S. Yamasaki

Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.

Clostridial neurotoxins inhibit neurotransmitter release by selective and specific intracellular proteolysis of synaptobrevin/VAMP, synaptosomal‐associated protein of 25 kDa (SNAP‐25) or syntaxin. Here we show that in binary reactions synaptobrevin binds weakly to both SNAP‐25 and syntaxin, and SNAP‐25 binds to syntaxin. In the presence of all three components, a dramatic increase in the interaction strengths occurs and a stable sodium dodecyl sulfate‐resistant complex forms. Mapping of the interacting sequences reveals that complex formation correlates with the presence of predicted alpha‐helical structures, suggesting that membrane fusion involves intermolecular interactions via coiled‐coil structures. Most toxins only attack the free, and not the complexed, proteins, and proteolysis of the proteins by different clostridial neurotoxins has distinct inhibitory effects on the formation of synaptobrevin‐syntaxin‐SNAP‐25 complexes. Our data suggest that synaptobrevin, syntaxin and SNAP‐25 associate into a unique stable complex that functions in synaptic vesicle exocytosis.

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

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)

Tetanus Toxin Action: Inhibition of Neurotransmitter Release Linked to Synaptobrevin Proteolysis

Tetanus toxin is a potent neurotoxin that inhibits the release of neurotransmitters from presynaptic nerve endings. The mature toxin is composed of a heavy and a light chain that are linked via a disulfide bridge. After entry of tetanus toxin into the cytoplasm, the released light chain causes block of neurotransmitter release. Recent evidence suggests that the L-chain may act as a metalloendoprotease. Here we demonstrate that blockade of neurotransmission by tetanus toxin in isolated nerve terminals is associated with a selective proteolysis of synaptobrevin, an integral membrane protein of synaptic vesicles. No other proteins appear to be affected by tetanus toxin. In addition, recombinant light chain selectively cleaves synaptobrevin when incubated with purified synaptic vesicles. Our data suggest that cleavage of synaptobrevin is the molecular mechanism of tetanus toxin action.

Inhibition of neurotransmitter release by clostridial neurotoxins correlates with specific proteolysis of synaptosomal proteins

Rat brain synaptosomes were used to study the effect of several clostridial neurotoxins on the neurotransmitter release. In this system the blockade of transmitter release correlated with the proteolytic activity of the toxins. Blockade of glutamate release was linked to selective proteolysis of one of the following synaptic proteins: synaptobrevin (BoNT/D, BoNT/F); SNAP-25 (BoNT/A, BoNT/E), or HPC-1/syntaxin (BoNT/C1). All the toxins used had an inhibitory effect on synaptosomes with the exception of BoNT/F. BoNT/F cleaved synaptobrevin in permeabilized synaptosomes but failed to produce the same effect on intact synaptosomes.

Exploring the functional domain and the target of the tetanus toxin light chain in neurohypophysial terminals

The tetanus toxin light chain blocks calcium induced vasopressin release from neurohypophysial nerve terminals. Here we show that histidine residue 233 within the putative zinc binding motif of the tetanus toxin light chain is essential for the inhibition of exocytosis, in the rat. The zinc chelating agent dipicolinic acid as well as captopril, an inhibitor of zinc-dependent peptidases, counteract the effect of the neurotoxin. Synthetic peptides, the sequences of which correspond to motifs present in the cytoplasmic domain of the synaptic vesicle membrane protein synaptobrevin 1 and 2, prevent the effect of the tetanus toxin light chain. Our results indicate that zinc bound to the zinc binding motif constitutes the active site of the tetanus toxin light chain. Moreover they suggest that cleavage of synaptobrevin by the neurotoxin causes the inhibition of exocytotic release of vasopressin from secretory granules.