Research Papers:

Anti-tumor activity of anthrax toxin variants that form a functional translocation pore by intermolecular complementation

Shihui Liu _, Qian Ma, Rasem Fattah, Thomas H. Bugge and Stephen H. Leppla

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Oncotarget. 2017; 8:65123-65131. https://doi.org/10.18632/oncotarget.17729

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Shihui Liu1,2, Qian Ma2, Rasem Fattah2, Thomas H. Bugge1 and Stephen H. Leppla2

1Proteases and Tissue Remodeling Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA

2Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Correspondence to:

Shihui Liu, email: [email protected]

Thomas H. Bugge, email: [email protected]

Stephen H. Leppla, email: [email protected]

Keywords: anthrax protective antigen, lethal toxin, MMPs, protein delivery, tumor targeting

Received: February 07, 2017     Accepted: April 16, 2017     Published: May 09, 2017


Anthrax lethal toxin is a typical A-B type protein toxin secreted by Bacillus anthracis. Lethal factor (LF) is the catalytic A-subunit, a metalloprotease having MEKs as targets. LF relies on the cell-binding B-subunit, protective antigen (PA), to gain entry into the cytosol of target cells. PA binds to cell surface toxin receptors and is activated by furin protease to form an LF-binding-competent oligomer-PA pre-pore, which converts to a functional protein-conductive pore in the acidic endocytic vesicles, allowing translocation of LF into the cytosol. During PA pre-pore-to-pore conversion, the intermolecular salt bridge interactions between Lys397 and Asp426 on adjacent PA protomers play a critical role in positioning neighboring luminal Phe427 residues to form the Phe-clamp, an essential element of the PA functional pore. This essential intermolecular interaction affords the opportunity to create pairs of PA variants that depend on intermolecular complementation to form a functional pore. We have previously generated PA variants with furin-cleavage site replaced by substrate sequences of tumor-associated proteases, such as urokinase or MMPs. Here we show that PA-U2-K397Q, a urokinase-activated PA variant with Lys397 residue replaced by glutamine, and PA-L1-D426K, a MMP-activated PA variant with Asp426 changed to lysine, do not form functional pores both in vitro or in vivo unless they are used together. Further, the mixture of PA-U2-K397Q and PA-L1-D426K displayed potent anti-tumor activity in the presence of LF. Thus, PA-U2-K397Q and PA-L1-D426K form a novel intermolecular complementation system with toxin activation relying on the presence of two distinct tumor-associated proteases, i.e., urokinase and MMPs.

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