title: Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics.
abstract: Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A-G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.
authors.affiliation: International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Jiafu
authors.initials: J
authors.lastname: Wang
authors.forename: Jianghui
authors.initials: J
authors.lastname: Meng
authors.forename: Gary W
authors.initials: GW
authors.lastname: Lawrence
authors.forename: Tomas H
authors.initials: TH
authors.lastname: Zurawski
authors.forename: Astrid
authors.initials: A
authors.lastname: Sasse
authors.forename: MacDara O
authors.initials: MO
authors.lastname: Bodeker
authors.forename: Marcella A
authors.initials: MA
authors.lastname: Gilmore
authors.forename: Ester
authors.initials: E
authors.lastname: Fernández-Salas
authors.forename: Joseph
authors.initials: J
authors.lastname: Francis
authors.forename: Lance E
authors.initials: LE
authors.lastname: Steward
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 / Q000737:chemistry*; D019274:Botulinum Toxins, Type A / Q000737:chemistry*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009474:Neurons / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D011522:Protons; D011993:Recombinant Fusion Proteins; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E