Exotoxin A (PE) is an ADP-ribosyltransferase produced by gram-negative bacterium Pseudomonas aeruginosa
. It transfers ADP-ribosyl group from NAD+ to diphthamide residue of the eukaryotic EF2 elongation factor [1
], thus permanently inhibiting translation in the attacked cell. Like many bacterial toxins, PE retains the “A-B” protein domain architecture composed of catalytic (A) and binding (B) domains [3
]. The latter comprises a receptor domain (Ia, residues 1–252; Ib, residues 365–404) and the inserted translocation domain (II, amino acids 253–364). The receptor domain recognizes and binds to the alpha 2-macroglobulin receptor exposed on the cell surface (LRP receptor) [5
], while the translocation domain is believed to participate in protein release from endocytotic vesicles to the cytoplasm after internalization, yet its detailed role remains unclear [2
]. The PE catalytic domain is located C
-terminally (III, residues 405–613) and performs ADP-ribosylation. The full-length toxin contains two sequence motifs critical for determination of its cellular trafficking and intoxication pathway: the C
REDLK sequence important for retention from the endoplasmic reticulum [7
], and the furin cleavage 276
RQPR motif (located at the C
-terminus of domain II) [8
], which is recognized by ubiquitous furin protease. Furin seems to play a significant role in PE activation by cleaving away almost the whole domain B from the toxin (cut between residues R279 and G280) [9
The natural potency of PE to cause cell death makes it a promising candidate for use in anticancer immunotoxins. These chimeric proteins are usually made of a modified antibody or antibody fragment, attached to a fragment of a toxin [11
]. The “A-B” modular domain architecture of PE is convenient for engineering toxin targeting specificity by exchanging domain B for a specific antibody, which recognizes receptors exposed on the surface of targeted cancer cells, allowing the toxic agent (domain A) to be delivered to the cells of interest [12
]. Therefore, the PE and closely related Diphtheria toxin (DT) are the most commonly tested toxins thus far, and numerous PE- and DT-based immunotoxins are currently subjected to various phases of clinical trials [13
A number of drawbacks has been observed for the tested immunotoxins: lack of specificity, high immunogenicity, vascular leak syndrome (VLS) or hepatogenicity being the most deleterious [15
]. The attempts to reduce the side effects focus mostly on: (i) improving the antibody specificity for antigens of interest and removing the cross-reactivity with others; and (ii) increasing toxicity of the effector moiety and thus lowering the administered dose of immunotoxin [16
]. Another approach, which is proposed in this work, is based on lowering nonspecific toxicity of potential immunotoxins by reducing the access of the toxin to its natural substrate in slowly dividing cells and changing it to a cancer-selective compound without interfering with its cytotoxic potency.
One of the most remarkable hallmarks of the cancer cells is their proliferative capacity [17
]. Therefore, in order to affect intensively dividing cancer cells, we propose a new strategy based on attaching the nuclear localization sequence (NLS) to the toxin and trapping it within the nucleus [18
]. In non-dividing cells, nuclear membrane maintains its integrity and effectively separates the nuclear interior from the cytoplasm, whilst, in dividing cells, a periodic disintegration of nuclear membrane is observed. Since NLS targets the toxin to the nucleus, the cytoplasmic protein synthesis machinery should be protected from the unintended activity of the toxin until the cell division. Consequently, as long as the cell is in an unproliferative state, the access of the modified toxin to its natural substrate (eEF2) is limited. In a proliferating cell, in prometaphase, the nuclear envelope disruption would lead to the release of the toxin into cytosol, hence promoting cell death. This newly proposed mechanism should make the toxin active mainly against highly proliferating cancer cells.
