Uniformly Cationized Protein Efficiently Reaches the Cytosol of

Sep 18, 2012 - and incubated with anti-GFP rabbit polyclonal antibody. (Invitrogen, USA) ... (Fuji Film, Japan) and the intensity was quantified using...
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Uniformly Cationized Protein Efficiently Reaches the Cytosol of Mammalian Cells Midori Futami,† Yasuyoshi Watanabe,§ Takashi Asama,§ Hitoshi Murata,‡ Hiroko Tada,§ Megumi Kosaka,§ Hidenori Yamada,§ and Junichiro Futami*,§ †

Department of Biomedical Engineering, Faculty of Engineering, Okayama University of Science, 1-1 Ridaicho, Okayama 700-0005, Japan § Department of Medical Bioengineering Science, Graduate School of Natural Science and Biotechnology, Okayama University, Japan ‡ Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikatachou, Okayama 700-8558, Japan S Supporting Information *

ABSTRACT: Protein cationization techniques are powerful protein transduction methods for mammalian cells. As we demonstrated previously, cationized proteins with limited conjugation to polyethylenimine have excellent ability to enter into cells by adsorption-mediated endocytosis [Futami, J., et al. (2005) J. Biosci. Bioeng. 99, 95−103]. In this study, we show that proteins with extensive and uniform cationization covering the protein surface reach the cytoplasm and nucleus more effectively than proteins with limited cationic polymers or proteins that are fused to cationic peptides. Although extensive modification of carboxylates results in loss of protein function, chicken avidin retains biotin-binding ability even after extensive amidation of carboxylates. Using this cationized avidin carrier system, the protein transduction ability of variously cationized avidins was investigated using biotinylated protein as a probe. The results revealed that cationized avidins bind rapidly to the cell surface followed by endocytotic uptake. Small amounts of uniformly cationized avidin showed direct penetration into the cytoplasm within a 15 min incubation. This penetration route seemed to be energy dependent and functioned under cellular physiological conditions. A biotinylated exogenous transcription factor protein that penetrated cells was demonstrated to induce target gene expression in living cells.

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that does not seriously affect protein function.13 We successfully transduced the proteins S100C, anti-S100C antibody,14 RNase A,13 p53,15 the N-terminal domain (1− 132 amino acids) of simian virus 40 large T-antigen (SVLTN),16 and β-catenin 17 by PEI cationization. Studies on the protein transduction of cationized RNase are a quantitative model for assessing the expression level of exogenously transduced functional protein in the cytosol, because degradation of cytosolic RNA is related to its cytotoxicity. In a comparison of the cytotoxicity of limited PEI-cationized RNase A (PEI-RNaseA) and extensively ethylenediamine-cationized RNase A (ED-RNaseA), ED-RNaseA showed 10-fold higher cytotoxicity, in spite of a 10-fold weaker enzymatic activity than PEI-RNaseA.11−13 These results suggest that ED-cationized RNase A showed more efficient cytosolic delivery than PEI-cationized RNase A. Because ethylenediamine protein cationization might be accomplished by extensive amidation of carboxyl groups, the functions of most proteins would be lost by the modification. Thus, this method

INTRODUCTION Cationic proteins electrostatically bind to the cell surface, which possesses abundant anionic molecules (e.g., glycoproteins). These proteins internalize into cells via adsorption-mediated endocytosis.1,2 Artificially cationized proteins are also internalized into cells by a similar route. On the basis of this concept, short cationic peptides known as protein transduction domains (PTD) are now widely exploited as delivery domains for moving molecular cargo into the cytoplasm and/or nucleus of living target cells.3−5 Because protein transduction technology has the potential for use in a variety of applications, more reliable technology is needed. Recent reports have demonstrated the successful generation of induced pluripotent stem (iPS) cells from fibroblasts by protein transduction of four PTD-fused reprogramming factor proteins (Oct4, Sox2, Klf4, and c-Myc). This means that protein transduction technology could be an alternative method for artificial regulation of cellular function in regenerative medicine.6,7 Chemical protein cationization by amidation of carboxyl groups with various diamines (e.g., ethylenediamine) or polyethylenimine (PEI) is a powerful protein transduction technology.8−13 PEI in particular provides a cationic domain sufficient for protein transduction with limited modification © XXXX American Chemical Society

