Streptavidin-Conjugated C3 Protein Mediates the Delivery of Mono

Jun 9, 2012 - adaptor protein for attaching biotinylated molecules to facilitate their subsequent ... Next, we prepared mono-biotinylated RNase A, whi...
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Streptavidin-Conjugated C3 Protein Mediates the Delivery of MonoBiotinylated RNAse A into Macrophages Maren Lillich,‡ Xi Chen,§ Tanja Weil,§ Holger Barth,*,‡ and Jörg Fahrer*,†,‡ ‡

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, D-89081 Ulm, Germany Institute of Organic Chemistry III, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany

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ABSTRACT: The C3 toxin produced by Clostridium botulinum (C3bot) catalyzes the mono-ADP-ribosylation of the small GTPases Rho A, B and C, resulting in their inactivation. Recently, a specific endocytotic uptake mechanism of C3bot was identified in macrophages and myeloid leukemia cells. Here, we present a novel delivery system based upon a mutant C3bot devoid of ADP-ribosylation activity (C3Mut) and wild-type streptavidin (Stv). The C3Mut moiety mediates endocytosis into macrophages, whereas Stv functions as an adaptor protein for attaching biotinylated molecules to facilitate their subsequent internalization. First, a bioconjugate consisting of recombinant C3Mut and Stv was generated via a thioether linkage that tightly interacted with biotinylated bovine serum albumin as demonstrated by dot blot analysis. We then showed the internalization of C3Mut-Stv into J774A.1 macrophages by confocal microscopy and observed translocation into the cytosol using cell fractionation. The C3Mut-Stv bioconjugate did not affect cell viability. Next, we prepared mono-biotinylated RNase A, which was attached to the C3Mut-Stv transporter, and demonstrated its C3Mut-Stv-mediated delivery into the cytosol of J774A.1 cells. Finally, C3Mut-Stv also promoted the efficient uptake of mono-biotinylated lysozyme into J774A.1 cells, highlighting its versatility. This study intriguingly demonstrates the use of the novel C3Mut-Stv delivery system for protein transduction and may provide a basis for future applications, in particular, of cytotoxic RNase A mutants.

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INTRODUCTION The C3-protein secreted by Clostridium botulinum (C3bot) belongs to the family of C3-like transferases that selectively catalyze the ADP-ribosylation of the small GTP-binding proteins RhoA, B, and C.1 Modification of these Rho GTPases by ADP-ribose results in their inactivation due to enhanced sequestration by RhoGDI in the cytoplasm and impaired interaction with Rho-GEF.2,3 Subsequently, Rho-mediated signaling is abolished and cellular processes governed by Rho such as control of the actin cytoskeleton are disturbed. One hallmark of Rho inactivation by C3 ADP-ribosyltransferases is a morphological change induced by a drastic rearrangement of the actin cytoskeleton.4−6 As Rho proteins are also master regulators of other cellular functions including cell cycle progression and cell migration, C3 transferases have been widely used as pharmacological tools to study Rho-mediated signaling cascades.7 Furthermore, C3bot was shown to stimulate neuronal outgrowth,8 and more recently, a C3botderived peptide was reported to promote the recovery after spinal cord injury by increased neuronal growth.9 In general, C3 proteins are internalized poorly into eukaryotic cells, most probably via unspecific pinocytosis, and have been considered as exoenzymes for a long time; no transport domain has been identified so far in C3-like ADP-ribosyltransferases. However, an efficient uptake of C3bot into macrophages and monocytes has been demonstrated recently.10 The uptake mechanism has © 2012 American Chemical Society

not yet been completely elucidated, but there is strong evidence for a specific entry pathway of C3bot via acidified endosomal compartments.10 Streptomyces avidinii produces streptavidin, a tetrameric protein, which is characterized mainly by its extraordinarily high affinity for biotin (KD = 10−15 M). Each streptavidin subunit forms an antiparallel β-barrel structure which is exceptionally stable even if exposed to heat, high or low pH, denaturants, or proteases.11 Due to its unique features, streptavidin as well as the related avidin from chicken egg white have been widely used in biochemical and pharmaceutical applications.12 Pharmaceutical applications comprise avidinfused monoclonal antibodies directed against cell surface receptors such as the human insulin receptor for targeted drug delivery.13 Conjugation of streptavidin to a monoclonal antibody directed against the transferrin receptor allowed the targeting of biotinylated siRNA to rat glial cells after intravenous application.14 In addition, biotinylated saporin, a plant-derived toxin that inactivates ribosomes, was successfully delivered into cancer cells using an anti-transferrin receptor− avidin fusion protein and was shown to potently induce cytotoxicity.15 Growth factors such as TGF-alpha have also Received: January 29, 2012 Revised: May 23, 2012 Published: June 9, 2012 1426

