Inhibition of the steroidogenic effects of cholera and heat-labile Escherichia coli enterotoxins by GM1 ganglioside: evidence for a similar receptor site for the two toxins

Donta, Sam T.; Viner, John P.

doi:10.1128/iai.11.5.982-985.1975

Cited by 69 publications

(23 citation statements)

References 16 publications

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“…GTl, Gfl1a,, GMI, and GM2 gangliosides (Supelco, Inc.) were chromatographed as controls. (1) GTI; (2) Gina; (3) ganglioside from horse erythrocyte membranes;…”

Section: Resultsmentioning

confidence: 99%

“…(4) ganglioside from human erythrocyte membranes; (5) GM,; (6) GM2-DISCUSSION GM1 ganglioside binds cholera toxin (choleragen) (5) and inhibits various biological activities of choleragen, such as lipolysis in isolated fat cells (1,16), skin permeability (8), and steroidogenesis in adrenal tumor cells (2). Diarrheogenic activity of choleragen is enhanced in rabbit small bowel by incubation of the bowel with GMI1 ganglioside (4).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Inactivation of the biological activities of the thermostable direct hemolysin of Vibrio parahaemolyticus by ganglioside Gt1

Takeda¹,

Takeda²,

Honda³

et al. 1976

Infect Immun

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Biological activities of the thermostable direct hemolysin produced by Vibrio parahaemolyticus, such as its hemolytic activity and lethal activity, were inhibited by neuraminidase-sensitive gangliosides, of which GT1 ganglioside was the most inhibitory. Neuraminidase-resistant gangliosides did not affect the activities of the hemolysin. Results showed that horse erythrocytes, which are resistant to the hemolysin, do not contain the neuraminidase-sensitive gangliosides GT, and GD1a. Therefore, we propose that neuraminidase-sensitive gangliosides, and especially GT1 ganglioside, may be the receptor sites on the membranes for the thermostable direct hemolysin of V. parahaemolyticus.In a previous paper, we reported that the hemolytic activity of the thermostable direct hemolysin of Vibrio parahaemolyticus was inhibited by a ganglioside mixture and that GM, ganglioside was not related to this inhibitory effect (14). To determine the tissue receptor for the thermostable direct hemolysin, we studied the inhibitory effects of various ganglioside components on biological activities of the thermostable direct hemolysin and found that neuraminidase-sensitive gangliosides, and especially GTI ganglioside, inhibited these biological activities. MATERIALS AND METHODSPurification of thermostable direct hemolysin. The thermostable direct hemolysin was isolated from culture filtrates of a Kanagawa phenomenonpositive strain, V. parahaemolyticus WP-1, and purified by successive column chromatographies on diethylaminoethyl-cellulose, hydroxylapatite, and Sephadex G-200, as described previously (7). The purified preparation of thermostable direct hemolysin contained a single protein with a molecular weight of about 42,000 (7).Assay of hemolytic activity. Hemolytic activity was assayed as described previously (7,13). The standard reaction mixture (2.5 ml) contained 10 mM tris(hydroxymethyl)aminomethane (Tris) -hydrochloride buffer (pH 7.2), 10 mM CaCl12, 0.9% NaCl, the thermostable direct hemolysin, and 1.25 ml of a 1% suspension of human erythrocytes (about 8.3 x 107 cells/ml). The reaction mixture was incubated at 37 C for 30 min and then centrifuged at 3,000 rpm for 5 min. The hemolytic activity was determined by measuring the absorbance of the resulting supernatant fluid at 540 nm.Assay of lethal toxicity. Lethal toxicity was assayed by injecting the thermostable direct hemolysin intravenously into 4-to 6-week-old mice (ddO strain) and measuring the survival time of the animals.Gangliosides. The ganglioside mixture used was type II from Sigma Chemical Co. Gangliosides GTI; GDIa, GM,, and GM2 (8). were obtained from Supelco Inc. GM, and GM2 were treated with V. cholerae neuraminidase (Calbiochem, B grade), as described below, before use. Treatment of gangliosides with neuraminidase.Ganglioside was incubated with V. cholerae neuraminidase (Calbiochem, B grade) in 0.2 ml of 0.01 M Tris-hydrochloride buffer (pH 7.2) for 48 h at 37 C. Then, the reaction mixture was dried in a rotary evaporator and the ganglioside was extracted into a mi...

