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An Oligoarginine-Bearing Tandem Repeat Penetration-Accelerating Sequence Delivers Protein to the Cytosol via Caveolae-Mediated Endocytosis Akiko Okuda, Shinya Tahara, Hisaaki Hirose, Toshihide Takeuchi, Ikuhiko Nakase, Atsushi Ono, Masanori Takehashi, Seigo Tanaka, and Shiroh Futaki Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01299 • Publication Date (Web): 20 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

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An Oligoarginine-Bearing Tandem Repeat Penetration-Accelerating Sequence Delivers Protein to the Cytosol via Caveolae-Mediated Endocytosis Akiko Okuda*‡§, Shinya Tahara‡§, Hisaaki Hirose#, Toshihide Takeuchi#, Ikuhiko Nakase¶, Atsushi Ono§, Masanori Takehashi○, Seigo Tanaka○, Shiroh Futaki# §Department

of Medical Technology, Graduate School of Health Sciences, Niigata University, Chuo-ku, Niigata, Niigata 951-8518, Japan

#Institute

¶Graduate

for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

School of Science, Osaka Prefecture University, Naka-ku, Sakai, Osaka 599-8570, Japan

○Laboratory

of Pathophysiology and Pharmacotherapeutics, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Osaka 584-8540, Japan

Corresponding Author *Address: Department of Medical Technology, Graduate School of Health Sciences, Niigata University, 746 Asahimachidori-2, Chuo-ku, Niigata, 951-8518, Japan; Phone & Fax: +81-25227-2387; E-mail: [email protected]

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ABSTRACT To facilitate the cytosolic delivery of larger molecules such as proteins, we developed a new CPP sequence, named Pas2r12, consisting of a repeated Pas sequence (FFLIG-FFLIG) and D-dodecaarginine (r12). This peptide significantly enhanced the cellular uptake and cytosolic release of enhanced green fluorescent protein (EGFP) and immunoglobulin G (IgG) as cargos. We found that simply mixing Pas2r12 with cargos could generate cytosolic introducible forms. The cytosolic delivery of cargos by Pas2r12 was found to be an energy-requiring process, relied on actin polymerization, and was suppressed by caveolae-mediated endocytosis inhibitors (genistein and methyl-β-cyclodextrin) and small interfering RNA against caveolin-1. These results suggest that Pas2r12 enhances membrane penetration of cargos without the need for crosslinking, and that caveolae-mediated endocytosis may be the route by which cytosolic delivery is enhanced.

Keywords: Cell-penetrating peptide; Oligoarginine; Cytosolic delivery; Penetration-accelerating sequence; Endocytosis; Enhanced green fluorescent protein


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INTRODUCTION Experimental techniques for introducing biomacromolecules, including deoxyribonucleic acid (DNA) and proteins, into cells have expanded the research possibilities available to those working in the fields of cellular biology and biomedical sciences. For example, gene-transfection systems using cationic lipids have been used for decades, and many related DNA-transfection or RNA-transfection reagents have been developed to allow researchers to select the optimal transfection method for their purposes.1,2 In contrast, research on transfection reagents for functional proteins, especially those to be introduced into cytosol, are still immature and should be developed for future therapies and diagnostics.3,4 Cell-penetrating peptides (CPPs) can deliver various cargos, including large molecules such as peptides and proteins, into cells. Examples of such carriers include TAT, derived from the HIV transactivator protein (Tat);5 penetratin, derived from the Drosophila antennapedia homeoprotein;6 VP22, derived from the herpes simplex virus VP22 protein;7 chimeric peptide transportan (TP);8 and various synthetic oligoarginine peptides.9,10 Recent studies using living cells have shown that endocytic pathways are the major routes for the cellular uptake of these peptides. CPPs interact strongly with cellsurface proteoglycans, leading to effective cellular uptake via macropinocytosis11–14 and other endocytic pathways.15–18 Therefore, a majority of CPPs and their cargo remains within endosomes, and enhancing their release from the endosomal compartment is a possible route toward cytosolic delivery. Takayama et al. reported that accelerated intracellular delivery of arginine-rich CPPs could be achieved by the introduction of a short peptide segment, Pas (penetration-accelerating sequence, amino-acid sequence: FFLIPKG), into arginine-rich CPPs.19 After fusion of the Pas sequence to octa-arginine (PasR8), the C-terminal 22 amino-acid segment of p53 was effectively delivered into malignant glioma cells, resulting in the effective inhibition of their growth.19 In addition,


