Chemoenzymatic Synthesis of Asymmetric Bisecting Bi-, Tri-, and Tetra-Antennary N-Glycans

Abstract

N-Acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase that can install a β1,4-linked N-acetylglucosamine (GlcNAc) residue at the central β-mannoside of N-glycans. The resulting so-called bisecting GlcNAc is not further extended by glycosyl transferases and has been implicated in a wide range of biological processes. The molecular mechanism by which bisection modulates the biosynthesis of N-glycans and influences molecular recognition is not well understood, which is due to a lack of well-defined N-glycans with and without bisection. We describe a chemoenzymatic methodology that can readily provide a wide range of asymmetrical bisecting bi-, tri-, and tetra-antennary N-glycans. It was found GnT-III can act on bi-, tri-, and tetra-antennary N-glycans and can also accept N-glycans having a β1,2GlcNTFA or GlcN 3 moiety at the α1,3- or α1,6-antenna making it possible to prepare panels of asymmetrical N-glycans with and without bisection and having different patterns of sialylation and fucosylation. Enzyme kinetic experiments showed GnT-III preferentially modifies biantennary glycans. The compounds were printed as a glycan microarray, which was screened for lectin binding. It was found that some lectins preferentially bind to bisecting glycans, whereas others do not tolerate or are not affected by this modification. We investigated receptor specificities of human H1N1 and H3N2 influenza viruses and animal H5N1 viruses that pose a pandemic threat, including a virus that has become endemic in cattle. The H1N1 and H3N2 viruses did not tolerate bisection, whereas it did not affect H5N1 viruses. A/bovine had the broadest receptor specificity, providing a rationale for its wide host range.