SYNAPTOBREVIN VESICLE-ASSOCIATED MEMBRANE-PROTEIN (VAMP) OF APLYSIA-CALIFORNICA - STRUCTURE AND PROTEOLYSIS BY TETANUS TOXIN AND BOTULINAL NEUROTOXINS TYPE-D AND TYPE-F

Synaptobrevin/vesicle-associated membrane protein (VAMP) and syntaxin are potential vesicle donor and target membrane receptors of a docking complex that requires N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment proteins as soluble factors for vesicle fusion with target membranes. Members of this docking complex are the target of clostridial neurotoxins that act as zinc-dependent proteases. Molecular cloning of the Aplysia californica synaptobrevin cDNA revealed a 180-residue polypeptide (M(r), 19,745) with a central transmembrane region and an atypically large C-terminal intravesicular domain. This polypeptide integrates into membranes at both the co- and posttranslational level, as shown by modification of an artificially introduced N-glycosylation site. The soluble and membrane-anchored forms of synaptobrevin are cleaved by the light chains of the botulinal toxins type D and F and by tetanus toxin involving the peptide bonds Lys(49)-Ile(50), Gln(48)-Lys(49), and Gln(66)-Phe(67), respectively. The active center of the tetanus toxin light chain was identified by site-specific mutagenesis. His(233), His(237), Glu(234), and Glu(270/271) are essential to this proteolytic activity. Modification of histidine residues resulted in loss of zinc binding, whereas a replacement of Glu(234) only slightly reduced the zinc content.