Thermodynamic Stability Is a Strong Predictor for the Delivery of DARPins to the Cytosol via Anthrax Toxin

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.

HOPS-Dependent Endosomal Escape Demands Protein Unfolding

“…We find that the efficiency of ZF5.3-mediated protein delivery to the cytosol is highest when the protein cargo readily unfolds under physiological conditions. Similar findings that low thermodynamic stability enhances intracellular delivery have been reported for toxin-mediated delivery of DARPins and even cytosolic penetration of antisense oligonucleotides, suggesting that the relationship between folding and endosomal escape may apply broadly to the passage of therapeutic macromolecules across cellular membranes.…”

DARPins bind their cytosolic targets after having been translocated through the protective antigen pore of anthrax toxin

“…Previously, we showed that the delivery of various DARPin cargoes to the cytosol of Flp-In 293-EpCAM-BirA cells is dependent on thermodynamic stability, probably due to the required unfolding step when traversing the pore. We carried out these experiments using an assay employing cells stably overexpressing the epithelial cell adhesion molecule (EpCAM) as the receptor to be targeted, and E. coli biotin ligase (BirA), stably expressed in the cytosol, to biotinylate the avi tag attached to the cargo once it has reached the cytosol 25 . We thus confirmed the presence of cytosolic DARPin cargoes, and thereby the successful translocation, by utilizing the BirA assay previously developed 24 .…”

Targeting the Inside of Cells with Biologicals: Toxin Routes in a Therapeutic Context

“…Unfolding of the cargo is required for delivery through anthrax pores. The ability to translocate to the cytosol was compared for DARPins with different thermodynamic stabilities and as a conclusion, delivery through anthrax pores was suggested to be probably limited to molecules that unfold easily [ 141 ]. High stability was not limiting for delivery of Pseudomonas exotoxin A-based cargoes in the study by Verdurmen et al from 2015 [ 118 ], although unfolding is also required for the route of Pseudomonas exotoxin A.…”

“…Although feasible in principle, the approach is limited to a selection of molecules that have to fulfil multiple criteria that are essential for an effect after passing toxin routes, for example, certain properties of cargo molecules as discussed previously [ 141 ]. This considerably narrows down the choice of cargo molecules.…”