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PasΔPKR8 (FFLIG-R8), which lacks the proline (P) and lysine (K) of Pas, was designed to facilitate attachment to biofunctional cargos using a bis(sulfosuccinimidyl)suberate (BS3) crosslinking reagent, resulting in an equivalent efficacy of intracellular delivery compared with PasR8.20 However, the efficacy of these molecules decreases with the increasing molecular mass of the cargo. Although PasΔPKR8 can deliver low molecular weight molecules, such as polyethylene glycol 5,000 (PEG5,000; 5 kDa), it is less effective in delivering higher molecular weight molecules, such as PEG30,000 (30 kDa).20 Therefore, PasR8 and PasΔPKR8 are likely to only be useful for intracellular delivery of molecules up to 5 kDa in size. In this study, we aimed to develop a method that can be performed to effectively introduce proteins into the cytosol, using common biological laboratory techniques and simple reagents. We used enhanced green fluorescent protein (EGFP, 27 kDa) as a model cargo because its intracellular localization is easily visualized. Previous reports have suggested that the use of the hydrophobic sequence in PasR8 produces a higher cell permeability than using R8 alone.20 Therefore, duplicating PasΔPK (FFLIG) to produce Pas2 (FFLIG-FFLIG) might further enhance penetration and hydrophobic intermolecular binding. Furthermore, we considered the possibility of increasing the protease resistance, and ultimately stability of the oligoarginine moiety, by using D-arginine instead of the L-isomer. In addition, Alexa488-labeled R12 has been shown to facilitate both cytosolic and nuclear localization after cellular uptake, whereas cytosolic localization decreases when using Alexa488-labeled R8.21 For the above reasons, we selected protease-resistant D-arginine and a longer sequence (L-R8 was changed to D-r12) couple to Pas2 (FFLIG-FFLIG) for the purposes of this study.


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MATERIALS AND METHODS

Cell Culture HEK293 (human embryonic kidney cell line, JCRB9068) cells were purchased from the JCRB cell bank. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under a 5% CO2 atmosphere at 37°C. Cells were passaged every 5–7 days.

Peptide Preparation All

peptides

(Figure

1a)

were

chemically

synthesized

by

Fmoc

(9-

fluorenylmethyloxycarbonyl)-solid-phase peptide synthesis on a Rink amide resin as described previously.22 Deprotection of the peptide and cleavage from the resin were performed by treating with a trifluoroacetic acid/ethanedithiol mixture (95:5) at room temperature for 3 h followed by reversed-phase high-performance liquid chromatography purification. The products’ masses was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS): (r12, rrrrrrrrrrrr-amide) 1,892.566 [calculated for (M+H)+: 1,892.28]; (PasΔPKr12, FFLIG-rrrrrrrrrrrr-amide) 2,469.958 [calculated for (M+H)+: 2,470.01]; and (Pas2r12, FFLIGFFLIG-rrrrrrrrrrrr-amide) 3,047.986 [calculated for (M+H)+: 3,047.73]. Lowercase letters denote D-type amino acids and uppercase letters L-type amino acids, respectively.

EGFP Preparation


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Hexahistidine (His6)-tagged EGFP22 was overexpressed in Escherichia coli strain BL21 (DE3). After cell lysis, the protein was purified using Ni-NTA Agarose (QIAGEN, Hilden, Germany), followed by protein concentration and buffer exchange with phosphate-buffered saline using a nanomembrane concentrator (VivaspinTM 6-PES 30,000 MWCO; Sartorius Stedim Biotech, Goettingen, Germany). EGFP concentration was determined from absorbance at 488 nm by using a UV mini-1240 single-beam spectrophotometer (Shimadzu, Kyoto, Japan). Purity was confirmed by Coomassie brilliant blue R-250 staining after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Confocal Microscopy HEK293 cells (4 × 105) in DMEM (2 mL) supplemented with 10% FBS were plated onto 35mm-diameter glass-based dishes (Iwaki, Tokyo, Japan) coated with 0.1% gelatin and incubated for 5 days at 37°C. The cargo and CPP mixture was added to the 12-mm diameter glass-based well (Figure 1b). Culture medium was then replaced with 2 mL of DMEM containing 2.5% FBS followed by further incubation for 24 h at 37°C. For analysis of EGFP and CPP, EGFP and CPP (Pas2r12, PasΔPKr12 or r12) were mixed in Opti-MEM (Thermo Fisher Scientific, Massachusetts, USA) at twice the final concentration (total volume 50 μL) and subsequently incubated for 1–90 min (for complex formation) at room temperature. After removing the culture medium, EGFP and CPP mixture (doubly diluted with 50 μL of Opti-MEM) was added to the cells, which were treated for 45 min at 37°C. Two milliliters of DMEM containing 10% FBS was then added to the cells without removing the EGFP and CPP mixture, and the cells were further incubated for 0 h, 3 h, or 16 h at 37°C. For analysis of full-length immunoglobulin G (IgG) antibody and Pas2r12, rabbit anti-mouse IgG (H+L) cross-adsorbed secondary antibody


