Structural Determinants of the Specificity for Synaptic Vesicle-associated Membrane Protein/Synaptobrevin of Tetanus and Botulinum Type B and G Neurotoxins

Abstract

Tetanus and botulinum neurotoxins type B and G are zinc-endopeptidases of remarkable specificity. They recognize and cleave a synaptic vesicle-associated membrane protein (VAMP)/synaptobrevin, an essential protein component of the vesicle docking and fusion apparatus. VAMP contains two copies of a nine-residue motif, also present in SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin, the two other substrates of clostridial neurotoxins. This motif was suggested to be a determinant of the target specificity of neurotoxins. Antibodies raised against this motif cross-react among VAMP, SNAP-25, and syntaxin and inhibit the proteolytic activity of the neurotoxins. Moreover, the various neurotoxins cross-inhibit each other's proteolytic action. The role of the three negatively charged residues of the motif in neurotoxin recognition was probed by sitedirected mutagenesis. Substitution of acidic residues in both copies of the VAMP motif indicate that the first one is involved in tetanus neurotoxin recognition, whereas the second one is implicated in binding botulinum B and G neurotoxins. These results suggest that the two copies of the motif have a tandem association in the VAMP molecule.Tetanus neurotoxin (TeNT) 1 and botulinum neurotoxins (BoNTs, seven types from A to G) are three-domain protein toxins that bind selectively to the neuronal presynaptic membrane. They are internalized inside intracellular compartments from which the amino-terminal 50-kDa domain (termed L chain) enters into the cytosol (1-4). The L chains of TeNT and BoNTs are zinc-endopeptidases that cleave specifically three proteins of the neuroexocytosis apparatus, thereby blocking neurotransmitter release (4 -7). TeNT and BoNT/B, BoNT/D, BoNT/F, and BoNT/G recognize and cleave specifically a synaptic vesicle-associated membrane protein (VAMP, also referred to as synaptobrevin) at different single peptide bonds (4, 8 -12). BoNT/A and BoNT/E specifically recognize and cut SNAP-25 (synaptosomal-associated protein of 25 kDa) at two different peptide bonds near the COOH terminus (10, 13, 14), whereas BoNT/C cleaves syntaxin (15, 16) and . VAMP, SNAP-25, and syntaxin are collectively termed SNARE proteins, because they act as receptors of soluble Nethylmaleimide-sensitive factor accessory proteins, involved in vesicle-membrane fusion (5-7).Sequence comparison of the L chains of the eight clostridial neurotoxins show strong similarities (20), which are even more extensive at the level of predicted secondary structure (21). These similarities suggest that they derive from a common ancestral metalloproteinase. On this basis, to account for their different substrate specificity, we considered the possibility that the three SNAREs contain a common neurotoxin recognition site in addition to the cleavage sites specific for each neurotoxin type. We identified a nine-residue-long motif (SNARE motif) present in eukaryotes only in the three proteins known to be proteolytic substrates of the neurotoxins (22). The SNARE motif is included within regions...

Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin

“…In cultured cells, SNARE-specific peptides and antibodies against the VAMP-2 V2 motif (32) blocked BoNT/A, B, and C cleavage of substrates, and mutations of charged residues within the SNARE motifs in VAMP-2 and SNAP-25 also inhibited substrate hydrolysis (33,34). The current mutational analysis observed that mutations of individual amino acids in the V1 SNARE motif of VAMP-2 inhibited LC/TeNT cleavage, whereas mutations of individual amino acids in the V2 SNARE motifs inhibited LC/B cleavage.…”

Identification and Characterization of an 18-Kilodalton, VAMP-Like Protein in Suspension-Cultured Carrot Cells

“…The 30-kD polypeptide is localized neither in clathrincoated nor in non-clathrin-coated vesicles; thus, it may perhaps be a SNARE protein cross-reacting with anti-VAMP-2 antibodies (Pellizzari et al, 1996). We have demonstrated that the 18-and 19.2-kD polypeptides are both enriched in purified non-clathrin vesicles (CV1).…”

“…Even the human synaptobrevin II 1-93 fragment (synthesized by the solid-phase peptide synthesis technique), is not completely soluble at the approximate concentration of 1 mm, which is the range of the V max for the digestion by tetanus toxin L chain (Cornille et al, 1994). On the other hand, the efficiency of the tetanus toxin for VAMP proteins, from different sources, vary in in vitro experiments (80%-25%) and depends on the amino acid sequences of the v-SNARE proteins (Yamasaki et al, 1994;Pellizzari et al, 1996). The 18-kD polypeptide was not degraded by activated tetanus toxin if CV1 vesicles were not washed with high pH (8.3) Tris-HCl buffer (Fig.…”

Structural Studies on Intact Clostridium botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design

“…In the case of TeNT, besides the actual cleavage site, amino acid residues of the SNARE recognition motif V1 (Figure 1), which is located N-terminal of the swapped region, were recognised as being essential for optimal cleavage. In contrast, residues of the SNARE recognition motif V2 were shown to be critically involved in substrate recognition by BoNT/B and G. 9,15 The failure of BoNT/F and BoNT/D to cleave this hybrid molecule matches the fact that V1 and V2 motif amino acid residues Botulinum Neurotoxin B Substrate Requirements are required for their optimal activity in addition to the cleavage site. 17 In order to narrow the interacting region, we next reset the VAMP-2 portion from either end to TI-VAMP residues, resulting in the hybrid constructs TI-VH27, TI-VH26, TI-VH19, and TI-VH10, in which the number specifies the size of the remained VAMP-2 segment (see Figure 2).…”

“…16 Amino acid substitutions 13,15,[17][18][19] and a recently determined cocrystal structure 20 confirmed that remote substrate sites upstream and downstream of the scissile peptide bonds do in fact interact with the L chains.…”