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 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.

Specific 13C reductive methylation of glycophorin A. Possible relation of the N-terminal amino acid and the lysine residues to MN blood group specificities.

Heterozygous and homozygous glycophorin A were partially and fully reductively methylated with 13C-enriched formaldehyde in the presence of sodium cyanoborohydride. Total reductive methylation modified the five lysine residues (to produce N epsilon,N-[13C]dimethyl lysine) and the N-terminal amino acid residues (N alpha,N-[13C]dimethyl serine and leucine) of glycophorins AM and AN, respectively. 13C-NMR spectra of these species indicated that the 13C-enriched methyl carbons of the five lysyl derivatives all occur at 44.1 ppm downfield from Me4Si. Titration results indicate that the pK alpha of these methylated lysines is greater than 10. The chemical shift equivalent methyl resonances of the 13C-enriched methylated N-terminal Leu derivative were found to occur at 42.8 ppm downfield from Me4Si and exhibited a normal pH titration behavior (pK alpha approximately 7.4). The methyl resonances of the N alpha,N-[13C]dimethyl Ser derivative, on the other hand, were found to exhibit chemical shift nonequivalence, indicating rotational constraints about the C alpha-N bond. The linewidths of the two methyl resonances were also found to be considerably different; this phenomenon could be eliminated by running spectra of the sample (pH approximately 5.0) at elevated temperatures (75 degrees C). This result suggested that for the N alpha,N-[13C]dimethyl Ser derivative of glycophorin AM, hindered rotation must occur about one of the N alpha-13CH3 bonds. This structural difference at the N-terminal residue of glycophorins AM and AN may be related to the MN blood group determinants displayed by these related glycoproteins.