Analysis of Cholera Toxin−Ganglioside Interactions by Flow Cytometry

Publisher Website
Google Scholar
Abstract
Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM1 in the cell membrane. We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes. A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants. In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions. From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM1 presented in bilayers of ∼8 × 107 M-1 while that for binding to soluble GM1-pentasaccharide was found to be ∼4 × 106 M-1. From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 ± 0.03) × 10-3 s-1, and the rate of association of a site on FITC-CTB in solution to a GM1 in the bilayer to be (2.8 ± 0.4) × 104 M-1 s-1. These values yield a lower estimate for the equilibrium binding constant of ∼1 × 107 M-1. We determined the equilibrium surface cross-linking constant [(1.1 ± 0.1) × 10-12 cm2] and from this value and the value for the rate constant for dissociation derived a value of ∼3.5 × 10-15 cm2 s-1 for the forward rate constant for cross-linking. We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits). Our results show that the whole toxin binds with ∼100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A2-subunit with the membrane surface. Interaction of cholera toxin B-subunit and whole cholera toxin with gangliosides other than GM1 revealed specific binding only to GD1b and asialo-GM1. These interactions, however, are marked by low avidity and require high receptor concentrations to be observed.