The idea of attacking only fast proliferating cells was already exploited in a traditional cytotoxic chemotherapy which works primarily through the inhibition of cell division [20
] with paclitaxel, cisplatin, doxorubicin and many others being frequently used in this type of treatment [21
]. In addition to cancer cells, other rapidly dividing cells (e.g., hair, gastrointestinal epithelium, and bone marrow) are affected by these drugs, which causes numerous side effects (e.g., neutropenia, cytopenia, anemia, hair loss, skin itch, nausea, vomiting or diarrhea, and damage to liver, kidney and bone marrow) [22
]. However, potential immunotoxins combining selective toxin, proposed in this work, with highly specific antibodies should be devoid of nonspecific toxicity.
Here, we report the cytotoxic activity and selected biochemical properties of a newly designed recombinant PE mutein enriched with NLS motif. We also present a novel protocol for PE purification developed in this study that allows obtaining highly active recombinant Exotoxin A and its sequence-modified variants.
In this this study, we designed a novel PE mutein by incorporating the NLS sequence at the C-terminus of the toxin [24
]. This modification was proposed to target the toxin to the nucleus and separate it from the cellular compartment, where its toxic activity is exerted [51
]. We found that NLS sequence does not influence the in vitro ADP-ribosylation activity as suggested by in silico analysis and is highly effective, leading to accumulation of the toxin in the nucleus in one of the cellular models employed in the study. Although we assumed that the toxin enriched with NLS should exhibit lower cytotoxicity because of its separation from its natural substrate eEF2, the results of the cytotoxicity assays were contradictory. Particularly, in HepG2 cells, where the toxin was triggered to preferentially accumulate within the cell nucleus, its toxicity was fully retained. One of the potential explanations is unexpected nuclease activity of PE detected in this study and its potential ability to cleave chromosomal DNA, which could be an alternative intoxication mechanism of the toxin accumulated in the nucleus. Consistently, when nuclear transport apparatus was inhibited by importazole, the viability of HepG2 cells increased. Furthermore, PE-NLS within hours of exposure of cells mediated significant damage of nuclear DNA in a caspase-independent fashion. This suggests that PE is another toxin which has both ADP-ribosylation and nuclease activities, what has been already reported for e.g., Diphtheria toxin from Corynebacterim diphtheriae
] and cytolethal distending toxin from Salmonella enterica
]. However, neither the molecular mechanism of nuclease activity of PE nor the catalytic amino acid residues responsible for this enzymatic activity have been identified yet. Our future investigations will address these issues to provide more insight into this newly detected activity of the toxin.
Additionally, we developed a novel and universal protocol for purification of PE and its muteins. It allowed purification of all analyzed toxins, which retain high biological activity, except of PE inactive triple mutant. Importantly, ADP-ribosylation activity of the toxins obtained using our newly developed method turned out to be higher both, in vitro and in vivo, as compared to commercially available PE and proteins purified using several purification protocols described in the literature (data not shown). In addition, in vitro ADP-ribosylation activities of independent batches of the analyzed PE muteins were comparable. Such reproducibility of the method combined with high activity of the obtained toxins is of great importance for its potential medical applications.
We also analyzed whether furin processing is necessary for toxin activation inside the cell. We detected only full-length toxins in the intoxicated cells and showed that the unnicked toxin (PE-furin) retains undiminished cytotoxicity. This indicates that furin cleavage is not critical for PE activity, at least in some cell types, which is consistent with several previous studies showing lack of correlation between proteolytic processing and PE toxicity [37
The general idea of modifying intracellular fate of PE protein by introducing NLS motif into its amino acid sequence was experimentally confirmed in this study. However, our main assumption that trapping the toxin inside the nucleus would lower the cytotoxicity of PE turned out to be incorrect, possibly due to an unknown alternative toxicity mechanism independent of cytoplasmic localization of the toxin. It suggests that further understanding of detailed mechanisms of PE action is needed for its successful application in cancer targeted therapies. For example, we do not know to what extent the observed nuclease activity participates in PE-mediated cytotoxicity. It is conceivable that by abolishing the nuclease activity (without affecting ADP-ribosylation potential) in combination with incorporation of NLS someone might engineer a PE mutein preferentially active against proliferating cells.