Received: January 19, 2012 Revised: August 26, 2012

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derivatives were purified by gel filtration on a Sephadex G-25 medium column (GE Healthcare, England, 1.6 × 36 cm) in PBS. The extent of modification of avidin derivatives was analyzed by mass spectrometry (Voyage-DE PRO; PerSeptive Biosystems, USA). Biotinylated eGFPNuc (biotin-eGFPNuc) was prepared by mixing purified eGFPNuc and GAL4-VP16 with Sulfo-NHS-SS-biotin (Pierce Biotechnology, USA) at a molar ratio of 1:3. SVLT-N was also biotinylated with biotinHPDP (Pierce Biotechnology, USA) at a molar ratio of 1:10 to give biotin-SVLT-N. After a 2 h incubation at room temperature, the biotinyl-proteins were purified by a PD10 desalting column (GE Healthcare, England) using PBS as an elution buffer. Cell Culture and Protein Transduction. HeLa S3 cells were cultured in RPMI1640 medium supplemented with 10% FBS and 70 μg/mL kanamycin. HeLa S3 cells were grown in ø35 mm dishes in growth medium at 37 °C. After 2 day culture, the medium was changed to Opti-MEM I reduced-Serum medium (Invitrogen, USA). After a 24 h culture, protein transduction experiments were performed as follows. Biotinylated protein and avidin derivative were premixed to form a complex at a molar ratio of 1:2 for 10 min at room temperature. The complex was added to culture medium at a concentration of biotinylated protein of 100 nM. TAT-eGFP was directly added to culture medium at a concentration of 100 nM. After incubation for the indicated time, cells were washed with PBS and observed under a confocal laser-scanning microscope (model LSM 510; Carl Zeiss, Germany). In the flow cytometric analysis, protein transduced cells were treated with or without 20 mM of DTT in PBS for 15 min at 37 °C, trypsinized, and analyzed with FACSCalibur (Becton Dickinson, USA). Western Blot Analysis. Avidin/biotinyl−protein complex and its dissociated components within cell were analyzed as follows. To assess total cellular components in avidin/biotinyl− protein complexes, samples were incubated at 95 °C for 5 min with 1% SDS and 50 mM dithiothreitol (DTT), and subjected to SDS-PAGE. Protein samples were also prepared in 1% SDS at 25 °C without DTT to analyze protein dissociated from the avidin/biotin−protein complex. Because avidin protein is stable and conserves its tetrameric conformation and biotin binding activity under the latter condition,18,19 only the dissociated protein was detected according to its molecular weight by SDSPAGE analysis. Since the biotinylated proteins used in this study have a disulfide bond in the linker region and are sensitive to cytosolic reduced glutathione, dissociated protein in cell lysates in the absence of DTT were assumed to be protein transduced into the cytosol. Using this assay, the cellular localization of biotinylated-protein transduced with cationized avidin was analyzed as follows. Cells treated with the avidin/ biotin protein complex were washed with PBS and then harvested by cell scraper in PBS. Cells were lysed with lysis buffer containing 10 mM Tris-HCl at pH 7.9, 1% Triton X-100, 150 mM NaCl, 25 mM iodoacetamide, and Complete Protease Inhibitor Cocktail (Roche Diagnostics, Switzerland) and clarified by centrifugation. Total protein concentration was determined by Bradford protein assay (Bio-Rad Laboratories, USA). To capture biotinylated protein completely, 1 μg of native avidin per 20 μg of total cellular protein was added to cell lysate before addition of Sample Buffer Solution (Wako Pure Chemical Industries) with or without 0.1 M DTT. 20 μg or 2.5 μg of cellular protein was separated by SDS-PAGE under nonreducing or reducing condition, and proteins were transferred to PVDF membranes. Membranes were blocked

is applicable only to robust proteins. Avidin from chicken egg white is a robust protein.18,19 Accordingly, we expected that extensively diamine-cationized avidin can retain biotin-binding function. Native avidin can have a highly cationic nature (pI > 10) and has cell penetrating activity.20 We already demonstrated that PEI cationized avidin promotes its cell-penetration activity.21 Streptavidin is also a robust protein and has the potential to be a biotinylated protein transduction carrier,21−23 but its pI is around 6.1. Because we have demonstrated that the cell penetrating activity of diamine-cationized RNase A appeared to relate to a cationic charge dependent manner,12 the cationic nature of a native protein is advantageous for creation of a cationized carrier. In this study, we prepared variously cationized chicken avidin derivatives to generate a superior carrier and assessed their ability for cytosolic delivery of biotinylated proteins.

