|Pellett, Sabine -|
|Tepp, William -|
|Band, Philip -|
|Johnson, Eric -|
|Ichtchenko, Konstantin -|
Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 26, 2011
Publication Date: February 1, 2011
Citation: Pellett, S., Tepp, W.H., Stanker, L.H., Band, P.A., Johnson, E.A., Ichtchenko, K. 2011. Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A. Biochemical and Biophysical Research Communications. 405:673-677. Interpretive Summary: Botulism is a serious, often fatal neuroparalytic disease in humans and animals caused by a protein toxin (botulinum toxin, BoNT) produced by the bacterium Clostridium botulinum. BoNT is considered the most toxic biological toxin known. Because of its high toxicity, the need for a long recovery period requiring extensive treatment, and the ease of producing BoNT, it is considered a class A bioterrorism agent. These same properties make the toxin difficult to study. In this report we describe development of an atoxic form of the molecule that is shown to be 100,000 times less toxic resulting in the Center for Disease Control and Prevention removal of this atoxic form from the select-agent list. The availability of this atoxic derivative will allow for detailed studies of the movement of the toxin in exposed animals, furthering our understanding of this foodborne toxin.
Technical 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 150kDa 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 demonstrate 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.