Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen

The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets, Genetically fusing the aminoterminal domain of LF (LF(N); residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol. We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery, Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis. The efficiency of translocation was dependent on tract length: (LF(N) > Lys(8) > Lys(6) > Lys(3)). Lys(6) was approximate to 100-fold more active than Arg(6) or His(6), whereas Glu(6) and (SerSerGly)(2) were inactive. Arg(6)DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys(6)DTA. The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs, Excess free LF(N) blocked the action of LF(N)DTA, but not of Lys(6)DTA, This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site, Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells.