Abstract
Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed.
Highlights
We showed the delivery of consensus designed ankyrin repeat proteins (DARPins) to the cytosol of FlpIn 293-EpCAM-BirA cells stably overexpressing the epithelial cell adhesion molecule (EpCAM) [6,9]
Consensus DARPins with two or three internal repeats could be efficiently translocated only upon introducing destabilizing mutations in the DARPin framework, as consensus DARPins were originally engineered for very high stability [6,22]
These findings indicate that all three internal repeats need to be mutated to restore efficient delivery for the very stable NI3 C consensus DARPin, but the intermediate unfolding curves show that a more subtle destabilization is possible, and a particular binder may be rescued by such a small change
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Anthrax toxin is an AB-type bacterial holotoxin of Bacillus anthracis. It comprises two protein components with distinct functions: a catalytically active and toxic A component that relies on the cell-binding and pore-forming B component, protective antigen (PA). There are two toxic A components: lethal factor (LF) and edema factor (EF). Upon cellular binding of PA, furin or furin-like proteases induce oligomerization of PA by cleaving off the 20 kDa domain 1. Three or four LF or EF moieties are able to bind to the oligomerized
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
