title: Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates.
abstract: Multi-domain bacterial protein toxins are being explored as potential carriers for targeted delivery of biomolecules. Previous approaches employing isolated receptor binding subunits disallow entry into the cytosol. Strategies in which catalytic domains are replaced with cargo molecules are presumably inefficient due to co-operation of domains during the endosomal translocation step. Here, we characterize a novel transport vehicle in which cargo proteins are attached to the amino terminus of the full-length botulinum neurotoxin type D (BoNT/D). The intrinsic enzymatic activity of the neurotoxin allowed quantification of the efficacy of cargo delivery to the cytosol. Dihydrofolate reductase and BoNT type A (BoNT/A) light chain (LC) were efficiently conveyed into the cytosol, whereas attachment of firefly luciferase or green fluorescent protein drastically reduced the toxicity. Luciferase and BoNT/A LC retained their catalytic activity as evidenced by luciferin conversion or SNAP-25 hydrolysis in the cytosol of synaptosomes, respectively. Conformationally stabilized dihydrofolate reductase as cargo considerably decreased the toxicity indicative for the requirement of partial unfolding of cargo protein and catalytic domain as prerequisite for efficient translocation across the endosomal membrane. Thus, enzymatically inactive clostridial neurotoxins may serve as effective, safe carriers for delivering proteins in functionally active form to the cytosol of neurones.
authors.affiliation: Institute für Biochemie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Bade
authors.forename: Andreas
authors.initials: A
authors.lastname: Rummel
authors.forename: Clemens
authors.initials: C
authors.lastname: Reisinger
authors.forename: Tino
authors.initials: T
authors.lastname: Karnath
authors.forename: Gudrun
authors.initials: G
authors.lastname: Ahnert-Hilger
authors.forename: Hans
authors.initials: H
authors.lastname: Bigalke
authors.forename: Thomas
authors.initials: T
authors.lastname: Binz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000235:genetics*; D019274:Botulinum Toxins, Type A / Q000235:genetics* / Q000506:poisoning; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D049408:Luminescent Agents; D051379:Mice; D009465:Neuromuscular Agents / Q000506:poisoning; D010791:Phrenic Nerve / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000506:poisoning; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000506:poisoning
chemical_list: D049408:Luminescent Agents; D009465:Neuromuscular Agents; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C096325:botulinum toxin type D; D008156:Luciferases; D013762:Tetrahydrofolate Dehydrogenase; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A