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EXPERIMENTAL PROCEDURES Materials. The preparation of eGFPNuc, which has a Histag sequence at the N-terminus and three tandem repeats of the nuclear localization signal (NLS) of the simian virus 40 large Tantigen at the C-terminus of eGFP, was as described previously.21 Preparations of His-tagged TAT-eGFP and SVLT-N fusion protein were described previously.13,16 PEI600, with a molecular mass of 600, was from Wako Pure Chemical Industries (Japan), and PEI300 was donated by Nippon Shokubai Co., Ltd. (Japan). 1,3-Diaminopropane and N-methyl-1,3-diaminopropane were from Tokyo Chemical Industry (Japan). N,N-Dimethyl-1,3-diaminopropane was from Sigma-Aldrich (USA). 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was from Pierce Biotechnology (USA). Preparation of Gal4-VP16 Fusion Protein. The DNAbinding domain (N-terminal 147 amino acids) of the yeast transcriptional activator GAL4 was genetically fused with the Cterminus of the activation domain of the VP16 protein herpes simplex virus (HSV) (C-terminal 130 amino acids) through a linker sequence with a His-Tag (GGSHHHHHHGGS) to make a GAL4-VP16 artificial transcription factor. cDNA encoding GAL4-VP16 was cloned as an Nde I/Not I fragment into the polylinker of pET-21b (Novagen, USA). The fusion protein was expressed in Escherichia coli BL21(DE3) (Novagen) and purified by TALON Metal Affinity Resin (TAKARA BIO, Japan) according to the method described previously.24 Chemical Modification. Coupling reactions of proteins with PEI or diamine by EDC were carried out as described previously.11,13 For preparation of PEI600-avidin, chicken avidin (Nakarai Tesque, Japan) was dissolved at 1 mg/mL in PEI600 (180 mg/mL, pH adjusted to 5.0 with HCl), and the coupling reaction was started by addition of EDC at a final concentration of 0.1 mg/mL. For preparation of diamine modified avidins, avidin was dissolved at 1 mg/mL in various diamine solutions (2 M, pH adjusted to 5.0 with HCl), and the coupling reaction was initiated by addition of EDC at a final concentration of 27.3 mg/mL. For preparation of PEI600 and diamine-modified avidin (PEI600, 1°-avidin), PEI600-avidin was further modified with 1,3-diaminopropane using EDC as described above. After overnight incubation at room temperature, modified avidins were treated with hydroxylamine for overnight at room temperature.25 Coupling reactions of avidin with PEI300 were started by addition of EDC at a final concentration of 0.5, 2.0, 10, 20, or 50 mg/mL. Avidin B

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and incubated with anti-GFP rabbit polyclonal antibody (Invitrogen, USA), anti-β-tubulin rabbit polyclonal antibody (Cell Signaling Technology, USA), or anti-SV40 large-T antigen antibody (Ab-1; Calbiochem, USA) for 1 h at room temperature, and detected by horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). Western blot signal was exposed for 600 s by LAS-4000 mini (Fuji Film, Japan) and the intensity was quantified using the Multi-Gauge software. To calculate eGFPNuc concentration in the HeLa S3 cell, the cellular volume was measured by flow cytometer Cell Lab Quanta SC (Beckman Coulter, USA). Reporter Gene Assay. The HeLa Luciferase Receptor (HLR) cell line, which has a GAL4-binding element-TATA sequence followed by the gene encoding firefly luciferase, was from Stratagene (USA). Biotinylated GAL4-VP16 was mixed with an avidin derivative at a molar ratio of 1:2 to form a complex. HLR cells in Opti-MEM I Reduced-Serum medium were treated with this complex at a GAL4-VP16 concentration of 1 μM. After cultivation for 4.5 h, cells were collected and luciferase activity was measured by the Steady-Glo Luciferase Assay System (Promega, USA) according to the manufacturer’s instructions.

