Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Abstract

Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity.

Solid‐phase synthesis, conformational analysis and in vitro cleavage of synthetic human synaptobrevin II 1–93 by tetanus toxin L chain

“…The inhibition by EDTA and ophenanthroline has already been reported by Schiavo et al (1992b) using an in vitro test and Sanders and Habermann (1992) in an in vivo test by preincubation of the toxin with different inhibitors and subsequent injection into permeabilized bovine adrenomedullary cells. The zinc contained in the protein has been shown to be necessary for activity (Schiavo et al, 1992a) and the L chain contains the consensus sequence HEXXH found in zinc endopeptidases (Devault et al, 1987;Jongeneel et al, 1989).…”

“…Even a 2.5-fold and 5-fold activation can be measured by addition of 1 mM Syb I1 73-81 or Syb I1 51 -93 peptides, respectively. Interestingly when Glu76 is replaced by Val as in the isoform I of rat synaptobrevin (which is not cleaved by TeTx) (Schiavo et al, 1992b), neither inhibition nor activation is found. Finally, 10% (by vol.)…”

“…ethanol in the incubation buffer of the TeTx L chain was found sufficient to inhibit 85 % of its peptidase*activity. Therefore the presence of ethanol to solubilize hydrophobic inhibitors of angiotensin-converting enzyme or neutral endopeptidase could explain their reported inhibition (Schiavo et al, 1992b) of TeTx L chain peptidase activity occurring at millimolar concentration.…”

“…The amino-acid sequence of the TeTx L chain presents the H-E-X-X-H sequence correspondi.ng to the signature of the zinc binding site of metallopeptidases (Devault et al, 1987;Matthews, 1988;Jongeneel et al, 1989;Vallee and Auld, 1990) and was recently found to contain a zinc atom (Schiavo et al, 1992b;Fraser-Wright et al, 1992). The deduced enzymic activity of TeTx L chain was formerly established by the demonstration that the toxin, but not its apoform, is able to cleave specifically rat synaptobrevin I1 at a single peptide bond (Gln76-Phe77) (Schiavo et al, 1992a).…”

Development of vaccines for prevention of botulism

“…Alternatively, the receptor may assist the toxin in transporting the light chain across the membrane. The final event of intoxication involves the catalytic hydrolysis of key synaptic vesicle proteins [3,[23][24][25] by the light chain [3,26]. 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.…”