An intact transmembrane helix 9 is essential for the efficient delivery of the diphtheria toxin catalytic domain to the cytosol of target cells.
To investigate structure/function relationships involved in the delivery of the diphtheria toxin (DT) catalytic (C) domain to the cytosol of target cells, we have constructed and characterized internal in-frame deletion mutants in the transmembrane (T) domain of the fusion toxin DAB389IL-2. This fusion protein is composed of the C and T domains of DT to which human interleukin-2 (IL-2) has been genetically fused. The mutant fusion toxins were compared to DAB389IL-2 with respect to cytotoxic potency, receptor binding affinity, channel formation in planar lipid membranes, and sensitivity to proteolytic digestion. We demonstrate that genetic fusion of human IL-2 sequences to a diphtheria toxin-related fragment that contains less than full-length transmembrane helix 9 results in a fusion protein that binds to the high affinity IL-2 receptor, but has lost > or = 3 logs of cytotoxic potency and has decreased ability to insert into planar membranes and form stable channels. These observations are consistent with the hypothesis that an intact transmembrane helix 9 is essential for the formation of stable channels that are required for the efficient delivery of the C domain to the cytosol of target cells.
vanderSpek
J
J
Evans Department of Clinical Research, Boston University Medical Center University Hospital, Massachusetts 02118.
Cassidy
D
D
Genbauffe
F
F
Huynh
P D
PD
Murphy
J R
JR
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D001692:Biological Transport; D002451:Cell Compartmentation; D003600:Cytosol / Q000378:metabolism; D004252:DNA Mutational Analysis; D004167:Diphtheria Toxin / Q000235:genetics / Q000494:pharmacology*; D016503:Drug Delivery Systems; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D007376:Interleukin-2 / Q000235:genetics / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D004167:Diphtheria Toxin; D000922:Immunotoxins; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; C078456:denileukin diftitox; D011065:Poly(ADP-ribose) Polymerases