Heterogeneity of bovine lactotransferrin glycans. Characterization of α- d-Gal p-(1 → 3)-β- d-Gal- and α-NeuAc-(2 → 6)-β- d-Gal pNAc-(1 → 4)-β- d-GlcNAc-substituted N-linked glycans

Abstract

Lactotransferrin isolated from a pool of mature bovine milk has been shown to contain N-glycosidically-linked glycans possessing N-acetylneuraminic acid, galactose, mannose, fucose, N-acetylglucosamine and N-acetylgalactosamine. The glycopeptides obtained by Pronase digestion were fractionated by concanavalin A-Sepharose affinity chromatography into three fractions: slightly retained (A), retained (B), and strongly retained (C). The structure of the glycans of the three fractions has been determined by application of methanolysis, methylation analysis, fast atom bombardment-mass spectrometry, and 1H NMR spectroscopy. Diantennary structures without GalNAc were present as partially sialylated and partially (1 → 6)-α- l-fucosylated structures in Fractions A and B. Sequences containing α- d-Gal p-(1 → 3)-β- d-Gal on the α- d-Man-(1 → 6) antenna, and β- d-Gal pNAc-(1 → 4)-β- d-GlcNAc and α-NeuAc-(2 → 6)-β- d-Gal pNAc-(1 → 4)-β- d-GlcNAc on the α- d-Man-(1 → 3) antenna were characterized in the oligosaccharide-alditols obtained by reductive cleavage of Fraction B. A series of Man 4 − 9-GlcNAc structures were identified in Fraction C after endo- N-acetyl-β- d-glucosaminidase digestion. These results show that the structures of bovine lactotransferrin glycans are more heterogeneous than those of previously characterized transferrin glycans.