Unmasking of actual and potential receptor sites for cholera toxin in intestinal mucosal homogenates.

Endongenous and exogenous sialidases appear to unmask sialidase-stable and sialidase-labile gangliosides in intestinal mucosal homogenates by attacking glycoproteins. Exogenous (but not endogenous) sialidase then converts sialidase-labile gangliosides into the cholera toxin-binding, sialidase-stable ganglioside GM1 (galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide). Since Vibrio cholerae produces sialidase, these observations may be relevant to the course of cholera.

Aspects of the interaction of Vibrio cholerae toxin with the pigeon red cell membrane.

The ganglioside galactosy-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide (GM1) is readily accumulated in pigeon red cell membranes soaked with [3H]GM1 (1-100 mug/ml) at 37 C for 30 min. This treatment enhances the activation of adenyl cyclase by the toxin of Vibrio cholerae. An attempt was made at correlation of the amount of incorporated GM1 with the increased binding of toxin and activation of adenyl cyclase. Cells with less than 2 mug of incorporated GM1 per 4 X 10(9) cells bind 5-10 mug more toxin than do untreated cells, which bind 0.25 mug per 4 X 10(9) cells. Cells with more than 2 mug of GM1 bound (per 4 X 10(9) CELls) which has been incorporated from micellar solutions of GM1 (greater than 20 mug/ml), do not bind any more extra toxin. In untreated cells, 0.1 mug of toxin is involved in the activation of adenyl cyclase. In GM1-treated cells 0.25-0.5 mug of toxin is involved, although at least 5 mug of toxin is bound. It is concluded that 90% of the extra toxin-binding sites on the GM1-treated cell are nonproductive.

The nature and action of cholera toxin.

Cholera diarrhoea is due to the action of a toxin that acts on all animal cells by stimulating the enzyme adenylate cyclase, which catalyses the production oc cyclic AMP from ATP. In intestinal brush border cells raised cyclic AMP levels result in increased secretion of chloride ions, leading to fluid accumulation in the gut. Escherichia coli produces a similar toxin. The receptor for cholera toxin on the cell membrane appears to be a complex containing the ganglioside GGnSLC (or GM1). Cholera toxin is a protein composed of two different kinds of subunits linked non-covalently. Each toxin molecule has one subunit A and four or more subunits B. Subunit B is inactive but binds to the ganglioside GGnSLC on the cell surface. Subunit A does not bind to cell membranes or gangioside and is slightly toxic to intact cells but strongly and instantly active in lysed cells. The binding of whole toxin through the B subunit to the cell is followed by a lag before subunit A penetrates the cell membrane (leaving subunit B on the surface) and stimulates the adenylate cyclase. The stimulation of adenylate cyclase depends on the presence of NAD and other co-factors present in the cell sap.

Binding of cholera toxin by various tissues.

Under certain conditions, it is possible to confirm the observation by Peterson (1974) that the cholera toxin-binding capacities of tissues from brain and colon mucosa, and from liver and small intestine mucosa, are comparable. Binding of toxin by all tissues except brain is very variable, but is roughtly proportional to their content of the toxin-binding ganglioside galactosyl-N-acetylgalactosaminyl (sialosyl) lactosyl ceramide. It appears that some toxin-binding sites of the mucosa of the small intestin and colon may be masked. It has also been confirmed that there may be some solubilization of toxin-binding material from brain on standing a few days at 4 C, but this is comparatively slight. Some disadvantages of measuring toxin binding by adding small amounts of radioactive toxin to compartively large amounts of tissue are discussed.

Evidence for the complex nature of the ganglioside receptor for cholera toxin.

Choleragenoid binds more slowly and less strongly than cholera toxin to intestinal mucosal cells, and even less strongly to free ganglioside in solution. However, binding to ganglioside is greatly enhanced when the ganglioside is in the form of an insoluble complex with cerebroside. These findings suggest that both the binding and the active components of the toxin molecule may be necessary for optimal binding of the toxin to the intact cell, and that the ganglioside in the cell receptor is in a complex form. Choleragenoid only partially blocks the action of the toxin on ruptured cells. This observation indicates that, while binding to a membrane receptor is necessary for the action of the toxin on the whole cell, it is possible to activate adenyl cyclase in a perforated cell by a process apparently independent of membrane binding; however, this activation may be possible only if the toxin preparation contains the active component dissociated from choleragenoid.