Research Paper
Subject Category: Biopharmaceuticals
British Journal of Pharmacology (2008) 153, 1143–1152; doi:10.1038/sj.bjp.0707678; published online 28 January 2008
Comparative study on transduction and toxicity of protein transduction domains
T Sugita1,2, T Yoshikawa1, Y Mukai1,2, N Yamanada1,2, S Imai1,2, K Nagano1,2, Y Yoshida1,3, H Shibata1, Y Yoshioka1,4, S Nakagawa2, H Kamada1,4, S-i Tsunoda1,4 and Y Tsutsumi1,3,4
- 1Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NIBIO), Ibaraki, Osaka, Japan
- 2Department of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan
- 3Department of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan
- 4The Center for Advanced Medical Engineering and Informatics, Osaka University, Suita, Osaka, Japan
Correspondence: Dr S-i Tsunoda, Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NIBIO), 7-6-8 Saito-Asagi, Ibaraki, Osaka 597-0085, Japan. E-mail: tsunoda@nibio.go.jp
Received 9 November 2007; Accepted 4 December 2007; Published online 28 January 2008.
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-
-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.
Keywords:
protein transduction domains, Tat, antennapedia, Rev, VP22, macropinocytosis
Abbreviations:
PTD, protein transduction domain; Antp, antennapedia