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RESULTS Visualization of Transduction by Chemically Cationized Avidin and TAT-eGFP. To assess cationic functional groups for protein transduction, avidin was cationized with 1,3diaminopropane, N-methyl-1,3-diaminopropane, or N,N-dimethyl-1,3-diaminopropane, by a carbodiimide reaction to generate 1°-, 2°-, or 3°-avidins, respectively (Figure 1a). The average numbers of conjugated carboxyl groups per monomeric avidin were evaluated to as 10.1 for 1°-avidin, 8.9 for 2°, and 10.0 for 3° by MALDI-TOF mass spectrometric analysis (Figure S1, Table 1). For PEI600-avidin, the modification ratio was estimated from the shifted band of the PEI-cationized monomer unit of avidin by SDS-PAGE (Figure S2, Table 1). At these conditions, part of the avidin derivatives showed limited intermolecular cross-linking reaction (Figure S2). The net charges were calculated to be +102 for 1°, +91 for 2°, +100 for 3°, and +51 for PEI600 avidins, per tetramer molecule indicating that all avidin derivatives were highly cationic. Protein transduction ability of these cationized avidins was visualized using biotinylated eGFPNuc (biotin-eGFPNuc) as a probe. The biotinylation level of eGFPNuc was controlled to be less than one biotin in each eGFPNuc (Figure S4 left panel), because multiply biotinylated protein forms a polymerized complex with avidin unsuitable for protein transduction. As shown in Figure 1b (laser scanning microscopy after 90 min incubation) and c (flow cytometric analysis after 15 min incubation), cationized avidin/biotin-eGFPNuc complex showed uniformly rapid binding to HeLa S3 cells, whereas both itself and native avidin/biotin-eGFPNuc complex showed weaker binding ability (Figure 1b). To estimate the quantity of eGFPNuc in the intracellular compartment, eGFPNuc on the cell surface was removed by reduction of disulfide bonds in the biotin spacer with 20 mM DTT for 15 min incubation (Figure S3). After treating cells with 20 mM DTT, the fluorescence intensity was drastically decreased because the exposed disulfide bond in the spacer of the biotin was reduced, removing eGFPNuc from the cell surface (Figure 1d). However, fluorescence from the eGFPNuc internalized within 15 min was retained. PEI600-avidin seemed to be the best carrier of biotin-eGFPNuc. Time-course analysis revealed that both the

Figure 1. Protein transduction of eGFPNuc into HeLa S3 cells by chemically cationized avidin or TAT cationic peptide. (a) Chemical cationization of avidin was performed by mixing with EDC in the presence of 1,3-diaminopropane, N-methyl-1,3-diaminopropane, N,Ndimethyl-1,3-diaminopropane, or PEI600. Avidin derivatives and biotin-eGFPNuc complex (at a molar ratio of 1:2) or TAT-eGFP was added to culture media to 100 nM of eGFP per experiment. (b,f) After a 90 min incubation, eGFP were visualized by confocal laser scanning microscopy. (c) Total eGFP protein including the surface adsorbed and internalized fraction after 15 min were analyzed by flow cytometer. (d) Time-course flow cytometry of biotin-eGFPNuc protein transduction using 1°-avidin carrier with or without reduction. (e) Internalized biotin-eGFPNuc by chemically cationized avidin after 15 min incubation was assessed after reduction of the disulfide bond in the biotinylated spacer by treatment of cell surface with 20 mM DTT to dissociate surface-adsorbed fraction. The dashed line indicates the cell adhesion area. The arrow heads indicated fluorescence-detected areas without cell adhesion.

Table 1. Properties of Cationized Avidin Derivatives wild type avidin Average number of modified carboxyl groups Total net-charge (tetramer) a b

1°avidin

2°avidin

3°avidin

PEI600avidin

PEI6001°avidin

-

10.3a

8.9a

10.0a

0.5b

N.D.

+20

+102

+91

+100

+51

N.D.