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was purchased from Sigma-Aldrich (Deisenhofen, Germany). SYBR Safe DNA gel stain was obtained from Invitrogen (Karlsruhe, Germany). Tris base and EDTA were purchased from first BASE (Singapore). 384-Well black microtiter plates for RNase A activity assay and sterile 96-well plates were received from Greiner Bio-One (Frickenhausen, Germany) and Nunc Surface (Roskilde, Denmark), respectively. Purification of Mutant C3bot Protein. The enzymatically inactive C3bot mutant E174Q (C3Mut) was overexpressed as GST-tagged fusion protein as described previously.10 Briefly, E. coli BL21 transformed with pGEX-2T-GL-C3Mut were grown in LB-medium to an optical density of 0.6−0.8. Isopropyl-β-Dthiogalactopyranoside (IPTG) was added to induce protein expression, and the culture was incubated at 29 °C for further 20 h. Cells were pelleted by centrifugation for 10 min at 4 °C at 4400 g. The pellet was resuspended in lysis buffer containing 1% Triton X-100 and sonicated. Cellular debris was pelleted (17 000 g, 15 min, 4 °C), and the supernatant was incubated with glutathione agarose beads (Macherey Nagel, Düren, Germany) for 20 h at 4 °C. Subsequently, the beads were washed several times and then incubated with thrombin (20 NIH units/L culture) for 1 h to remove the GST-tag. Glutathione beads were pelleted by centrifugation for 30 s at 10 000 g at 4 °C, and the supernatant containing C3Mut protein was incubated with benzamidin sepharose beads (GE Healthcare, Uppsala, Sweden) to remove thrombin. Isolated C3Mut was checked for purity by SDS-PAGE followed by Coomassie staining. Bioconjugation of C3Mut and Streptavidin. C3Mut protein in borate buffer containing 2 mM EDTA, pH 8.0, was incubated with 5-fold molar excess of 2-iminothiolane (Sigma, Deisenhofen, Germany) for 90 min at 25 °C. Thiolation efficiency was then monitored using Ellman’s reagent.27 Briefly, 12.5 μL of the protein solution were incubated with Ellman’s reagent (4 mg/mL in buffer containing 100 mM Na2HPO4 and 1 mM EDTA, pH 8.0) for 15 min at RT. Absorbance was measured at 405 nm, and the number of thiol groups was determined by comparison with a standard curve. Wild-type streptavidin (Pierce, Rockford, USA) was dissolved in buffer containing 50 mM NaH2PO4, 2 mM EDTA (pH 7.2), and activated for 30 min at 25 °C using a 10-fold molar excess of Sulfo-SMCC (Pierce, Rockford, USA) dissolved in DMF. Afterward, nonreacted 2-iminothiolane and Sulfo-SMCC, respectively, were removed by desalting spin columns (BioRad, Munich, Germany). Equal amounts of both activated proteins were then incubated together for 2 h at 25 °C. The coupling reaction was then monitored by SDS-PAGE and Western Blot analysis using anti-C3- and antistreptavidin antibodies. SDS-PAGE and Immunoblot Analysis. Proteins were separated by SDS-PAGE and subsequently transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). 5% nonfat dry milk in PBS containing Tween-20 (0.1%, PBS-T) was used to block the membrane for 1 h at room temperature (RT). Subsequently, the membrane was probed with the respective primary antibody diluted in PBS-T for 1 h at RT. After washing the membrane 3 times with PBS-T, it was incubated with the appropriate secondary antibody coupled to horseradish peroxidase for 1 h (1:2500 in PBS-T). After further washing steps, proteins were detected using enhanced chemiluminescence. Dot Blot Analysis. Decreasing amounts of biotinylated and native BSA, RNase A, or lysozyme were vacuum-aspirated onto

been used as streptavidin fusion proteins to promote the uptake of biotin-labeled DNA into cells via the EGF receptor.16 Furthermore, streptavidin fused to importin-β promoted the transport of biotinylated DNA into the nucleus after microinjection into the cytoplasm of cells.17 Recently, we generated a novel delivery system based upon the binary C2 toxin of C. botulinum and streptavidin.18 The C2-streptavidin transporter allowed the specific uptake of small biotinylated compounds into various mammalian cell lines.18,19 Interestingly, a C3bot mutant is available that is devoid of ADP-ribosylation20 and is efficiently internalized into macrophages allowing the generation of a molecular Trojan horse for drug delivery.21 RNase A is a small (14 kDa) bovine pancreatic ribonuclease that exhibits endonuclease activity catalyzing the cleavage of single-stranded RNA.22 RNase A displays high thermal stability,23 can be completely refolded after denaturation,24 and is commercially available in huge amounts. Interestingly, RNase A is internalized poorly into mammalian cells,25 which renders it an attractive model cargo protein for cellular delivery systems. Native RNase A is not cytotoxic in mammalian cells due to inactivation by ribonuclease inhibitor (RI) protein that binds with femtomolar affinity to RNase A.26 The present study builds on the uptake of an inactive C3 mutant (C3Mut) linked to wild-type-streptavidin (Stv) to deliver biotinylated ligands into macrophage-like cells. The synthesized C3Mut-Stv bioconjugate exhibits strong biotin binding and is internalized into J774A.1 macrophage-like cells without affecting cell viability. Importantly, we demonstrate the C3Mut-Stv-mediated uptake of macromolecules into the cytosol of J774A.1 cells using both RNase A and lysozyme, which carry single biotin groups at their N-terminus, as protein cargos.

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EXPERIMENTAL PROCEDURES Material. Cell culture medium and fetal calf serum (FCS) were obtained from Invitrogen (Karlsruhe, Germany). Cell culture dishes and plates were bought from TPP (Trasadingen, Switzerland). Trypsin and digitonin were purchased from Sigma-Aldrich (Munich, Germany). Streptavidin and SulfoSMCC were from Pierce (Rockford, USA). Page Ruler prestained Protein Ladder was obtained from Fermentas (St. Leon-Rot, Germany). Streptavidin-POD and staurosporine were purchased from Roche (Mannheim, Germany). Immobilon Western Chemiluminescent HRP Substrate was obtained from Millipore (Schwalbach, Germany). Biotin−fluorescein was obtained from Sigma (Deisenhofen, Germany) and fluorophorcoupled secondary antibodies were from Invitrogen (Karlsruhe, Germany). DyLight649-NHS was bought from Pierce (Rockford, USA). Biotin-LC-NHS was received from Sigma-Aldrich (Deisenhofen, Germany). Ribonuclease A (RNase A) and lysozyme were obtained from MP Biomedicals (Illkirch, France) and Sigma (Deisenhofen, Germany), respectively. 10× phosphate buffered saline was purchased from first BASE (Singapore). Monomeric Avidin Kit for affinity chromatography and Sephadex G-15 for gel filtration were from Pierce (Rockford, USA) and Sigma-Aldrich (Deisenhofen, Germany), respectively. BCA assay kit for protein concentration determination was obtained from Merck Biosciences (Darmstadt, German) and absorbance was recorded by a BioTEK Synergy 4 Microplate Reader (Bad Friedrichshall, Germany). All MALDI-ToF-MS spectra of protein samples were recorded on Applied Biosystems 4700 Proteomics Analyzer 88 using sinapinic acid solution as matrix. Baker’s yeast ribonucleic acid 1427

