Proteolytic cleavage of synthetic fragments of vesicle‐associated membrane protein, isoform‐2 by botulinum type B neurotoxin

Abstract

Recent data suggest that botulinum type-B neurotoxin is a protease which acts on vesicleassociated membrane protein, isoform 2 (VAMP-2). In this report, botulinum type-B neurotoxin is shown to cleave a synthetic fragment (HV62) of VAMP-2, corresponding to the bulk of the hydrophillic domain (amino acids 33-94). The neurotoxin acts at a single site between Gln76 and Phe77. Little or no proteolytic activity by botulinum type-B neurotoxin was observed with peptides containing 7, 10 or 20 amino acids spanning the site of cleavage. The proteolytic action of neurotoxin was strongly inhibited by EDTA and o-phenanthroline whereas captopril and phosphoramidon were ineffective. A series of model peptide substrates were synthesised in order to define the smallest VAMP-2 fragment to be cleaved by botulinum type-B neurotoxin. Data obtained from these substrates suggest that the neurotoxin belongs to a novel class of zinc-endoprotease; more than 12 amino acid residues are required on both the NH,-and COOH-terminal side of the cleavage site for optimal proteolytic activity. The results demonstrate that no other components of cellular vesicles are required for the specific action of the neurotoxin on VAMP-2. The data further show that the highly specific action of the neurotoxin is not dictated solely by the properties of the amino acid residues at the cleavage site but is also dependent on amino acid sequences distal to its site of action.

A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate

“…SNARE proteins are thought to be the primary and perhaps only intracellular target for the BoNT͞LCs, and each serotype cleaves a different peptide bond within its corresponding SNARE substrate. This remarkable specificity results from the LCs' recognition of unusually long SNARE sequences (Ϸ30-50 residues, depending on the serotype), suggesting that substrate residues distal to the cleavage site are recognized (3)(4)(5). This hypothesis is supported by the recently elucidated crystal structure of serotype A-LC in complex with a fragment of SNAP-25 (6,7).…”

Substrate residues N‐terminal to the cleavage site of botulinum type B neurotoxin play a role in determining the specificity of its endopeptidase activity

“…Previous studies have shown that a VAMP(55-82) peptide which contains both the motif and cleavage site is poorly cleaved by BoNT/B [12]. This observation clearly suggests that substrate residues flanking the C-terminus of the cleavage site also play some role in substrate recognition.…”

“…Peptides were purified by reverse-phase high pressure liquid chromatography on C8 columns (2.2 × 25 cm, Vydac), and characterised as described previously [12].…”

“…Final concentrations of BoNT/B were between 3 and 100 nM and were chosen so that less than 15% of the total peptide was cleaved during the reaction time. The extent of peptide cleavage was estimated from HPLC analysis of reaction mixtures on C8 columns (4.6x45 mm, Ultrasphere, Beckman) as described [12]. Initial rates of peptide cleavage were estimated from the integral analysis of the two fragment peak areas as well as that of the parent peak.…”

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.…”