Abstract
Superoxide dismutase (SOD) plays important roles in the balance of oxidation and antioxidation in body mostly by scavenging superoxide anion free radicals (O2.−). Previously, we reported a novel Cu/Zn SOD from jellyfish Cyanea capillata, named CcSOD1, which exhibited excellent SOD activity and high stability. TAT peptide is a common type of cell penetrating peptides (CPPs) that efficiently deliver extracellular biomacromolecules into cytoplasm. In this study, we constructed a recombinant expression vector that combined the coding sequences of TAT peptide and CcSOD1, and then obtained sufficient and high-purity TAT-CcSOD1 fusion protein. Compared with some reported SODs/CPP-SODs, TAT-CcSOD1 possessed stronger tolerance to heat and acid–base environment. TAT-CcSOD1 efficiently penetrated cell membrane and significantly enhanced the O2.− scavenging ability in cells, and attenuated H2O2-induced cytotoxicity and NO generation in HaCaT cells. This study serves as a critical step forward for the application of TAT-CcSOD1 as a potential protective/therapeutic agent against oxidative stress-related conditions in the future.
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Introduction
Reactive oxygen species (ROS) mainly includes peroxide, superoxide, hydroxyl radicals, and singlet oxygen1. At low concentrations, ROS acts as an important signaling molecule involved in physiological activities of cell metabolism2,3. However, excessive ROS due to the imbalance of generation and scavenging can attack cellular molecules and cause a variety of pathological damage2,4, leading to many diseases including cancer, diabetes, heart disease, and some neurodegenerative diseases3,5,6.
Antioxidant enzymes can efficiently scavenge ROS, and have been used to treat various oxidative stress-associated diseases. Among them, SOD, which can irreversibly catalyze the dismutation of superoxide free radicals to generate oxygen and hydrogen peroxide, is a key antioxidant metalloenzyme and plays an important role in body’s antioxidant system7,8,9. As shown in Fig. 1, there are mainly three isoforms of SODs including cytoplasmic Cu/Zn SOD (SOD1), mitochondrial Mn SOD (SOD2) and extracellular Cu/Zn SOD (SOD3 or EcSOD). SODs require catalytic metal (Cu/Zn or Mn) for activation and exhibit specific biological activities10,11. SODs can attenuate the occurrence and development of many diseases including skin aging, psoriasis, Alzheimer’s disease, cancer and etc.12,13,14. As the only type of SOD in cytoplasm, Cu/Zn SOD (SOD1) plays an important role in multiple ROS scavenging processes and is critical for the oxidation and antioxidation balance in cytoplasm.
Roles of SOD in the regulation of intracellular and extracellular O2.−. NOX NADPH oxidase family, ETC mitochondrial electron transport chain, GPX glutathione peroxidase, PRX peroxidase, CAT catalase.
Jellyfish are an important component of the marine plankton with abundant resources. Studies have shown that jellyfish contained a wide variety of bioactive substances which possessed multiple pharmacological potentials including anti-inflammatory, anti-microbial, anti-tumor and antioxidant activities15,16,17,18. We previously identified a novel Cu/Zn SOD from jellyfish Cyanea capillata, named CcSOD1, and confirmed its specific SOD activity19. The CcSOD1 protein was found to markedly inhibit superoxide anions and retain highly stable activity in a wide range of pH and temperature intervals, suggesting a great potential of this enzyme as therapeutic agent for the treatment of oxidative stress-related conditions. However, due to the biological barrier of cell membrane, most biomacromolecules cannot enter the cells and perform their biological effects20.
TAT peptide21,22, a specific sequence from HIV-1 transactivator protein, is a classic type of CPPs which can facilitate the delivery of proteins and other biomacromolecules into cells20,23,24. TAT peptide has been reported to deliver proteins into cells efficiently25,26. In this study, we generated a recombinant TAT-CcSOD1 fusion protein, named TAT-CcSOD1. Transmembrane ability of TAT-CcSOD1 and its enhancement effect on intracellular SOD activity were investigated. The fusion protein also exhibited protective effect on H2O2-induced oxidative stress in HaCaT cells. To our knowledge, this is the first study on a cell-permeable marine-derived SOD. The current study demonstrates an effective method for the delivery of marine SOD into cells, and lays the foundation for future clinical application of marine antioxidant enzymes.
