Expression of influenza A virus glycan receptor candidates in mallard, chicken, and tufted duck

Abstract

Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Receptor incompatibility due to differently expressed glycan structures between species has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative sialic acid-containing glycan receptors for IAV in mallard, chicken and tufted duck; three bird species with different roles in the zoonotic ecology of IAV. The methodology used pinpoints specific glycan structures to specific glycosylation sites of identified glycoproteins and was also used to successfully discriminate α2-3- from α2-6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides in tandem MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex N-glycans including α2-3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both α2-3- and α2-6-linked Neu5Ac. We also found the recently identified putative IAV receptor structures, Man-6P N-glycopeptides, in all tissues of the three bird species. Furthermore, we found many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures, previously anticipated to be mammalian specific, in all three bird species may have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV.

Keywords: birds; glycoproteomics; influenza virus; interspecies transmission; sialic acid receptor.

Fig. 1

Glycoproteomic analysis of sialylated and fucosylated N-glycopeptides found in mallard. A) HCD-MS² of a disialylated complex biantennary N-glycopeptide including a bisecting GlcNAc and a Lewis x fucose (boxed structure) from epithelial cell adhesion molecule (A0A8B9TTX1_ANAPL) with the sequence R.LNVSIDNEVVQLEK.A. The asterisk marks the presence of an ion corresponding to [peptide + GlcNAc + Fuc] possibly due to fucose migration or to co-elution of two glycoforms B) MS³ of the m/z 657 ion provides identification of the Neu5Acα2-3Gal linkages on both antennas (Pett et al. 2018). C) HCD-MS² spectrum of a disialylated complex biantennary N-glycopeptide (boxed structure) from signal recognition particle receptor subunit beta (R0JEC8_ANAPL) and D) the m/z 100–400 region of the expanded MS² spectrum shows the presence of several oxonium ions used to identify the Neu5Acα2-6-linkage of the sialic acids. Similar glycan structures were also detected in chickens and tufted ducks (Supplementary Table S1). Glycan symbols are according to the SNFG format (Varki et al. 2015).

Fig. 2

Glycoproteomic analysis of sialylated core 1 O-glycopeptides from mallard samples. A) HCD-MS² of a Neu5Acα2-6 sialylated core 1 O-glycopeptide from fibrinogen beta chain (A0A8B9SHX9_ANAPL) with the R.ETAPTLRPVAPPISGTGYQPR.P amino acid sequence. B) HCD-MS² of two Neu5Acα2-3 and Neu5Acα2-6 disialylated core 1 O-glycans of a glycopeptide from dystroglycan 1 (A0A493T5Q5_ANAPL) with the R.VISEATPTLAAGKDPEK.S sequence. Glycan symbols are according to the SNFG format (Varki et al. 2015).

Fig. 3

Identification of Man-6P N-glycopeptides. The example is taken from Man-6P glycopeptides, originating from chicken aminopeptidase (F1NB92_CHICK) with the R.ENHTVVSSNDR.A sequence, and the MS2 spectra show A) the (phosphate)₁(Hex)₆(HexNAc)₂ glycoform and B) the (phosphate)₁(Hex)₆(HexNAc)₃ glycoform. Glycan symbols are according to the SNFG format (Varki et al. 2015).

Fig. 4

Overview of Neu5Ac modified N-glycopeptides from glycoproteins in different tissues in mallard, tufted duck and chicken. The figure displays the number and identity of all Neu5Ac isomers located on membrane or secreted glycoproteins for all species and tissue types. Orange color indicates Neu5Ac expressed on secreted glycoproteins, gray indicates expression on membrane bound glycoproteins, purple indicates expression on glycoproteins bound to the golgi apparatus membrane, yellow—glycoproteins bound to the endosome membrane, green—glycoproteinsfound in the endoplasmatic reticulum and secreted and blue indicates proteins found in the cytoplasm. The numbers on the X-axis denote the linkage between the Neu5Ac and the penultimate glycan on single or multi-antennary glycopeptides.

Fig. 5

Glycan structure and protein information on avian sialylated N-glycopeptides. The heat maps display glycan structure including linkage conformation on terminal Sia and protein information for all N-linked glycopeptides detected in A) mallards, B) tufted ducks and C) chickens. The top X-axis indicates the linkage conformation on the Sia linkage and the bottom X-axis indicates the glycan structure including the linkage position. The right Y-axis indicates the name of the glycoprotein and the left Y-axis indicates whether the glycoprotein is membrane bound (orange), secreted (blue), located to the Golgi apparatus membrane (yellow), endosome membrane (brown), endoplasmic reticulum and secreted (green), cytoplasm (dark blue) or if glycoprotein location was not specified (white). Heatmaps were generated using the pheatmap v. 1.0.12 package (Kolde 2019) and the ComplexHeatmap v. 2.12.1 (Gu et al. 2016) in R.

Fig. 6

Shared and unique avian sialylated N-glycans. The UpSet plot indicates the intersection of sialylated N-glycans detected in trachea, lung, ileum and colon of mallard, tufted duck and chicken. Vertical bars visualize the number of shared and unique sialylated N-glycans to each species and tissue type. Horizontal bars represent the total number of sialylated N-glycans found in each tissue and species. The nature of a given intersection is indicated by the dots below the bar plot, with mallard shown in green, tufted duck in black and chicken in purple. For instance, the eight sialylated N-glycans in the first column were only detected in trachea of tufted duck, while the single sialylated N-glycan in the last column was detected in all tissues of all species. For information on which specific sialylated N-glycans that were shared or unique between species and tissue types, see Supplementary Table S6.