Kinetic analysis of binding between shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surfa ce and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonanc e analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrysta lline hydrophobic layer was used to immobilize an amphipathic lipid layer t hat mimics the plasma membrane surface. Dose responsiveness was observed wi th both isoforms when either the toxin concentration or the Gb3Cer concentr ation was increased. In addition, this assay was shown to be specific, beca use neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the " bivalent analyte" model was found to best fit the interaction between Stxs and Gb3Cer. This shows that,the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, althoug h kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more sl owly than Stx1 but, once bound, is difficult to dissociate. The data descri bed herein clearly demonstrate differences between the binding properties o f Stx1 and Stx2 and may facilitate understanding of the differences in clin ical manifestations caused by these toxins.