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  • TCEP treatment reduces proteolytic activity of BoNT/B in human neuronal SHSY-5Y cells.

TCEP treatment reduces proteolytic activity of BoNT/B in human neuronal SHSY-5Y cells.

Journal of cellular biochemistry (2009-06-06)
Xuerong Shi, Gregory E Garcia, Roger J Neill, Richard K Gordon
ABSTRACT

The light chain (LC) of botulinum neurotoxin B (BoNT/B) is unable to enter target neuronal cells by itself. It is brought into the cell in association with the BoNT/B heavy chain (HC) through endocytosis. The BoNT HC-LC subunits are held together by a single disulfide bond. Intracellular reduction of this bond and separation of the two subunits activates the endopeptidase activity of the LC. This requirement suggests a strategy to prevent uptake by prophylactic reduction to disrupt the disulfide bond prior to endocytosis of the complex. We examined the utility of tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP), a relatively non-toxic, non-sulfur containing disulfide bond reducing agent that lacks the undesirable properties of mercapto-containing reducing agents. We found that TCEP was as effective as DTT with maximal LC endopeptidase activation occurring at 1 mM, a concentration not toxic to the human neuronal cell line, SHSY-5Y. In these cells, 1 mM TCEP maximally protected against BoNT/B inhibition of [(3)H]-NA release, achieving 72% of the release from un-intoxicated controls. This effect appears to be due to the sparing of SNARE proteins as the levels of VAMP-2, the specific target of BoNT/B, were protected. These results show that TCEP disrupts the structure of BoNT/B by reduction of the LC and HC bridging disulfide bond and prevents neuronal intoxication. Since disulfide bond coupling between toxin subunits is a general motif for many toxins, e.g., ricin, snake venom, and all BoNT serotypes, this suggests that TCEP is a promising means to protect against these toxins by preventing cell penetration.

MATERIALS
Product Number
Brand
Product Description

Supelco
Tris(2-carboxyethyl)phosphine hydrochloride solution, 0.5 M, pH 7.0(aqueous solution; pH was adjusted with ammonium hydroxide)