Material and methods
Construction of plasmids expressing TAT-CcSOD1 fusion protein
The TAT-CcSOD1 expression vectors were constructed by incorporating the coding sequences of HIV-1 TAT and CcSOD1 into the pET-24a vector. First, the coding sequence of the TAT-CcSOD1 recombinant protein, including NdeI and XhoI restriction sites, was synthesized by Beijing Syngentech Co., LTD. Then, the obtained sequence was inserted into the pET-24a vector (Novagen, Madison, WI, USA) through DNA digestion and ligation. The recombinant vectors were transformed into TOP-10 E. coli. Afterwards, the appropriate recombinant plasmids of TAT-CcSOD1/pET-24a were identified through selection on a solid culture medium plate containing 100 μg/mL kanamycin. The TAT-CcSOD1 expression plasmids were extracted using the Plasmid Extraction Kit (Solarbio, Beijing, China) and then subjected to sequencing by Berry Genomics Co., Ltd. Additionally, a CcSOD1 expression vector devoid of the TAT sequence was constructed as a control for future experiments.
Exploration of the optimal inducible expression conditions
Briefly, the sequenced TAT-CcSOD1/pET-24a vectors were transformed into BL21 competent E. coli., and the positive vectors were picked out from culture plates and amplified in LB liquid medium at 37 °C, 250 rpm until the OD600 reaching 0.8–1.0. Subsequently, the bacterial cells were incubated with different concentrations of IPTG (0.2, 0.4, 0.6, and 0.8 mM) at 12 °C, 25 °C and 37 °C, respectively. Following these procedures, the cell pellets of each sample were collected by centrifugation at various durations (6, 12, 24, 48 h) and resuspended in binding buffer (20 mM NaH2PO4, 500 mM NaCl, 30 mM imidazole, pH 7.4). The resuspended pellets underwent sonication for 2 min, and then centrifuged at 13,000×g for 10 min at 4 °C. Subsequently, the sonicated supernatants and sediments were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained with the Coomassie Blue Staining Kit (Sigma, USA). The temperature, duration of induction, and concentration of IPTG that resulted in the highest expression of the recombinant protein in the supernatants were identified as the optimal conditions for inducible expression.
Preparation and identification of fusion protein TAT-CcSOD1
The expression of the fusion protein TAT-CcSOD1 was induced under optimal conditions. Following sonication of cell pellets, the supernatant containing TAT-CcSOD1 was harvested and subjected to purification using a HisTrap affinity column. Briefly, the supernatant was allowed to flow slowly through the column, facilitating binding of the fusion protein TAT-CcSOD1. Subsequent washing of the column was carried out using plenty of binding buffer. The His-tagged TAT-CcSOD1 was then eluted from the column by employing a gradient of elution buffer concentrations (20 mM NaH2PO4, 500 mM NaCl, 500 mM imidazole, pH 7.4).
The eluted TAT-CcSOD1 was identified through western blot analysis27. Following electrophoresis on a 12% SDS polyacrylamide gel, the proteins were subsequently transferred to a PVDF membrane and blocked using 5% non-fat dry milk for a duration of 1 h at 25 °C. The transferred proteins were then subjected to treatment with a His-tag primary antibody (dilution 1:2000, Beyotime, China) overnight at 4 °C, followed by incubation with HRP-labeled secondary antibody (dilution 1:1000, Beyotime, China) for 1 h at 25 °C. Subsequently, the membranes underwent three washes lasting 30 min before visualization utilizing a multifunctional imaging system (Thermo Fisher Scientific, USA).
The eluted fluid containing TAT-CcSOD1 was subjected to lyophilization following desalination. The resulting lyophilized powder of TAT-CcSOD1 was then reconstituted in PBS at a concentration of 1 mg/mL to serve as the working solution for subsequent assays.