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“…GTl, Gfl1a,, GMI, and GM2 gangliosides (Supelco, Inc.) were chromatographed as controls. (1) GTI; (2) Gina; (3) ganglioside from horse erythrocyte membranes;…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Inactivation of the biological activities of the thermostable direct hemolysin of Vibrio parahaemolyticus by ganglioside Gt1

Takeda¹,

Takeda²,

Honda³

et al. 1976

Infect Immun

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“…Donta has investigated both toxins in adrenal tumour cells with a similar conclusion. Both toxins had the same effect on the cells and these effects were prevented by preincubation of either toxin with ganglioside G, , (about the same amounts), but in neither case by preincubation of the cells with choleragenoid (Donta, 1974(Donta, , 1976; Donta & Viner, 1975), perhaps for the reasons described above.…”

Section: (I) Hormonesmentioning

confidence: 90%

Cholera Toxin

Heyningen¹

1977

Biological Reviews

101

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Summary 1. Death in several infectious diseases is caused by protein toxins secreted by invading bacteria. Cholera toxin is a simple protein secreted by Vibrio cholerae colonizing the gut; it is responsible for the massive diarrhoea that is cholera. 2. The primary action of cholera toxin is an activation of adenylate cyclase, an enzyme found on the inner membrane of eukaryotic cells that catalyses the conversion of ATP to cyclic AMP. Consequent increases in the intracellular concentration of cyclic AMP are responsible for other manifestations of cholera toxin including the diarrhoea. The toxin is active on almost all eukaryotic cells. 3. The toxin can be purified from culture filtrates of V. cholera. It has a molecular weight of 82000; and is composed of one subunit A (itself two polypeptide chains joined by a disulphide bond: AI (22000) and A2 (5000)) and five subunits B (11500). These can be separated in dissociating solvents such as detergents or 6 M guanidine hydrochloride. An amino‐acid sequence of subunit B has been published. The five B subunits (sometimes found by themselves in the filtrate and known as ‘choleragenoid’) are probably arranged in a ring with the subunit A in the middle joined to them non‐covalently by peptide A2. 4. The first action of cholera toxin on a cell is to bind to the membrane strongly and irreversibly. Several thousand molecules of toxin bind to each cell and the binding constants are of the order of 10‐10 M. The binding is rapid, but is followed by a lag phase of about an hour before the intracellular cyclic AMP concentration begins to increase. 5. Ganglioside GM1, a complex amphiphilic lipid found in cell membranes, binds tightly to the toxin which shows an enzyme‐like specificity for this particular ganglioside. Toxin that has already bound ganglioside can no longer bind to cells and is therefore inactive. This and other experiments using cells depleted of endogenous ganglioside suggest that ganglioside GM1 is the natural receptor of the toxin on the cell surface. The binding is followed by a lateral movement of the toxin‐ganglioside complex in the cell surface forming a ‘cap’ at one pole of the cell. 6. The binding of ganglioside by toxin is a function exclusively of subunit B; Subunit A does not bind and can be eluted with 8 M urea from an insolubilized toxin‐ganglioside complex. Subunit B is not by itself active, and so preincubation with B can protect cells or even whole gut from the action of toxin by occupying all the ganglioside binding sites. 7. Subunit A is responsible for activation of adenylate cyclase. Purified subunit A or just peptide AI is active by itself and this activity is not inhibited by ganglioside or by antisera to subunit B. In intact cells the activity is low and shows the characteristic lag phase but in lysed cells the subunit (or the whole toxin) is much more active and there is no lag phase. This suggests that the lag phase represents the time that subunit A takes to cross the cell membrane and get to its target. 8. Several cofactors are needed for t...

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“…While thus differing from choleragen, the E coli enterotoxin does have immunologic cross-reactivity [62,224,227,235,238,, probably with the B or ganglioside-binding subunit of choleragen [241,243] . There is some evidence that the E coli enterotoxin will interact with gangliosides [62,63,71,235,246]. Although enterotoxin seems to have a lower affinity than that of choleragen for G M~ in some systems [ 50, 531 ,t its action can be inhibited by the ganglioside [ 621 .…”

Section: Mechanistic Similarities Between Choleragen a N D Eschermentioning

confidence: 99%

Mechanism of action of choleragen

Vaughan

Moss

1978

J Supramol Struct

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Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside GM1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A1 fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A1 fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.

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Inhibition of the steroidogenic effects of cholera and heat-labile Escherichia coli enterotoxins by GM1 ganglioside: evidence for a similar receptor site for the two toxins

Cited by 69 publications

References 16 publications

Inactivation of the biological activities of the thermostable direct hemolysin of Vibrio parahaemolyticus by ganglioside Gt1

Inactivation of the biological activities of the thermostable direct hemolysin of Vibrio parahaemolyticus by ganglioside Gt1

Cholera Toxin

Mechanism of action of choleragen

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