4. Materials and Methods
4.1. Proteins Expression and Purification
DNA constructs encoding the analyzed toxins were cloned into modified Champion pET SUMO vector using AgeI and HindIII restriction sites. The proteins were overexpressed as N
-terminal his-tagged SUMO fusions in Escherichia coli
strain NiCo21(DE3) under the control of inducible T7 promoter activated by 0.5 mM IPTG using TB as growth medium [55
]. In each case, pellet was lysed in buffer A (50 mM NaH2
, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 12.0) with addition of 2 M urea, 0.5 mM PMSF, 5 mM β-mercaptoethanol, benzonase (5 U/mL), lysozyme (0.1 mg/mL) and Triton X100 (0.05%). Sonicated lysate was diluted 4 times with buffer A at pH 8.0. After centrifugation, crude extract was loaded on chitin resin. Flow through from the chitin column was passed through NiNTA column (Qiagen, Hilden, Germany). The column was washed with buffer containing high salt concentration to remove unspecific binding (50 mM NaH2
, 2 M NaCl, 20 mM imidazole, 10% glycerol, pH 8.0). Toxin was eluted in gradient elution mode with buffer 50 mM NaH2
, 300 mM NaCl, 500 mM imidazole, 10% glycerol, pH 8.0. SUMO protease was added to the eluate (to remove his-tagged SUMO) and left at 4 °C overnight. The protein was loaded on size exclusion column (HiLoad Superdex 200, GE Healthcare Life Sciences, Marlborough, MA, USA) equilibrated with phosphate buffer saline amended with 10% glycerol, pH 7.4. The collected fractions were concentrated using Vivaspin Turbo. Protein concentrations were measured using Bradford protein assay (BioRad, Hercules, CA, USA). The proteins were subsequently analyzed by Western blot and using SDS-PAGE gels to identify their purity.
His-tagged eEF2 was expressed in Saccharomyces cerevisiae strain TKY675 in 12 L of yeast extract peptone dextrose medium for 22 h at 30 °C. Pellets obtained after centrifugation were frozen in liquid nitrogen and rapidly disrupted in stainless-steel mill. The disrupted cells were suspended in 1 L of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0). The lysate was sonicated, centrifuged and filtered, and then loaded on a NiNTA column. The protein was further purified and analyzed as described above.
4.2. ADP-Ribosylation Assays
4.2.1. Western Blotting with Anti-Biotin
1 μg of toxin was mixed with 600 ng of eEF2, 0.5 μL of biotinylated NAD+ (250 μM, Trevigen, Gaithersburg, MD, USA) in a total volume of 24 μL reaction buffer containing 50 mM Tris, 1 mM EDTA, 1 mM DTT, 500 mM NaCl, pH 7.6. The reaction mix was incubated for 1 h at 37 °C. The samples were separated by electrophoresis and transferred to PVDF membrane. The detection was performed using HRP-conjugated streptavidin (Sigma Aldrich, St. Louis, MO, USA) at concentration 1:7000.
4.2.2. Solid-Phase Assay
U16 MaxiSorp plate (Nunc A/S, Roskilde, Denmark) was precoated with goat anti-mouse antibody (GAM) (Agilent Technologies, Santa Clara, CA, USA) .Antibody was diluted 1:100 in PBS and 100 μL of the mixture was placed in each well for 2 h. To avoid cross-contamination, every second column of wells was coated. The wells were washed three times with fresh PBST (PBS with 0.05% Tween 20), soaked for 30 min with the same buffer and washed again. GAM precoated wells were incubated overnight with 100 μL of PBS with penta-his antibody (Qiagen, Hilden, Germany) (final concentration of antibody 5 μg/mL), and then washed with PBST.
