Determination of the Carbohydrate-Binding Specificity of Lectin-Like Domains in Vibrio Cholerae Cytolysin

Abstract

Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin that contains two putative sugar-binding domains: the β-trefoil domain and the β-prism domain. Previous hemagglutinating studies suggest that VCC binds glycoproteins with terminal β1-galactosyl moieties [1]; however, crystallization of monomeric VCC with β-octyl glucoside suggests the β-prism domain may have an affinity for glucosides. We aim to characterize the carbohydrate-binding specificity of the β-trefoil and β-prism domains. The crystal structure of VCC indicates that Asp617 within the β-prism domain forms hydrogen bonds with the bound glucoside moeity. Hemolysis assays demonstrated that the removal of the β-prism domain causes a 1000-fold decrease in lytic activity toward rabbit erythrocytes, while conversion of Asp617 to alanine and lysine decreases the hemolytic activity100-fold, indicating that the sugar-binding activity of VCC may play a role in cell lysis. The β-prism domain was expressed as a separate construct and isothermal titration calorimetry (ITC) was used to determine the binding affinity for the interaction of the β-prism domain with monosaccharides. The binding of the isolated β-prism domain reflected the binding affinities observed using ITC for full length VCC. The β-prism domain showed an affinity for mannose, but not galactose, indicating that recognition of terminal β1-galactosyl moieties does not occur at the β-prism domain. The carbohydrate-binding activity of the β-prism domain may explain its role in cell lysis.[1] Saha, N; Banerjee, K.K. Carbohydrate-mediated Regulation of Interaction of Vibrio cholerae Hemolysin with Erythrocyte and Phospholipid Vesicle, J. Biol. Chem. (1997) 272, 162-167.