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labeled with Alexa fluor 488 (Alexa488-IgG; Thermo Fisher Scientific) and Pas2r12 were mixed in Opti-MEM at twice the final concentration (total volume 50 μL) and then incubated for 60 min (for complex formation) at room temperature. After removing the culture medium, the Alexa488-IgG and Pas2r12 mixture (doubly diluted with 50 μL of Opti-MEM) was added to the cells, which were treated for 45 min at 37°C. Two milliliters of DMEM containing 10% FBS was then added without removing the Alexa488-IgG and Pas2r12 mixture, and the cells were further incubated for 16 h at 37°C. For EGFP overexpression, HEK293 cells were transfected with pcDNA3-EGFP (Addgene, Massachusetts, USA) using Lipofectamin 3000 transfection reagent (Thermo Fisher Scientific). EGFP and Alexa488-IgG were imaged within cells using a FluoView FV1200 confocal laser scanning microscope (CLSM; Olympus, Tokyo, Japan) without fixing the cells to avoid artifactual localization of the internalized CPP.12,23 The average percentage of cells with cytosolic distribution of EGFP or Alexa488-IgG was determined from more than three visual fields, and experiments were conducted three times independently. The total number of cells and number of cytosol-introduced cells were counted visually from merged images of fluorescence and differential interference contrast (DIC). Data are presented as mean ± SD.

Experiments with Endocytosis Inhibitors HEK293 cells were prepared in the same manner for the CLSM analysis. For complex formation, EGFP (30 μM) and Pas2r12 (40 μM) were mixed in Opti-MEM at twice the final concentration (total volume 50 μL), and subsequently incubated for 60 min at room temperature. For ATP-synthesis inhibition, the cells were pretreated for 60 min with both 5-mM sodium azide (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 5-mM 2-deoxyglucose (Sigma-Aldrich, Missouri, USA) (NaN3/DOG) in 2 mL of DMEM without FBS. The complex mixture and 50 μL


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of NaN3/DOG were mixed. After removing the culture medium, the cells were treated with twofold diluted EGFP (15 μM) and Pas2r12 (20 μM) in the presence of NaN3/DOG (total volume 100 μL) for 45 min at 37°C. For experiments at 4°C, cells were pretreated at 4°C for 30 min. After removing the culture medium the cells were treated with complex mixture (total volume 100 μL) for 45 min at 4°C. For F-actin polymerization inhibition, cells were pretreated with 500nM cytochalasin D (Sigma-Aldrich)24 for 10 min; the subsequent procedures were as described for NaN3/DOG treatment. To establish that Pas2r12 was involved in endocytic cytosolic delivery of EGFP, cells were treated with biochemical inhibitors of clathrin-mediated endocytosis (chlorpromazine),

macropinocytosis

(5-(N-Ethyl-N-isopropyl)-amiloride

[EIPA]

and

wortmannin), and caveolae-mediated endocytosis (genistein and methyl- β -cyclodextrin [M β CD]). Chlorpromazine decreases the transportation of clathrin-coated pits inside cells.25,26 EIPA, an inhibitor of the Na+/H+ exchanger NHE 1, inhibits macropinocytosis as a result of secondary acidification.27–29 Wortmannin, specifically inhibits PI3-kinase; therefore, also inhibits signaling pathways that promote membrane ruffling and macropinosome formation.30,31 Genistein, a tyrosine kinase inhibitor, rapidly blocks the actual internalization.32 M β CD is a sterol-binding compound that removes cholesterol from the cell membrane, leading to a significant reduction in caveolar density.33,34 For the endocytosis-inhibitor experiments, cells were pretreated with 50μM EIPA (Sigma-Aldrich), 100-nM wortmannin (Adoq Bioscience, California, USA), 5-μM chlorpromazine (LKT Laboratories, Minnesota, USA), 500-μM genistein (Sigma-Aldrich) or 2mM MβCD (Sigma-Aldrich) at 37°C. The medium for MβCD pretreatment was 2 mL of DMEM lacking FBS, whereas for others it was 2 mL of DMEM containing 2.5% FBS. The pretreatment duration for EIPA was 10 min, whereas it was 30 min for the others. Subsequent operations were carried out as for the NaN3/DOG treatment. After treatment, cells were washed with fresh


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medium and analyzed by CLSM. The proportion of cells with cytosolic distribution of EGFP was determined from three visual fields, and experiments were conducted independently three times. The total number of cells and number of cytosol-introduced cells were counted visually from merged images of fluorescence and DIC. Data are presented as mean ± SD.

Cell-Viability Assay HEK293 cells (1 × 104) in DMEM (100 μL) containing 10% FBS were plated onto 96-well microplates (Iwaki) coated with 0.1% gelatin and were incubated for 5 days at 37°C. Medium was changed for DMEM containing 2.5% FBS and followed by a further 24 h incubation at 37°C. Cargo and Pas2r12 were mixed in Opti-MEM at twice the final concentration (total volume 25 μL) and then incubated for 60 min (for complex formation) at room temperature. After removing the culture medium, the cargo and Pas2r12 mixture (doubly diluted with 25 μL of Opti-MEM) was added to the cells, which were treated for 45 min at 37°C. After a 45-min treatment, 40 μL of the cargo and Pas2r12 mixture was removed and 200 μL of DMEM containing 10% FBS was added and incubated for 16 h. As a result, the cargo and Pas2r12 (10 μL) was diluted 20-fold with DMEM during this step and the final concentration of the cargo and Pas2r12 was the same as in the 16-h incubation for the CLSM analysis described above. Cell viability was assayed using a CellQuanti-MTTTM cell-viability assay kit (Bioassay Systems, California, USA). The diluted reagents (95 μL) were added to each well and incubated for 4 h at 37°C. Cells were then lysed for 1 h, and absorbance was measured at 570 nm using model 550 microplate reader (Bio-Rad, California, USA). Data were presented as the mean ± SD of three experiments.


