Summary
Choleragen (cholera toxin) activates adenylate cyclase in HeLa cells, which contain less than 15,000 toxin receptors per cell, in a time-and concentration-dependent manner. Activation is blocked by the addition of the oligosaccharide chain of the ganglioside GM1, the receptor for the toxin. When the cells are preincubated with choleragen at 4°C and then incubated with oligosaccharide at 37°C, adenylate cyclase is activated less than 10%. When the preincubation phase is above 18°C, adenylate cyclase becomes activated and the amount of activation depends on the time of preincubation. This inhibitory effect of the oligosaccharide is also observed with human lymphocytes and rat glial C6 cells but not with Friend erythroleukemic and mouse neuroblastoma N18 cells. The latter two cell lines have large numbers of toxin receptors, whereas the former two cell lines have few receptors. When the number of toxin receptors in HeLa and C6 cells is increased by treating the cells with GM1, activation of adenylate cyclase by choleragen is no longer blocked by the oligosaccharide. The oligosaccharide has a corresponding effect on the displacement of bound125I-choleragen. When bound to cells at 4°C, most of the radiotoxin is displaced from HeLa, C6, and lymphocytes but not from Friend, N18, or HeLa cells pretreated with GM1. In untreated HeLa cells, dissociation of toxin-receptor complexes by the oligosaccharide depends on the time and temperature of complex formation; above 18°C, the toxin rapidly becomes stably bound to the cells. The inhibitory effect of GM1 oligosaccharide is reversible, as, once it is removed, the small amount of toxin that remains bound can activate adenylate cyclase. These results are consistent with a model in which choleragen, which is multivalent, must bind to several GM1 molecules on the cell surface in order to subsequently activate adenylate cyclase. Lateral mobility of toxin-receptor complexes may be required only to achieve multivalent binding in cells with few receptors.
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Abbreviations
- GM1 :
-
galactosyl (β1-3)N-acetylgalactosaminyl(β1-4)[N-acetylneuraminyl(α2-3)]galactosyl(β1-4)glucosyl(β1-1)ceramide
- HEPES:
-
N-2-hydroxyethylpiperazine-N′-ethylsulfonic acid
- MEM:
-
minimal essential medium
- cAMP:
-
adenosine 3′∶5′-monophosphate
References
Bennett, V., Craig, S., Hollenberg, M.D., O'Keefe, E., Sahyoun, N., Cuatrecasas, P. 1976. Structure and function of cholera toxin and hormone receptors.J. Supramol. Struct. 4:99
Boyum, A. 1968. Separation of leucocytes from blood and bone marrow.Scand. J. Clin. Lab. Invest 21:31 (Suppl. 97)
Cassel, D., Pfeuffer, T. 1978. Mechanism of cholera toxin action: Covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system.Proc. Nat. Acad. Sci. USA 75:2669
Craig, S., Cuatrecasas, P. 1975. Mobility of cholera toxin receptors on rat lymphocyte membranes.Proc. Nat. Acad. Sci. USA 72:3844
Cuatrecasas, P. 1973a. Interaction ofVibrio cholerae enterotoxin with cell membranes.Biochemistry 12:3547
Cuatrecasas, P. 1973b. Cholera toxin-fat cell interaction and the mechanism of activation of the lipolytic response.Biochemistry 12:3567
Dawson, G., Kemp, S.F., Stoolmiller, A.C., Dorfman, A. 1971. Biosynthesis of glycosphingolipids by mouse neuroblastoma (NB41A), rat glia (RGC-6) and human glia (CHB-4) in cell culture.Biochem. Biophys. Res. Commun. 44:687
Dawson, G., Stoolmiller, A.C. 1976. Comparison of the ganglioside composition of established mouse neuroblastoma cell strains grownin vivo and in tissue culture.J. Neurochem. 26:225
Duffard, R.O., Fishman, P.H., Bradley, R.M., Lauter, C.J., Brady, R.O., Trams, E.G. 1977. Ganglioside composition and biosynthesis in cultured cells derived from CNS.J. Neurochem. 28:1161
Fishman, P.H. 1980. Mechanism of action of cholera toxin: Studies on the lag period.J. Membrane Biol. 54:61
Fishman, P.H., Atikkan, E.E. 1979. Induction of cholera toxin receptors in cultured cells by butyric acid.J. Biol. Chem. 254:4342
Fishman, P.H., Brady, R.O. 1976. Biosynthesis and function of gangliosides.Science 194:906
Fishman, P.H., Moss J., Manganiello, V.C. 1977. Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts.Biochemistry 16:1871
Fishman, P.H., Moss, J., Osborne, J.C., Jr. 1978. Interaction of choleragen with the oligosaccharide of ganglioside GM1: Evidence for multiple oligosaccharide binding sites.Biochemistry 17:711
