Kinetic Analysis of Binding between Shiga Toxin and Receptor Glycolipid Gb3Cer by Surface Plasmon Resonance

Abstract

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface 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 resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because 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, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins.

Hemolytic Uremic Syndrome

“…Stx-1 and Stx-2 bind to different epitopes on the Gb3 molecule, and they also differ in binding affinity and kinetics (62). Surface plasmon resonance analysis showed that Stx-1 easily binds to and detaches from Gb3, in contrast to Stx-2, which binds slowly but also dissociates very slowly, thus staying on the cells long enough to be incorporated (62). The latter could explain why Stx-2 is 1000-fold more toxic than Stx-1 on human endothelial cells in vitro (63).…”

Structural and molecular basis for hyperspecificity of RNA aptamer to human immunoglobulin G

“…The affinity of the selected aptamers to hIgG1-Fc was examined by surface plasmon resonance (SPR) using BIAcore 2000. Divalent kinetic parameters for each aptamer were estimated by curve fitting of the sensorgrams (Table 2; Nakajima et al 2001), reflecting the nuclear magnetic resonance (NMR) observation that two aptamers bind to one Fc (homodimer) unit (described below).…”

Determination of Glycolipid–Protein Interaction Specificity

“…SPR is performed by immobilizing glycolipids on a commercial SPR sensor chip, followed by exposure to unlabeled soluble GBPs (Kuziemko et al, 1996;MacKenzieet al, 1997;MacKenzie and Hirama, 2000;Nakajima et al, 2001). Binding is performed in a biosensor designed to detect mass bound to the sensor chip surface.…”