Botulinum neurotoxins serotypes A and E cleave SNAP‐25 at distinct COOH‐terminal peptide bonds

Abstract

SNAP-25 a membrane-associated protein of the nerve terminal, is specifically cleaved by botulinurn neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo-endopeptidase toxins cleave SNAP-25 at two distinct carboxyl-terminal sites. Serotype A catalyses the hydrolysis of the Gln'97-Arg'98 peptide bond, while serotype E cleaves the Arg'80-Ile'8' peptide linkage. These results indicate that the carboxyl-terminal region of SNAP-25 plays a crucial role in the multi-protein complex that mediates vesicle docking and fusion at the nerve terminal.

Botulinum neurotoxin light chains inhibit both Ca2+‐induced and GTP analogue‐induced catecholamine release from permeabilised adrenal chromaffin cells

“…The requirement for the other two neurotoxin sensitive substrates for secretion due to Ca 2+ or GppNHp in chromaffin cells was examined using recombinant light chains of the botulinum C1 neurotoxin, which was shown to preferentially cleave syntaxin [42,43] and E neurotoxin which cleaves SNAP-25 [44,45]. The maximum concentration of these light chains that could be used was limited by their instability following dialysis.…”

A peptide that mimics the carboxy‐terminal domain of SNAP‐25 blocks Ca2+‐dependent exocytosis in chromaffin cells

“…The blocking effect mimics that exerted by the catalytically-active light chains ofBoTx A and E [12][13][14], that cleave SNAP-25 single sites in the C-terminal domain [23][24]. This cleavage is sufficient to disable the fusion process that leads to secretion by inhibiting vesicle priming [2,30].…”

“…Specifically, we focused on the carboxy-terminus of SNAP-25 because it is a target of clostridial neurotoxins A and E (BoTx A and E), known potent inhibitors of exocytosis [2,23,24], and it has been shown to interact with synaptobrevin and syntaxin (Fig. 3A).…”

Development of vaccines for prevention of botulism

“…The light chains are zinc-dependent endoproteases that selectively inactivate three essential proteins involved in the docking and fusion of acetylcholinecontaining synaptic vesicles to the plasma membrane. The light chains of BoNT serotypes A, C 1 , and E cleave SNAP-25 (synaptosomal-associated protein of 25 kDa) [27][28][29][30]; serotypes B, D, F, and G cleave VAMP/ synaptobrevin (synaptic vesicle-associated membrane protein) [31]; and serotype C 1 cleaves syntaxin [32]. Inactivation of SNAP-25, VAMP, or syntaxin by BoNT leads to an inability of the nerve cells to release acetylcholine, resulting in neuromuscular paralysis.…”