100 μL reaction mix contained 1.2 μg of His-tagged eEF2, 5 μM of biotinylated NAD+, 50 mM Tris (pH 7.6), 10 μL BSA (3 mg/mL), 10 mM l-arginine, 0.5 mM NaCl, 1 mM EDTA, 1 mM DTT and 1 μL of toxin (concentration ranging from 0.1 ng/mL to 100 μg/mL). The reaction was incubated for 2 h at 37 °C and transferred to freshly washed coated wells. Each toxin was tested in triplicate on a separate plate with PE-inactive as a control and background indication.
The reaction mixture was incubated on a 96-well plate for 2 h at constant 21 °C without shaking. Precoated wells were washed and soaked with PBST between all steps. After incubation and washing, the wells were filled with blocking solution (50 mg/mL dry milk powder in 200 μL PBS) for 30 min at 21 °C, followed by 30 min at 37 °C. After incubation with HRP-conjugated streptavidin (Sigma Aldrich) for 30 min at 21 °C (1 μg/mL of antibody in 100 μL PBS containing 100 μL/mL FBS and 30 mg/mL BSA), the wells were washed again and finally incubated with 100 μL of 3,3’,5,5′-tetramethylobenzidine (0.1 mg/mL TMB resuspended in 0.05 M citric-phosphate buffer, 0.01% H2O2, pH 5.0) for color development. The reaction was stopped after 30 min by addition of 50 μL of 2 M H2SO4. The absorbance in each well was measured at 450 nm and 490 nm, using Enspire microplate reader (Perkin Elmer, Waltham, MA, USA). The EC50 values were calculated from dose–response curves using GraphPad Prism 5 software (version 5.0 for Windows, GraphPad Software, Inc., San Diego, CA, USA, 2010).
4.3. Cytotoxicity Measurements
The HepG2 cells and A549 cells were obtained from ATCC (Manassas, VA, USA) and were maintained under standard conditions in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2. The passage number range for both cell lines was maintained between 20 and 25.
To estimate the cytotoxicity of the investigated toxins, the neutral red uptake assay was used [56
]. HepG2 and A549 cell lines were seeded into 96-well plates at a density of 1.5 × 104
cells per well. After 24 h, the cells were treated with increasing concentrations of the toxins and incubated for another 48 h. Cells treated with CHX (final concentration 20 μg/mL) and SDS (final concentration 200 μg/mL) were used as additional controls for sensitivity of cells to inhibition of protein synthesis and to standard cell membrane damaging cytotoxic agent, respectively. After incubation, the medium was removed and the cells were washed with cold PBS. The washed cells were incubated with 50 μg/mL neutral red in HBSS for 3 h. Following incubation, the neutral red solution was removed, the cells were washed with PBS and the cell-bound dye was extracted using a solution containing 50% ethanol and 1% acetic acid by gentle shaking for 10 min. Absorbance at 550 nm was determined using a Sunrise microplate reader (Tecan, Männedorf, Switzerland). The IC50
values were calculated based on linear dose–response curves using GraphPad Prism 5 software (version 5.0 for Windows, GraphPad Software, Inc., San Diego, CA, USA, 2010).
4.4. Viability Measurements
HepG2 and A549 cells were seeded into 96-well plates at a density of 1.5 × 104 cells per well, and after 24 h cells were preincubated for 1 h with the following inhibitors: MG132, lactacystin and importazole (all inhibitors from Sigma Aldrich). The inhibitors were added at a final concentration of 5 μM. Medium was not removed after incubation and cells were supplemented with pure medium (control cells) or treated with selected toxins at concentrations equal to IC50 values for another 48 h. After that time, the incubation medium was removed, and the viability was assessed as described above for cytotoxicity measurements.
