Processing of tetanus and botulinum A neurotoxins in isolated chromaffin cells

Abstract

Tetanus and botulinum A neurotoxins were introduced into the cytosol of chromaffin cells by means of an electric field in which the plasma membrane is forced to form pores of approximately 1 micron at the sites facing the electrodes. As demonstrated by electron microscopy, both [125I] and gold-labelled tetanus toxin (TeTx) diffuse through these transient openings. Dichain-TeTx, with its light chain linked to the heavy chain by means of a disulfide bond, causes the block of exocytosis to develop more slowly than does the purified light chain. The disulfide bonds, which in both toxins hold the subunits together, were cleaved by the intrinsic thioredoxin-reductase system. Single chain TeTx, in which the heavy and light chains are interconnected by an additional peptide bond, was far less effective than dichain-TeTx at blocking exocytosis, which indicates that proteolysis is the rate-limiting step. The toxins were degraded further to low-molecular weight fragments which, together with intact toxins and subunits, were released by the cells. The intracellular half-life of [125I] dichain-TeTx was approximately three days. The number of light-chain molecules required to maintain exocytosis block in a single cell, as calculated by two different methods, was less than 10. The long duration of tetanus poisoning may result from the persistence of intracellular toxin due to scarcity of free cytosolic proteases. This may also hold for the slow recovery from botulism.

Evaluation of the Therapeutic Usefulness of Botulinum Neurotoxin B, C1, E, and F Compared with the Long Lasting Type A

“…Attempts by others to examine the t1 ⁄2 of the LC of the closely related Clostridial neurotoxin, TeTx, in cultured spinal neurons, found that a highly radio labeled toxin disappeared long before even an initial onset of recovery from blockade of neurotransmission (44); the authors correctly suggest that degradation of TeTx LC (t1 ⁄2 ϭ ϳ6 days) may underlie the slow recovery from neuroinhibition. Indeed, it has been estimated that only 10 -100 intracellular toxin molecules are required to inhibit exocytosis (45), precluding straightforward radiolabeled detection; furthermore, this approach does not distinguish between relevant functional toxin protease in the cytosol and that which may reside in other cellular locations (i.e. endosomes).…”

Persistence of Botulinum Neurotoxin Inactivation of Nerve Function

“…Most significant is the inability to quantify cytosolic BoNT proteases within intoxicated cells using current detection methods. Although the number of cytosolic protease molecules necessary to inactivate exocytosis is not known, it has been estimated to be no more than about a thousand [34] for BoNT proteases, and less than ten molecules for the closely related tetanus toxin protease [35]. Even these remarkably low numbers may be overestimations as for other toxins such as ricin or diphtheria toxin, a single molecule has been shown sufficient to elicit a measurable effect (cell death) [36,37].…”

Conformational analysis and design of cross‐strand disulfides in antiparallel β‐sheets

“…Indeed, it has been shown that, although the majority of disulfides are structurally inert in proteins, there are several disulfides that act as redox‐sensitive regulators 15, 16. The oxidation status of cross‐strand disulfides is believed to be important for the activity of toxins 17–19. Conserved cross‐strand disulfides occurring in viral fusion proteins and surface glycoproteins are believed to play a role in viral entry 20–24.…”