Average value per avidin monomer estimated by mass spectrometry. Value per avidin monomer estimated by SDS-PAGE.

adsorption and internalization levels of the cationized avidin/ eGFPNuc complex were at a maximum after 30 min (Figure C

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Figure 2. Schematic model of internalized biotin-conjugated protein with a disulfide bond in the biotinylated spacer, in complex with cationized avidin carrier. Biotin-protein and cationized avidin complexes outside the cell, on the cell surface and in endocytic vesicles. Complexes dissociated after entry of the complex into the cytosolic reducing environment.

As shown in Figure 3a, eGFPNuc reaching cytosol was detected in cell lysates after incubation with cationized avidin/ biotin-eGFPNuc complex for 15 min at 37 °C. The intensity of each bands were quantified using Multi-Gauge software (Fuji Film). To calculate the quantity of eGFPNuc protein, known quantities of purified eGFPNuc were used as standards (Figure S5, S6), and their signal intensity were corrected by β-tubulin signal as loading control on the reprobing membrane. As shown in Figure 3b, 1°-avidin and 2°-avidin showed superior cytosolic transduction of biotin-eGFPNuc, whereas PEI-avidin showed highest efficiency of total biotin-eGFPNuc transduction to whole cell (including endosome, cell surface, and cytosolic fraction). The cytosolic concentration of eGFPNuc delivered with 1°-avidin was estimated to be 4.4 × 10−8 M, when the cytosolic volume was assumed as cellular volume (3.4 × 10−12 L, analyzed with flow cytometer). It also appeared that about 2.5% of eGFPNuc included in whole cell was delivered by 1°avidin to cytosol and or nucleus. Next, we tested the transduction efficiency of 1°-avidin/biotin-eGFPNuc under various conditions of cell density, indicating that the amount of eGFPNuc delivered to the cytosol under the three cell density conditions was nearly equivalent (Figures 3d, S7, S8). This result suggested that cationized avidin carriers can deliver biotinylated proteins into the cytosol at both logarithmic and stationary growth phases. We also showed temperature dependency of eGFPNuc transduction with 1°-avidin. eGFPNuc delivered to the cytosol with 1°-avidin was not detectable when cells were incubated at 4 °C (Figure 3e upper panel), while binding of 1°-avidin/ biotin-eGFPNuc to cells for 15 min at either 4 or 37 °C was equal (Figure 3e upper panel). This penetration of cationized protein seemed to be an energy-dependent physiological process. Quantitative Estimation of SVLT-N Transduced to the Cytoplasm by Cationized Avidin. Using an oncogene product of the N-terminal domain of simian virus 40 large-T antigen (SVLT-N), we further compared biotinylated protein transduction efficiency between diamine-cationized avidin and PEI-cationized avidin. Previously, we demonstrated that BiotinHPDP modified SVLT-N protein is successfully transduced into normal fibroblasts using PEI600-avidin as a carrier, and is functional.16 Because Biotin-HPDP was conjugated with cysteine side-chain of the protein surface through a disulfide bond, and biotin−avidin complex is stable even in SDS (Figure S9), biotinylated SVLT-N reaching the cytosol with cationized avidins could be assessed from the monomeric band after