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Transfection of siRNA HEK293 cells in DMEM (200 μL) supplemented with 10% FBS were plated onto the well in 35mm-diameter glass-based dishes and incubated for 24 h to be 50% confluent at 37°C. Small interfering RNA (siRNA) against caveolin-1 (cav-1) (AGACGAGCUGAGCGAGAAG) was designed by Soraj et al.35 Both siRNA cav-1 (si-cav-1) and siRNA control (si-NC) (MISSION siRNA Universal Negative Control) were purchased from Sigma-Aldrich. The siRNA procedure was performed according to the manufacturer’s protocol using the following volumes and concentrations for each dish: 2.4 pmol of siRNA in 20 μL of Opti-MEM and 0.4 μL Lipofectamine RNAiMAX (Thermo Fisher Scientific) in 20 μL of Opti-MEM. The diluted siRNA and diluted Lipofectamine RNAiMAX were then combined, gently mixed and incubated at room temperature for 10 min. The siRNA- Lipofectamine RNAiMAX complex was then added dropwise to the 12-mm diameter glass-based well and incubated for 48 h. Culture medium was then replaced with 2 mL of DMEM containing 2.5% FBS followed by further incubation for 24 h at 37°C. After the incubation cells were utilized for analysis.

SDS-PAGE and Western blotting Cells were washed and sample buffer solution (2×) containing 2-mercaptoethanol (Cosmo Bio, Tokyo, Japan) was added. This mixture was then incubated at 95°C for 5 min. Samples were separated by using 4%–20% Mini-PROTEAN TGX gel (Bio-Rad) and SDS-PAGE electrophoresis buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS) at a constant voltage of 200 V for 30 min at room temperature. Proteins were transferred to PVDF membrane using the trans-blot turbo transfer system (Bio-Rad). The membrane was blocked with Blocking One


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(Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature. Membrane was incubated with primary antibody against Cav-1 (Proteintech, Wuhan, China) and β-actin (Sigma-Aldrich) for 1 h at room temperature and then incubated with HRP-conjugated secondary antibody (GE Healthcare, Illinois, USA) for 45 min at room temperature. Blots were subsequently developed using ImmunoStar LD (FUJIFILM Wako Pure Chemical).

Statistical Analysis Statistically significance differences (*p < 0.05, **p < 0.01, and ***p < 0.001) were determined by Student’s t test.

RESULTS Pas2r12 is necessary to efficiently introduce EGFP into the cytosol. The experimental scheme for treatment of cells by CPP is shown in Figure 1b. It consisted of three steps: first, complex formation, where the cargo protein and CPP were incubated in a tube; second, treatment by addition of the doubly diluted mixture to cells and incubation; and third, further incubation of the cells after adding fresh cell culture medium. The design of CPPs used here was validated (see Supporting Information: Peptide design and Figure S1–S3). The ability of these CPPs to deliver EGFP cargo was measured by flow cytometry. EGFP uptake efficacy in the presence of Pas2r12 was approximately 60 times higher than that of EGFP alone (Figure S4), and the amount of EGFP taken up by cells co-treated with r12 or PasΔPKr12 was as low as that with EGFP alone (Figure S4). Subsequently, fluorescent signals of internalized EGFP


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were assessed by CLSM. Cells treated with EGFP and Pas2r12 contained EGFP in the cytosol and nucleus to an extent similar to EGFP expressed by plasmid DNA (Figure 1c, Figure S5). In contrast to Pas2r12, EGFP was not delivered to the cytosol by r12 or PasΔPKr12, as also observed in the absence of any CPP (Figure 1c). Among the peptides examined, therefore, only Pas2r12 could deliver EGFP to the cytosol.


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Figure 1. Cytosolic EGFP delivery by Pas2r12. (a) Sequences of CPPs used in this study. Lowercase letters denote D-amino acids and uppercase letters L-amino acids. (b) Experimental procedure of introducing the cargo into HEK293 cells using CPPs. The first step involves the complex formation, where the cargo and CPP are mixed and incubated at room temperature. In


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the treatment step, cells are treated with mixture of the cargo and CPP doubly diluted by OptiMEM at 37°C. In the final incubation step, DMEM containing 10% FBS is added to the cells, which are further incubated at 37°C. (c) Thirty-micromolar EGFP and 40-µM CPP (Pas2r12, PasΔPKr12, or r12) were incubated for 60 min to form their complex as according to our experimental procedure illustrated at the upper panel. In the second step, which was the treatment step, cells were treated with mixture of EGFP and CPP doubly diluted by Opti-MEM at 37°C. Cellular uptake of EGFP was analyzed by CLSM. EGFP overexpressing cells were transfected with an EGFP expression plasmid. Scale bars = 20 µm.