SOD activity assay in cell-free system
The SOD activity of the fusion protein TAT-CcSOD1 was assessed following the experimental procedure outlined in the Total Superoxide Dismutase Assay Kit (Beyotime, China). Initially, the fusion protein TAT-CcSOD1 was diluted in SOD sample preparation solution at a gradient of concentrations (0, 15.6, 31.25, 62.5, 125, 250 or 500 μg/mL). Subsequently, WST-8 enzyme working solution (160 µL), sample (20 µL), and reaction start solution (20 µL) were successively added to a 96-well plate and incubated at 37 °C for 30 min. The control group was established by substituting PBS for the sample, while the blank group lacked the reaction start solution. Finally, the absorbance at 450 nm was detected using a multifunctional microplate detector (Thermo Fisher Scientific, USA).
pH and thermal stability assay
Thermal stability of TAT-CcSOD1 was evaluated by measuring the residual activity after heat treatment. First, 0.5 mL of TAT-CcSOD1 (200 μg/mL) was incubated at 30 °C, 50 °C and 70 °C for 10, 20, 30, 40, 50 and 60 min, respectively. Then, SOD activity of each sample was detected and measured by the standard method as described in Material and methods 2.4. The SOD activity of TAT-CcSOD1 before incubation was defined as 100%.
pH stability of TAT-CcSOD1 was determined by measuring the residual activity in buffers with different pH values (ranging from 3.0 to 11.0). First, TAT-CcSOD1 (200 μg/mL) was dissolved in various buffers including 50 mM acetate buffer (pH 3.0–6.0), sodium phosphate buffer (pH 6.0–8.0), Tris–HCl buffer (pH 8.0–9.0) and glycine–NaOH buffer (pH 9.0–11.0) and incubated at 37 °C for 2 h. Then, SOD activity of each sample was detected as described above. The SOD activity of TAT-CcSOD1 (200 μg/mL) dissolved in PBS was defined as 100%.
Western blot analysis
HaCaT cells (Anweisci, Shanghai, China) were cultured in petri dishes until the cell density reached 80%, and then the cells were exposed to 100 μg/mL TAT-CcSOD1 for varying durations of 0, 15, 30, 60, and 120 min. PBS and 100 μg/mL CcSOD1 served as the blank and negative controls, respectively. Subsequent to treatment with TAT-CcSOD1, the HaCaT cells were washed twice with ice-cold PBS and then lysed with cell lysis buffer (Beyotime, China) at 4 °C for 15 min. Following centrifugation at 4 °C and 12,000×g for 15 min, the resulting supernatants were collected for western blot analysis, as described in Materials and Methods section "Preparation and identification of fusion protein TAT-CcSOD1".
Immunofluorescence assay
HaCaT cells were cultured on treated coverslips in a dish until reaching approximately 80% confluency, at which point they were exposed to 100 μg/mL TAT-CcSOD1 for various durations of 0, 15, 30, 60, and 120 min. PBS and 100 μg/mL CcSOD1 were used as the blank and negative controls, respectively. The coverslips containing the cells were then washed with ice-cold PBS three times, fixed with 4% paraformaldehyde at 4 °C for 30 min, permeabilized with Triton X-100 for 10 min, and subjected to three 5-min washes. After being blocked with 3% BSA, the cells were subjected to incubation with a His-tag antibody (dilution 1:200, Beyotime, China) overnight at 4 °C. Subsequently, the cells were treated with FITC (fluorescein isothiocyanate) fluorescent antibody (dilution 1:500, Beyotime, China) for 90 min at 25 °C in the absence of light following three 5-min washes. Finally, the cells were examined using a fluorescence microscope (LEICA DM2500, Germany), and fluorescence intensity values were determined using GraphPad software.
Determination of intracellular SOD activity
HaCaT cells were cultured in a 6-well plate until the cell density reached 80% and then treated with TAT-CcSOD1 (1, 10, 100 and 1000 μg/mL), 100 μg/mL CcSOD1 or PBS for 2 h at 37 °C in a 5% CO2 atmosphere. The cells were then washed with ice-cold PBS twice and added 100 μL of SOD sample preparation solution to lyse cells entirely. After centrifugation at 4 °C, 12,000×g for 5 min, the lysed supernatant was collected for determination of the SOD activity by the standard method as described in Material and Methods 2.4.
Cell viability measurement and NO level assay
The cells were incubated with PBS, 100 μg/mL CcSOD1 or 100 μg/mL TAT-CcSOD1 for 2 h, respectively. Glutathione (GSH) and epigallocatechin-3-gallate (EGCG) were used as positive controls in 100 μg/mL. After rinsed with PBS, the cells were exposed to 400 µM H2O2 for 4 h. Cell viability was detected using a CCK-8 Assay Kit (Dojindo, China). The cell supernatant was collected, and NO level was determined using a Total Nitric Oxide Assay Kit (Beyotime, China) according to the manufacturer’s instructions.
