title: Polylysine-mediated translocation of the diphtheria toxin catalytic domain through the anthrax protective antigen pore.
abstract: The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores in the endosomal membrane, which translocate the effector proteins of the toxin to the cytosol. Effector proteins bind to oligomeric PA via their respective N-terminal domains and undergo N- to C-terminal translocation through the pore. Earlier we reported that a tract of basic amino acids fused to the N-terminus of an unrelated effector protein (the catalytic domain diphtheria toxin, DTA) potentiated that protein to undergo weak PA-dependent translocation. In this study, we varied the location of the tract (N-terminal or C-terminal) and the length of a poly-Lys tract fused to DTA and examined the effects of these variations on PA-dependent translocation into cells and across planar bilayers in vitro. Entry into cells was most efficient with ∼12 Lys residues (K12) fused to the N-terminus but also occurred, albeit 10-100-fold less efficiently, with a C-terminal tract of the same length. Similarly, K12 tracts at either terminus occluded PA pores in planar bilayers, and occlusion was more efficient with the N-terminal tag. We used biotin-labeled K12 constructs in conjunction with streptavidin to show that a biotinyl-K12 tag at either terminus is transiently exposed to the trans compartment of planar bilayers at 20 mV; this partial translocation in vitro was more efficient with an N-terminal tag than a C-terminal tag. Significantly, our studies with polycationic tracts fused to the N- and C-termini of DTA suggest that PA-mediated translocation can occur not only in the N to C direction but also in the C to N direction.
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School , 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.
authors.forename: Onkar
authors.initials: O
authors.lastname: Sharma
authors.forename: R John
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D004167:Diphtheria Toxin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D008051:Lipid Bilayers; C030325:anthrax toxin; D011107:Polylysine