TCEP treatment reduces proteolytic activity of BoNT/B in human neuronal SHSY-5Y cells.
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.
Shi
Xuerong
X
Department of Regulated Laboratories, Division of Regulated Activities, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland 20910-7500, USA. xuerong.shi@amedd.army.mil
Garcia
Gregory E
GE
Neill
Roger J
RJ
Gordon
Richard K
RK
D001905:Botulinum Toxins / Q000737:chemistry / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004220:Disulfides / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D010720:Phosphines / Q000494:pharmacology*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D004220:Disulfides; D006918:Hydroxyurea; D009638:Norepinephrine; D010720:Phosphines; D021122:Protein Subunits; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; C080938:tris(2-carboxyethyl)phosphine