Gill, D.M. 1977. The mechanism of action of cholera toxin.Adv. Cyclic Nucleotide Res. 8:85
Gill, D.M., Meren, R. 1978. ADP-ribosylation of membrane proteins catalyzed by cholera toxin: Basis of the activation of adenylate cyclase.Proc. Nat. Acad. Sci. USA 75:3050
Hansson, H.-A., Holmgren, J., Svennerholm, L. 1977. Ultrastructural localization of cell membrane GM1 ganglioside by cholera toxin.Proc. Nat. Acad. Sci. USA 74:3782
Henneberry, R.C., Fishman, P.H., Freese, E. 1975. Morphological changes in cultured mammalian cells: Prevention by the calcium ionophore A23187.Cell 5:1
Holmgren, J., Lonnroth, I. 1976. Cholera toxin and the adenylate cyclase-activating signal.J. Infect. Dis. 133:S64
Holmgren, J., Lonnroth, I., Svennerholm, L. 1973. Tissue receptor for cholera exotoxin: postulated structure from studies with GM1 ganglioside and related glycolipids.Infect. Immun. 8:208
Johnson, G.L., Kaslow, H.R., Bourne, H.R. 1978. Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase.J. Biol. Chem. 253:7120
Joseph, K.C., Steiber, A., Gonatas, N.K. 1979. Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 gangkioside.J. Cell. Biol. 81:543
Kanfer, J.N., Carter, T.P., Katzen, H.M. 1976. Lipolytic action of cholera toxin on fat cells. Re-examination of the concept implicating GM1 ganglioside as the native membrane receptor.J. Biol. Chem. 251:7610
King, C.A., van Heyningen, W.E. 1973. Deactivation of cholera toxin by a sialidase-resistant monosialoganglioside.J. Infect. Dis. 127:639
Moss, J., Fishman, P.H., Manganiello, V.C, Vaughan, M., Brady, R.O. 1976a. Functional incorporation of ganglioside into intact cells: Induction of choleragen responsiveness.Proc. Nat. Acad. Sci. USA 73:1034
Moss, J., Fishman, P.H., Richards, R.L., Alving, C.R., Vaughan, M., Brady, R.O. 1976b. Choleragen-mediated release of trapped glucose from liposomes containing ganglioside GM1.Proc. Nat. Acad. Sci. USA 73:3480
Moss, J., Manganiello, V.C., Fishman, P.H. 1977. Enzymatic and chemical oxidation of gangliosides in cultured cells: Effects of choleragen.Biochemistry 16:1876
Moss, J., Richards, R.L., Alving, C.R., Fishman, P.H. 1977. Effect of the A and B protomers of choleragen on release of trapped glucose from liposomes containing or lacking ganglioside GM1.J. Biol. Chem. 252:797
Pacuszka, T., Moss, J., Fishman, P.H. 1978. A sensitive method for the detection of GM1 ganglioside in rat adipocyte preparations based on its interaction with choleragen.J. Biol. Chem. 253:5103
Reidler, J., Elridge, C., Schlessinger, Y., Elson, E., Wiegandt, H. 1978. Lateral mobility of a fluorescent ganglioside GM1 analog in cell membranes.J. Supramol. Struct. 124 (Suppl.):2
Revesz, T., Greaves, M. 1975. Ligand-induced redistribution of lymphocyte membrane ganglioside GM1.Nature (London) 257:103
Robert, J., Rebel, G., Mandel, P. 1977. Glycosphingolipids from cultured astroblasts.J. Lipid. Res. 18:517
Sattler, J., Schwartmann, G., Staerk, J., Ziegler, W., Wiegandt, H. 1977. Studies of the ligand binding to cholera toxin. II. The hydrophilic moiety of sialoglycolipids.Hoppe Seyler's Z. Physiol. Chem. 358:159
Schlessinger, J., Barak, L.S., Hammes, G.G., Yamada, K.M., Pastan, I., Webb, W.W., Elson, E.L. 1977. Mobility and distribution of a cell surface glycoprotein and its interaction with other membrane components.Proc. Nat. Acad. USA 74:2909
Sedlacek, H.H., Stark, J., Seiler, F.R., Ziegler, W., Wiegandt, H. 1976. Cholera toxin induced redistribution of sialoglycolipid receptor at the lymphocyte membrane.FEBS Lett. 61:272
Simmons, J.L., Fishman, P.H., Freese, E., Brady, R.O. 1975 Morphological alteractions and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells.J Cell Biol. 66:414
Tallman, J.F., Smith, C.C., Henneberry, R.C. 1977. Induction of functional beta-adrenergic receptors in HeLa cells.Proc. Nat. Acad. Sci. USA 74:873
Van Heyningen, S. 1977. Cholera toxin.Biol. Rev. 52:509
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Developmental and Metabolic Neurology Branch.
Laboratory of Molecular Biology
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Fishman, P.H., Atikkan, E.E. Mechanism of action of cholera toxin: Effect of receptor density and multivalent binding on activation of adenylate cyclase. J. Membrain Biol. 54, 51–60 (1980). https://doi.org/10.1007/BF01875376
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DOI: https://doi.org/10.1007/BF01875376