Entry of diphtheria toxin-protein A chimeras into cells.
Fusion proteins consisting of diphtheria toxin and a duplicated Fc-binding domain of protein A were made in vitro after amplification of the DNA template by the polymerase chain reaction. The fusion proteins bound avidly to Vero cells coated with antibodies. A fusion protein containing full-length diphtheria toxin was toxic at lower concentrations than diphtheria toxin alone, apparently due to more efficient binding. The enzymatic part of the fusion protein was translocated across the surface membrane upon exposure to low pH. Like authentic diphtheria toxin, the fusion protein formed cation selective channels at low pH. Excess amounts of unlabeled diphtheria toxin inhibited formation of pronase-protected fragments derived from radiolabeled fusion protein. Furthermore, conditions that down-regulate the diphtheria toxin receptors reduced the sensitivity of the cells to the fusion protein, supporting the notion that authentic diphtheria toxin receptors are required. At temperatures below 18 degrees C the toxicity of the fusion protein was strongly reduced, whereas there was no temperature block for authentic diphtheria toxin. Brefeldin A protected Vero cells against the fusion protein but not against diphtheria toxin. The results indicate that the diphtheria toxin receptor is required for efficient toxin translocation even under conditions where the toxin is bound by an alternate binding moiety, and they suggest that the intracellular routing of the fusion protein is different from that of diphtheria toxin.
Madshus
I H
IH
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Stenmark
H
H
Sandvig
K
K
Olsnes
S
S
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D020126:Brefeldin A; D003513:Cycloheximide / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D003854:Deoxyribonucleotides; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D015536:Down-Regulation; D004705:Endocytosis; D006367:HeLa Cells; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D013696:Temperature; D014709:Vero Cells
D003517:Cyclopentanes; D003854:Deoxyribonucleotides; D004167:Diphtheria Toxin; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D020126:Brefeldin A; D008985:Monensin; D003513:Cycloheximide