Abstract
α-Hemolysin (αHL), a β-barrel pore-forming toxin (βPFT), is secreted as a water-soluble monomer by Staphylococcus aureus. Upon binding to receptors on target cell membranes, αHL assembles to form heptameric membrane-spanning pores. We have previously engineered αHL to create a protease-activatable toxin that is activated by site-specific proteolysis including by tumor proteases. In this study, we redesigned αHL so that it requires 2-fold activation on target cells through (i) binding to specific receptors, and (ii) extracellular proteolytic cleavage. To assess our strategy, we constructed a fusion protein of αHL with galectin-1 (αHLG1, αHL-Galectin-1 chimera). αHLG1 was cytolytic toward cells that lack a receptor for wild-type αHL. We then constructed protease-activatable mutants of αHLG1 (PAMαHLG1s). PAMαHLG1s were activated by matrix metalloproteinase 2 (MMP-2) and had approximately 50-fold higher cytolytic activity toward MMP-2 overexpressing cells (HT-1080 cells) than toward non-overexpressing cells (HL-60 cells). Our approach provides a novel strategy for tailoring pore-forming toxins for therapeutic applications.
Highlights
Engineered pore-forming toxins (PFTs) have been extensively studied and applied in biotechnology.[1,2] A few PFTs have been successfully employed in the field of biomolecule sensing, such as DNA sequencing.[2]
The susceptibility of HT-1080 cells to PAMs (PLHL, 9.8%; PLHLG1, 25%; IGHL, 8.0%; and IGHLG1, 19%) was higher than that of HL-60 cells
PLHLG1, 1.9%; IGHL, 0.47%; and IGHLG1, 0.44%), αHL was fused with galectin-1, and divided into two suggesting that matrix metalloproteinase 2 (MMP-2) activated cytotoxicity (Figure 6A). complementary fragments, which are cotranslated in vitro
Summary
Engineered pore-forming toxins (PFTs) have been extensively studied and applied in biotechnology.[1,2] A few PFTs have been successfully employed in the field of biomolecule sensing, such as DNA sequencing.[2]. Terminus or N-terminus to generate cytolytic activity toward target cells by receptor binding and pore-formation.[11] For example, furin activates proaerolysin[8] and proanthrax toxin[9] by cleaving peptides from the C-terminus and N-terminus, respectively, and A Disintegrin and Metalloprotease 17. Pore assembly mechanisms of αHL and its distinct pore properties have been studied using biochemical and genetic approaches.[16−18] αHL lyses rabbit erythrocytes at concentrations 1000-fold lower than those required for human erythrocytes.[19] αHL has been shown to bind to target cell membranes via direct interactions with specific membrane lipids and binding to specific receptors, such as phosphocholine headgroups[20,21] and A Disintegrin and Metalloprotease 10 (ADAM 10).[22] These results account for the high αHL cytolytic activity toward rabbit erythrocytes, which express ADAM 10 highly by comparison with human erythrocytes.[22]
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