ABSTRACT
Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.
Subject(s)
Blood Group Antigens , Caliciviridae Infections , Fucose , Glycoproteins , Histocompatibility Antigens , Jejunum , Organoids , Glycomics , Proteomics , Genotype , Phenotype , Glycoproteins/chemistry , Glycoproteins/genetics , Glycoproteins/metabolism , Fucose/metabolism , Glycosylation , Blood Group Antigens/chemistry , Blood Group Antigens/genetics , Blood Group Antigens/metabolism , Histocompatibility Antigens/chemistry , Histocompatibility Antigens/genetics , Histocompatibility Antigens/metabolism , Humans , Glycopeptides/chemistry , Caliciviridae Infections/blood , Caliciviridae Infections/immunology , Caliciviridae Infections/metabolism , Organoids/metabolism , Jejunum/metabolism , Jejunum/virologyABSTRACT
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.