title: Retargeting the Clostridium botulinum C2 toxin to the neuronal cytosol.
abstract: Many biological toxins are known to attack specific cell types, delivering their enzymatic payloads to the cytosol. This process can be manipulated by molecular engineering of chimeric toxins. Using toxins with naturally unlinked components as a starting point is advantageous because it allows for the development of payloads separately from the binding/translocation components. Here the Clostridium botulinum C2 binding/translocation domain was retargeted to neural cell populations by deleting its non-specific binding domain and replacing it with a C. botulinum neurotoxin binding domain. This fusion protein was used to deliver fluorescently labeled payloads to Neuro-2a cells. Intracellular delivery was quantified by flow cytometry and found to be dependent on artificial enrichment of cells with the polysialoganglioside receptor GT1b. Visualization by confocal microscopy showed a dissociation of payloads from the early endosome indicating translocation of the chimeric toxin. The natural Clostridium botulinum C2 toxin was then delivered to human glioblastoma A172 and synchronized HeLa cells. In the presence of the fusion protein, native cytosolic enzymatic activity of the enzyme was observed and found to be GT1b-dependent. This retargeted toxin may enable delivery of therapeutics to peripheral neurons and be of use in addressing experimental questions about neural physiology.
authors.affiliation: Department of Chemical and Biomolecular Engineering, 207 Othmer Hall, University of Nebraska-Lincoln, Lincoln, NE 68588-0643, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Pavlik
authors.affiliation: School of Biological Sciences, 1901 Vine Street, University of Nebraska-Lincoln, Lincoln, NE 68588-0665, USA.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Hruska
authors.affiliation: Department of Chemical and Biomolecular Engineering, 207 Othmer Hall, University of Nebraska-Lincoln, Lincoln, NE 68588-0643, USA.
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Van Cott
authors.affiliation: Department of Chemical and Biomolecular Engineering, 207 Othmer Hall, University of Nebraska-Lincoln, Lincoln, NE 68588-0643, USA.|School of Biological Sciences, 1901 Vine Street, University of Nebraska-Lincoln, Lincoln, NE 68588-0665, USA.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Blum
mesh_terms: D001905:Botulinum Toxins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D016503:Drug Delivery Systems; D006367:HeLa Cells; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D001905:Botulinum Toxins; C096324:botulinum toxin type C