Temperature‐Mediated Interaction of Tetanus Toxin with Cerebral Neuron Cultures: Characterization of a Neuraminidase‐Insensitive Toxin‐Receptor Complex

Abstract

Energy-dependent internalization of 125I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0-4 degrees C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0-4 degrees C; the effect is further amplified at 37 degrees C. Addition of disialoganglioside GD1b (1-5 micrograms/ml) also lowers the amount of cell-associated toxin. The fraction of 125I-labeled toxin retained by the cells after exposure to high-salt medium at 0-4 degrees C or after addition of GD1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37 degrees C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.

Affinity purified tetanus toxin binds to isolated chromaffin granules and inhibits catecholamine release in digitonin‐permeabilized chromaffin cells

“…These values are about ten times higher than the Kd values for the actual binding of the toxin to the granules and are consistent with our notion of some irreversible interaction between the bound toxin and its sites, similar to that previously described for toxin sequestration in neuronal systems [16,17,19].…”

“…By contrast, the toxin-binding site complex formed at 37°C was stable and refractive to neuraminidase treatment (open bars, A). This finding might reflect a process similar to TT sequestration at 37°C that has previously been reported with cultures of neuronal cells [19].…”

“…After two rinses, the granule-associated radioactivity was counted. In all experiments nonspecific binding of 12~I-TeToB was defined as the amount of radioactivity found in the presence of excess (1 unit of antitoxin) [19]. This value represents less than 15070 of the largest value determined and was subtracted from all data presented.…”

“…However, in neuronal cells which avidly bind this toxin, the toxin affects the neurotransmitter release following internalization and retrograde transport [4,11]. Once the toxin molecules reach their site of action on the granule membranes or, alternatively, on the cytoplasmic face of the neuronal cell membrane [19], they might interfere directly with the fusion of the secretory granule with the neuronal membrane. While the chemical nature of tetanus toxin-binding sites on chromaffin granule membranes requires further elucidation, the present report provides an explanation for the inhibitory potency of tetanus toxin and of botulinum D toxin (which recognizes the same type of gangliosides) to block exocytosis [5,[7][8][9].…”

GT1b Ganglioside Prevents Tetanus Toxin‐Induced Protein Kinase C Activation and Down‐Regulation in the Neonatal Brain In Vivo

“…The strong affinity of TeTx for nervous system tissue has been classically attributed to a particular group of gangliosides (van Heyningen, 1974;Dimpfel et al, 1977;Helting et al, 1977;Yavin, 1984;Critchley et al, 1986) containing two adjacent sialyl residues attached to an inner galactose, and termed, according to the nomenclature of Svennerholm (1963), GDlb and GTlb. Participation of other cellular constituents, e.g., sialoglycoproteins, in the interaction between the toxin and the TeTx-receptor complex has been suspected but not unequivocally established (Schmitt et al,198 1; Yavin et al, 1983;Lazarovici and Yavin, 1986;Yavin and Nathan, 1986;Montecucco, 1988).…”