Interaction of Ganglioside G_Gtet1 and Its Derivatives with Choleragen

Abstract

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...

Cholera toxin induced redistribution of sialoglycolipid receptor at the lymphocyte membrane

“…The DANSyl gangliosidoi'de or ganglioside GGt,tl, both at concentrations of 0.1 pmol/ml of the above-mentioned buffer were mixed with aliquots of the solution of protein I. Mixtures at molar ratios of 0.1: 1, 1: 1, 2: 1, and 4: 1 for the glycolipid and protein I complexes were prepared. Precipitate formation was visually estimated 20 h later and the inhibition of immunoprecipitates was evaluated by immunodiffusion against antiserum to protein I, essentially as described earlier [9] .…”

“…The glycolipid ganglioside GG,J* which occurs localised in the plasma membrane of mammalian cells is known to interact strongly and specifically with cholera toxin [8,9]. Using fluoresceine-labelled cholera toxin, Craig and Cuatrecasas [lo] have demonstrated a redistribution and cap formation on the surface of lymphocytes which was temperaturedependent and sensitive for metabolic inhibitors.…”

Role of Cholera Toxin in <i>Vibrio cholerae</i> Infection in Humans - A Review

“…Experiments with purified NANase and 125I-labeled CTX showed that isolated rabbit small-intestine brush borders or cultured mouse neuroblastoma cells exposed to NANase bound 2 to 7 times more CTX than did untreated cells (Griffiths et al, 1986;Miller-Podraza et al, 1982). In ligated canine ileal loops injected with CTX, secretion increased fourfold in loops pretreated with NANase compared with untreated control loops (Staerk et al, 1974). Using isogenic V. cholerae strains specifically mutagenized in the gene encoding NANase (nanH) and flow cytometry, Galen et al, (Galen et al, 1992) showed that fluorescence due to binding of fluoresceinconjugated CTX to mouse fibroblasts exposed to NANase-positive culture filtrates increased five-to eightfold relative to binding to cells exposed to NANase-negative filtrates.…”

Choleragen (cholera toxin): a bacterial lectin.

“…Choleragen also bound to liposomes containing GM1; this binding resulted in membrane damage (22,23), and membrane damage occurred only with the B protomer (24). Because choleragen could bind specifically to the oligosaccharide portion of GM1 (17,(25)(26)(27) and each toxin molecule could bind more than one molecule of this oligosaccharide (26, 27), we hypothesized that choleragen might exhibit lectinlike behavior at a membrane surface. In this paper we show that choleragen can agglutinate cells and liposomes containing GM1, as would be expected of a lectin or lectinlike protein.…”

“…Previous studies have demonstrated that the receptor for choleragen is ganglioside GM1 (11)(12)(13)(14)(15)(16)(17)(18). Binding of choleragen to lymphocytes resulted in patching and capping of membrane GM1 (19)(20)(21).…”