Mosquito-larvicidal BinA toxin displays affinity for glycoconjugates: Proposal for BinA mediated cytotoxicity.

Abstract

Lysinibacillus sphaericus parasporal BinAB toxin displays mosquito larvicidal activity against Culex and Anopheles, but several Aedes species are refractory. Recently reported crystal structure of BinAB revealed the presence of N-terminal lectin-like domain in BinA. Hemagglutination and hemolytic activities were not observed for BinA in the present studies. We attempted to characterize carbohydrate specificity of BinA by high-throughput approaches using extrinsic fluorescence and thermofluor shift assay. A total of 34 saccharides (mono-, di- and polysaccharides, and glycoproteins) were used for initial high-throughput screening. The promising glycans were identified based on significant change in the fluorescence intensity. Surface plasmon resonance revealed differential binding of BinA with glycoproteins (fetuin, asialofetuin and thyroglobulin) and affinity for simple sugars, l-fucose and l-arabinose. In the limited carbohydrate competition assay, arabinose, fucose and fetuin inhibited BinA toxicity towards Culex larvae. This study for the first time provides direct evidence that BinA is competent to bind diverse and structurally different glycosylated proteins. This activity may be linked to its intracellular cytotoxicity, as protein N-glycosylation is thought to be critical for development and survival of insect larvae. The glycoproteins do not form stable complexes with BinA, however, as observed in the pull-down assay using affinity immobilized BinA and in native-PAGE analysis. As BinA displays only mild affinity with receptor polypeptide, we hypothesize that toxin-receptor specificity of BinA in Culex may be mediated by dual interaction of BinA with glycan core of GPI anchor and receptor polypeptide. The study shall be useful for refining strategies for improving larvicidal activity and for broadening target specificity of BinAB toxin.