Anthrax toxin protective antigen facilitates cellular internalization of a heterologous protein fused to short polycationic peptides

The anthrax protective antigen (PA) binds to the edema factor (EF) and lethal factor (LF) of anthrax toxin at the surface of target cells, and delivers these proteins into the cytoplasm. The amino-terminal 255 residues of LF (LFN) comprise the PA-binding domain, and fusion proteins of LFN with the diphtheria toxin catalytic domain (DTA) are readily internalized in the presence of PA. In these studies, we found that when fused to DTA- short polycationic peptides are nearly as efficient as LFN in promoting PA-dependenl translocation. Peptides containing lysine residues facilitated DTA cell entry more efficiently than those with arginine or histidine residues. Peptides containing acidic or hydrophilic residues appeared not to be functional substitutions for LFN. In addition, increasing the number of lysine residues correlated with a stimulation in DTA translocation. In contrast to LFN-mediated entry, a molar excess of free LFN did not block Lys-6-mediated entry of DTA. Although these investigations did not rule out a possible mechanistic role for LFN, they indicate that PA-mediated translocation of a heterologous protein does not require LFN. Furthermore, these studies suggest that binding at the LFN-binding site is not a prerequisite for PA-mediated translocation. These data have implications for the mechanism of toxin entry into cells, and the development of a heterologous protein delivery system.