Statistical analysis
GraphPad Prism 8.0.1 was used to analyze the experimental data. Comparison between two groups was processed in independent samples t test, and three or more groups were compared by one way ANOVA test. It was considered statistically significant when p-value less than 0.05. Experiments were conducted in triplicate and all data represented as mean ± SD.
Results
Preparation and characterization of fusion protein TAT-CcSOD1
The full-length coding sequence of TAT-CcSOD1, containing cDNA sequence encoding HIV-TAT basic domain and the CcSOD1 gene (Fig. 2A), was first obtained via chemical synthesis. A TAT-CcSOD1/pET-24a expression vector was then constructed (Fig. 2B), and sequencing result confirmed that the coding sequence of TAT-CcSOD1 was correctly inserted into pET-24a vector.
(A) The full-length coding sequence and deduced amino acids of TAT-CcSOD1. The restriction enzyme sites were bolded and underlined, the coding sequence of TAT gene was marked in grey, and the characteristic metal-binding sites were bolded and boxed. (B) Construct of TAT-CcSOD expressing plasmid.
In order to obtain sufficient fusion protein with high activity, the optimal inducible expression conditions for TAT-CcSOD1 were explored. The induction temperature is critical to the soluble expression of recombinant protein. As shown in Fig. 3A,B, the ratio of TAT-CcSOD1 in soluble form increased as the induction temperature decreased from 37 to 12 °C. Nearly 80% TAT-CcSOD1 existed in form of soluble protein at 12 °C that was determined as the optimal temperature for TAT-CcSOD1 expression in subsequent experiments. The expression levels of TAT-CcSOD1 were almost the same under IPTG concentrations of 0.2 mM, 0.6 mM and 0.8 mM, but were higher than that at 0.4 mM IPTG (Fig. 3C). As shown in Fig. 3D, with the prolongation of induction time, the expression of TAT-CcSOD1 in soluble form gradually increased, and there were no significant differences between 24 and 48 h. Based on these results, 12 °C of induction temperature, 0.2 mM of IPTG concentration and 24 h of induction time were determined as the optimal induction conditions, which could guarantee the maximum soluble expression of fusion protein TAT-CcSOD1.
Gel electrophoretic analysis of inducible expression of fusion protein TAT-CcSOD1. (A) TAT-CcSOD1 expression under different induction temperatures. SN, supernatant; SD, sediment. 0.2 mM IPTG was added into each bacterial solution for 6 h. (B) Ratio of TAT-CcSOD1 in soluble form was quantified using densitometric analysis by image J 1.8.0. (C) TAT-CcSOD1 expression under different IPTG concentrations. IPTG were added into each bacterial solution at 12 °C for 6 h, and the sonicated supernatant in each group was collected for gel electrophoresis analysis. (D) TAT-CcSOD1 expression under different induction time. 0.2 mM IPTG was added into each bacterial solution at 12 °C for 6, 12, 24 and 48 h. The sonicated sediment and supernatant in each group were collected for gel electrophoresis analysis. The quantification data were from at least three independent repetitions and the data were presented as means ± SD (n = 3) (ns stands for no significance, **P < 0.01).
Fusion protein TAT-CcSOD1 was then purified using a HisTrap HP Chelating column, and was subsequently detected by SDS-PAGE and western blot analysis with a His-tag primary antibody (Fig. 4). The actual molecular weight of fusion protein TAT-CcSOD1 was almost identical to the estimated molecular weight (18.3 kDa).
Purification and identification of fusion protein TAT-CcSOD1. (A) SDS-PAGE analysis of the samples obtained during protein purification. (B) Western blot analysis of the samples obtained during protein purification. Lane 1, supernatant of sonicated lysates after induction; lane 2, the flow through peak; lane 3–5, the eluted peaks under 15%, 30% and 50% elution buffer (20 mM NaH2PO4, 500 mM NaCl, 500 mM imidazole, pH 7.4), respectively. The position corresponding to TAT-CcSOD1 protein is indicated by red arrow.