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Preparation of Mono-Biotinylated RNase A. Monobiotinylated RNase A was prepared based on a procedure reported very recently.29 A single Biotin-LC (biotinamidohexanoyl) group was introduced at the side chain of Lys 1 residue of RNase A via an amide bond, thus yielding Biotin-LC(K1)RNase A. The corresponding MALDI-ToF-MS spectrum is depicted in Figure 5 revealing that monofunctionalized RNase A was obtained. The location of the biotin substituent has been identified via the LCMS2-XIC method. The protein concentration of mono-biotinylated RNase was determined by BCA assay as described by the manufacturer. Conjugation of DyLight 649 Fluorophore to MonoBiotinylated RNase A. 50 μg of DyLight 649 (49.5 nmol) was added to 250 μL of mono-biotinylated RNase A solution (250 μg, 17.5 nmol) in PBS buffer (0.1 M, pH 7.2) and incubated at RT overnight. Thereafter, the labeled protein was desalted via gel filtration using Sephadex G-15 gel and successful labeling was confirmed by MALDI-ToF-MS analysis. According to the mass spectrum, about 2−3 chromophores were attached to mono-biotinylated RNase A, yielding a clear blue solution. MALDI-ToF-MS (SA): m/z 15716 (dual labeled), m/z 16547 (triple labeled). In Vitro RNase A Activity Test. The enzymatic activity of native and modified RNase A was assessed using yeast RNA (yRNA), which forms a fluorescent complex in the presence of SYBR dye. RNase A catalyzes the cleavage of yRNA to ribonucleotides, resulting in the release of free SYBR dye and a concomitant drop in fluorescence, which is proportional to the RNase A activity. The in vitro assay was performed in a sterile 384-well plate under light exclusion. Briefly, 10 μL of SYBR dye (10× in TE buffer pH 8.0) was transferred into each well and supplemented with 50 μL TE buffer (blank) or native RNase A (100 ng/mL), mono-biotinylated RNase A (100 ng/mL), C3Mut-Stv (400 ng/mL), and C3Mut-Stv/mono-biotinylated RNase A complex (400 ng/mL and 100 ng/mL), respectively. Subsequently, 40 μL of freshly prepared yRNA substrate (100 μg/mL) was added into each well. The relative fluorescence units (RFU) of the samples were then monitored for 7 min in a BioTEK Synergy 4 Microplate Reader (excitation 485 nm; emission 535 nm). C3Mut-Stv-Mediated Internalization of Mono-Biotinylated RNase A. Internalization of mono-biotinylated RNase A (B-RNase A) was analyzed with confocal microscopy. To this end, J774A.1 cells seeded on coverslips were treated with 8 μg/ mL C3Mut-Stv and 4 μg/mL DyLight649-labeled B-RNase A or each protein alone for 4 h. Afterward, cells were fixed, permeabilized, and processed as described above. C3Mut-Stv was visualized using an anti-C3-antibody (1:5000) in conjunction with an anti-rabbit-IgG-Alexa488 (1:400). Finally, confocal images were recorded in 1 μm sections. To analyze the intracellular localization of B-RNase A in more detail, digitonin-based cell fractionation was used. J774A.1 cells grown in 12-well plates were treated with 4 μg/ mL C3Mut-Stv, 2 μg/mL B-RNase A, or both proteins for up to 24 h. Control cells were left untreated. Afterward, cells were washed and digitonin-based cell fractionation was performed as described before. Recovered fractions were then subjected to SDS-PAGE followed by Western Blot analysis. Internalized BRNase A was detected using Streptavidin-POD and enhanced chemoluminescence. In addition, the C3Mut-Stv transporter was visualized using an anti-C3-antibody. Equal protein loading was confirmed by an anti-Hsp90-immunoblot, and the absence

a nitrocellulose membrane using a dot-blot manifold (Bio-Rad, Munich, Germany). The membrane was then stained with Ponceau S and blocked with 5% nonfat dry milk in PBS-T for 1 h. Subsequently, the membrane was incubated with C3Mut-Stv conjugate (5 μg/mL in PBS-T) or C3Mut (5 μg/mL in PBS-T) for 1 h. After three washing steps in PBS-T, the membrane was incubated with a polyclonal anti-C3-antibody. Following additional washing steps in PBS-T, the membrane was incubated with peroxidase-coupled anti-rabbit−antibody. Bound proteins were finally detected using the ECL system. In addition, biotinylated proteins were detected with streptavidin−peroxidase. Cell Culture and Cell Viability Assay. J774A.1 cells were cultured in DMEM containing 10% heat-inactivated FCS, Lglutamate (4 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C with 5% CO2. Cells were scraped off and reseeded three times a week. To check for possible cytotoxic effects of the C3Mutstreptavidin delivery system, cell viability was measured over a period of 48 h. To this end, J774A.1 cells were grown in 96-well plates and incubated with up to 16 μg/mL C3Mut−streptavidin bioconjugate. Staurosporine, a strong inducer of apoptosis, was used as a positive control. Cell viability was then determined with Cell Titer 96 Aqueous One Solution Proliferation Assay (Promega, Mannheim, Germany) according to the manufacturer’s instructions. Confocal Microscopy of C3Mut-Stv Uptake. J774A.1 cells grown on coverslips were incubated for 4 h with C3MutStv (8 μg/mL) that was conjugated to biotin−fluorescein. Furthermore, cells were treated with C3Mut/biotin−fluorescein or biotin−fluorescein. Subsequently, the medium was removed and cells were washed twice with PBS. After fixation with 4% PFA and permeabilization by 0.4% Triton X-100, the cells were blocked in 5% nonfat dry milk in PBS-T. To stain early endosomes, cells were then treated with an anti-EEA1antibody (1:250), washed with PBS, and incubated with an Alexa-647-coupled secondary antibody (1:400). Finally, cells were mounted on microscope slides with Prolong Gold Antifade Solution (Invitrogen, Karlsruhe, Germany) and analyzed by confocal microscopy using a LSM510 Meta confocal system connected to a Zeiss Axiovert 200 M microscope (Zeiss, Oberkochen, Germany). Z-Stack images were acquired in optical sections of 1 μm and processed with ImageJ (NIH, USA). Digitonin-Based Cell Fractionation. Cell fractionation of J774A.1 macrophages was performed as described earlier.18,28 Briefly, cells were seeded in 12-well plates and incubated with C3Mut or C3Mut-Stv for the time points indicated. The medium was then removed and cells were washed 3 times with PBS. Subsequently, the cells were permeabilized by incubation with digitonin (20 μg/mL in PBS) for 5 min at RT. Cells were incubated for additional 25 min on ice to obtain the cytosolic supernatant. Afterward, the supernatant was collected carefully and the extracted cells were scraped off. Both fractions were subjected to SDS-PAGE followed by Western Blot analysis. C3Mut-Stv was detected using an anti-C3-antibody. To confirm equal protein amounts, the cytosolic marker protein Hsp90 was probed using a monoclonal anti-Hsp90 antibody (Santa Cruz, Heidelberg, Germany). The endosomal protein EEA1 was visualized by a polyclonal antibody (Acris, Herford, Germany) to check for cross-contamination of the cytosol with endosomal vesicles. 1428

