Enzymatic transfer of a β1,6-linked N-acetylglucosamine to the α-galactose of globo-N-tetraose: in vitro synthesis of a novel hybrid pentasaccharide of lacto-globo type

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Abstract
A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAcβ1–3Galα1–4Galβ1–4Glc, and UDP-[3H]GlcNAc with hog gastric mucosal microsomes, known to contain β1,6-N-acetylglucosaininyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAcβ1–3([3H]GlcNAcβ1–6)Galal-4Galβ1–4Glc. The new enzyme activity possesses substrate specificity features common to a purified β1,6-GlcNAc-transferase from bovine tracheal epithelium, which forms branches at the subterminal β1,3-substituted galactose and accepts both GlcNAc- and Gal-configuration at the terminal residue of the acceptor (Ropp et al. (1991) J. Biol. Chem., 266, 23863–23871). The new β1,6-GlcNAc-branch was readily galactosylated by bovine milk β1,4-galac-tosyltransferase, revealing a pathway to novel hybrid type glycans with N-acetyllactosamine chains on globotype sac-charides. This pathway may lead to the rare IP blood-group antigen and to globoside-like molecules mediating cell adhesion.

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