Studies of the Ligand Binding to Cholera Toxin, I. The Lipophilic Moiety of Sialoglycolipids

1981

Mol Cell Biochem

SummaryCholeragen exerts its effects on cells through the activation of adenylate cyclase. The initial event appears to be the binding of the B subunit of the toxin to ganglioside GM1 on the cell surface, following which there is a delay prior to activation of adenylate cyclase. Patching and capping of the toxin on the cell surface, perhaps involved in the internalization of the enzymatically active subunit, may be occuring during this time. The activation of adenylate cyclase, which is catalyzed by the A! peptide of choleragen, does not require the B subunit or ganglioside GMI. The A 1 peptide catalyzes the transfer of ADP-ribose from NAD to an amino acid, probably arginine, in a 42 000 dalton membrane protein. This protein appears to be the GTP-binding component (or G/F factor) of the adenylate cyclase system and is crucial to the regulation of cyclase activity by hormones such as epinephrine. ADP-ribosylation of the G/F factor is enhanced by GTP and, in some systems, by a cytosolic factor. GTP is also required for stabilization and optimal catalytic function of the choleragen-activated cyclase. Calmodulin, a calcium-binding protein, is necessary for expression of catalytic activity of the toxin-activated adenylate cyclase in brain and other tissues.The ADP-ribosyltransferase activity required for activation of the cyclase is an intrinsic property of the A 1 peptide of choleragen which is expressed only after the peptide is released from the holotoxin by reduction of a single disulfide bond. In the absence of cellular components, choleragen catalyzes the ADP-ribosylation of small guanidino compounds such as arginine as well as peptides and proteins that contain arginine. It is assumed, therefore, that the site of ADP-ribosylation in the natural acceptor protein is an arginine or similar amino acid. When guanidino compounds are not present as ADP-ribose acceptors, choleragen hydrolyzes NAD to ADP-ribose and nicotinamide at a considerably slower rate.E. coli heat-labile enterotoxin (LT) is very similar to choleragen in structure and function. It consists of two types of subunits, A and B, with sizes comparable to those of the A and B subunits of choleragen. Binding of LT to the cell surface is enhanced by prior incorporation of GM1 but not other gangliosides; the oligosaccharide of GMI specifically interacts with LT and its B subunit. The A subunit of LT exhibits ADP-ribosyltransferase activity following activation by thiol to release the A 1 peptide. The A subunit of LT can be isolated in an 'unnicked' form and thus requires, in addition to reduction by a thiol, proteolytic cleavage to generate the active A 1 peptide. Like choleragen, LT uses guanidino compounds as model ADP-ribose acceptors and catalyzes the ADP-ribosylation of a 42 000 dalton protein in cell membrane preparations.ADP-ribosyltransferases that use arginine as ADP-ribose acceptors are not restricted to bacterial systems; such an enzyme has been purified to apparent homogeneity (>500 000-fold) from turkey erythrocytes. Based on a subunit molecular...

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Section: Specificity Of Interaction Of Choleragen and Ganglioside Gm1mentioning

confidence: 99%

Section: Interaction Of Choleragen and B Subunits With G M1 And G mentioning

confidence: 99%

Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

1981

Mol Cell Biochem

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“…In several instances, it has been shown that GMl is much more effective than other gangliosides which differ only in the structure of the oligosaccharide moiety [47,51,52,62,83]. Indeed, although it has a lower affinity for choleragen than does the ganglioside, the oligosaccharide of GMl will bind to the toxin and inhibit its precipitation by G M~ [59,60,81,90].Treatment of cells with neuraminadase, which converts trisialo-and disialogangliosides to G M~, produced increases in GM1 content [83], choleragen responsiveness [57,66] and choleragen binding [52,54,80,83]. It was further found that prior incorporation of GMl increased the capacity of cells or membrane preparations to bind choleragen [48,67, 7 0 , 7 6 , 8 0 , 8 3 ] and enhanced the responsiveness of cells to the toxin [48,65,67,68,74,75, 891 .…”

mentioning

confidence: 99%

Mechanism of action of choleragen

1978

J Supramol Struct

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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Adp‐Ribosylation of Guanyl Nucleotide‐Binding Regulatory Proteins by Bacterial Toxins

Advances in Enzymology - And Related Areas of Molecular Biology

1988

140