Comparative study on transduction and toxicity of protein transduction domains

Abstract

Background and purpose: Protein transduction domains (PTDs), such as Tat, antennapedia homeoprotein (Antp), Rev and VP22, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. There is little known, however, about the relative transduction efficacy, cytotoxicity and internalization mechanism of individual PTDs.

Experimental approach: We examined the cargo delivery efficacies of four major PTDs (Tat, Antp, Rev and VP22) and evaluated their toxicities and cell internalizing pathways in various cell lines.

Key results: The relative order of the transduction efficacy of these PTDs conjugated to fluorescein was Rev>Antp>Tat>VP22, independent of cell type (HeLa, HaCaT, A431, Jurkat, MOLT-4 and HL60 cells). Antp produced significant toxicity in HeLa and Jurkat cells, and Rev produced significant toxicity in Jurkat cells. Flow cytometric analysis demonstrated that the uptake of PTD-fluorescein conjugate was dose-dependently inhibited by methyl-beta-cyclodextrin, cytochalasin D and amiloride, indicating that all four PTDs were internalized by the macropinocytotic pathway. Accordingly, in cells co-treated with 'Tat-fused' endosome-disruptive HA2 peptides (HA2-Tat) and independent PTD-fluorescent protein conjugates, fluorescence spread throughout the cytosol, indicating that all four PTDs were internalized into the same vesicles as Tat.

Conclusions and implications: These findings suggest that macropinocytosis-dependent internalization is a crucial step in PTD-mediated molecular transduction. From the viewpoint of developing effective and safe protein transduction technology, although Tat was the most versatile carrier among the peptides studied, PTDs should be selected based on their individual characteristics.

Figure 1

Comparison of the cellular uptake of protein transduction domains (PTDs). FAM-labelled Tat (white column), antennapedia (Antp; hatched column), Rev (grey column) and VP22 (black column) were incubated with six cell lines: HeLa (a), HaCaT (b), A431 (c), Jurkat (d), MOLT-4 (e) and HL60 (f) at 10 μM for 3 h. After trypsin treatment to digest PTDs adsorbed on the cell surface, the PTD-transduced cells were harvested and analysed by flow cytometry. Note that the y axis scales for the adherent cell lines are markedly different from that for the nonadherent cell lines. Data shown are the mean±s.d. of triplicate assays.

Figure 2

Viability of protein transduction domain (PTD)-treated cells. HeLa cells (a) and Jurkat cells (b) were incubated with serially diluted biotin-conjugated Tat, antennapedia (Antp), Rev and VP22 at 37 °C. After 24 h, cell viability was analysed using a WST-8 assay. Data shown are the mean±s.d. of triplicate assays.

Figure 3

Membrane integrity of protein transduction domain (PTD)-treated cells. HeLa cells (a) and Jurkat cells (b) were incubated with serially diluted biotin-conjugated Tat, antennapedia (Antp), Rev and VP22 at 37 °C. After 3 h, the release of lactate dehydrogenase (LDH) was analysed. Data shown are the mean±s.d. of triplicate assays.

Figure 4

Effects of temperature on protein transduction domain (PTD) transduction efficiency. HeLa cells were preincubated at 37 or 4 °C for 1 h prior to adding FAM-labelled PTDs or fluorescein isothiocyanate-labelled transferrin for 3 h. Cells were washed in trypsin and analysed by flow cytometry. Data shown are the mean±s.d. of triplicate assays.

Figure 5

Effects of endocytosis inhibitors on transduction efficiency of protein transduction domains (PTDs). HeLa cells were pretreated with a range of concentrations of (a) methyl-β-cyclodextrin, (b) cytochalasin D or (c) amiloride for 30 min prior to adding FAM-labelled PTDs or fluorescein isothiocyanate-labelled transferrin for 1 h (a and b) or 30 min (c). Cells were washed in trypsin and analysed by flow cytometry. Data shown are the mean±s.d. of triplicate assays.

Figure 6

Intracellular behaviour of protein transduction domain (PTD)-FAM in living cells. HeLa cells were treated with 10 μM PTD-FAM for 3 h. Fluorescence images were acquired using confocal laser scanning microscopy and the signals were merged electronically. The nucleus was counterstained with Hoechst 33342 (blue). From top to bottom: Tat-, antennapedia (Antp)-, Rev- and VP22-FAM. From left to right: FAM (green), nucleus (blue), merged fluorescence and transmission image. Scale bars in each microphotograph indicate 20 μm.

Figure 7

Intracellular behaviour of protein transduction domain (PTD)-Venus in living cells. HeLa cells were treated with 10 μM PTD-Venus alone (a) or 10 μM HA2-Tat (b) for 3 h. Fluorescence images were acquired using confocal laser scanning microscopy and the signals were merged electronically. The nucleus was counterstained with Hoechst 33342 (blue). From top to bottom: Tat-, antennapedia (Antp)-, Rev- and VP22-Venus. From left to right: Venus (green), nucleus (blue), merged fluorescence and transmission image. Scale bars in each microphotograph indicate 20 μm.