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

Abstract

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.

• Authors: Shihui Liu, Qian Ma, Rasem Fattah, Thomas H Bugge, Stephen H Leppla
• Journal: Oncotarget (Oncotarget) Vol. 8 Issue 39 Pg. 65123-65131 (Sep 12 2017) ISSN: 1949-2553 [Electronic] United States
• PMID: 29029417 (Publication Type: Journal Article)