1e). To compare protein transduction efficiency, TAT, a widely used short cationic peptide protein transduction domain fused with eGFP (TAT-eGFP, net-charge: +1) was used. By microscopic analysis, TAT-eGFP-treated cells showed clearly higher fluorescence intensity than cationized avidin/biotineGFPNuc-treated cells (Figure 1f). However, by high magnification analysis, we found differences between the cellular shape and fluorescence of TAT-eGFP. The strong fluorescence came from the surface of the glass where the cells had adhered (Figure 1f). Flow cytometric analysis supported this observation, since most of the TAT-eGFP was not associated with trypsinized HeLa S3 cells (Figure 1c). These results indicated that TAT-eGFP mainly bound to the extracellular matrix. However, chemically cationized avidin showed rapid internalization into cells within 30 min. Quantitative Analysis of eGFPNuc Internalized into the Cytoplasm. For cellular proteins that perform functions in the cytoplasm, protein transduction techniques require efficient cytoplasmic delivery of exogenous protein into living cells. To assess the amount of eGFPNuc that reached the cytoplasm or nucleus, whole lysates of HeLa S3 cells treated with avidin/ biotin-eGFPNuc complexes were subjected to SDS-PAGE under nonreducing and reducing conditions, and eGFPNuc was detected by Western blotting. Because avidin is extraordinarily stable, it has a tetramer conformation that binds biotin even in the presence of SDS and hardly moved in polyacrylamide gel, if the sample is not heated.18 Using this stability, eGFPNuc-biotin complexed with avidin could discriminate as a broad band with high molecular mass on SDS-PAGE under nonreducing/ nonheating conditions (Figure S4 left panel). The cytosol of living cells is a reducing environment,26 and we demonstrated that accessible disulfide bonds are easily reduced in living cytosol.15,16,21 Additionally, we confirmed that the disulfide bond in the biotinylated spacer was uncleavable in cell lysate (Figure S4 right panel). Thus, dissociated eGFPNuc observed in the HeLa S3 cell lysate can be assumed to reflect the amount in the cytosol (Figure 2). In other words, when avidin/biotineGFPNuc-treated HeLa S3 lysates were separated by SDSPAGE under nonreducing conditions, monomeric eGFPNuc detected by Western blotting was presumed to be delivered from eGFPNuc that had reached cytoplasm and was dissociated from cationized avidins in the cytoplasmic reducing environment. By SDS-PAGE under reducing conditions, the eGFPNuc monomer band indicated eGFPNuc molecules in whole cells (including the cell surface, endosomal vesicles, cytosol, and nucleus). D

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Figure 4. Evaluation of cytosolic protein transduction efficiency of SVLT-N by cationized avidin. All experiments were conducted by addition of 100 nM concentration of biotinylated cationized avidin/ biotin-SVLT-N complex to HeLa S3 cells, with incubation at 37 °C. Cell lysates were subjected to SDS-PAGE under nonreducing and nonheating conditions, and detected by anti-SV40 large-T antigen antibody. (a) Comparison of cytosolic protein transduction by 1°avidin or PEI600-avidin at the indicated incubation times. (b) Comparison of protein transduction efficiency with 1°, 2°, or 3°avidin for 15 min. (c) Comparison of protein transduction efficiency with variously cationized avidins with PEI300 for 15 min.

Next, we prepared variously cationized avidins with PEI whose molecular mass was 300 (PEI300). Coupling reactions of avidin with PEI300 were started by addition of EDC at a final concentration of 0.5, 2.0, 10, 20, or 50 mg/mL, resulting in preparation of PEI300-avidin E0.5, 2.0, 10, 20, and 50. The average numbers of modified carboxyl groups per avidin monomer were estimated to be 3.4, 3.8, 7.8, 8.5, and 9.3, respectively, by mass spectrometry (Figure S10). The maximum net charges of PEI300-avidin were calculated to be +132 for E0.5, +146 for 2.0, +279 for 10, +302 for 20, and +329 for 50 per tetramer. For comparison, biotinylated SVLT-N was introduced into HeLa S3 cells with PEI600-avidin, 1°-avidin, and PEI300-avidins, and the cytosolic transduction efficiency estimated by Western blotting. As shown in Figure 4c, 1°-avidin was the best carrier for cytosolic transduction of SVLT-N in spite of the relatively small cationic net-charge (+102). Furthermore, extensive modification of avidin with PEI300 was found to increase cytosolic delivery of cargo protein. Nuclear Delivery of Artificial Transcription Factor by Cationized Avidin Carrier. Because the expression of function following protein transduction into living cells is important for this technology, we used the artificial transcription factor GAL4-VP14 fusion protein as the third model for this study. This fusion protein consists of the DNA-binding domain of yeast GAL4 (residues 1−147) and the C-terminal activation domain of HSV VP16 (128 amino acids), which binds to GAL4-binding elements (5′-CGGRNNRCYNYNCNCCG-3′) and activates transcription.24 The HLR cell line contains a stably integrated reporter gene with a GAL4binding element-TATA sequence followed by the firefly luciferase gene for evaluating the amount of GAL4-VP16 fusion protein functioning in living cells. To enhance release from the cationized avidin carrier in cytosol, GAL4-VP16 was biotinylated with a linker with a disulfide bond that would be cleaved in the reducing cytosolic environment. As shown in Figure 5, the fusion protein was successfully transduced into the cytosol of HRL cells by 1°-avidin carrier within 4.5 h.