4.5. Intracellular Localization Studies
4.5.1. Western Blotting with Anti-Exotoxin A
For Western blotting, cells were pretreated with toxins (PE-native and PE-NLS) for 3 h, 4.5 h and 6 h, and then cytoplasmic and nuclear fractions were prepared: cells were lysed for 10 min at room temperature in a buffer containing 0.4% IGEPAL CA-630, 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, and 1× Roche protease inhibitor mixture. Lysates were centrifuged at 15,000× g for 3 min at 4 °C. Supernatants (cytosolic fractions) were collected and used directly in Western blots or stored at −80 °C. The pellets were suspended in a buffer containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 10% glycerol and 1 × Roche protease inhibitor mixture; shaken vigorously for 2 h at 4 °C; and centrifuged at 15,000× g for 5 min at 4 °C. The resulting supernatants (nuclear extracts) were collected and used directly in Western blots or stored at −80 °C. Protein concentrations were determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
The fractions were separated using electrophoresis and transferred to a membrane using the iBlot Dry Blotting System (Life Technologies, Carlsbad, CA, USA). After overnight incubation with blocking agent (SuperBlock (TBS) Blocking Buffer, Thermo Fisher Scientific), the membrane was incubated with anti-Pseudomonas Exotoxin A (Sigma Aldrich, St. Louis, MO, USA) for 1 h (1:5000). The primary antibody was detected with goat anti-mouse peroxidase conjugated secondary antibody (Agilent Technologies, Santa Clara, CA, USA) (1:2000) and visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) Quantification of individual signals was normalized to the level of α-actin (SC-1616 HRP antibody, Santa Cruz, Santa Cruz, CA, USA).
4.5.2. Confocal Microscopy
For confocal microscopy, cells were pretreated for 3 h with toxins (PE-native and PE-NLS) labeled with Alexa Fluor 488 Dye (Thermo Fisher Scientific) according to manufacturer protocol. The cells were fixed (4% formaldehyde, for 10 min) and stained with fluorescent dyes (NucRed Live 647 ReadyProbes Reagent for nuclei visualization and Alexa Fluor 594 Phalloidin for actin visualization; Thermo Fisher Scientific) in standard conditions, and visualized under a Nikon C1 confocal microscope.
4.6. Furin Digestion Assay
The efficacy of furin cleavage was tested in two buffers: B1 (10 mM HEPES, 0.05% Triton X100, 1 mM CaCl2, 1 mM β-mercaptoethanol, pH 7.5) and B2 (0.05 mM citric acid, 100 mM NaH2PO4, 1 mM CaCl2, 1 mM β-mercaptoethanol, pH 5.5). Two thousand five hundred nanograms of each toxin was diluted to 100 μL in B1 or B2 and incubated for 24 h at 37 °C with 2 U of recombinant furin (NEB). The cleavage efficiency was assessed using both, silver stained SDS-PAGE and Western blot.
4.7. Agarose Gel Nuclease Assays
4.7.1. DNA/RNA Degradation
The analyzed toxins (2 μg) were mixed with nucleotide substrates: 200 ng of dsDNA (Xho I—linearized pBSK plasmid treated with alkaline phosphatase and circular pBSK), 20 ng of ssDNA (synthetic oligonucleotide GACTGGAGCACGAGGACACTGACATGGACTGAAGGAGTAGAAA) and 10 ng of RNA (synthetic oligonucleotide CGACUGGAGCACGAGGACACUG) in 20 μL of digestion buffer (10 mM Tris, 10 mM MgCl2, 1 mM CaCl2, pH 7.5). Reaction was incubated for 16 h at 37 °C and terminated by mixing with gel loading buffer (6× DNA Loading Dye, Thermo Fisher Scientific). Digestion reactions were also prepared with addition of 20 mM EDTA simultaneously with PE-native or 2 μg of SUMO protease. For positive controls, 2 U of DNase I (Thermo Fisher Scientific, Waltham, MA, USA ) and 2 U of RNase I (Thermo Scientific) were used for DNA and RNA degradation, respectively. Samples were analyzed on 1% agarose gels stained with SYBR Gold (DNA) or SYBR Green II (RNA) and visualized under UV light.
