Journal Article

Comparative study on transduction and toxicity of protein transduction domains

Yamanada, N, Nakagawa, S, Shibata, H, Yoshioka, Y, Mukai, Y, Yoshikawa, T, Tsunoda, S‐i, Tsutsumi, Y, Yoshida, Y, Kamada, H, Sugita, T, Imai, S, Nagano, K

2008

Overview

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‐β‐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. British Journal of Pharmacology (2008) 153, 1143–1152; doi:10.1038/sj.bjp.0707678; published online 28 January 2008

• Publisher: Blackwell Publishing Ltd,Nature Publishing,Nature Publishing Group
• Subject: Amiloride - administration & dosage / Amiloride - pharmacology / antennapedia / Antennapedia Homeodomain Protein - adverse effects / Antennapedia Homeodomain Protein - metabolism / beta-Cyclodextrins - administration & dosage / beta-Cyclodextrins - pharmacology / Biological and medical sciences / Cell Line, Tumor / Cytochalasin D - administration & dosage / Cytochalasin D - pharmacology / Flow Cytometry / Fluoresceins - metabolism / Gene Products, rev - adverse effects / Gene Products, rev - metabolism / Gene Products, tat - adverse effects / Gene Products, tat - metabolism / Humans / macropinocytosis / Medical sciences / Pharmacology. Drug treatments / Pinocytosis - physiology / protein transduction domains / Protein Transport / Research Papers / Rev / Tat / Viral Structural Proteins - adverse effects / Viral Structural Proteins - metabolism / VP22