Figure 3. Transduction efficiency to the cytosol of eGFPNuc by cationized avidin estimated by Western blotting. (a) HeLa S3 cells incubated with various cationized avidin/biotin-eGFPNuc complexes (molar ratio 2:1, final concentration 100 nM of eGFPNuc moiety) for 15 min at 37 °C, and were analyzed in this assay. The whole cell lysates were prepared after washing with PBS, collected by cell scraper, and lysed with lysis buffer. The lysate analyzed under reducing and heating conditions (right panel) eGFPNuc band (indicated by arrow) contains both surface bound and internalized fractions including endocytic vesicle and cytosol, whereas the band at nonreducing and nonheating condition (left panel) presumably means cytosolic reaching fraction. The loading control of β-tubulin was detected by reproving of membranes with anti-β-tubulin antibody. (b,c) The intensity of each eGFPNuc band in Figure 3a was quantified using Multi-Gauge software (Fuji Film). Standard curves of eGFPNuc and loading control β-tubulin were used to calculate the quantity of eGFPNuc protein in cytosol or whole cell (Figure S6, S7). (d) The cytosolic protein transduction efficiency of biotin-eGFPNuc with 1°avidin to four different cellular density of HeLa S3 cells was assessed by the same procedures described above. (e) Temperature dependency of cytosolic protein transduction of biotin-eGFPNuc with 1°avidin to HeLa S3 cells.

reduction in cytosol (Figure 4). When HeLa S3 cells were treated with various cationized avidin/biotin-SVLT-N complexes from 15 to 180 min at 37 °C, 1°-avidin gave the highest delivery to the cytosol of SVLT-N, and the efficiency of cytosolic delivery decreased, in order, for 1°-, 2°-, 3°-, and PEI600-avidins (Figure 4a,b). According to densimetic analysis of the SVLT-N band after 15 min with 1°-avidin, the cytosolic concentration of SVLT-N was estimated to be 2.1 × 10−6 M. E

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by electrostatic interactions, they had different postadsorptive cellular responses. This difference suggested that chemically cationized protein and TAT cationic peptide-fused protein interact with different molecules on the cellular surface, with the former inducing efficient cellular uptake following adsorption. Among the chemically cationized carriers, intracellular uptake with the limited cationized PEI600-avidin was superior to the more uniformly cationized avidins (1°-, 2°-, and 3°-avidins), suggesting that PEI-cationized proteins have a strong ability to induce adsorption-mediated endocytosis (Figure 1d). Technique for cytosolic delivery of exogenous protein into living cells is important for this technology. We successfully evaluated the fraction delivered to the cytosol by various cationized avidin carriers using a cleavable disulfide biotin linker sensitive to the cytosolic reducing environment (Figures 2, 3, and 4). These studies revealed that uniformly cationized avidin with diamines showed the highest ability for cytosolic delivery of cargo proteins. Especially, 1°- and 2°-avidin were the best as cargo of both biotin-GFPNuc and biotin-SVLT-N (Figures 3a, 4b). This cytosolic delivery was energy dependent and rapid, and seemed to be by direct penetration of the plasma membrane (Figure 3e). This unique penetration route was not observed when limited cationized PEI600-avidin was used as carrier for delivery of the biotinylated SVLT-N and GAL4VP16 fusion proteins (Figures 4a, 5), but the more uniformly cationized PEI300-avidins and the PEI600−1°-avidin carrier showed a similar ability to directly penetrate with the cargo protein (Figures 4c, 5). Previous reports showed that ethylenediamine-cationized RNase A (RNaseA-NH3+) internalizes into cells and is more cytotoxic than PEI600-RNaseA,13 suggesting the contribution of the direct penetration ability of uniformly cationized proteins. Furthermore, the taurinemodified RNase A (RNaseA-SO3−) with the same net charge as native RNase A showed enhanced hydrophobic adsorption to the cellular surface,13 suggesting that extensive modification of the protein carboxyl groups also enhances protein hydrophobicity. Taken together, the most reasonable explanation is that the protein surface with a concentrated cationic net charge and moderate hydrophobicity contributes to the ability to directly penetrate living cells. Although the detailed cellular mechanism of this penetration route is unclear, rapid and virtually nontoxic protein transduction techniques are highly valuable for biotechnological applications. We are optimistic that protein cationization techniques will further accelerate the methodology for artificial regulation of cellular function.

