Probing Altered Receptor Specificities of Antigenically Drifting Human H3N2 Viruses by Chemoenzymatic Synthesis, NMR and Modeling

Abstract

Prototypic receptors for human influenza viruses are cell surface N-glycans carrying 2,6-linked sialosides. Under immune pressure, A/H3N2 influenza viruses have emerged with altered receptor specificities that appear to recognize 2,6-linked sialosides presented on extended N-acetyl-lactosamine (LacNAc) moieties. Here, molecular recognition features of such drifted hemagglutinins (HAs) are examined by chemoenzymatic synthesis of complex N-glycans having 13C-labeled monosaccharides at strategic positions. The labeled glycans were employed in 2D STD-1H and 13C-HSQC NMR experiments to pinpoint which monosaccharides of the extended LacNAc chain engage with evolutionarily distinct HAs. The NMR data in combination with computational and mutagenesis studies demonstrate that mutations distal to the receptor binding domain of recent HAs have created an extended binding site that can directly interact with the extended LacNAc chain. A fluorine containing sialyl-LacNAc derivative is used as NMR probe to derive relative binding affinities and confirmed the contribution of the extended LacNAc chain for binding.