Enzyme activity of TAT-CcSOD1 in cell-free system
TAT-CcSOD1 protein exhibited a specific inhibition effect towards superoxide anions in a concentration-dependent manner. When the concentration of TAT-CcSOD1 reached 500 μg/ml, the inhibition of superoxide anion was close to 100% (Fig. 5). There was no statistical difference between the activity of TAT-CcSOD1 and that of CcSOD1, indicating that the fusion expression of TAT has no significant effect on SOD activity.
SOD activity of TAT-CcSOD1 protein. 20 μL of sample (0, 15.6, 31.25, 62.5, 125, 250 or 500 μg/mL) was mixed with 160 μL of SOD enzyme assay working solution and 20 μL of reaction starter solution at 37 °C for 30 min, and then the absorbance values were measured at 450 nm using a microplate reader. Inhibition (%) = (Acontrol − Asample)/(Acontrol − Ablank) × 100%. Three independent experiments were used to obtain quantitative data, and the data were presented as means ± SD (n = 3) (ns, no significance).
Effects of temperature and pH on stability of TAT-CcSOD1
In our previous study, CcSOD1 exhibited a distinct thermostability and a wide alkalic and acerbic tolerance range. To explore the stability of fusion protein TAT-CcSOD1, the residual activity of TAT-CcSOD1 was assayed at different incubation temperatures and pH value. As shown in Fig. 6A, after incubation at 30 °C and 50 °C for 1 h, TAT-CcSOD1 retained activity nearly up to 100%, and retained 75% activity after incubation at 70 °C for 1 h, indicating that TAT-CcSOD1 was highly thermostable. TAT-CcSOD1 also exhibited a remarkable stability in pH ranging from 5.0 to 9.0, and retained approximately 50% activity in pH 10.0 for 2 h (Fig. 6B). These results is similar to the previous result of CcSOD1, indicating that TAT fusion expression had no significant effect on the stability of CcSOD1.
Stability of TAT-CcSOD1. (A) Thermal stability. TAT-CcSOD1 dissolved in PBS (pH 7.0) was incubated at 30 °C, 50 °C and 70 °C. The residual SOD activities were detected at different time points. (B) pH stability. TAT-CcSOD1 was incubated in buffers with different pH values (ranging from 3.0 to 11.0) at 37 °C for 2 h. SOD activity was measured and the activity before incubation was defined as 100%. The quantification data were from at least three independent repetitions and the data were presented as means ± SD (n = 3).
Noteworthy, compared with some reported SODs and CPP-SODs derived from various species, TAT-CcSOD1 showed stronger thermal and acid–base stability. As shown in Table 1, SOD1 derived from Anoxybacillus gonensis, a thermophilic bacterium, retained approximately 50% activity after incubation at 50 °C for 1 h. SOD1 identified from Benthodytes marianensis, a new species of deep-sea sea cucumbers, had 70% activity after incubation at 40 °C for 1 h. SOD1 of Sonneratia alba and Rosa roxburghii, both derived from plant, had 70% activity after incubation at 55 °C and 70 °C for 1 h, respectively. While fused with TAT peptide, human SOD1 and SOD2 retained approximately 60% activity after 1 h of incubation at 60 °C and 50 °C, respectively. These results indicated that, compared with some reported SODs/CCP-SODs, TAT-CcSOD1 possessed stronger tolerance to heat and acid–base environment.
Transduction of TAT-CcSOD1 into HaCaT cells
As shown in Fig. 7A, TAT-CcSOD1 could efficiently penetrate the cell membrane after 15 min of incubation, and the amount of intracellular TAT-CcSOD1 protein gradually increased in a time-dependent manner (15–120 min). This result is consistent with that of immunofluorescence assay (Fig. 7B,C). However, fluorescence intensity was hardly observed in the cells treated with CcSOD1. These results confirmed the efficient transmembrane ability of TAT-CcSOD1.
Transmembrane ability of TAT-CcSOD1 in HaCaT cells. (A) Western-blot analysis of intracellular TAT-CcSOD1 and CcSOD1 detected by His-tag antibody. (B) Transmembrane amount analysis of TAT-CcSOD1 and CcSOD1 by immunofluorescence assay. (C) The intensity of fluorescence was quantified using image J 1.8.0 software. The quantification data were from at least three independent repetitions and the data were presented as means ± SD (n = 3) (**P < 0.01).
