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

Abstract

Glycan binding properties of respiratory viruses have been difficult to probe due to a lack of biologically relevant glycans for binding studies. Here, a stop-and-go chemoenzymatic methodology is presented that gave access to a panel of 32 asymmetrical biantennary 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 GlcNH₂ at one of the antennae, which temporarily blocks extension by glycosyl transferases. The N-glycans were printed as a microarray that was probed for receptor binding specificities of the 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 are 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 again agglutinate erythrocytes, 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 indicate that an asymmetric N-glycan having 2,6-sialoside at a di-LacNAc moiety is a commonly employed receptor by human influenza A viruses.

Scheme 1. Stop-and-Go Strategy for the Synthesis of Asymmetrical Biantennary N-Glycans

a) Modification of symmetrical glycan 1, which was derived from egg yolk powder, with MAGT4 and MAGT5 using the unnatural donor UDP-GlcNTFA, followed by removal of the TFA moiety and chemical functionalization of the resulting amines gave 3, which is an appropriate starting material to prepare asymmetrical glycans such as 4. b) The biosynthesis of N-glycan involves trimming of high mannose N-glycans to 5 that can be modified by MGAT1 to provide 6 that after further mannoside trimming provides a substrate MGAT2. c) Compound 9, which is assessable from 1, was expected to be an appropriate substrate for MGAT1 and 2 and in combination with the natural and unnatural donor UDP-GlcNAc and UDP-GlcNTFA should give access to asymmetrical glycans 12 and 15. d) Asymmetrical glycans 12 and 15 were expected to be appropriate starting materials for the preparation of asymmetrical biantennary N-glycans having extended LacNAc moieties typical of the respiratory glycome.

Scheme 2. Chemoenzymatic Synthesis of Asymmetrical N-Glycans Having an Extended LacNAc Moiety at the MGAT1 or MGAT2 Antenna

a) Preparation of tri-mannoside 9 from a sialoglycopeptide isolated from egg yok powder. b) Preparation of asymmetrical N-glycans having an extended LacNAc moiety at the MGAT1 antenna. c) Preparation of asymmetrical N-glycans having an extended LacNAc moiety at the MGAT2 antenna.

Scheme 3. Selective Modification of Termini of the MGAT1 and MGAT2 Antennae of Glycans 20–22

Figure 1

Probing receptor binding specificities of A(H3N2) and A(H1N1)pdm09. a) Collection of glycans printed on succinimide reactive microarray slides. Glycan binding data of b) early A(H3N2) and A(H3N2) 3C.2 viruses; c) A (H3N2) 3C.3 viruses; and d) A(H1N1)pdm09 viruses. Whole viruses were exposed to a glycan microarray, and binding was visualized antistalk antibodies. Bars represent the average relative fluorescence units (RFU) of four replicates ± SD.