In vitro synthesis of mucin-type O-glycans using saccharide primers comprising GalNAc-Ser and GalNAc-Thr residues

Abstract

Mucin-type O-glycosylation of serine or threonine residue in proteins is known to be one of the major post-translational modifications. In this study, two novel alkyl glycosides, Nα-lauryl-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-l-serineamide (GalNAc-Ser-C12) and Nα-lauryl-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-l-threonineamide (GalNAc-Thr-C12) were synthesized as saccharide primers to prime mucin-type O-glycan biosynthesis in cells. Upon incubating human gastric cancer MKN45 cells with the saccharide primers, 22 glycosylated products were obtained, and their structures were analyzed using liquid chromatography-mass spectrometry and enzyme digestion. The amounts of glycosylated products were dependent on the amino acid residues in the saccharide primers. For example, in vitro synthesis of T antigen (Galβ1-3GalNAc), fucosyl-T (Fucα1-2Galβ1-3GalNAc), and sialyl-T (NeuAcα2-3Galβ1-3GalNAc) preferred a serine residue, whereas sialyl-Tn (NeuAcα2-6GalNAc) preferred a threonine residue. Furthermore, the glycosylated products derived from GalNAc-Ser/Thr-C12 and Gal-GalNAc-Ser/Thr-C12 using cell-free synthesis showed the same amino acid selectivity as those in the cell experiments. These results indicate that glycosyltransferases involved in the biosynthesis of mucin-type O-glycans distinguish amino acid residues conjugated to GalNAc. The saccharide primers developed in this study might be useful for comparing mucin-type oligosaccharides in cells and constructing oligosaccharide libraries to study cell function.