W. E. van Heyningen

Gangliosides as membrane receptors for tetanus toxin, cholera toxin and serotonin

Tetanus toxin is bound by a sialidase-labile disialosyl ganglioside, serotonin by another, and cholera toxin by a sialidase-stable monosialosyl ganglioside. There is good evidence that the monosialosyl ganglioside may be the receptor for cholera toxin in cell membranes; what of the disialosyl ganglioside and tetanus toxin?

The fixation of tetanus toxin by ganglioside.

SUMMARY: The fixation of tetanus toxin by brain ganglioside has been confirmed. A method has been developed for assaying the toxin-fixing capacity of ganglioside in the analytical ultracentrifuge. Under appropriate conditions ganglioside will fix many times its own weight of toxin. Ganglioside preparations from brain contain at least three gangliosides; at least two of these differ in their sialic acid contents and toxin-fixing capacities. The sialic acid residues (and their free carboxyl groups) are essential for toxin-fixation. Tetanus toxin does not appear (so far) to bring about any change in the ganglioside molecule.

The fixation of tetanus toxin, strychnine, serotonin and other substances by ganglioside.

SUMMARY: Although tetanus toxin is fixed by brain ganglioside, it is not fixed by a number of substances more or less closely related to brain ganglioside, or by naturally occurring brain ganglioside not containing hexosamine, or by a hexosamine-containing ganglioside from Tay-Sachs brain. Isolated brain gangliosides may vary considerably in their toxin-fixing capacities. In chloroform + methanol extracts of brain a number of gangliosides which differ in ability to fix toxin can be separated chromatographically. Complexes of ganglioside with phrenosine and sphingomyelin have diminished toxin-fixing capacity. Tetanus toxin and ganglioside have a high binding capacity for calcium, but calcium does not affect the fixation of toxin by ganglioside. Ganglioside fixes strychnine, brucine and thebaine, drugs which have the same neurophysiological activity as tetanus toxin. Ganglioside does not fix γ-aminobutyric acid, β-hydroxy-γ-aminobutyric acid, histamine, adrenaline, noradrenaline or dopamine, but it does fix serotonin and a number of related compounds. It has been confirmed that albumin is fixed to a small extent by ganglioside at low salt concentration, but (unlike tetanus toxin) this fixation is practically abolished at physiological salt concentration.

Tentative Identification of the Tetanus Toxin Receptor in Nervous Tissue

SUMMARY: The tetanus toxin receptr in nervous tissue appears to be a ganglioside (mucolipid, strandin, containing: fatty acid, sphingosine, glucose, galactose, acetylgalactosamine, acetylneuraminic acid and amino acid (?) residues). The water-soluble ganglioside occurs in ervous tissue in the form of labile water-insoluble complexes with cerebrosides and sphingomyelines. The solubility of the complexes in water increases with increasing ganglioside content, but decreases when calcium is incorporated. Standing with ganglioside inactivates the toxin.

The Effect of Cerebroside and Other Lipids on the Fixation of Tetanus Toxin by Gangliosides

SUMMARY: Water-insoluble complexes of ganglioside with cerebroside fixed tetanus toxin at low concentrations (a few LD 50/ml.) of toxin. A complex containing 25% ganglioside with cerebroside was 50 times better at fixing toxin than complexes containing either 2% or 50% ganglioside. A complex containing 25% of a mixture of the gangliosides Giii and Giv was 12 times better at fixing toxin than a similar complex with gangliosides Gi and GII. Complexes of ganglioside with sphingomyelin and lecithin fixed toxin to a slight extent, while complexes with tripalmitin and cholesterol did not fix toxin. The complex of cerebroside and ganglioside, containing 25% ganglioside, did not fix strychnine, serotonin, botulinum toxin or plasma albumin.

The fixation of tetanus toxin by nervous tissue.

SUMMARY: The fixation of tetanus toxin by nervous tissue (Wassermann-Takaki phenomenon, 1898) has been re-investigated. Fixation is greatly dependent on the concentration of toxin and of receptor. Toxin is fixed in preference to toxoid, but its toxicity does not appear to be markedly diminished by fixation. A biological assay of the toxin receptor in nervous tissue and its components has been devised. The receptor activity is not due to a cerebroside, as thought by Landsteiner & Botteri (1906), but to some substance (present mainly in grey matter) that tends to associate with it.

Chemical Assay of the Tetanus Toxin Receptor in Nervous Tissue

SUMMARY: The fixation of tetanus toxin by the receptor in nervous tissue is highly specific in so far as tetanus toxic protein (and toxoid to a much smaller extent), and apparently nothing else, is adsorbed from solutions of ionic strength greater than 0·15. At lower ionic strengths certain other proteins may be adsorbed by a mechanism that is probably different. The specific adsorption of toxic protein has been used as the basis of a chemical method of assay of the receptor which depends on the estimation of the amount of protein adsorbed under prescribed conditions by the unknown amount of receptor from a fixed amount of crude tetanus toxin.

A Note on the Specific Fixation, Specific Deactivation and Non-Specific Inactivation of Bacterial Toxins by Gangliosides

SummaryThe fixation of tetanus toxin and the deactivation of cholera toxin by gangliosides are toxin-specific and ganglioside-specific, and not prevented by protective colloid, whereas the inactivation of a number of toxins by gangliosides which is prevented by protective colloid is a non-specific phenomenon.

Ganglioside as a prophylactic agent in experimental tetanus in mice.

SUMMARY: The symptoms of tetanus in mice, resulting from intramuscular injection of either purified tetanus toxin or vegetative bacilli of Clostridium tetani, can be partially prevented if the mice are injected at the same time, or a few hours before or afterwards, with a preparation of mixed gangliosides or with a suspension of ganglioside/cerebroside complex. Injection at the same site as the toxin injection is most effective, although intravenous injection of ganglioside (but not of the complex) also has some action; 0.5 mg. of ganglioside when complexed with 1.5 mg. cerebroside is as effective in protection as 5 mg. ganglioside alone. Protection by complexes containing different proportions of ganglioside reflects their ability to fix tetanus toxin in vitro. It is tentatively suggested that injection of ganglioside/cerebroside complex at a site of injury might be of prophylactic value in human tetanus.

The Role of Gangliosides in the Action of Cholera Toxin

Cholera toxin (choleragen, cholera exotoxin, cholera entero-exotoxin) is the toxin produced by Vibrio cholerae which is responsible for the increased secretion of chloride ions into the gut lumen resulting in the severe diarrhoea of cholera. The toxin (molecular weight 84,000 daltons) which is entirely protein in nature, consists of two types of subunit A and B (also referred to by Holmgren and Lonnroth (1975) as H and L subunits). The toxin has one copy of the A subunit (molecular weight about 28,000 daltons), and between 4 and 6 copies of the B subunit (each of molecular weight around 10,000 to 14,000 daltons). An aggregate of the 4 to 6 B subunits is known as choleragenoid (molecular weight 56,000 daltons), and this biologically inert “natural toxoid” can be isolated during the process of purification of cholera toxin from V. cholerae culture filtrates (Finkelstein and LoSpalluto 1970). Subunit A, which is connected to choleragenoid by only weak forces, can be dissociated from it by acid or by urea.