Novel Class of Human Milk Oligosaccharides Based on 6′-Galactosyllactose Containing N-Acetylglucosamine Branches Extended by Oligogalactoses

Human milk oligosaccharides (HMOs) have attracted much attention in recent years not only as a prebiotic factor but also in particular as an essential component of infant nutrition in relation to their impact on innate immunity. The backbone structures of complex HMOs generally contain single or repetitive lacto-N-biose (type 1) or lactosamine (type 2) units in either linear or branched chains extending from a lactose core. While all known branched structures originate from the 3,6-substitution of the lactosyl core galactose, we here describe a new class of HMOs that tentatively branch at the terminal galactose of 6′-galactosyllactose. Another novel feature of this class of HMOs was found in linear oligo-galactosyl chains linked to one of the N-acetylglucosamine (GlcNAc) branches. The novel structures exhibit general formulas with hexose versus hexosamine contents of 5/2 to 8/2 and can be designated as high-galactose (HG)-HMOs. In addition, up to three fucosyl residues are linked to the octa- to dodecasaccharides, which were detected in two human milk samples from the Lewis blood-group-defined donors. Structural analyses of methylated glycans and their alditols comprised matrix-assisted laser desorption ionization mass spectrometry, electrospray-(collision-induced dissociation) mass spectrometry and linkage analyses by gas chromatography–mass spectrometry of the derived partially methylated alditol acetates. Enzymatic degradation by the application of β1–3,4-specific galactosidase supported the presence of terminal galactose-linked β1–6 to one of the two GlcNAc branches. The mass spectrometry glycomic data have been deposited at the GlycoPOST archive with the data set identifier GPST000191 (Username: franz.hanisch@uni-koeln.de; Password: Soma1Dita2Carb. Watanabe, Y. GlycoPOST realizes FAIR principles for glycomics mass spectrometry data. Nucleic Acids Res. 2021, 49, D1523–D1528)