Peptides fused to the amino-terminal end of diphtheria toxin are translocated to the cytosol.
Diphtheria toxin belongs to a group of toxic proteins that enter the cytosol of animal cells. We have here investigated the effect of NH2-terminal extensions of diphtheria toxin on its ability to become translocated to the cytosol. DNA fragments encoding peptides of 12-30 amino acids were fused by recombinant DNA technology to the 5'-end of the gene for a mutant toxin. The resulting DNA constructs were transcribed and translated in vitro. The translation products were bound to cells and then exposed to low pH to induce translocation across the cell membrane. Under these conditions all of the oligopeptides tested, including three viral peptides and the leader peptide of diphtheria toxin, were translocated to the cytosol along with the enzymatic part (A-fragment) of the toxin. Neither hydrophobic nor highly charged sequences blocked translocation. The results are compatible with a model in which the COOH-terminus of the A-fragment first crosses the membrane, whereas the NH2-terminal region follows behind. The possibility of using nontoxic variants of diphtheria toxin as vectors to introduce peptides into the cytosol to elicit MHC class I-restricted immune response and clonal expansion of the relevant CD8+ cytotoxic T lymphocytes is discussed.
Stenmark
H
H
Institute for Cancer Research, Norwegian Radium Hospital, Oslo.
Moskaug
J O
JO
Madshus
I H
IH
Sandvig
K
K
Olsnes
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012503:Saponins; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014357:Trypsin; D014709:Vero Cells
D004167:Diphtheria Toxin; D015345:Oligonucleotide Probes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012503:Saponins; D014357:Trypsin