TAT-CcSOD1 enhanced the intracellular SOD activity in HaCaT cells
HaCaT cells were treated with various concentrations of TAT-CcSOD1 (0.1–1000 μg/ml) to explore its effect on cell viability. As shown in Fig. 8A, the cell viability exceeded 80%, indicating that TAT-CcSOD1 almost exerted no cytotoxicity.
Effect of TAT-CcSOD1 on intracellular SOD activity. (A) Cell viability after treatment with TAT-CcSOD1 was assessed by CCK-8 assay. (B) Effect of TAT-CcSOD1 on intracellular SOD activity in HaCaT cells. SOD activity (U) = inhibition%/(1 − inhibition%) units. Quantification data were obtained from three independent experiments and the data were shown as means ± SD (n = 3) (ns, no significance, #P < 0.05, ##P < 0.01 vs. control group, **P < 0.01).
The intracellular SOD activity after incubation with TAT-CcSOD1 was further detected. As shown in Fig. 8B, compared with CcSOD1, TAT-CcSOD1 could significantly enhance the intracellular SOD activity in HaCaT cells in a concentration-dependent manner, and when treated with 100 μg/ml of TAT-CcSOD1, the intracellular SOD activity increased nearly 2 times.
Effects of TAT-CcSOD1 on H2O2-induced cytotoxicity and NO generation in HaCaT cells
To further explore the protective effect of TAT-CcSOD1 in oxidative stress, HaCaT cells were exposed to H2O2, and the cell viability was evaluated. GSH and EGCG were chosen as positive controls. Antioxidant peptide GSH is a common antioxidant. EGCG is an active component of green tea polyphenols, exhibiting strong antioxidant properties due to the presence of phenolic groups in its molecular structure.
As shown in Fig. 9A, cell viability in the presence of 400 μM H2O2 was significantly reduced, while pretreatment with TAT-CcSOD1 could significantly increase cell viability. A similar result was obtained in EGCG pretreatment group, but not for CcSOD1 and GSH which might relate to their weak ability to penetrate membranes. NO level is closely associated with inflammation, which has been implicated in the pathogenesis of various inflammatory diseases. As shown in Fig. 9B, 400 µM H2O2 caused a significant increase in NO content in HaCaT cells. After pretreated with TAT-CcSOD1 or EGCG, a significant decrease of NO generation was observed. These results indicated that TAT-CcSOD1 could attenuate H2O2-induced cytotoxicity and NO generation, which showed a similar protective effect with that of EGCG, exhibiting both antioxidant and anti-inflammatory potentials in HaCaT cells.
Effects of TAT-CcSOD1 on H2O2-induced cytotoxicity and NO generation in HaCaT cells. (A) HaCaT cells were pretreated with 0.1 mg/ml TAT-CcSOD1, CcSOD1, EGCG and GSH for 2 h before exposure to 400 μM H2O2. Cell viabilities were determined using CCK-8 assay kit. (B) Effects of pretreated with TAT-CcSOD1, CcSOD1 and EGCG on NO level in the H2O2-induced cells model. Nitric oxide secretion in the supernatant of cell cultures was measured by NO Assay Kit. Three independent experiments were used in the quantification, and the data were presented as means ± SD (n = 3) (ns, no significance, #P < 0.05, ###P < 0.001 vs. control group, ***P < 0.001 vs. H2O2 alone group).
Discussion
Many studies have demonstrated that the occurrences of some diseases including degeneration, inflammation and even cancer were closely related to cellular oxidative stress33,34,35,36,37. Superoxide dismutases can irreversibly scavenge O2.-, and are the core antioxidant enzymes38,39,40,41. In addition, increasing evidences have indicated that SODs are also actively involved in diverse cellular processes. Studies have also found that the expression level or catalytic activity of SODs altered in various pathological conditions42,43,44. In recent years, the clinic application of exogenous SODs has drawn increasing attention. However, exogenous SODs can hardly enter cells which limited their therapeutic application.
