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.
Highlights
Anthrax lethal toxin has become one of the bestcharacterized protein delivery systems, one that can be modified to selectively target cancer [1,2,3,4,5,6,7,8]
We show that protective antigen (PA)-U2-K397Q, a urokinase-activated PA variant with Lys397 residue replaced by glutamine, and PA-L1-D426K, a matrix metalloproteinase (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
We show that PA-U2-K397Q, a urokinase activated PA variant with Lys397 residue replaced by glutamine, and PA-L1D426K, a matrix metalloproteinase (MMP)-activated PA variant with Asp426 changed to lysine, could not form functional pore when used alone, but were able to complement to form an active PA oligomer when used together both in vitro and in vivo
Summary
Anthrax lethal toxin has become one of the bestcharacterized protein delivery systems, one that can be modified to selectively target cancer [1,2,3,4,5,6,7,8]. The toxin is an A-B type toxin, with protective antigen (PA, the B subunit) as the cell binding moiety and lethal factor (LF, the A subunit) as the effector component. To intoxicate a target cell, PA binds to its specific cell surface receptors, tumor endothelium marker-8 (TEM8) and/or capillary morphogenesis protein-2 (CMG2) [9, 10]. An aminoterminal fragment (PA20) from receptor-bound PA is proteolytically removed by ubiquitous furin or furin-like proteases, resulting in formation of an LFbinding-competent PA oligomer (heptamer or octamer), called PA pre-pore [11]. The acidic environment causes conformational changes of the PA oligomer, resulting in membrane insertion and conversion of the PA pre-pore to a functional pore [12,13,14], which actively translocates LF into the cytosol to exert its cytotoxic effects [11, 15,16,17]
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