Quantifying the binding stoichiometry and affinity of histo-blood group antigen oligosaccharides for human noroviruses.

Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis. Many HuNoVs recognize histo-blood group antigens (HBGAs) as cellular receptors or attachment factors for infection. It was recently proposed that HuNoV recognition of HBGAs involves a cooperative, multistep binding mechanism that exploits both known and previously unknown glycan binding sites. In this study, binding measurements, implemented using electrospray ionization mass spectrometry (ESI-MS) were performed on homodimers of the protruding domain (P dimers) of the capsid protein of three HuNoV strains [Saga (GII.4), Vietnam 026 (GII.10) and VA387 (GII.4)] with the ethyl glycoside of the B trisaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-OC2H5) and free B type 1 tetrasaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-(1→3)-d-GlcNAc) in an effort to confirm the existence of new HBGA binding sites. After correcting the mass spectra for nonspecific interactions that form in ESI droplets as they evaporate to dryness, all three P dimers were found to bind a maximum of two B trisaccharides at the highest concentrations investigated. The apparent affinities measured for stepwise binding of B trisaccharide suggest positive cooperativity. Similar results were obtained for B type 1 tetrasaccharide binding to Saga P dimer. Based on these results, it is proposed that HuNoV P dimers possess only two HBGA binding sites. It is also shown that nonspecific binding corrections applied to mass spectra acquired using energetic ion source conditions that promote in-source dissociation can lead to apparent HuNoV-HBGA oligosaccharide binding stoichiometries and affinities that are artificially high. Finally, evidence that high concentrations of oligosaccharide can induce conformational changes in HuNoV P dimers is presented.