Figure 5. Transduction of biotinylated artificial transcription factor GAL4-VP16 fusion protein by cationized avidin and induction of target gene expression. After incubation with 1 μM of cationized avidin/biotin-GAL4-VP16 complex for 4.5 h, induced luciferase activity was measured. Control indicates nontreated HLR cells. Graph represents three individual experiments using triplicate wells. Each value represents the mean ± SD.

Furthermore, doubly cationized avidin, conjugated with PEI600 followed by 1,3-diaminopropane (PEI600−1°-avidin) showed superior transduction to PEI600-avidin. This result suggested that PEI600 did not interfere with protein penetration into the cytosol, and further cationization with 1,3-diaminopropane allowed cytosolic penetration under physiological conditions.

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DISCUSSION Artificial regulation of mammalian cell functions is a potent, highly valuable technology for biotechnological applications. Protein transduction strategies are an alternative approach for gene transfection. Although several approaches, including cationic peptides or lipid-based protein transduction methodologies, are proposed,4,5,27−29 in all cases the initial step for protein transduction is adsorption onto the cellular surface. Because the cellular surface is negatively charged, cationized proteins show rapid adsorption onto the cellular surface by electrostatic interaction. Following adsorption, intracellular uptake appears to proceed through two distinct energydependent pathways: rapid and direct penetration into cytosol, or an endocytotic and/or pinocytotic pathway. In this study, we successfully discriminated between these two routes, estimated their contributions, and evaluated the best cationized carrier for cytosolic delivery of proteins. We prepared several cationized avidin modified with diamines, PEI600, and PEI300 as biotinylated protein transduction carrier. These avidin derivatives had slight intermolecular cross-linking without forming aggregation (Figure S2). Because these chemical modifications were conducted with a high concentration of diamine (2 M) or PEI (180 mg/mL), most amidation sites did not contribute to intermolecular crosslinking. Additionally these derivatives retained biotin-binding activity (Figure 1). Electrostatic binding of chemically cationized proteins to the cellular surface is rapid, and endocytotic and/or pinocytotic uptake occurs immediately (Figure 1b−d). However, the cationic peptide of TAT-fused eGFP showed strong adsorption onto the extracellular matrix (Figure 1f), but was not accompanied with cellular uptake within 15 min (Figure 1c). Although both chemically cationized protein and TAT cationic peptide-fused protein seemed to be attracted to the cell surface

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ASSOCIATED CONTENT

S Supporting Information *

Mass spectra of the conjugates, binding of biotinylated protein with avidin derivatives and stability of the disulfide bond in biotin spacer assessed by SDS-PAGE, standard curves for quantitative Western blotting analysis, photograph indicating cell density observed before protein transduction. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone and fax: +81-86-251-8217; E-mail: futamij@cc. okayama-u.ac.jp. Notes

The authors declare no competing financial interest. F

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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (B) to J.F. from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and NEDO Industrial Technology Research Grant Program in 2008 to J.F.

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ABBREVIATIONS: DTT, dithiothreitol; EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride; eGFP, enhanced green fluorescent protein; eGFPNuc, eGFP with three tandem repeats of the nuclear localization signal of the simian virus 40 large T-antigen at the C-terminus; FBS, fetal bovine serum; HLR cell, HeLa luciferase receptor cell; PEI, polyethylenimine; HSV, herpes simplex virus; PBS, phosphate-buffered saline; PTD, protein transduction domain; PVDF, poly(vinylidene difluoride); RNase A, bovine ribonuclease A; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SVLT-N, N-terminal domain of simian virus 40 large Tantigen; TBS, Tris-buffered saline

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dx.doi.org/10.1021/bc300030d | Bioconjugate Chem. XXXX, XXX, XXX−XXX