title: Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen.
abstract: 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 amino-terminal domain of LF (LFN; 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: (LFN > Lys8 > Lys6 > Lys3). Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive. Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA. 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 LFN blocked the action of LFNDTA, but not of Lys6DTA. 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.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Blanke
authors.forename: J C
authors.initials: JC
authors.lastname: Milne
authors.forename: E L
authors.initials: EL
authors.lastname: Benson
authors.forename: R J
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000008:administration & dosage* / Q000737:chemistry; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D003600:Cytosol; D004167:Diphtheria Toxin / Q000008:administration & dosage*; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism; D010446:Peptide Fragments / Q000008:administration & dosage*; D010455:Peptides / Q000737:chemistry*; D011107:Polylysine / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; C030325:anthrax toxin; C029795:diphtheria toxin fragment A; D000246:Adenosine Diphosphate Ribose; D011107:Polylysine