PROTEOLYTIC CLEAVAGE OF SYNTHETIC FRAGMENTS OF VESICLE-ASSOCIATED MEMBRANE-PROTEIN, ISOFORM-2 BY BOTULINUM TYPE-B NEUROTOXIN

Recent data suggest that botulinum type-B neurotoxin is a protease whi ch acts on vesicle-associated membrane protein, isoform 2 (VAMP-2). In this report, botulinum type-B neurotoxin is shown to cleave a synthet ic fragment (HV62) of VAMP-2, corresponding to the bulk of the hydroph illic domain (amino acids 33-94). The neurotoxin acts at a single site between Gln76 and Phe77. Little or no proteolytic activity by botulin um 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 pept ide 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 c lass of zinc-endoprotease; more than 12 amino acid residues are requir ed on both the NH2- and COOH-terminal side of the cleavage site for op timal proteolytic activity. The results demonstrate that no other comp onents of cellular vesicles are required for the specific action of th e 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.