Complex formation time and mixing concentration are important for cytosolic EGFP delivery by Pas2r12. To determine the optimal incubation time for complex formation, the cytosolic EGFP introduction experiments were conducted with different complex formation times (1–90 min). Cytosolic delivery was observed after incubation for >15 min, and was most effective after a 60min incubation (Figure 2a). Therefore, we performed subsequent experiments using a 60-min incubation period. For further confirmation of the optimal mixing ratio for complex formation, incubated mixture in different concentrations of EGFP (30 µM) and Pas2r12 (20 µM, 40 µM, and 60 µM) was doubly diluted by Opti-MEM and added to cells. The percentage of cells with cytosolic distribution of EGFP was highest (35%) with the mixture of 15-µM EGFP and 20-µM Pas2r12 (final concentration to cells) (Figure 2b). Furthermore, MTT-reducing activity decreased in a Pas2r12 concentration-dependent manner compared with the untreated control cells (10 µM: 94%, 20


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µM: 85%, 30 µM: 68%; Figure 2c). MTT-reducing activity of cells treated with hydrogen peroxide as positive controls was significantly reduced (2 mM, 44%; 20 mM, 9%) (Figure S6). These results showed that a mixture of 15-µM EGFP and 20-µM Pas2r12 (final concentration to cells) has a mild effect on cell viability but is the most effective for cytosolic EGFP delivery. We therefore used these concentrations of EGFP and Pas2r12 in subsequent experiments.


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Figure 2. Incubation time and concentration optimization of EGFP and Pas2r12 complex formation. (a) EGFP (30 µM) and Pas2r12 (40 µM) were incubated for 1–90 min at room temperature to optimize incubation time of EGFP and Pas2r12 for complex formation. In the treatment step, cells were treated with doubly diluted mixture of EGFP (15 µM) and Pas2r12 (20 µM). Scale bars = 20 µm. (b) EGFP (30 µM) and Pas2r12 (20 µM, 40 µM, or 60 µM) were mixed and incubated for 60 min at room temperature. In the treatment step, cells were treated with a doubly diluted mixture of EGFP (15 µM) and Pas2r12 (10 µM, 20 µM, or 30 µM). Final concentration to cells is as shown in the figure. Scale bars = 20 µm. The bar graph depicts the percentage of cells with cytosolic distribution of EGFP. (c) Cells were treated with EGFP and Pas2r12 as in (b) and assessed using an MTT-assay. Differences from untreated control cells were determined by Student’s t test (*p < 0.05 and ***p < 0.001).

Pas2r12 delivers EGFP into the cytosol via energy-dependent endocytosis. Next, the incubation time required for cytosolic EGFP delivery by Pas2r12 was investigated. After complex formation, the mixture was added to cells and examined after various incubation periods. Cytosolic EGFP uptake was scarcely detectable after 10 min (Figure 3a), and after 45min, 3-h, and 16-h the proportions of cells with cytosolic distribution of EGFP were 13%, 16%, and 36% of total cells, respectively (Figure 3a). We next focused on the routes associated with cytosolic EGFP uptake by Pas2r12 after 45-min incubation. To investigate this, cells were pretreated with NaN3/DOG to inhibit ATP synthesis36,37 or at low temperature (4°C) to inhibit all energy-dependent processes.38 After the pretreatment, cells were treated with mixture of EGFP and Pas2r12 for 45 min in the presence of


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NaN3/DOG or at 4°C. Treatment with NaN3/DOG and 4°C significantly decreased the proportion of cells with cytosolic distribution of EGFP [to 6% (p < 0.001) and 0% (p < 0.001), respectively] compared to cells without inhibitor at 37°C (Figure 3b). Introduction of transferrin, a clathrindependent endocytosis marker that was used as a control, was inhibited both by NaN3/DOG and 4°C treatment (Figure S7a). Actin polymerization plays key roles in energy-dependent endocytic processes, such as macropinocytosis,39 clathrin-mediated endocytosis,40,41 and caveolae-mediated endocytosis.42–44 To investigate the involvement of F-actin, cells were treated with cytochalasin D, which depolymerizes F-actin. Cytochalasin D decreased the proportion of cells with cytosolic distribution of EGFP to only 4% (p < 0.001) compared to cells without inhibitor at 37°C (Figure 3b). Loss of actin filaments was observed at the same concentration (Figure S7b). These results indicate that cytosolic delivery of EGFP by Pas2r12 is an energy-requiring process and is also reliant on actin polymerization.


