Enzymatic synthesis and functional characterization of site-specific S/O-linked N-glycopeptides: Elucidating the impact of glycosidic linkage patterns on bioactivity

Glycoengineering of therapeutic peptides/proteins has been hindered by the fact that no cost-effective and scalable strategy is currently available to install a β-N-acetylglucosamine (GlcNAc) residue to a pre-determined Asn site in peptides/proteins, to be further glycosylated by N-glycan. In this study, we developed a two-step enzymatic method to prepare N-glycan-modified peptides with natural β-S/O-glycosylation linkage by sequentially catalyzed by the S/O-HexNAc-transferase from Streptomyces venezuelae ATCC 15439 (SvGT) and the N180H mutant of endoglycosidase from Coprinopsis cinerea (EndoCC^N180H). To unravel how glycosylation linkage patterns and glycosylation extent impact the bioactivity of the glycopeptides, cell-based receptor activation potency and in vivo hypoglycemic activity were assayed for β-S/O/N-glycosylated glucagon-like peptide-1 (GLP-1) analogues. The comparable bioactivity exhibited by the S-glycosylated and N-glycosylated products presented S-glycosylation as an efficient and effective strategy for future glycoengineering of peptide/protein pharmaceuticals.