Interaction of cholera toxin with ganglioside GM1 receptors in supported lipid monolayers

Abstract

Lipid monolayers formed at the air-water interface containing the ganglioside GMi in egg yolk phosphatidylcholine have been transferred according to the to the Langmuir-Blodgett technique to (a) glass cover slips coated with octadecyl- or hexadecyltrichlorosilane and (b) carbon-coated electron microscope grids. Monolayer transfer has been demonstrated with fluorescence microscopy, by the transfer of a fluorescent phospholipid analogue, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine or Lucifer yellow labeled GM1 (LY-GM1), incorporated into the lipid monolayer. Incubation of supported monolayers with solutions of fluorescein-labeled cholera toxin (FITC cholera toxin) resulted in specific binding of the toxin to monolayers containing GM1, as revealed by fluorescence microscopy. Lateral diffusion coefficients were measured for both the receptor (LY-GM1) [(3.9 .+-. 2.1) .times. 10-8 cm2/s] and the receptor-ligand complex (GM1-FITC cholera toxin) [(8.9 .+-. 3.2) .times. 10-9cm2/s] according to the technique of fluorescence recovery after photobleaching. In separate studies, GM1-containing monolayers transferred to electron microscope grids were incubated with solutions containing unlabeled cholera toxin, followed by negative staining with uranyl acetate. Electron microscopy revealed patches of stained cholera toxin molecuels (diamter .apprx. 70 .ANG.) in crystalline, two-dimensional hexagonal arrays. Optical diffraction and image reconstruction showed the arrangement of the cholera toxin molecules in a planar hexagonal cell, a = 81 .ANG.. These initial reconstructions give structural information to a resolution of .apprx. 30 .ANG. and indicate a doughnut-shaped molecule with a central aqueous channel.