Abstract
Zwitterionic modifications of glycans, such as phosphorylcholine and phosphoethanolamine, are known from a range of prokaryotic and eukaryotic species and are recognized by mammalian antibodies and pentraxins; however, defined saccharide ligands modified with these zwitterionic moieties for high-throughput studies are lacking. In this study, we prepared and tested example mono- and disaccharides 6-substituted with either phosphorylcholine or phosphoethanolamine as bovine serum albumin neoglycoconjugates or printed in a microarray format for subsequent assessment of their binding to lectins, pentraxins, and antibodies. C-Reactive protein and anti-phosphorylcholine antibodies bound specifically to ligands with phosphorylcholine, but recognition by concanavalin A was abolished or decreased as compared with that to the corresponding nonzwitterionic compounds. Furthermore, in array format, the phosphorylcholine-modified ligands were recognized by IgG and IgM in sera of either non-infected or nematode-infected dogs and pigs. Thereby, these new compounds are defined ligands which allow the assessment of glycan-bound phosphorylcholine as a target of both the innate and adaptive immune systems in mammals.
Highlights
Zwitterionic modifications of glycans, such as phosphorylcholine and phosphoethanolamine, are known from a range of prokaryotic and eukaryotic species and are recognized by mammalian antibodies and pentraxins; defined saccharide ligands modified with these zwitterionic moieties for highthroughput studies are lacking
PC is present on various fungal glycoconjugates such as N-glycans of Penicillium nordicum, the peptidophosphogalactomannan of Penicillium charlesii, and glycolipids of Aspergillus f umigatus or Acremonium sp.[10−13] PC is a modification of annelid glycolipids[14] but of the lipopolysaccharides of bacteria such as Haemophilus inf luenzae and Pasteurella multocida, pilin glycans of Neisseria meningitidis, or the teichoic acid of Streptococcus pneumoniae.[15−18] In Neisseria and Haemophilus species, on−off switching of PC expression occurs depending on whether the bacteria reside in the upper respiratory tract or in systemic sites.[19,20]
We describe the synthesis of the respective 6-O-PC- and 6-O-PE-substituted mannoside ligands as well as the disaccharide ligand 6-O-PC-βGlcpNAc(1→2)-α-Manp, which correspond to part of the structures identified, e.g., in N-glycans of various fungi or protists (PE or PC modifications of mannose residues) or of free-living and parasitic nematodes [PC modification of GlcNAcβ1,2Man motifs]; these conjugates were either printed in a glycan microarray format or converted into BSA neoglycoconjugates, prior to subsequent testing with antibodies and pentraxins
Summary
Zwitterionic modifications of glycans, such as phosphorylcholine and phosphoethanolamine, are known from a range of prokaryotic and eukaryotic species and are recognized by mammalian antibodies and pentraxins; defined saccharide ligands modified with these zwitterionic moieties for highthroughput studies are lacking. In array format, the phosphorylcholine-modified ligands were recognized by IgG and IgM in sera of either non-infected or nematode-infected dogs and pigs Thereby, these new compounds are defined ligands which allow the assessment of glycan-bound phosphorylcholine as a target of both the innate and adaptive immune systems in mammals. Thereby, in comparison to conjugates such as PC directly conjugated to BSA,[33] these novel reagents better mimic the interaction of components of the innate and adaptive immune systems to glycans carrying zwitterionic modifications than those based on the PC or PE alone These zwitterionic glycosides were tested for binding to either lectins, pentraxins, monoclonal antibodies, or antibodies in sera of infected animals
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