Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A-fragment.
A number of proteins are able to enter cells from the extracellular environment, including protein toxins, growth factors, viral proteins, homeoproteins, and others. Many such translocating proteins, or parts of them, appear to be able to carry with them cargo into the cell, and a basic sequence from the HIV-1 Tat protein has been reported to provide intracellular delivery of several fused proteins. For evaluating the efficiency of translocation to the cytosol, this TAT-peptide was fused to the diphtheria toxin A-fragment (dtA), an extremely potent inhibitor of protein synthesis which normally is delivered efficiently to the cytosol by the toxin B-fragment. The fusion of the TAT-peptide to dtA converted the protein to a heparin-binding protein that bound avidly to the cell surface. However, no cytotoxicity of this protein was detected, indicating that the TAT-peptide is unable to efficiently deliver enzymatically active dtA to the cytosol. Interestingly, the fused TAT-peptide potentiated the binding and cytotoxic effect of the corresponding holotoxin. We made a fusion protein between VP22, another membrane-permeant protein, and dtA, and also in this case we detected association with cells in the absence of a cytotoxic effect. The data indicate that transport of dtA into the cell by the TAT-peptide and VP22 is inefficient.
Falnes PØ
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Wesche
J
J
Olsnes
S
S
D000818:Animals; D001693:Biological Transport, Active / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D004357:Drug Synergism; D015696:Gene Products, tat / Q000235:genetics / Q000502:physiology*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D015497:HIV-1 / Q000502:physiology; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000502:physiology; D006801:Humans; D026563:LDL-Receptor Related Protein-Associated Protein; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism* / Q000502:physiology* / Q000633:toxicity; D010957:Plasmids / Q000138:chemical synthesis / Q000633:toxicity; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D014709:Vero Cells; D015678:Viral Structural Proteins / Q000502:physiology*; D054322:tat Gene Products, Human Immunodeficiency Virus
D002352:Carrier Proteins; D004167:Diphtheria Toxin; D015696:Gene Products, tat; D006023:Glycoproteins; D026563:LDL-Receptor Related Protein-Associated Protein; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C029795:diphtheria toxin fragment A; C073413:herpes simplex virus type 1 protein VP22; D054322:tat Gene Products, Human Immunodeficiency Virus; D006493:Heparin