Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.

Abstract

The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc‐dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017‐1023; Schiavo et al., Nature, 359, 832‐835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC‐1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC‐1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC‐1/syntaxin and the toxin light chain which acts as a metallo‐endoprotease. Toxin‐induced cleavage resulted in the generation of a soluble fragment of HPC‐1/syntaxin that is 2‐4 kDa smaller than the native protein. When HPC‐1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full‐length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane‐free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC‐1/syntaxin, which lacked the putative transmembrane domain at the C‐terminus, was soluble and resistant to toxin action. We conclude that HPC‐1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…Our results provide functional evidence for the presence of the SNAP receptor (SNARE) proteins in neuroendocrine cells [17,18,32], which together with action of clostridial neurotoxins on secretion [2,[12][13][14], strongly support the hypothesis of a common secretory machinery present in neural and neuroendocrine cells consisting of the described SNARE elements of the fusion complex [33].…”

“…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].…”

“…Poignant evidence implicating these components in neurotransmision emerged from the action of clostridial tetanus and botulinum neurotoxins that block secretion by cleaving synaptobrevin, syntaxin and SNAP-25 [12][13][14]. The participation of *Corresponding author.…”

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.…”

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.…”