Asymmetrical Bi-antennary Glycans Prepared by a Stop-and-Go Strategy Reveal Receptor Binding Evolution of Human Influenza A Viruses

Glycan binding properties of respiratory viruses have been difficult to probe due to a lack of biological relevant glycans for binding studies. Here, a stop-and-go chemoenzymatic methodology is presented that gave access to a panel of 32 asymmetrical bi-antennary N-glycans having various numbers of N-acetyl lactosamine (LacNAc) repeating units capped by α2,3- or α2,6-sialosides resembling structures found in airway tissues. It exploits that the branching enzymes MGAT1 and MGAT2 can utilize unnatural UDP-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNTFA) as donor. The TFA moiety of the resulting glycans can be hydrolyzed to give GlcNH(2) at one of the antennae that temporarily blocks extension by glycosyl transferases. The N-glycans were printed as a microarray that was probed for receptor binding specificities of evolutionary distinct human A(H3N2) and A(H1N1)pdm09 viruses. It was found that not only the sialoside type but also the length of the LacNAc chain and presentation at the α1,3-antenna of N-glycans is critical for binding. Early A(H3N2) viruses bound to 2,6-sialosides at a single LacNAc moiety at the α1,3-antenna whereas later viruses required the sialoside to be presented at a tri-LacNAc moiety. Surprisingly, most of the A(H3N2) viruses that appeared after 2021 regained binding capacity to sialosides presented at a di-LacNAc moiety. As a result, these viruses agglutinate erythrocytes again, commonly employed for antigenic characterization of influenza viruses. Human A(H1N1)pdm09 viruses have similar receptor binding properties as recent A(H3N2) viruses. The data indicates that an asymmetric N-glycan having 2,6-sialoside at a di-LacNAc moiety is a commonly employed receptor by human influenza A viruses.