Epitope tagging of DAB389IL-2: new insights into C-domain delivery to the cytosol of target cells.
The fusion toxin DAB389IL-2 is composed of the catalytic (C) and transmembrane (T) domains of native diphtheria toxin to which human interleukin-2 (IL-2) has been genetically fused (1,2). Following binding to the IL-2 receptor, the fusion toxin is internalized by receptor mediated endocytosis, and upon acidification of the endocytic vesicle, the T domain spontaneously inserts into the membrane, and facilitates the delivery of the C domain to the cytosol (3,4). In order to further study the process by which the C domain is delivered to the target cell cytosol, we genetically fused an eleven amino acid epitope derived from the vesicular stomatitis virus (VSV) G protein to the N-terminal end of DAB389IL-2. The epitope labelled fusion toxin, VSV-G-DAB389IL-2, was found to retain IL-2 receptor specific binding and cytotoxic activity. Target cells were incubated for various times in the presence of VSV-G-DAB389, fixed and then treated with anti-VSV G and FITC conjugated secondary antibody. Laser scanning confocal microscopy was used to determine the location of the fluorescent signal. The VSV-G epitope tagged fusion toxin was found only to be associated with small vesicles that were situated adjacent to the plasma membrane. These results suggest that the C domain of the fusion toxin is associated with an early intracellular compartment and is rapidly delivered to the cytosol. Since channel formation by the T domain is necessary for the delivery of the C domain, it follows that T domain insertion into the membrane also occurs early in the intoxication pathway.
van der Spek
J
J
Evans Department of Clinical Research, University Hospital, Boston University Medical Center, Massachusetts 02118.
Hémard
A
A
Dautry-Varsat
A
A
Boquet
P
P
Murphy
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D001692:Biological Transport; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000276:immunology / Q000493:pharmacokinetics*; D000939:Epitopes; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology / Q000493:pharmacokinetics*; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics*; D014759:Viral Envelope Proteins / Q000276:immunology / Q000493:pharmacokinetics
D004167:Diphtheria Toxin; D000939:Epitopes; C028391:G protein, vesicular stomatitis virus; D007376:Interleukin-2; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C078456:denileukin diftitox