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Infection and Immunity, October 2006, p. 5617-5624, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00552-06

Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins

Fang Cai, Carrie B. Adrion, and James E. Keller^*

Laboratory of Bacterial Toxins, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

Received 4 April 2006/ Returned for modification 30 May 2006/ Accepted 28 July 2006

Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn²⁺ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn²⁺-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn²⁺ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn²⁺ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn²⁺ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn²⁺ is not required for toxin entry into neurons, and removal of Zn²⁺ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn²⁺.

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* Corresponding author. Mailing address: Laboratory of Bacterial Toxins, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892. Phone: (301) 402-4418. Fax: (301) 402-2776. E-mail: james.keller{at}fda.hhs.gov.

Editor: J. T. Barbieri

F.C. and C.B.A. contributed equally to this work.

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Infection and Immunity, October 2006, p. 5617-5624, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00552-06

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