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Figure 1. Generation of C3Mut−streptavidin bioconjugate and functional properties. C3Mut protein was treated with 2-iminothiolane to introduce sulfhydryl groups. Wild-type streptavidin (Stv) was maleimide-activated with sulfo-SMCC. Subsequently, activated proteins were incubated together to yield the C3Mut-Stv bioconjugate. The C3Mut domain facilitates cellular uptake, while Stv provides a versatile platform for the attachment of biotinylated ligands such as proteins or siRNA.

performed, in which biotinylated and non-biotinylated bovine serum albumin (BSA) was vacuum aspirated onto a nitrocellulose membrane in decreasing amounts. The efficiency of the blotting was monitored with Ponceau S staining of the

of endosomal vesicles in the cytosolic fractions was confirmed using an anti-EEA1-antibody. To quantify the cytosolic internalization, the signal intensity of B-RNase A in the cytosolic fractions was determined by Adobe Photoshop CS3 software, corrected for background using untreated control cells and evaluated by GraphPad Prism 4 software. Preparation and C3Mut-Stv-Mediated Internalization of Mono-Biotinylated Lysozyme. Site-selective monobiotinylation of lysozyme was performed as described very recently.29 A single Biotin-LC group was attached to the side chain of Lys 1 via an amide bond, resulting in Biotin-LC-(K1) lysozyme. N-Terminal monofunctionalization of lysozyme was confirmed by MALDI-ToF-MS and nanoLC-MS2 analysis.29 The protein concentration of mono-biotinylated lysozyme was determined by BCA assay. Internalization of mono-biotinylated lysozyme into J774A.1 macrophages was monitored by digitonin-based cell fractionation as described above for BRNase A. Reproducibility of the Experiments and Statistics. All experiments were performed independently at least twice. Results from representative experiments are shown in the figures. Values (n ≥ 3) are calculated as mean ± standard error of the mean (SEM) using GraphPad Prism 4 Software.

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RESULTS Generation and Characterization of the C3Mut− Streptavidin Bioconjugate. To generate the novel transport system, we have selected an enzymatically inactive mutant of C3botC3botE174Qwhich lacks ADP-ribosyltransferase activity, but is internalized into target cells comparable to wild-type C3bot. This mutant C3 protein (C3Mut) was chemically linked to wild-type streptavidin (Stv), which provides a platform for biotin binding (Figure 1). Therefore, thiol groups were introduced into C3Mut, while Stv was activated using the amine-reactive linker Sulfo-SMCC. Equal amounts of activated proteins were then incubated to allow bioconjugation via a stable thioether linkage. The coupling reaction was analyzed by immunoblotting and showed the formation of multiple C3Mut−Stv coupling products of varying molecular weight (Figure 2A). Interestingly, Stv was efficiently converted into bioconjugate molecules, whereas a substantial amount of free C3Mut was detected after the reaction. However, further attempts to optimize the coupling procedure, e.g. increasing the stoichiometry of Stv versus C3Mut, increasing thiolation of C3Mut, or application of a long amine-reactive PEG-linker to minimize steric hindrance, did not result in a higher yield of bioconjugate molecules. Subsequently, the C3Mut−Stv bioconjugate was characterized regarding its biotin-binding ability. Dot Blot analysis was

Figure 2. Analysis of C3Mut−streptavidin conjugate. (A) Immunoblot analysis of the resulting bioconjugation products. Detection with antiC3-antibody and anti-streptavidin-antibody, respectively, showed the formation of C3Mut−streptavidin bioconjugates with different molecular weight. (B) C3Mut−streptavidin specifically binds to biotin-labeled BSA in vitro. Biotinylated BSA (B-BSA) and nonbiotinylated BSA were vacuum-aspirated onto a nitrocellulose membrane using a dot-blot manifold (500, 250, 125, 63, 31, and 16 ng of each protein). The membrane was blocked and afterward incubated with either C3Mut-streptavidin (5 μg/mL, upper panel) or native C3Mut (5 μg/mL, lower panel). Bound C3Mut(-streptavidin) was detected using a C3-antibody. Binding is only observed for C3Mut−streptavidin conjugate but not for native C3Mut protein, and is specific for biotinylated BSA. Ponceau S staining at the bottom shows the membranes spotted with BSA and biotinylated BSA. 1429

