Possible role of the carbohydrate residues in the display of the MN blood group determinants by glycophorin A.

Heterozygous glycophorin AM,N and homozygous glycophorin AM were reductively methylated with 13C-enriched formaldehyde in the presence of cyanoborohydride. Total reductive methylation modified the five lysine residues, and the N-terminal amino acid residues (serine and leucine) of glycophorins AM and AN, respectively. The 13C resonances of the incorporated labels were monitored as a function of the degree of glycosylation of the glycoprotein. While minimal, if any, structural changes were observed near the N-terminal amino acid upon removal of alpha-D-N-acetylneuraminic acid residues, gross structural changes were observed when most of the oligosaccharide chains were removed. We also found that progressive methylation of the lysine residues of glycophorin AM may influence either the chemical shift of one of the nonequivalent methyl groups of the N alpha, N-[13C]dimethyl serine residue, or one of the two states of glycophorin AM.

13C-N.M.R.-spectral study of the binding of Gd3+ to glycophorin.

Natural-abundance, 13C-n.m.r. spectroscopy was used to study the binding of Gd3+ to glycophorin, and also to the tetrasaccharides isolated from glycophorin after treatment of the glycoprotein with NaOH-NaBH4. Gd3+ binds to the tetrasaccharide (both in the isolated, reduced form and when still attached to the native glycoprotein), and, especially, to the alpha-NeuAc residues. In order to cause severe line-broadening of the 13C resonances of alpha-NeuAc, the ratios of the alpha-NeuAc residues of glycophorin, and of the isolated, reduced tetrasaccharide, to Gd3+ were much higher than that needed for causing similar broadening for 2-O-methyl-alpha-NeuAc-Gd3+ solutions. These results indicate that the other carbohydrate residues of the tetrasaccharide may be involved in the binding of Gd3+, producing a stronger metal-ion-binding effect.

13C-N.m.r.-spectral study of the mode of binding of Gd3+ to various glycopeptides.

Natural-abundance, 13C-n.m.r. spectroscopy was used to study the mode of binding of Gd3+ to mono-O-glycosylated L-serine and tripeptides variously composed of Gly and L-Thr. When the amino and carboxyl groups of the amino acid are not blocked, strong interaction of Gd3+ with them is observed; this is also readily apparent with some related, nonglycosylated peptides. When the amino and carboxyl groups of the amino acid are blocked, noticeable interaction of Gd3+ with the glycosidic oxygen atom (O-3) and O-2' for the glycopeptide containing alpha-D-Galp, and with O-3 and N-2' for the glycopeptide containing alpha-D-GalpNAc, is observed. Weak interactions are also possible with O-4' and O-6' of the glycosyl groups. Although the amino acids were protected, these metal ion-carbohydrate interactions may still be mediated, to some extent, by the acetyl protecting the amino group and by the ester group on the amino acid.