Electrophoresis
Electrophoresis
3 years ago

Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate-based cholera toxin inhibitors

Geert Jan Boons, Javier Sastre Torano, Aliaksei Pukin, Roland J. Pieters, Gerhardus J. Jong, Oier Aizpurua-Olaizola, Ou Fu
Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help to fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimized and applied. The method uses unlabeled cholera toxin B-subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1-oligosaccharides (GM1os). In an optimized method at pH 4, adsorption of the protein to the capillary walls was prevented by a polybrene-dextran sulfate-polybrene coating. Different concentrations of the ligands were added to the BGE. CTB binding was observed by a mobility shift that could be used for dissociation constant (Kd) determination. The Kd values of two GM1 derivatives differed by close to an order of magnitude (600 ± 20 nM and 90 ± 50 nM) which was in good agreement with the differences in their reported nanomolar IC50 values of an ELISA-type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen-induced secretory diarrhea.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/elps.201700207

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