Nuclease assays with 2 μg of PE-native toxin and dsDNA fragment were performed in various conditions: (i) using different cations—MgSO4, MnCl2, NaCl, CaCl2, ZnCl2, FeCl3, NiSO4, CoCl2, KCl, and MgCl2 salts were added to the Tris buffer (10 mM salt, 10 mM Tris, pH 7.5); and (ii) in a broad temperature range (4 °C–54 °C) in Tris buffer (2.5 mM MgSO4, 10 mM Tris, pH 7.5). All samples were incubated for 24 h at 37 °C (except for ii) and analyzed on 1% agarose gels stained with Midorii Green.
Analyzed toxins (10 μg) and 60 μL of total lysate of NiCo21 (DE3) were mixed with 6× Laemmli buffer without heating and separated on 10% SDS-PAGE. The gel bands of interest (toxins), visualized by Ponceau S, were excised and placed in dialysis tubes with 250 μL of TG buffer. In addition, fragment of the gel containing proteins ranging from ~55 kDa to 75 kDa were excised from the separated mock E. coli
extract. Tubes were placed in horizontal electrophoresis chamber with constant 10 mV for 24 h [57
]. Thirty microliters of the eluted and filtered toxins were mixed with 200 ng of circular DNA (circular pBSK plasmid) in buffer 10 mM Tris, 10 mM MgCl2
, 1 mM CaCl2
, pH 7.5 (total volume 50 μL). Digestion reaction was also mixed with 30 μL of TG used for electroelution or PE-native with addition of 20 mM EDTA.
For positive control, 2 U of DNAse I (Thermo Scientific) was used. Samples were incubated in 37 °C and analyzed after 96 h on 1% agarose gels stained with SYBR Gold.
4.8. Alkaline Comet Assay
Assessment of DNA damage was carried out using the alkaline CometAssay® Kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer protocol. Briefly, HepG2 cells (1 × 105 cells per well) were exposed to PE-NLS (230 ng/mL) in the presence of Pan-Caspase Inhibitor Z-VAD-FMK (50 μM) in 24-well plates for 3 h and 6 h at 37 °C. The H2O2 (50 μM) was used as a positive control. After washing twice with PBS, the cells were suspended in 1 mL of PBS and scraped. The cell suspension was centrifuged at 3000 rpm for 5 min and the pellet was suspended in 100 μL of PBS. After that, 1 × 105 cells were combined with molten LMAgarose (at 37 °C) at a ratio of 1:10 (v/v), placed (50 μL) on CometSlide and kept for 10 min at 4 °C. The cells were lysed in a lysing solution for 60 min. After the lysis, the slides were placed in alkaline solution for 20 min to allow DNA unwinding, and then electrophoresed for 30 min with 21 V. All preparative steps were conducted in dark to prevent secondary DNA damage. Cells were stained with 100 μL of diluted (1:10,000 in TE Buffer, pH 7.5) SYBR Green I for 30 min (room temperature) in the dark. The slides were analyzed at 20× magnification using a fluorescence microscope (Olympus BH2-RFCA with Hamamatsu ORCAII BT-1024 camera (Hamamatsu, Hamamatsu City, Japan). Comets were quantitatively analyzed using Comet Assay Software Project casp-1.2.2 (University of Wroclaw, Wroclaw, Poland). Each treatment was carried out in duplicate and 20 randomly selected comets from two microscope slides were analyzed.
4.9. Statistical Analysis
IC50 were calculated using nonlinear regression of log (concentration of toxin) vs. normalized response using variable slope model. In analysis of statistical significance of differences in dose response to PE and PE muteins, the area under the curve for dose response curves obtained in individual experiments were determined and used in multiple t-test comparison (with Holmak–Sidak adjustment). One way unpaired ANOVA followed by Dunnett’s multiple comparisons test was employed in other analyses. All statistical analyses were performed using GraphPad Prism 7.02 software (version 7.02 for Windows, GraphPad Software, Inc., San Diego, CA, USA, 2016).