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Figure 3. Internalization of C3Mut−streptavidin into the cytosol of J774A.1 cells without affecting cell viability. (A) Uptake of C3Mut−Stv into macrophages using immunofluorescence analysis. J774A.1 cells seeded on coverslips were treated with 8 μg/mL of C3Mut−Stv conjugated with biotin−fluorescein. After 4 h of incubation, cells were fixed and stained with an EEA1-antibody to visualize early endosomes. Z-Stack images were acquired by confocal microscopy in 1 μm sections and processed by ImageJ. Representative single optical sections thereof are displayed. Scale bar: 10 μm. (B) C3Mut−Stv bioconjugate is translocated into the cytosol of J774A.1 cells. Cells were incubated with C3Mut (1 μg/mL) or C3Mut− streptavidin (12 μg/mL), respectively, for up to 6 h. Subsequently, cell fractionation was performed and fractions were subjected to SDS-Page followed by Western Blot analysis with a C3-antibody. Equal amounts of proteins were confirmed by an anti-Hsp90 immunoblot. (C) Treatment of J774A.1 cells with C3Mut−Stv exerts no cytotoxic effect. J774A.1 cells were seeded into 96 well plates and treated with C3Mut−Stv (16 μg/mL) or staurosporine (1 μM). After 5, 24, and 48 h, cell viability was measured using MTS cell proliferation assay. Data are given as mean ± SEM, n = 2 (each experiment performed in triplicate).

panel). Confocal microscopy using an anti-C3 antibody revealed the same cytoplasmic distribution of C3Mut−Stv (data not shown). It is important to note that BF was not observed in J774A.1 cells without C3Mut−Stv. As expected, neither native C3Mut nor wt-Stv promoted the internalization of BF (data not shown). To analyze the internalization of C3Mut−Stv in more detail, J774A.1 cells were incubated for up to 6 h with C3Mut−Stv and native C3Mut, respectively. Thereafter, digitonin-based cell fractionation was performed as described previously19,28 to monitor the cytosolic delivery of the C3Mut−Stv bioconjugate. Both the cytosolic fraction and the extracted cells were subjected to SDS-PAGE followed by Western Blot analysis. Using an anti-C3-antibody, C3Mut as well as C3Mut−Stv were detected in the cytosolic fraction already after 30 min of incubation, and protein levels in the cytosol remained stable up to 6 h, which is in agreement with previous studies on the uptake kinetics of native C3 protein.10 However, we observed a strong decrease of the bioconjugate in the cytosol after 24 h, most likely due to protein degradation (data not shown and Figure 5B). As expected, high amounts of C3Mut−Stv were detected in extracted cells corresponding to membrane-bound or vesicular internalized bioconjugate (Figure 3B). Comparable protein amounts and successful cytosol preparation were confirmed with an immunoblot against cytosolic Hsp90, which was found in both fractions likewise. The endosomal marker protein EEA130 was visualized only in extracted cells, excluding contaminations of the cytosolic fraction with

membrane (Figure 2B). The membrane was blocked and then incubated with C3Mut−Stv. After extensive washing steps, bound C3Mut−Stv was visualized using an anti-C3-antibody. As expected, C3Mut−Stv binds to biotinylated BSA in a concentration-dependent manner, but not to non-biotinylated BSA. Importantly, native C3Mut protein binds neither biotinylated nor non-biotinylated BSA (Figure 2B). C3Mut−Stv was also capable of binding an end-biotinylated oligonucleotide with high affinity as assessed by an electrophoretic mobility shift assay in solution (data not shown). Notably, a huge excess of C3Mut did not bind the biotinylated oligonucleotide, underscoring the binding specificity. Taken together, the C3Mut−Stv bioconjugate interacts with biotinylated macromolecules such as proteins and oligonucleotides in vitro, which is crucial for further cell-based delivery studies. C3Mut−Stv is Internalized into J774A.1 Macrophages without Affecting Cell Viability. First, we used confocal laser-scanning microscopy to study the uptake of C3Mut−Stv into J774A.1 macrophages. Cells were grown on coverslips and were treated with C3Mut−Stv labeled with biotin−fluorescein (BF) for 4 h. Thereafter, cells were thoroughly washed, fixed, and stained for EEA1, a specific marker for early endosomes.30 Strikingly, the BF-conjugated C3Mut−Stv was clearly detectable in the cytoplasm of J774A.1 cells and displayed perinuclear staining (Figure 3A, upper panel). In addition, C3Mut−Stv showed some overlap with early endosomal vesicles as attested by the yellow dots in the merged channel (Figure 3A, lower 1430

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Figure 4. Characterization of Biotin-LC-(K1)RNase A. (A) MALDI-ToF MS spectrum of Biotin-LC-(K1)RNase A. Biotin-LC-(K1)RNase A displays a peak at m/z 14 021 [M+H]+ in the recorded MS spectrum. The increase in molecular weight from native RNase A to mono-biotinylated RNase A (339.5 Da) exactly matches the molecular weight of the Biotin-LC moiety. B. C3Mut−Stv binds to Biotin-LC-(K1)RNase A with high affinity. Mono-biotinylated RNase A (b-RNase A) and native RNase A were vacuum-aspirated onto a nitrocellulose membrane in decreasing amounts. After blocking, the membrane was incubated with either C3Mut−Stv (5 μg/mL, upper panel) or native C3Mut (5 μg/mL, middle panel). Bound C3Mut(-Stv) was detected using a C3-antibody and revealed a strong and specific interaction of the C3Mut−Stv conjugate with monobiotinylated RNase A. In addition, biotin-modified RNase A was detected with a streptavidin−peroxidase conjugate (lower panel). (C) Monobiotinylated RNase A retains its functional activity upon binding to C3Mut−Stv. Enzymatic degradation of substrate RNA was determined in the presence of SYBR dye and measured as decay in fluorescence.

