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J. Biol. Chem., Vol. 283, Issue 25, 16993-17002, June 20, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/25/16993    most recent M710442200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Wang, J. Articles by Dolly, J. O. PubMed PubMed Citation Articles by Wang, J. Articles by Dolly, J. O. Social Bookmarking                      What's this?

Novel Chimeras of Botulinum Neurotoxins A and E Unveil Contributions from the Binding, Translocation, and Protease Domains to Their Functional Characteristics^*

Jiafu Wang, Jianghui Meng, Gary W. Lawrence, Tomas H. Zurawski, Astrid Sasse, MacDara O. Bodeker, Marcella A. Gilmore, Ester Fernández-Salas, Joseph Francis, Lance E. Steward, K. Roger Aoki, and J. Oliver Dolly¹

From the International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland and Allergan Incorporated, Irvine, California 92623

Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A–G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.

Received for publication, December 21, 2007 , and in revised form, April 4, 2008.

^* This work was supported in part by a Science Foundation Ireland research professorship award (to J. O. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 353-1-700-7757; Fax: 353-1-700-7758; E-mail: oliver.dolly{at}dcu.ie.

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