No abstract

Two kinds of tetravalent double-headed sialo-glycosides with short/long spacers between the Neu5Acα2,6Galβ1,4GlcNAc unit and ethylene glycol tetraacetic acid (EGTA) scaffold were found to be capable of binding to virus-like particles of Merkel cell polyomavirus (MCPyV-LP). The binding process and time course of interaction between the tetravalent ligand and MCPyV-LP were assessed by dynamic light scattering (DLS). On the addition of increasing concentrations of ligand to MCPyV-LP, larger cross-linked aggregates formed until a maximum size was reached. The binding was stronger for the tetravalent ligand with a short spacer than for that with a long spacer. The binding of the former ligand to the virus was observed to proceed in two stages during agglutination. The first step was the spontaneous formation of small aggregates comprising the cross-linked ligand–virus complex. In the second step, the aggregates grew successively larger by cooperative binding among the initially produced small aggregates. In transmission electron microscopy, the resulting complex was observed to form aggregates in which the ligands were closely packed with the virus particles. The cross-linked interaction was further confirmed by a simple membrane filtration assay in which the virus-like particles were retained on the membrane when complexed with a ligand. The assay also showed the effective capture of particles of pathogenic, infectious human polyomavirus JCPyV when complexed with a ligand, suggesting its possible application as a method for trapping viruses by filtration under conditions of virus aggregation. Collectively, these results show that the tetravalent glycocluster serves as a ligand not only for agglutinating MCPyV-LP but also for trapping the pathogenic virus.

An in situ measurement method of monitoring protein aggregation in precrystalline solutions is presented. The method is based on a small-angle forward static light scattering (F-SLS) technique. This technique uses an accurate optical arrangement of a combination of a collimating lens and a CCD to obtain an F-SLS pattern from an aggregate-containing protein solution in one shot. The real-time observation of a crystallizing lysozyme captured the formation of fractal aggregates in the initial formation stage.

We present real-time observations of a structurally variable process for cross-linking agglutination between multivalent lectins and glycoclusters using a small-angle forward static light scattering (F-SLS) technique. In this study, a cross-linking agglutination reaction was carried out using a tetravalent Neu5Acα2,6LacNAc-glycocluster and Sambucus sieboldiana agglutinin (SSA). The scattering intensity of time-resolved F-SLS increased with formation of the Neu5Acα2,6LacNAc-glycocluster–SSA cross-linked complex. Using this approach, fine sequential cross-linking agglutination between glycoclusters and lectins was observed in real-time. The rate of increase in intensity of time-resolved F-SLS increased with the concentration of sialo-glycoclusters and SSA. Structural analysis based on the fractal dimension using time-resolved F-SLS patterns revealed that the density of the aggregates changed with progression of the cross-linking reaction until equilibrium was reached. This is the first report to evaluate the cross-linking agglutination reaction between glycoclusters and lectins and analysis of the subsequent structure of the obtained aggregates using time-resolved measurements of F-SLS.