Immunochemical analysis shows all three domains of diphtheria toxin penetrate across model membranes.
Diphtheria toxin undergoes membrane insertion and translocation across membranes when exposed to low pH. In this study, the translocation of the toxin has been investigated by the binding of antibodies to two preparations of model membrane-inserted toxin. In one preparation, toxin was added externally to model membrane vesicles and then inserted by exposure to low pH. In the other preparation, toxin was entrapped in the vesicles at neutral pH, and then inserted by decreasing pH. At neutral pH, externally added antibodies could not bind to entrapped toxin, although they could bind to externally added native toxin. However, after low pH exposure, antibodies against all three toxin domains (catalytic (C), transmembrane (T), and receptor-binding (R)) could bind to entrapped toxin, and also to externally added membrane-inserted toxin. The binding to the entrapped toxin shows that all three domains of the toxin translocate to the trans face of the membrane after exposure to low pH. The observation that antibodies bind to both external and entrapped preparations of toxin after low pH exposure shows that toxin inserts in a mixed orientation. A difference in antibody binding to low pH-treated toxin in which the C domain is folded (Lr' conformation) or unfolded (Lr" conformation) was also observed. An increase in antibody binding to C and T domains in the Lr" conformation relative to binding to the Lr' conformation was found for entrapped toxin, suggesting that more of the C and T domains translocate across the bilayer in the Lr" conformation. These results suggest all three toxin domains insert in the membrane bilayer and participate in translocation in vitro. The C and R domains lack classical transmembrane hydrophobic sequences. However, they possess sequences that have the potential to form membrane-inserting beta-sheets.
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006041:Goats / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers; D051379:Mice / Q000276:immunology; D008961:Models, Structural; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D004167:Diphtheria Toxin; D008051:Lipid Bilayers