title: Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A.
abstract: Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Dr, Madison, WI 53706, United States.
authors.forename: Sabine
authors.initials: S
authors.lastname: Pellett
authors.forename: William H
authors.initials: WH
authors.lastname: Tepp
authors.forename: Larry H
authors.initials: LH
authors.lastname: Stanker
authors.forename: Philip A
authors.initials: PA
authors.lastname: Band
authors.forename: Eric A
authors.initials: EA
authors.lastname: Johnson
authors.forename: Konstantin
authors.initials: K
authors.lastname: Ichtchenko
mesh_terms: D000818:Animals; D001681:Biological Assay; D019274:Botulinum Toxins, Type A / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D003600:Cytosol / Q000378:metabolism; D005260:Female; D051379:Mice; D008813:Mice, Inbred ICR; D009469:Neuromuscular Junction / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25; D019274:Botulinum Toxins, Type A