Tetanus and botulinum neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans

The clostridial neurotoxin (CNT) family is composed of tetanus neurotoxin (TeNT) and seven different serotypes of botulinum neurotoxins (BoNT) (Minton, 1995). The active holotoxin is comprised of two fragments, termed heavy (H, 100 kDa) and light (L, 50 kDa) chains (Fig. 1A) and blocks neurotransmitter release in vitro and in vivo (Schiavo and Montecucco, 1997). The L chain is responsible for the intracellular activity of CNTs and contains the catalytic domain of the neurotoxins. CNTs are zinc-endoproteases which specifically cleave a family of proteins, named SNAREs (soluble NSF attachment protein receptor) (Söllner et al., 1993). These proteins, whose integrity is necessary to sustain regulated secretion, are involved in multiple steps leading to the docking and fusion of small synaptic vesicles (SSVs) with the presynaptic plasma membrane and in a variety of other intracellular trafficking events (Hanson et al., 1997; Hay and Scheller, 1997; Robinson and Martin, 1998). Vesicle-associated membrane protein (VAMP)/synaptobrevin is localised on SSVs and other vesicular compartments and is cleaved by TeNT and four serotypes of BoNT (B, D, F and G) (Schiavo et al., 1992, 1993a,c, 1994; Yamasaki et al., 1994a,b,c). BoNT/A and E instead cleave synaptosomal-associated protein of 25 kDa (SNAP-25) (Blasi et al., 1993a; Schiavo et al., 1993a,b), whilst BoNT/C has a dual-specificity for SNAP-25 and syntaxin 1 (Blasi et al., 1993b; Schiavo et al., 1995; Foran et al., 1996; Osen Sand et al., 1996; Williamson et al., 1996; Vaidyanathan et al., 1999). Both syntaxin and SNAP-25 are mainly localised on the plasma membrane (Garcia et al., 1995). They are therefore defined as target SNAREs or t-SNAREs, whilst VAMP/synaptobrevin is a vesicular SNARE or vSNARE. v-and t-SNAREs form a very stable complex, possibly promoting a strict apposition of the vesicular and target lipid bilayers which is likely to drive membrane fusion (Sutton et al., 1998; Weber et al., 1998). CNTs alter the formation and assembly of this complex by reducing its stability (Hayashi et al., 1994, 1995) and in the case of syntaxin and VAMP/ synaptobrevin, by detaching the cytoplasmic portion from their membrane anchors (Schiavo and Montecucco, 1997). Despite the widespread distribution of the cleavable isoforms of VAMP, SNAP-25 and syntaxin, the action of CNTs in vivo is absolutely neurospecific. All the clinical symptoms of botulism can be ascribed to the inhibition of acetylcholine 2715 Journal of Cell Science 112, 2715-2724 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS4674