title: Therapeutic effectiveness of botulinum neurotoxin A: potent blockade of autonomic transmission by targeted cleavage of only the pertinent SNAP-25.
abstract: In search of a basis for the impressive potency of an endoprotease that cleaves SNAP-25, botulinum neurotoxin type A (BoNT/A), in treating numerous diseases due to hyper-active autonomic nerves, truncation of its target and inhibition of neurotransmission were studied in rat sympathetic neurons. Tetrodotoxin-sensitive spontaneous cholinergic neurotransmission was blocked >80% by 1 pM BoNT/A despite cleaving <20% of the SNAP-25. A maximum cleavage of ∼60% SNAP-25 could be achieved with >1 nM BoNT/A, despite an absence of non-cleavable SNAP-25 in the detergent-solubilised neurons. In contrast, BoNT/E (100 nM) truncated nearly all the SNAP-25 in the intact cells, but was unable to block neurotransmission at low concentrations like BoNT/A. Chimeras created by inserting the acceptor-binding HC domain of BoNT/A into BoNT/E still cleaved all the SNAP-25, indicating ubiquitous expression of BoNT/A acceptors. Accordingly, SV2 and SNAP-25 were found to be co-expressed and broadly co-localised in neurons, but absent from non-neuronal cells. On the other hand, partial cleavage by the BoNT/A protease persisted upon replacing its HC with counterparts from BoNT/E or BoNT/B. Moreover, limited cleavage of SNAP-25 was conferred onto the protease from BoNT/E when fused to the N-terminus of BoNT/A. Thus, the BoNT/A protease is uniquely well-adapted for selectively inactivating the SNAP-25 directly involved in neurotransmission; this together with the toxin's acceptor and its target being localised on the peri-somatic boutons likely contribute to its exceptional therapeutic utility in the clinic.
authors.affiliation: International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland. gary.lawrence@dcu.ie
authors.forename: Gary W
authors.initials: GW
authors.lastname: Lawrence
authors.forename: Saak V
authors.initials: SV
authors.lastname: Ovsepian
authors.forename: Jiafu
authors.initials: J
authors.lastname: Wang
authors.forename: K Roger
authors.initials: KR
authors.lastname: Aoki
authors.forename: J Oliver
authors.initials: JO
authors.lastname: Dolly
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000493:pharmacokinetics / Q000494:pharmacology; D019274:Botulinum Toxins, Type A / Q000493:pharmacokinetics / Q000494:pharmacology*; D002478:Cells, Cultured; D002799:Cholinergic Fibers / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D008562:Membrane Glycoproteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D051381:Rats; D055366:Synaptic Potentials / Q000187:drug effects; D009435:Synaptic Transmission / Q000187:drug effects*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C494210:Snap25 protein, rat; C489618:Sv2a protein, rat; D050825:Synaptosomal-Associated Protein 25; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E