Among several strategies used to deliver molecules into cells, conjugation with CPPs is a common approach45,46. CPPs, composed of 6–30 amino acids, are a class of short peptides with high transmembrane activity and can be used for intracellular delivery of various molecules, including nucleic acids, proteins and small molecule drugs. In recent years, due to their high efficiency and low toxicity, CPPs have been widely explored for transmembrane delivery of therapeutic drugs to treat various diseases44,47,48.
CPP-SODs have been studied since 199349. It was found that the fusion protein CPP-SODs could effectively penetrate the membranes of various types of cells, alleviating oxidative stress-related diseases, such as Parkinson, diabetes, and cancer50,51,52. Pan et al. reported that SOD-TAT pretreatment could effectively increase pulmonary antioxidant ability and improve the living quality of irradiated mice53. PEP-1-SOD1 have been reported to alleviate spinal cord injury in rats50,54. Chen et al. reported that topically applied TAT-SOD1 significantly attenuated UVB-induced skin damage55. These studies demonstrated that CPPs-SOD exhibited a great potential as possible therapeutic agents. So far, the exogenous SODs used in these studies are mainly human-derived SODs.
The ocean is a special environment with high pressure, high salinity, low temperature and weak illumination, but with local high temperature and high ultraviolet radiation in the ocean surface. Therefore, the unique living conditions endow marine organisms with more bioactive substances, including antitumor molecules, antimicrobial peptides, antioxidant and other biosubstances with promising prospects for drug development. Previously, we identified a novel Cu/Zn SOD, named CcSOD1, from the jellyfish C. capillata. Cu/Zn SOD, also known as SOD1, is a widely distributed antioxidant enzyme in many types of cells. SOD1 can extensively eliminate reactive oxygen species and have important physiological functions. Studies have shown that SOD1 dysfunction is closely related to a variety of diseases, such as hypertension, liver disease and tumor56. Our previous results indicated that CcSOD1 could markedly scavenge superoxide anions, and most notably, retained high activities within a wide range of pH and temperatures, exhibiting an excellent stability.
So far, there are few studies on the fusion expression of CPPs and marine-derived antioxidant. In the present study, we fused CcSOD1 with a cell-penetrating peptide TAT in order to deliver CcSOD1 protein into cells and explore its therapeutic prospects. The full-length coding sequence of TAT-CcSOD1 was subcloned into pET-24a plasmid to generate recombinant TAT-CcSOD1 expression vector. As previous reported, the protein will be in the form of inactive inclusion bodies due to the failing fold under a relatively high induction temperature57,58,59. So we set a series of induction temperatures and time points to determine the optimal expression condition of TAT-CcSOD1. The results demonstrated that TAT-CcSOD1 protein was highly expressed as a major component of the total soluble proteins at an induction temperature of 12 °C, and the fusion protein could be obtained sufficiently at 24 h after the addition of IPTG. What’s more, there was no significant correlation between the concentration of IPTG (0.2, 0.4, 0.6 and 0.8 mM) and TAT-CcSOD1 expression. Thereafter, fusion protein TAT-CcSOD1 was purified through a HisTrap HP Chelating column and subsequently confirmed by SDS-PAGE and western blot analysis, indicating that cell-permeable CcSOD1 was successfully generated.
The activity of TAT-CcSOD1 in cell-free system was further confirmed, and the results showed that, compared with natural CcSOD1, modification in its N-terminus by TAT peptide didn’t affect the intrinsic SOD activity. The stability of CPPs-SOD is a key point for ensuring its efficiency. In addition, for therapeutic application, it is preferable that an enzyme should have both structural and functional stabilities under severe conditions. As is shown by our previous work27, CcSOD1 could retain stable activity within a wide range of temperatures (10–70 °C), and was also highly stable at a wide pH range (pH 4–9), exhibiting a higher stability than some reported SODs from other organisms. In the present study, the stability of recombinant TAT-CcSOD1 was further detected, and the results showed that TAT-CcSOD1 could retain high activity under a wide range of pH (5.0–10.0) and stayed stable in temperature 70 °C for at least 1 h, exhibiting a distinct thermostability and a wide alkali and acerbic tolerance range, which is similar to that of CcSOD1. These results clearly illustrated that TAT fusion expression had no significant effect on the stability of CcSOD1. Compared with some reported SODs/CPP-SODs derived from other species including several extreme environmental species, TAT-CcSOD1 showed stronger heat resistance and acid alkaline tolerance.