A and native RNase A were spotted onto a nitrocellulose membrane. After blocking, the membrane was incubated with C3Mut−Stv and binding was revealed by immunoblot detection using a polyclonal C3 antibody. C3Mut−Stv showed a concentration-dependent binding to Biotin-LC-(K1)RNase, whereas no interaction was observed with native RNase A (Figure 4B, upper panel). As an important negative control, spotted proteins were also probed with C3Mut that did bind neither to native RNase A nor to mono-biotinylated RNase A (Figure 4B, middle panel). In addition, the biotin moiety of the synthesized Biotin-LC-(K1)RNase A was also detected with a streptavidin−peroxidase conjugate (Figure 4B, lower panel). Next, we analyzed the influence of the C3Mut−Stv transporter on the enzymatic activity of biotin-substituted RNase A upon preincubation of both components, since tight interaction with C3Mut−Stv may affect RNA substrate accessibility and degradation. The enzymatic activity of Biotin-LC-(K1)RNase bound to C3Mut−Stv was assessed using a functional in vitro assay. Here, yeast RNA (yRNA) forms a fluorescent complex with the SYBR dye and serves as substrate. Degradation of yRNA leads to the release of the SYBR dye, which is nonfluorescent in its free form, resulting in decay of the fluorescence. As a control, the reactions were performed in TE buffer and revealed autohydrolysis of yRNA over time (Figure 4C, squares). In contrast, the addition of both native RNase A (triangles) and Biotin-LC-(K1)RNase A (data not shown)

endosomal vesicles (data not shown). It should also be mentioned that C3Mut−Stv was detectable in CHO-K1 fibroblasts (data not shown), which has not been observed for C3Mut in previous studies.10 To exclude cytotoxic effects of the novel transport system, cell viability was measured in J774A.1 cells after treatment with C3Mut−Stv. Staurosporine was applied as a positive control, which strongly induces apoptosis by inhibiting protein kinases. Cells incubated with up to 16 μg/mL C3Mut−Stv showed no reduction in cell viability compared to untreated cells for up to 48 h, whereas the viability in staurosporine-challenged cells was decreased to 25% after 24 h (Figure 3C). Collectively, we demonstrated the internalization of C3Mut− Stv into the cytosol of J774A.1 macrophages without affecting cell viability over a period of 48 h. Generation and Characterization of Mono-Biotinylated RNase A. Mono-biotinylated RNase A serves as an attractive model protein due to the availability of a single biotin group at a precisely defined position (K1), which was introduced via a procedure developed very recently,29 as well as its high homology with clinically relevant RNase variants such as Onconase.31 The structure of the prepared Biotin-LC(K1)RNase A was determined by MALDI-ToF (Figure 4A), and the position of the biotin substituent was identified via the LCMS2-XIC method (data not shown). Subsequently, a dot blot analysis was performed, in which mono-biotinylated RNase 1431

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Figure 5. C3Mut−streptavidin-dependent delivery of mono-biotinylated RNase A into J774A.1 cells. (A) J774A.1 cells were treated with C3Mut− streptavidin (8 μg/mL), mono-biotinylated RNase A-DyLight649 (4 μg/mL), or both proteins. After 4 h, cells were fixed and C3Mut−streptavidin was visualized using C3-antibody. Finally, z-stack images were recorded by confocal microscopy with an optical section of 1 μm and processed with ImageJ. Representative single optical sections thereof are shown. Scale bar: 10 μm. Ph = phase contrast. (B) Mono-biotinylated RNase A (B-RNase A) is taken up into the cytosol of J774A.1 cells. Macrophages were treated with C3Mut−streptavidin (4 μg/mL), B-RNase A (2 μg/mL), or both proteins for up to 24 h. Subsequently, cell fractionation was performed and samples were subjected to SDS-PAGE and Western Blot analysis. C3Mut−streptavidin and B-RNase A were visualized using a C3-antibody and streptavidin-POD, respectively. Equal protein amounts were confirmed by Hsp90-immunoblot. EEA1 was detected as marker for early endosomes. (C) Quantification of B-RNase A uptake into the cytosol of J774A.1 cells. Intensity of B-RNase A signal in cytosolic fractions was determined using Adobe Photoshop CS3 software. Signal intensity is given as mean ± SEM of three independent experiments (a.u., arbitrary units). (D) Influence of C3Mut−Stv-mediated uptake of B-RNase A on J774A.1 cells. Cells were seeded on 96-well plates and treated for 5, 24, or 48 h with C3Mut−Stv, B-RNase A, or both proteins. As a positive control, staurosporine was used to induce apoptosis. Cell viability was determined using MTS cell proliferation assay. Data are given as mean ± SEM, n = 3 (each experiment performed in triplicate).

resulted in a fast drop of the fluorescence intensity of the yRNA-SYBR complex due to RNA degradation. Importantly, Biotin-LC-(K1)RNase A attached to the C3Mut−Stv transporter (circles) causes a similar decay of the fluorescence intensity as native RNase A, indicating that the complex retained its functional activity in vitro. C3Mut−Stv Promotes the Internalization of MonoBiotinylated RNase A into J774A.1 Cells. To monitor the uptake of Biotin-LC-(K1)RNase A into J774A.1 cells by the C3Mut−Stv transporter, laser-scanning microscopy was performed. Cells seeded on coverslips were treated with DyLight649-labeled Biotin-LC-(K1)RNase alone or together with C3Mut−Stv. Subsequently, cells were fixed, permeabilized, and incubated with an anti-C3-antibody followed by an Alexa488-coupled secondary antibody. Negligible amounts of Biotin-LC-(K1)RNase A-DyLight649 were detectable in the macrophages in the absence of the transporter (Figure 5A, middle panel). In turn, the C3Mut−Stv delivery system strongly promoted the uptake of Biotin-LC-(K1)RNase A-

