Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.
Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].
D000818:Animals; D001894:Borohydrides; D002460:Cell Line; D002772:Cholera Toxin / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005677:G(M1) Ganglioside / Q000378:metabolism; D005692:Galactose Oxidase; D005732:Gangliosides / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D007163:Immunosorbent Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*
D001894:Borohydrides; D005732:Gangliosides; D006023:Glycoproteins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C023552:choleragen receptor; C017336:galactoproteins; D005677:G(M1) Ganglioside; C025364:sodium borohydride; D002772:Cholera Toxin; D005692:Galactose Oxidase