Heterogeneity of bovine lactotransferrin glycans. Characterization of α-D-Galp-(1→3)-β-D-Gal- and α-NeuAc-(2→6)-β-D-GalpNAc-(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)-alpha-L-fucosylated structures in Fractions A and B. Sequences containing alpha-D-Galp-(1 ¨ 3)-beta-D-Gal on the alpha-D-Man-(1 ¨ 6) antenna, and beta-D-GalpNAc-(1 ¨ 4)-beta-D-GlcNAc and alpha-NeuAc-(2 ¨ 6)-beta-D-GalpNAc-(1 ¨ 4)-beta-D-GlcNAc on the alpha-D-Man-(1 ¨ 3) antenna were characterized in the oligosaccharide-alditols obtained by reductive cleavage of Fraction B. A series of Man4 − 9-GlcNAc structures were identified in Fraction C after endo-N-acetyl-beta-D-glucosaminidase digestion. These results show that the structures of bovine lactotransferrin glycans are more heterogeneous than those of previously characterized transferrin glycans.