DyLight649 into J774A.1 cells, which displayed almost complete colocalization with the C3Mut−Stv transporter as visualized by the yellow staining in the merge channel (Figure 5A, bottom panel). Due to the high biotin-binding affinity of the C3Mut−Stv bioconjugate, the cargo molecule should not be released, but remains attached to the transporter inside the cell. To study the intracellular localization of mono-biotinylated RNase A in more detail, digitonin-based cell fractionation was used. J774A.1 cells were incubated with C3Mut−Stv complexed to Biotin-LC-(K1)RNase A for up to 24 h. As controls, cells were left untreated or incubated only with the single components. Cell fractionation was then performed to obtain the cytosol and extracted cells. Both fractions were subjected to SDS-PAGE followed by immunoblot analysis (Figure 5B). The majority of Biotin-LC-(K1)RNase A was detected in extracted cells, but was also found in the cytosol of J774A.1 cells (Figure 5B). Biotin-LC-(K1)RNase A accumulated in the cytosol after 5 h and disappeared after 24 h, most likely due to protein 1432

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degradation, which is further illustrated by quantification of three independent time course experiments (Figure 5C). This paralleled the uptake kinetics of the C3Mut−Stv transporter that colocalized with Biotin-LC-(K1)RNase A to a large extent as shown above by confocal microscopy. It should also be noted that the early endosomal marker EEA1 was visualized exclusively in extracted cells, ruling out a putative contamination of the cytosol with early endosomal vesicles (Figure 5B). Equal protein amounts were confirmed by immunoblot detection of the cytosolic protein Hsp90. Importantly, BiotinLC-(K1)RNase A was not internalized into J774A.1 cells in the absence of C3Mut−Stv, highlighting the specificity of the C3Mut−Stv-mediated delivery. Internalization of Biotin-LC(K1)RNase A into J774A.1 cells did not influence cell viability over a period of 48 h as measured by MTS assay (Figure 5D). This finding was anticipated due to the known inactivation of wild-type RNase A by its highly potent inhibitor protein RI that is abundantly expressed in mammalian cells.26 C3Mut−Stv Facilitates the Delivery of Mono-Biotinylated Lysozyme into J774A.1 Cells. To establish the novel C3Mut−Stv delivery vehicle for a more general purpose, we used the protein lysozyme from hen egg white. At first, lysozyme was site-selectively labeled with biotin as outlined very recently,29 yielding Biotin-LC-(K1)-lysozyme. The structure was confirmed by MALDI-ToF-MS and subsequent nanoLC-MS2 analysis (data not shown). Next, the specific highaffinity binding of C3Mut−Stv to immobilized monobiotinylated lysozyme was shown by dot blot analysis (Figure 6A), which is instrumental for subsequent uptake studies. Finally, we were able to demonstrate the C3Mut−Stvdependent uptake of Biotin-LC-(K1)-lysozyme into J774A.1 cells. Five hours after treatment, mono-biotinylated lysozyme was detected in extracted J774A.1 cells and, to a lower extent, in the cytosol obtained after cell fractionation (Figure 6B). It is noteworthy that streptavidin on its own does not facilitate the uptake of Biotin-LC-(K1)-lysozyme into the cytosol of J774A.1 cells. Furthermore, the endosomal marker protein EEA1 was observed only in extract cells, indicating successful separation of endosomal compartments and the cytosol. In summary, the novel C3Mut−Stv delivery system mediated the specific uptake of both mono-biotinylated RNase A and lysozyme into the cytosol of J774A.1 macrophage-like cells.

Figure 6. C3Mut−Stv interacts with biotinylated lysozyme (B-Lys) and promotes its internalization into J774A.1 cells. (A) C3Mut−Stv binds specifically to B-Lys. B-Lys and native lysozyme (Lys) were vacuum-aspirated onto nitrocellulose membrane in decreasing concentrations (500, 250, 125, 62.5, 31.3, and 15.7 ng). After blocking of the membrane, overlay with C3Mut−Stv or uncoupled C3Mut (5 μg/mL each) was performed. Bound protein was detected using a C3antibody and the ECL system, revealing a specific binding of the bioconjugate C3Mut−Stv to B-Lys. (B) C3Mut−Stv promotes the internalization of B-Lys into the cytosol of J774A.1 macrophages. C3Mut, Stv, or C3Mut−Stv (4 μg/mL each) was preincubated with BLys (2 μg/mL) for 30 min at room temperature. Proteins were then added to J774A.1 cells grown in 12-well plates and further incubated for 5 h. Control cells were treated with PBS, C3Mut−Stv (4 μg/mL), or B-Lys (2 μg/mL) alone. Subsequently, digitonin-based cell fractionation was performed, and the fractions obtained were subjected to SDS-PAGE and Western Blot. B-Lys was visualized using streptavidin−POD, while the bioconjugate was detected using antiC3-antibody. Equal protein loading was confirmed with Hsp90 immunostaining. Detection of EEA1 was used to exclude endosomal contamination of the cytosolic fraction.

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DISCUSSION Owing to their unique features such as efficient and specific cellular uptake, bacterial protein toxins are of growing interest in the field of drug delivery,32 which led to the development of cellular delivery vehicles based upon the clostridial C2 toxin and diphtheria toxin.18,33 In the present study, we have engineered a C3bot-derived transport system by covalent linkage of wt-streptavidin (wt-Stv) to the enzymatically inactive C3Mut protein. Wt-Stv is commercially available and recombinant C3Mut protein can be purified at huge amounts after expression in E. coli. The bioconjugate obtained was heterogeneous, comprising several species of varying molecular weight and still contained free C3Mut protein, which could interfere with the subsequent entry of C3Mut−Stv into cells. Nevertheless, we observed efficient intracellular uptake of C3Mut−Stv in the presence of nonreacted C3Mut (Figures 3 and 5). Owing to its homogeneity and site-directed character, genetic engineering of a C3Mut−Stv fusion protein would offer some advantages over the bioconjugation procedure reported herein. However, genetic fusions in general are more time-

consuming and, in case of C3 protein, have to be performed under elevated biological safety level S2. We have very recently succeeded in the construction of a genetic fusion protein that displays comparable biochemical and cell-biological features as the C3Mut−Stv bioconjugate (H. Christow, unpublished results). The bioconjugate prepared was functionally active in vitro with regard to biotin binding as attested by its high-affinity interaction with immobilized biotin-labeled BSA and a biotinylated oligonucleotide in solution. The strong interaction of wt-Stv and its biotinylated ligands is crucial for complex stability in vivo and has also enabled the use of streptavidin for pretargeting of cancer cells in radioimmunotherapy.34 In addition, we could demonstrate an efficient uptake of the C3Mut−Stv conjugate into J774A.1 cells where it translocated 1433

