No abstract

Clostridial neurotoxins (CNTs) 2 (i.e. tetanus neurotoxin (TeNT) and seven serotypes of botulinum neurotoxins (BoNTs A-G)) interfere with neurotransmitter release. BoNTs act in extremely low doses in motoneurons at the neuromuscular junction and thereby cause muscle paralysis. TeNT operates in inhibitory interneurons of the spinal cord that down-regulate the activity of motoneurons and thus causes the opposite physiological effect of BoNTs. CNTs are synthesized as single chain protein toxins by bacteria of the genus Clostridium and later become proteolytically activated. They consist of a catalytic domain (designated the L chain (LC)), a translocation domain that transfers the L chain subsequent to receptor-mediated endocytosis across the membrane of the endosomal compartment, and a cell binding subunit that mediates the selective binding to nerve cells. Upon delivery to the cytosol, the L chain is released from the rest of the molecule by reduction of the disulfide bridge by which it is tethered to the translocation domain. The L chains finally attack their intracellular substrates by acting as zinc endoproteases of high individual substrate specificity. BoNT/C hydrolyzes syntaxins 1 and 3 in certain species. BoNT/A, C, and E cleave SNAP-25 (synaptosome-associated protein of 25 kDa) and SNAP-23 in certain species, and all other CNTs proteolyze several vesicle-associated membrane proteins (VAMPs)/synaptobrevins. Except for BoNT/B and TeNT, all CNTs hydrolyze unique peptide bonds in their substrates (1). The members of the VAMP/synaptobrevin, SNAP-25, and syntaxin families are collectively termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) (2) and constitute core components of the vesicular fusion machinery. Individual sets of SNAREs are responsible for discrete intracellular vesicular fusion events, and CNTs thus became valuable tools for studying vesicular trafficking routes (3, 4). The set consisting of syntaxin 1A, SNAP-25, and VAMP-2, which are all cleaved by CNTs, is best investigated. It accomplishes the fusion of synaptic vesicles with the presynaptic membrane. Many other SNARE proteins like the TeNT-insensitive VAMP (TI-VAMP; also known as VAMP-7) (5, 6) or syntaxin 4, which together with SNAP-23 carry out membrane fusion of secretory vesicles with the plasma membrane (7), are not hydrolyzed by CNTs (6,[8][9][10].A peculiarity of CNT L chains which distinguishes them from conventional proteases is that they require an extended substrate segment for optimal catalytic activity, as evidenced by prior studies employing truncated substrates (8,(11)(12)(13)(14)(15) or amino acid substitutions (8, 16 -22). The interaction of BoNT/A, C, and E with SNAP-25 has been most thoroughly analyzed. A recently determined co-crystal structure of a BoNT/A-SNAP-25 complex (23), along with structural studies on mutated L chains (24, 25) and the enzymatic characteriza-

The clostridial neurotoxin family consists of tetanus neurotoxin and seven distinct botulinum neurotoxins which cause the diseases tetanus and botulism. The extreme potency of these toxins primarily relies not only on their ability to specifically enter motoneurons but also on the activity their catalytic domains display inside presynaptic motoneuronal terminals. Subsequent to neurotoxin binding and endocytosis the catalytic domains become translocated across endosomal membranes and proteolyze unique peptide bonds of one of three soluble N-ethylmaleimide-sensitive fusion protein attachment receptors (SNAREs), vesicle associated membrane protein/synaptobrevin, synaptosome associated protein of 25 kDa, or syntaxin. As these substrate proteins are core components of the vesicular membrane fusion apparatus, cleavage of any of the substrate molecules results in the blockade of neurotransmitter release. This review summarizes the present knowledge about the molecular basis of the specific substrate recognition and cleavage mechanism and assesses the feasibility of reengineering catalytic domains to hydrolyze non-substrate members of the three SNARE families in order to expand the therapeutic application of botulinum neurotoxins.