So far, the research on cell-permeable SOD mainly focuses on human SOD. Notably, TAT-CcSOD1 is significantly more stable than human SOD and shows better resistance to heat. This distinct thermostability of TAT-CcSOD1 may be related to the source species. As typical marine plankton, jellyfish mainly live in the sea surface, a relative harsh environment with strong ultraviolet radiation and high temperature. Thermal denaturation which causes enzyme inactivation is of great concern in industrial production, thus the excellent thermal and acid-alkaline stability of TAT-CcSOD1 may provide a great potential for its therapeutic application.
Compared with CcSOD1, TAT-CcSOD1 protein could be rapidly and efficiently transported into HaCaT cells. It took only 15 min for the recombinant protein to enter the cells, and the cell viability was not significantly influenced when TAT-CcSOD1 was at the maximum concentration of 1000 μg/ml. SOD without CPP has been reported to exhibit protective effects for the treatment of many diseases, such as cardiovascular, respiratory, and inflammation-related diseases60. However, some studies showed that CPPs-SOD has a stronger antioxidant activity and higher therapeutic efficiency than SOD61. In this study, the results confirmed that, compared with CcSOD1-pretreated group, the level of intracellular SOD activity was markedly enhanced by pretreatment with TAT-CcSOD1. TAT-CcSOD1 also resulted in a significant increase of cell viability after H2O2 treatment, indicating that TAT-CcSOD1 markedly enhanced the antioxidant capacity of the cells, exhibiting protective effects on cellular oxidative stress. Increased levels of ROS can lead to various pathophysiological conditions including inflammatory responses. Nitric oxide (NO) level has a close association with inflammation. Our results showed that TAT-CcSOD1 pretreatment could efficiently inhibit H2O2-induced NO generation in HaCaT cells The protective effect of TAT-CcSOD1 on oxidative stress in cells was similar to that of EGCG, the major component of green tea polyphenols which were reported to have strong antioxidant properties62. These results indicate that TAT-CcSOD1 may have strong antioxidant activity and anti-inflammatory potential. However, the specific underlying mechanism remains to be investigated. In addition, as a non-human-derived exogenous SOD, whether TAT-CcSOD1 affects the normal physiological processes in cells is indeed a question, which needs to be considered in the next step of research.
Conclusion
In summary, a jellyfish-derived Cu/Zn SOD with cell-permeable activity, TAT-CcSOD1, was successfully produced. TAT-CcSOD1 exhibited efficient scavenging ability towards free radicals, and showed strong heat resistance and acid alkaline tolerance. Compared with previous reported natural CcSOD1, TAT-CcSOD1 efficiently entered HaCaT cells in a dose-dependent manner, and significantly enhanced SOD activity in cells. Furthermore, pretreatment of TAT-CcSOD1 resulted in a markedly increase in cell viability and a decrease in NO generation after H2O2 treatment. This study will serve as a critical step forward for the application of TAT-CcSOD1 as a potential protective/therapeutic agent against oxidative stress-related conditions in the future.
Data availability
The datasets generated and/or analysed during the current study are available in the [National Center for Biotechnology Information] repository, [https://www.ncbi.nlm.nih.gov/]. However, there is no restriction on the availability of materials and data from the corresponding author on reasonable request.
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This research was funded by the National Key R&D Program of China (2019YFC0312605) and National Natural Science Foundation of China (32201251).
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B.W., G.L. and L.Z Conceptualization; B.W., Y.H. and X.C methodology; software, J.S.; Q.W. and Y.H validation; B.W. and Y.H formal analysis; B.W., Y.H. and X.C investigation; Q.W data curation; B.W and Y.H writing—original draft preparation; G.L., L.Z and Y.Z writing—review and editing; G.L. and Y.Z visualization; G.L. and L.Z supervision; Q.W project administration; B.W., G.L., L.Z funding acquisition. All authors have read and agreed to the published version of the manuscript.
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Wang, B., Huang, Y., Cheng, X. et al. Transduction of jellyfish superoxide dismutase mediated by TAT peptide ameliorates H2O2-induced oxidative stress in HaCaT cells. Sci Rep 14, 31037 (2024). https://doi.org/10.1038/s41598-024-82261-6
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DOI: https://doi.org/10.1038/s41598-024-82261-6
