It was confirmed that ganglioside GGtetl, i.e. GalPl + 3GalNAcPl + 4[NeuNAca2 + 3lGalB1 + 4Glc-ceramide, specifically interacts with choleragen (cholera-exoenterotoxin) as shown by precipitate formation and inhibition of the toxicity. The isolated carbohydrate moiety of ganglioside GGtetl, i.e. GGtetl minus ceramide, neither precipitated choleragen nor interferred with the reaction between the toxin and its antibody. However, polyacrylamide gel electrophoresis revealed a specific interaction between the sialo-oligosaccharide and choleragen. Identical results were obtained with a sialo-sugar derivative prepared from the carbohydrate moiety of ganglioside GGtetl by reductive amination followed by N-acetylation of its glucose unit at the reducing end.Variations in the lipophilic moiety of ganglioside GGtetl yielded compounds (gangliosidoides) which completely retained the specific ability to precipitate choleragen, but did not interfere with its toxicity. The choleragenicity of gangliosidoide . choleragen complexes could still be abolished by ganglioside GGtetl . Polyacrylamide gel electrophoresis of ganglioside . choleragen as well as gangliosidoide . choleragen complexes, in the presence of sodium dodecylsulphate, showed only the protein subunits (approx. 8000) molecular weight of the toxin to be irreversibly aggregated. When incubating canine ileal loops with gangliOside-GGtetl . choleragen complex or with ganglioside GGtetl in presence of sodium deoxycholate, followed by washing of the loops with buffer after a certain period of time, an inhibition of the secretory response to a subsequent challenge with choleragen was observed. Similar treatment of ileal loops with Vibrio cholerae neuraminidase dissolved in buffer without sodium deoxycholate followed by washing of the loops, caused an enhancement as well as an inhibition of the secretory response to the subsequent challenge with choleragen depending upon the time of incubation with neuraminidase.Monosialoganglioside GGtetl neutralizes the various biological activities of choleragen (cholera-exoenterotoxin), as has been demonstrated in vivo and with models in vitro [l -31. Furthermore, choleragen is specifically precipitated by the ganglioside GGtetl ~~4 1 .Abbreviations. Ganglioside G,,,,I = GalPl+ 3GalNAcPI + 4WeuNAca2 + 3]Gal/31+ 4Glc-ceramide where NeuNAc = N-acetylneuraminic acid and other symbols are standard for carbohydrates. Other gangliosides are designated according to Wiegandt (1973) This paper describes biochemical, biological and immunological properties of complexes formed by choleragen and ganglioside GGtetl. The effect of variations in the sialo-oligosaccharide, as well as the lipid moiety of gangliosides, for the interaction with the toxin are reported. Some animal experiments will be described, which should contribute to the elucidation of the role of ganglioside in the pathogenesis of cholera. MATERIALS AND METHODS Materialsprepared as described by Kuhn and Wiegandt [5].Gangliosides GGtetl, GGtet2a and GGtet2b were Eur. J. Biochem. 48 (197...

The two choleragen protein constituents were isolated and characterized. Protein I has a molecular weight of approximately 54 000. It consists of subunits of approximate molecular weight 10 000. Protein I1 with molecular weight of approximately 32000 is cleaved by 2-mercaptoethanol into two fragments, protein 11, (N-terminal Asx, M , = 25000) and protein 11, (N-terminal Ser, M , = 7000).Proteins 111 and 112 could be recombined by oxidation to yield protein 11. Upon treatment of choleragen with 2-mercaptoethanol protein 11, precipitates quantitatively. The remaining protein consisting of proteins I and II,, was quantitatively precipitated by ganglioside GGtetl. Of the separated choleragen subunit proteins, only protein I and not protein I1 complexed specifically with ganglioside GGtetl. The isolated proteins I and I1 were considerably less toxic in the skin test but almost full toxicity was recovered after mixing the two proteins I and 11. Antisera against protein I and protein 11 revealed no immuno-cross reactivity between the two proteins. Both antisera inhibited the biological effects of choleragen in the skin and ileal loop tests. A molecular model for the constitution of choleragen is proposed.The pathogenic effects of cholera bacteria in vivo are caused by the action of a secreted toxin upon the mucosal cells. This cholera exo-entero-toxin, choleragen, induces major irreversible membrane permeability changes including the massive outflow of fluid into the intestine. Choleragen also acts on numerous other cell types by way of stimulation of adenylate cyclase (for review see [3]).The work of van Heyningen and collaborators first identified ganglioside to be the cell receptor for choleragen [l 1. These authors discovered that choleragen complexes very specifically with the major monosialo-ganglioside of brain, ganglioside GGtetl .In an earlier investigation it was shown that both the lipophilic and the hydrophilic moiety of the glycolipid contribute to the strength of the binding [Z]. It was also reported that the precipitation of choleragen with ganglioside GGtetl leads to the formation of an apparently high-molecular-weight aggregate of one of the two protein subunits of the toxin. Theother protein subunit, however, showed a less tight binding to the toxin-ganglioside complex and could be readily dissociated by detergents.The ganglioside complexing ability does not appear to be directly related to the toxic activity of the choleragen in vivo. This is shown by the fact that ganglioside is also bound to choleragenoid, which itself is nontoxic and consists only of one type protein of the original choleragen. Furthermore, gangliosidoides i.e. analogues of the ganglioside GGtetl, display comparable binding properties to choleragen, but in contrast to the natural ganglioside still do not inhibit its toxicity in vivo.In order to study the role of the subunits of choleragen in the binding to ganglioside GGtJ as well as choleragenicity, the two proteins were separated and isolated in pure form.The two choleragen prot...

The binding between cholera toxin or its B-protein subunit and various ganglioside-related oligosaccharides was studied by equilibrium displacement dialysis. At low concentrations of ligand, the binding of monosialo-gangliotetraitol exceeded that of the parent ganglioside II 3 NeuAcGgOse 4 -Cer, the possible cell surface receptor for the toxin. The terminal galactose residue and an intact carboxyl group of the sialic acid moiety of monosialo-gangliotetraose were found to be necessary for strong binding to the toxin. Untersuchungen über die Bindung von Liganden an Choleratoxin, II: Der hydrophile Anteil von SialoglykolipidenZusammenfassung: Die Bindung von verschiedenen Gangliosidzuckern an Choleratoxin bzw. dessen B-Protein-Untereinheit wurde mittels Verteilungsdialyse untersucht. Bei geringen Ligandkonzentrationen bindet Monosialogangliotetrait an das Toxin stärker als das entsprechende Gangliosid II 3 NeuAc-GgOse 4 -Cer, das mögli-cherweise der Zelloberflächen-Rezeptor des Toxins ist. Der terminale Galaktoserest sowie eine intakte Carboxylgruppe der Sialinsäure der Monosialogangliotetraose ist Voraussetzung für eine starke Bindung an das Choleratoxin.

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