EPITOPE TAGGING OF DAB(389)IL-2 - NEW INSIGHTS INTO C-DOMAIN DELIVERYTO THE CYTOSOL OF TARGET-CELLS

The fusion toxin DAB(389) IL-2 is composed of the catalytic (C) and tr ansmembrane (T) domains of native diphtheria toxin to which human inte rleukin-2 (IL-2) has been genetically fused (1,2). Following binding t o the IL-2 receptor, the fusion toxin is internalized by receptor medi ated 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 st udy the process by which the C domain is delivered to the target cell cytosol, we genetically fused an eleven amino acid epitope derived fro m the vesicular stomatitis virus (VSV) G protein to the N-terminal end of DAB(389) IL-2. The epitope labelled fusion toxin, VSV-G-DAB(389) I L-2, was found to retain IL-2 receptor specific binding and cytotoxic activity. Target cells were incubated for various times in the presenc e of VSV-G-DAB(389), fixed and then treated with anti-VSV G and FITC c onjugated secondary antibody. Laser scanning confocal microscopy was u sed to determine the location of the fluorescent signal. The VSV-G epi tope tagged fusion toxin was found only to be associated with small ve sicles that were situated adjacent to the plasma membrane. These resul ts suggest that the C domain of the fusion toxin is associated with an early intracellular compartment and is rapidly delivered to the cytos ol. Since channel formation by the T domain is necessary for the deliv ery of the C domain, it follows that T domain insertion into the membr ane also occurs early in the intoxication pathway.