Bovine Milk Whey for Preparation of Natural N-glycans: Structural and Quantitative Analysis

Glycans exhibit enormous structural diversity in nature and are of particular importance for self-cell survival and are often targeted by microbes. In this study, N-glycans (374.9 pmol/mg in dry delipidated weight) were enzymatically released from bovine milk whey protein concentrate, and they were isolated and analyzed by a two-dimensional HPLC mapping technique and/or by MALDI-TOF mass spectrometry. A total of 39 identified N-glycans are biand tri-antennary sugar chains terminated with multiple mannose residues (Man-Man; molar ratio of 39.5%), N-acetyl-lactosamine (LacNAc; Gal1-4GlcNAc; molar ratio of 17.9), di-N-acetylated lactosamine (LacdiNAc; GalNAc1-4GlcNAc; molar ratio of 22.8), GlcNAc (molar ratio of 7.05), Neu5Ac2-6Gal1-4GlcNAc (molar ratio of 5.3), Neu5Ac2-6GalNAc1-4GlcNAc (molar ratio of 1.25), Neu5Gc2-6Gal1-4GlcNAc (molar ratio of 2.5), and Neu5Gc2-3Gal1-4GlcNAc (molar ratio of 0.25), in which some are fucosylated on the proximal core GlcNAc1-N-Asn. Terminal Neu5Ac2-3Gal/GalNAc and Neu5Gc2-3/2-6GalNAc were not detected in the bovine whey protein concentrate. Among the 39 glycans, GalNAcβ14GlcNAcβ1-2Manα1-3(GalNAcβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβand Manα1-2Manα1-2Manα 1-3(Manα1-2Manα1-3(Manα1-2Manα1-6)Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβwere the most abundant types found with molar ratios of 11.3 and 10, respectively. Elucidation of glycan molecular structures will lead to an understanding of their biological roles and functions. Whey contains a variety of glycans and is inexpensive, and it is thus considered to be source of glycans for array glycan libraries to be used for investigations of specific glycan-protein interactions, enabling not only analysis of biological roles of the glycan-binding proteins but also development of molecules affecting these interactions. Furthermore, these natural glycans may have therapeutic value in prevention and inhibition of infection of microbes that recognize them.