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Figure 3. Involvement of endocytosis on cytosolic EGFP delivery by Pas2r12. (a) Time-lapse observation of EGFP-introduced cells. Cells were treated with EGFP (15 µM) and Pas2r12 (20 µM) and observed at 10 min, 45 min, 3 h, and 16 h as according to our experimental procedure illustrated at the upper right panel. Scale bars = 20 µm. The bar graph (lower right) indicates the


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proportion of cells with cytosolic distribution of EGFP assessed by CLSM imaging. (b) Influence of NaN3/DOG, 4°C treatment, and cytochalasin D (CytD) on cytosolic EGFP delivery. Cells were treated with EGFP (15 µM) and Pas2r12 (20 µM) for 45 min under each inhibitory condition. For the control, EGFP and Pas2r12 were added to cells without inhibitors and then cultured for 45 min at 37°C. The bar graph (lower right) indicates the proportion of cells with cytosolic distribution of EGFP compared to control assessed by CLSM imaging. Scale bars = 20 µm. Significant difference from control was assessed by Student’s t test (***p < 0.001).

Pas2r12 delivers EGFP into the cytosol via caveolae-mediated endocytosis. To determine the type of endocytosis involved in the delivery of EGFP by Pas2r12, cells were treated with various endocytosis inhibitors. We used a clathrin-mediated endocytosis inhibitor (chlorpromazine), macropinocytosis inhibitors (EIPA and wortmannin), and caveolae-mediated endocytosis inhibitors (genistein and MβCD). After pretreatment with each inhibitor, cells were treated with the mixture of EGFP and Pas2r12, and then incubated in the presence of the same inhibitor for 45 min. The proportions of cells with cytosolic distribution of EGFP were markedly decreased to 1% (p < 0.001) and 7% (p < 0.001) of control without inhibitor by treatment with genistein and MβCD, respectively (Figure 4). In contrast, chlorpromazine and wortmannin had no significant effect on cytosolic delivery of EGFP compared with control (Figure 4), whereas EIPA treatment produced a slight increase in the proportion of cells with cytosolic distribution of EGFP. Uptake of transferrin (a clathrin-dependent endocytosis marker), 70,000 MW dextran (a macropinocytosis marker) and bovine serum albumin (a caveolae-mediated endocytosis marker) was inhibited by treatment with chlorpromazine, EIPA and wortmannin, and genistein and Mβ


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CD, respectively (Figure S7c). We also confirmed that caveolae-dependent endocytosis was involved in cellular uptake using flow cytometry by showing that the proportion of EGFP uptake was markedly inhibited by genistein and MβCD (13% and 16%, respectively), compared with a control without inhibitors (Figure S8). MTT-reducing activity of cells with Pas2r12 and EGFP was measured under inhibitory conditions. Compared to control cells (untreated cells), marked decreases were observed in cells with Pas2r12 and EGFP in the presence of genistein (Figure S9). Furthermore, to investigate the involvement of caveolae-dependent endocytosis, expression of cav-1, a major constituent of caveolae-mediated endocytosis, was repressed by siRNA.35,45 It was confirmed by western blotting that expression of cav-1 was completely suppressed (Figure 5a). CLSM analysis was performed to si-cav-1 cells by adding a mixture of Pas2r12 and EGFP. In the si-cav-1 cells, the proportion of cells with cytosolic distribution of EGFP remarkably decreased as compared with that in si-NC (to 26%, p < 0.001), and the cytosolic EGFP amount was very low (Figure 5b). Moreover, suppression of intracellular introduction of bovine serum albumin, a marker of caveolae-mediated endocytosis, was observed in si-cav-1 cells (Figure S10).


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Figure 4. Caveolae-mediated endocytosis inhibitors decrease cytosolic EGFP delivery by Pas2r12. Cells were treated with EGFP (15 µM) and Pas2r12 (20 µM) in the presence of each inhibitor for 45 min at 37°C. For the control, EGFP and Pas2r12 were added to cells without inhibitors and then incubated for 45 min at 37°C. The bar graph indicates the proportion of cells


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with cytosolic distribution of EGFP compared to control assessed by CLSM imaging. Scale bars = 20 µm. Significant difference from control was assessed by Student’s t test (*p < 0.05 and ***p < 0.001).

(a)

anti-Cav-1

anti-β-actin

(b) si-cav-1

EGFP + Pas2r12

cav-1 knockdown

Treatment 45 min CLSM

si-NC

Cytosolic EGFP+ cells (% of negative control)

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si-cav-1

120 100 80 60 ***

40 20 0

si-NC

si-cav-1

Figure 5. Cav-1 knockdown cells decrease cytosolic EGFP introduced by Pas2r12. (a) Western blotting. (b) Cav-1 knockdown cells were treated with EGFP (15 µM) and Pas2r12 (20 µM) for 45 min at 37°C. The bar graph indicates the proportion of cells with cytosolic distribution of EGFP compared to control (si-NC) assessed by CLSM imaging. Scale bars = 20 µm. Data are


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presented as the mean ± SD of three samples. Significant difference from si-NC was assessed by Student’s t test (***p < 0.001).

