereby enhancingthe (micro)heterogeneity. It should be noted that information about the type andposition of the non-carbohydrate substituents can be obtained by 1D- or 2D-1H-NMRspectroscopy. For the analysis of phosphate-containing oligosaccharides we developed a 1H(31P} relayed spin-echo difference spectroscopy technique to characterize the residue to whichthe phosphateis attached and to detect the C-atom that is substituted. By meansof this analytical procedure we were able to determine the structure of glycoprotein-derived carbohydrate chains for quite a numberof glycoproteins.
Citation
Protein glycosylation, 137 - 146
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Book chapter
conference paper
conference paper
Language
en
Description
Series/Report no.
GBF monographs ; Volume 15
ISSN
0930-4320
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ISBN
3527283676
1560811846
1560811846
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Attribution-NonCommercial-ShareAlike 4.0 International