PMID:15584922

Citation	Bade, S, Rummel, A, Reisinger, C, Karnath, T, Ahnert-Hilger, G, Bigalke, H and Binz, T (2004) Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates. J. Neurochem. 91:1461-72
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.
Links	PubMed Online version:10.1111/j.1471-4159.2004.02844.x
Keywords	Amino Acid Sequence; Animals; Botulinum Toxins/genetics; Botulinum Toxins, Type A/genetics; Botulinum Toxins, Type A/poisoning; Cells, Cultured; Cytosol/metabolism; Gene Transfer Techniques; Green Fluorescent Proteins/genetics; Luciferases/genetics; Luminescent Agents; Mice; Neuromuscular Agents/poisoning; Phrenic Nerve/drug effects; Rats; Rats, Wistar; Recombinant Fusion Proteins/metabolism; Recombinant Fusion Proteins/poisoning; Tetrahydrofolate Dehydrogenase/genetics; Tetrahydrofolate Dehydrogenase/poisoning
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CLOBO:BXD		GO:0044164: host cell cytosol	ECO:0000314:		C		Figure 6a. Shows that Baf A1pre-treatment drastically decreased cleavage of synaptobrevin 2 by BoNT/D and GFP-BoNT/D evidencing that both proteins pass through acidic compartments en route delivering the catalytic domain to the cytosol. Figure 6b. Shows that GFP-BoNT/D co-localizes with transferrin	complete CACAO 11142

References

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