Pas2r12 can deliver Immunoglobulin G into the cytosol. The ability of Pas2r12 to deliver IgG to cytosol was next investigated. Alexa488-IgG was incubated with Pas2r12 at several concentrations. Twenty-micromolar Pas2r12 was most effective at deliver 0.3-μM Alexa488-IgG to the cytosol, and fluorescent signal was detectable in the cytosol of approximately 14% cells (Figure 6a) with no significant decrease in cell viability (Figure 6b). No signal was detectable in the nuclei of Alexa488-IgG-positive cells, suggesting that Alexa488-IgG was not transported into the nucleus (Figure S11). A mixture of Pas2r12 and IgG was added to the cells, and the proportion of cells with cytosolic IgG was measured. At 45 min and 16 h after addition of Pas2r12 and IgG, the proportion of cells with cytosolic distribution of IgG remained unchanged (15% and 18%) (Figure S12).


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(a)

60 min IgG + Pas2r12 45 min Treatment

Incubation 16 h CLSM

0.3

Pas2r12 (µM)

10

3

*

* 16 h

*

*

*

*

30

20

*

*

* * (b) 120

MTT-reducing activity (%)

25

Cytosolic IgG + cells (% of total cells)

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20 15 10 5

100 80 60 40 20 0

0

0.3

3

10

20

Pas2r12 (µM)

30

control 0.3

3

10

20

30

Pas2r12 (µM)

Figure 6. Cytosolic Alexa488-IgG delivery by Pas2r12. (a) Alexa488-IgG (0.6 µM) and Pas2r12 (0.6 µM, 6 µM, 20 µM, 40 µM, or 60 µM) were mixed and incubated for 60 min. In the treatment step, cells were treated with a doubly diluted mixture of IgG (0.3 M) and Pas2r12 (0.3 µM, 3 µM, 10 µM, 20 µM, or 30 M). The final concentrations to cells are shown in the


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figure. The asterisks indicate the nuclei with cytosolic distribution of Alexa488-IgG and the graph shows the percentage of cells with cytosolic distribution of Alexa488-IgG at each concentration. Scale bars = 20 m. (b) MTT-reducing activity of cells were treated with Alexa488-IgG and Pas2r12 as in (a).

Pas2r12 delivers IgG into the cytosol via caveolae-mediated endocytosis. Next, to clarify the pathway of cytosolic introduction of IgG by Pas2r12, the introduction experiment was performed using cells subjected to the inhibition of energy-dependent processes and endocytosis, as well as by the experiment of EGFP. The proportion of cells with cytosolic distribution of IgG was significantly decreased by NaN3/DOG, low temperature (4oC), cytochalasin D, genistein, and MβCD, compared with the levels in controls without inhibitor (2%, 0%, 5%, 6%, and 36%, respectively) (Figure S13). In addition, the proportion of cells with cytosolic distribution of IgG was decreased by EIPA and wortmannin, macropinocytosis inhibitors (70% and 75%) (Figure S13). In contrast, chlorpromazine, a clathrin-mediated endocytosis inhibitor, had no significant effect on cytosolic delivery of IgG compared with control (Figure S13). Furthermore, to confirm that caveolae-mediated endocytosis was involved in IgG cytosolic introduction of IgG into the cytosol by Pas2r12, a mixture of Pas2r12 and IgG was added to the si-cav-1 cells (Figure S14a). The results showed that the proportion of cells with cytosolic distribution of IgG was suppressed compared with that in si-NC cells (39%) (Figure S14b).


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DISCUSSION This study aimed to introduce proteins into the cytosol of living cells using a CPP without the need for genetic or chemical manipulation nor the requirement for specialized equipment and techniques. We showed that addition of r12 to Pas2 (FFLIG-FFLIG), a tandem repeat of PasΔPK (FFLIG), produced a CPP that could deliver not only EGFP (27 kDa) but also IgG (150 kDa) to the cytosol. We also found that Pas2r12 could form complexes with the cargo by simple mixing and incubation without the need for a crosslinking reagent or genetic manipulation. We hypothesize that Pas2r12 delivers its cargo into the cytosol as follows. First, Pas2r12 forms complexes with the cargo. The hydrophobicity of the Pas2 sequence may contribute to its interaction with the cargo. Second, once the complexes are in the vicinity of a cell surface, the positively charged oligoarginine stretch interacts with negatively charged membrane proteoglycans such as heparan sulfate.14,46 Third, complexes are taken up by caveolae-mediated endocytosis. After uptake into the cells, the cargo diffuses into the cytosol. The hypothesis described above is based on our experiments that the proportion of cells with cytosolic distribution of cargo was visually counted under microscope. Therefore, the quantity of protein delivered into the cytosol of cells and the quantity that remains in the vesicular compartments is unclear. Furthermore, diffusion into the nucleus depends on a certain molecular weight threshold. In this study, diffusion into the nucleus was observed with EGFP but not with Alexa488-IgG, and it has been previously reported that molecules up to 40 kDa can passively diffuse into the nucleus through the nuclear pore.47 Therefore, it is likely that IgG (150 kDa) could not pass through the nuclear pores because of its high molecular weight, and that 40 kDa is the threshold that determines whether or not nuclear localization will occur. To understand the mechanism of cargo delivery into organelles in further detail, quantification studies should be performed in the future.


