Abstract
The Epstein-Barr virus basic leucine zipper transcriptional activator ZEBRA was shown recently to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. We investigated the potential application of the Epstein-Barr virus trans-activator ZEBRA as a transporter protein to facilitate transduction of cargo proteins. Analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization spans residues 170-220. MD efficiently transported reporter proteins such as enhanced green fluorescent protein (EGFP) and beta-galactosidase in several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by beta-galactosidase activity in transduced cells, and no MD-associated cell toxicity was detected. Translocation of MD through the cell membrane required binding to cell surface-associated heparan sulfate proteoglycans as shown by strong inhibition of protein uptake in the presence of heparin. We found that internalization was blocked at 4 degrees C, whereas no ATP was required as shown by an only 25% decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors such as nystatin, chlorpromazine, and wortmannin had no significant impact on MD-EGFP uptake. Only methyl-beta-cyclodextrin inhibited MD-EGFP uptake by 40%, implicating the lipid raft-mediated endocytotic pathway. These data suggest that MD-reporter protein transduction occurs mostly via direct translocation through the lipid bilayer and not by endocytosis. This mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins and renders ZEBRA-MD a promising candidate for therapeutic protein delivery applications.
Highlights
Only the uptake of non-polar molecules Ͻ500 Da into the cell [1]
Fusion of ZEBRA truncations to enhanced green fluorescence protein (EGFP) resulted in a higher yield of soluble purified protein compared with -galactosidase fusions
Because the truncated forms Z6 and Z9 (MD) were shown that cell-surface heparan sulfate proteoglycans (HSPGs) play a key role in the cellular internalto transduce into cells, we further investigated the DNA ization of cell-penetrating peptides (CPPs) [14, 35]
Summary
Only the uptake of non-polar molecules Ͻ500 Da into the cell [1]. Coupling of CPPs to molecules such as small interfering RNA, peptide nucleic acids, and bioactive proteins facilitates delivery of therapeutic agents into live cells [2,3,4,5]. Cells were incubated for 30 min prior to EGFP fusion protein addition in serum-free medium containing the indicated concentrations of individual drugs (20 g/ml heparin, 100 nM wortmannin, 50 g/ml nystatin, 30 M chlorpromazine hydrochloride, and 5–10 mM methyl--cyclodextrin).
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