Novel Serine/Threonine-O-glycosylation with N-Acetylneuraminic acid and 3-Deoxy-D-manno-octulosonic acid by Maf glycosyltransferases

Abstract

Some bacterial flagellins are O-glycosylated on surface-exposed Serine/Threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid, and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called Motility associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, Serine/Threonine-O-linked N-Acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and Serine/Threonine-O-linked 3-Deoxy-D-manno-octulosonic acid (Ser/Thr-O-KDO), both catalysed by Geobacillus kaustophilus Maf (putative flagellin sialyltransferase) and Clostridium botulinum Maf (putative flagellin legionaminic acid transferase). We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were co-expressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. The glycosylation of G. kaustophilus flagellin with KDO, and that of C. botulinum flagellin with Neu5Ac and KDO indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering. Significance StatementGlycosylation, the modification of proteins with sugars, is one of the most common post-translational modifications observed in proteins. While glycosylation is versatile, the most common forms of glycosylation are N-glycosylation, where the N atom of Asparagine is modified with a glycan, and O-glycosylation where the O atom of serine or threonine residues is modified with a glycan. Here, we report a novel type of O-glycosylation in the bacterial flagellin proteins of two Gram-positive bacteria, Geobacillus kaustophilus and Clostridium botulinum. We demonstrate for the first time that the enzyme flagellin Maf glycosyltransferase is capable of transferring the monosaccharides, N-acetylneuraminic acid and 3-Deoxy-D-manno-octulosonic acid, on to serine and threonine residues of these proteins.