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The delivery efficacy of EGFP and IgG by Pas2r12 was remarkably changed by the incubation times. EGFP increased the proportion of cells with cytosolic distribution of EGFP for 16 h (45 min, 13%; 16 h, 36%) (Figure 3a), but IgG did not increase the proportion even after 16 h (45 min, 15%; 16 h, 18%) (Figure S12). The main cytosolic pathway (at 45 min) of both EGFP and IgG by Pas2r12 was shown to be caveolae-mediated endocytosis by experiments with pharmacological inhibitors and cav-1 knockdown. Regarding IgG, the cytosolic distribution was reduced by EIPA and wortmannin, macropinocytosis inhibitors, so the involvement of macropinocytosis cannot be ruled out. In the cytosolic delivery of cargo by Pas2r12, the cytosolic introduction efficiency and introduction route may change depending on various features of cargo such as the molecular weight, isoelectric point, and concentration. There is an interesting report on the difference between caveolae-mediated endocytosis and uptake via macropinocytosis.48 In that study, GM3-binding peptide, a CPP, and a cargo were intracellularly introduced by caveolae-dependent endocytosis, but they did not fuse with the lysosome and remained within the endosome for 4–24 h. In contrast, endosomes containing TAT peptide their cargo introduced into the cells by macropinocytosis fused with lysosomes within approximately 1 h. This example of long-term storage in endosomes derived from caveolae may be associated with the increase in the time-dependent EGFP diffusion observed in this study (Figure 3a). EGFP and Pas2r12 might switch to a state susceptible to release from the endosome while residing within that compartment for an extended time (~16 h). Caveolae are 50–100-nm-diameter flasks or omega-shaped plasma membrane invaginations.49 It has been reported that simian virus 40 is transported to the cytosol via the endoplasmic reticulum (ER)50 and that cholera toxin shifts from the trans-Golgi to ER.51 Because these molecules are transported to the cytosol via the Golgi apparatus and ER lumens, the cargo of Pas2r12 might


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also be able to access the Golgi apparatus and ER lumens as well as the cytosol. Furthermore, caveolae are not present in all types of cells; they are abundant in muscle and adipocytes, and absent in lymphocytes and neurons.52 By taking advantage of these features, Pas2r12 could be used for a variety of applications, such as drug delivery to specific tissues in vivo. Currently, many DNA-transfection reagents have been developed,1,2 allowing researchers to choose the optimal transfection method for their particular application. In contrast, development of delivery reagents that are effective in protein delivery is still required. Our method for introducing the cargo to the cytosol using Pas2r12 is very simple. There is no need to perform a chemical reaction to conjugate the cargo to CPP or to produce recombinant fusion proteins. The usefulness of this simple mixing strategy has also been exemplified by its use with other endosome perturbing peptides.53 Although EGFP and IgG were able to be adapted for use as a cargo of Pas2r12, we need to consider whether other molecules will function similarly. Therefore, it is necessary to investigate the properties of molecules that could be introduced as the cargo. In addition, introducing cargos to various cells, including primary cultured cells, must be confirmed. If Pas2r12 can be applied to other macromolecules and cells, it will be a very promising and efficient protein-delivery tool in the fields of cell biology and biomedical science.

CONCLUSIONS Here we describe the successful cytosolic delivery of two model cargos—EGFP and IgG—using Pas2r12. This peptide can form complexes with cargo upon simple mixing and without the need for genetic engineering or chemical crosslinking to couple them covalently. Furthermore, cytosolic delivery of EGFP and IgG by Pas2r12 was found to be due mainly to caveolae-


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mediated endocytosis. Pas2r12 could be used in the future for targeting both specific tissues and the subcellular organelles by taking advantage of caveolae-mediated endocytosis. On the basis of this research, Pas2r12 is a candidate for a protein-delivery tool for various cargos with varied molecular characteristics.

Supporting Information Materials and Methods, Peptide design, Figures, References (PDF)

AUTHOR INFORMATION Acknowledgments We thank Professor Miwako Narita and Ms. Wakana Goto for the technical help for the flow cytometer. We also thank Enago (www.enago.jp) for the English language review. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was supported by JSPS KAKENHI (Grant Numbers JP25860400 and 17K08947) and by the Collaborative Research Program of Institute for Chemical Research, Kyoto University


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(Grant Numbers 2015-66 and 2016-78) to A.O. This work was also supported in part by JSPS KAKENHI (15H02497 and 16H01145) to S.F and (16H02612) to I.N.

Conflict of Interest The authors declare no competing financial interest.

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Table of Contents Graphic

Protein

Pas2r12

Caveolae-mediated endocytosis

Cytosolic protein delivery

HEK293 cell


39

Oligoarginine-Bearing Tandem Repeat ... - ACS Publications

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