Structural studies of a hexasaccharide from a blood-group-active glycoprotein and evidence for the binding of its terminal non-reducing N-acetyl-D-glucosamine residues to wheat germ agglutinin.

Abstract

We have isolated blood‐group substance H from pig stomach mucosa and subjected the glycoprotein to two consecutive cycles of mith degradation. The wheat germ agglutinin hemagglutination was strongly inhibited by the native glycoprotein and its first Smith degradation product. A second cycle of Smith degradation resulted in the loss of most of the inhibitory activity. Methylation analyses of undegraded and Smith‐degraded glycoprotein demonstrated a strong correlation between the number of terminal N‐acetylglucosamine residues and the wheat germ agglutinin reactivity.An oligosaccharide (Hf) which inhibited the wheat germ agglutinin hemagglutination more strongly than N,N′,N″‐triacetylchitotriose was isolated from the mixture of degradation products obtained upon alkaline sodium borohydride treatment of blood‐group substance H. We have previously shown that oligosaccharide Hf contains galactose, N‐acetylglucosamine and N‐acetylgalactosaminitol in a molar ratio of 2:3:1 and has inhibitory activity sensitive to N‐acetylglucosaminidase of Trichomonas foetus [Tuppy, H. and Wrann, M., Monatsh. Chem. 109 (1978) 703–710].The methylation analyses described in this paper indicate that: firstly, oligosaccharide Hf is a branched hexasaccharide, 3,6‐linked N‐acetylgalactosaminitol being the branching point. The mass spectrum of 1,4,5‐tri‐O‐methyl‐3,6‐di‐O‐acetyl‐2‐deoxy‐2‐(N‐methylacetamido)‐galactitol is shown. Secondly, both nonreducing termini of oligosaccharide Hf are occupied by N‐acetylglucosamine, the third N‐acetylglucosamine residue is 4‐linked. One of the galactose residues is substituted in position 4, the other in position 2. Thirdly, the loss of inhibitory activity of oligosaccharide Hf after treatment with N‐acetylglucosaminidase of Trichomonas foetus is a result of the removal of a terminal N‐acetylglucosamine residue from the 2‐position of the respective subterminal galactose residue.The structure of oligosaccharide Hf and the significance of terminal N‐acetylglucosamine residues for the wheat germ agglutinin inhibition are discussed.