Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet， the synthesis of structurally well-defined， core-fucosylated glycoproteins remains a challenging task due to the complexity in multistep chemical synthesis or the inability of the biosynthetic α1，6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1，6-fucosynthase and fucoligase for direct core fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme， but se... More
Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet， the synthesis of structurally well-defined， core-fucosylated glycoproteins remains a challenging task due to the complexity in multistep chemical synthesis or the inability of the biosynthetic α1，6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1，6-fucosynthase and fucoligase for direct core fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme， but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1，6-fucosidase， including E274A， E274S， and E274G， acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using α-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the α1，6-fucosidase mutants could introduce an α1，6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide but also to high-mannose and complex-type N-glycans in the context of N-glycopeptides， N-glycoproteins， and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous， core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.