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The novel C3Mut−Stv delivery system described in this study is a versatile tool for the efficient cytosolic internalization of biotin-labeled macromolecules into macrophages as exemplified by the use of Biotin-LC-(K1)RNase A. Moreover, the established delivery system is not restricted to transduction of mono-biotinylated RNase A, but also efficiently promoted the uptake of biotin-labeled lysozyme into J774A.1 cells, suggesting a more general applicability. This approach should also prove to be useful for the internalization of biotin-labeled siRNA to silence gene expression. This is supported by a study of Xia and co-workers that demonstrated the efficient siRNAmediated knockdown of target gene expression by means of biotin−streptavidin technology.45 Another promising application for C3Mut−Stv represents the delivery of peptides spanning BH3-domains into malignant leukocytes to induce mitochondrial apoptosis via cytochrome c release.46 This is illustrated by the finding that a cell-permeable Bax peptide was able to trigger apoptosis in cancer cells.47 Collectively, we have generated a novel delivery vehicle consisting of clostridial C3 protein and streptavidin that directs the uptake of biotin-labeled molecules into macrophages. Moreover, we demonstrated the C3Mut-Stv-mediated internalization of mono-biotinylated RNase A into the cytosol of J774A.1 cells, providing a framework for future applications of cytotoxic RNase A variants to kill cancer cells.

into the cytosol. As mentioned above, C3Mut−Stv internalization was also detectable in CHO-K1 cells (data not shown), which is in contrast to native C3 protein that is predominantly internalized into macrophages/monocytes.10 This change in cell-type selectivity could arise from the covalent bioconjugation to streptavidin molecules, influencing the interaction with cell surface proteins. Streptavidin was reported to harbor an RYD sequence that is similar to the RGD motif of fibronectin involved in cell adhesion.35 It is conceivable that the chemical engineering used herein may have modulated the cell surface binding properties of the C3Mut-coupled streptavidin. On the other hand, streptavidin on its own does not facilitate the internalization of biotinyated (macro)molecules into J774A.1 cells as confirmed above. This altered cell-type selectivity may broaden the general applicability of the bioconjugate and allows addressing other cell types of interest. Furthermore, the C3Mut−Stv transporter did not affect cell viability, which is in accordance with other streptavidin-based delivery vehicles.18,36 We then used Biotin-LC-(K1)RNase A as protein cargo to analyze the internalization of biomacromolecules. To this end, RNase A was mono-biotinylated at the N-terminal Lys residue by site-directed chemistry, not affecting its ribonuclease activity. This specific labeling is very important, since modification of other Lys residues, in particular, Lys41, can strongly reduce its activity.37 Mono-biotinylation of RNase A is also beneficial with regard to complex formation with C3Mut−Stv, since high degrees of biotin modification could favor aggregation. In addition, C3Mut−Stv did not affect the degradation of RNA catalyzed by Biotin-LC-(K1)RNase A as demonstrated above by an in vitro RNase A activity assay. Biotin-LC-(K1)RNase A was then successfully delivered into J774A.1 macrophages using the C3Mut−Stv delivery system. Immunoblot analysis revealed partial translocation into the cytosol of J774A.1 cells after cell fractionation. To further increase the cytosolic translocation of RNase A, it is conceivable to use pH-responsive polymers cocomplexed with C3Mut−Stv that disrupt endosomal membranes upon acidification.38,39 In line with this, the cytosolic release of RNase A could be promoted by endosome-disruptive peptides such as the HA2 peptide derived from influenza virus.40 Future studies may also involve cleavable biotin linkers with disulfide bonds that undergo reduction in the intracellular environment, leading to the release of the cargo molecule.41 Native RNase A used in the present study is a good model cargo protein for delivery studies, but is known not to be cytotoxic in mammalian cells, which was also confirmed by our delivery studies using biotin-substituted native RNase A (Figure 5D). This is attributable to the presence of ribonuclease inhibitor (RI) protein which was found at micromolar concentrations in the cytosol of all mammalian cells studied so far26 and binds with femtomolar affinity to RNase A,42 resulting in its inactivation. Hence, RNase A variants with a reduced binding to RI and related ribonucleases such as onconase that does not interact with RI are of great interest in tumor therapy.31,43 On the basis of the findings presented herein, the promising C3Mut−Stv delivery strategy could be transferred to mono-biotinylated cytotoxic RNase A mutants such as G88R-RNase A that evades RI binding and inhibits cell proliferation.44 Enhanced cellular uptake of these mutants mediated by the C3Mut−Stv delivery system should potentiate their cytotoxic effect, which would be of great clinical relevance in tumor therapy.

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AUTHOR INFORMATION

Corresponding Author

*Dr. Holger Barth: Tel. +49-731-50065503; Fax +49-73150065502; E-mail [email protected]. Dr. Jörg Fahrer: Tel. +49-6131-179218; Fax +49-6131-230506; E-mail fahrer@ uni-mainz.de. Present Address †

Institute of Toxicology, University Medical Center Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Angelika Rück, ILM, University of Ulm, Germany, for kindly providing the confocal microscope facility and to Ulrike Binder for excellent technical assistance. This work was funded by grant of the Medical Faculty, University of Ulm (Bausteinprojekt L.SBN.0060 to J.F.) and supported by the International Graduate School in Molecular Medicine Ulm.

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REFERENCES

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