Development of Targeted Therapeutic Agents for Botulism.
Annual rept. 25 Aug 97-24 Aug 98
IMPERIAL COLL OF SCIENCE TECHNOLOGY ANDMEDICINE LONDON (UNITED KINGDOM)
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Although the proteolysis of synaptobrevin by botulinum neurotoxin BoNT type B has been well characterised, much less is known about the efficient cleavage of SNAP-25 by BoNTA and E. Therefore, an ELISA was optimised and standardised for measuring their proteolysis of immobilised, bacterially-expressed SNAP-25, using purified IgGs reactive solely with full-length or BoNTA-truncated SNAP-25. Polystyrene-attached SNAP-25 proved an excellent substrate for BoNTA relative to the soluble or native protein. A small SNAP-25 C-terminal peptide residues 181-206 was cleaved 200-fold less efficiently than the intact molecule, establishing that sequences distant from the scissile bond are a prerequisite for optimal activity. P2, 1, 1 or 2 residues in SNAP-25 were altered by site-directed mutagenesis one- and two-step PCR procedures or the efficient Dpn-1 nuclease-quick-change method. Olutatltione-S-transferase-linked SNAP-25 variants - Incorporating single, double or triple mutations, addition or deletions at the C- and N- termini - have been isolated by affinity chromatography. Their susceptibilities to proteolysis by BoNTA are currently being examined to reveal residues distant from, and adjacent to, the cleavage site that are needed for the toxins efficient action. Eventually, both these domains will be incorporated into a down-sized ideal substrate before its modification to make an effective inhibitor. An additional strategy will entail creation of a toxin-resistant SNAP-25 variant that is still able to support transmitter release and, thus, have the potential to rescue botulinised nerve endings when targetted with an available cholinergic transporter.