In Vivo/In Vitro Properties of Novel Antioxidant Peptide from Pinctada fucata
1South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences; Key Lab of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Guangzhou 510300, P. R. China
2Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang, P.R. China
3School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, P.R. China
4Guangzhou Maritime University, Guangzhou 510725, P.R. China
J. Microbiol. Biotechnol. 2021; 31(1): 33-42
Published January 28, 2021
Copyright © The Korean Society for Microbiology and Biotechnology.
Free-radical theory that endogenous oxygen radicals are generated from endogenous metabolic processes in cells and result in a pattern of cumulative damage was proposed by Denham Harman in the mid-1950s . Antioxidant peptides play an important role in our lives because they scavenge free radicals, like superoxide anions or hydroxyls, which damage protein and cause DNA mutations that trigger certain illnesses including coronary heart diseases and diabetes . Typically, macro-molecular proteins do not have antioxidant activity; thus, proteases need to be applied to digest these proteins first. After the protein is enzymatically hydrolyzed, it is decomposed into various small bioactive peptides . Several preparation methods have been developed to obtain antioxidant peptides of high purity and activity including chemical synthesis, enzymatic hydrolysis and bio-fermentation [4, 5]. However, the enzymatic hydrolysis technique has a low extraction rate and is too expensive to be used for antioxidant peptide purification, thereby preventing this method from being widely for industrial production. Meanwhile, the types and amounts of synthesized antioxidant peptides used in food or cosmetics production are very limited because of their potential toxic effects. Thus, most peptides with antioxidant properties are still at their laboratory-scale research stage [3, 5].
Nowadays, marine-derived antioxidant peptides are attracting a lot of attention because of their excellent antioxidant activity, quick physiological absorption, safety, and promising economic value when applied as functional ingredients in cosmetics or health foods [3, 6, 7]. Good example sources of antioxidant peptides include those obtained through the digestion of proteins from marine organisms [8-10]. Meanwhile, several strategies are typically used to enhance antioxidant peptide activity. The first example includes two peptides designed based on glutathione and prepared using L-conformation of each amino acid by solid-phase synthesis. The resulting peptides exhibit high antioxidant activity because of site-directed mutagenesis of the active sites . The second example involves glycosylation used to purify and characterize peptides from
Recently, an antioxidant peptide from
Materials and Methods
Glutathione and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) were acquired from Sigma-Aldrich (USA). Expression vector pET-30a (pET) and Rosetta (DE3) strains were preserved in our laboratory. DNA markers 2000, restriction endonucleases (Xho I and Bam HI), DNA Fragment and Plasmid Purification Kits 4.0, DNA gel extraction kits and BCA protein assay were purchased from Takara (Japan). Precast-GL gel Hepes SDS-PAGE, kanamycin, tryptone, isopropyl β-D-1-thiogalactopyranoside (IPTG) and yeast extracts were obtained from Sangon (China). Fetal bovine serum (FBS), protein markers, penicillin-streptomycin solution, Dulbecco’s modified Eagle medium (DMEM) and trypsin-EDTA were purchased from Thermo Fisher Scientific (USA). All commercially purchased chemicals were of analytical grade.
Bioinformatics Analysis of Antioxidant Peptide Sequence
PFAOP sequence structure was analyzed according to the SWISS-MODEL method by Bertoni, Kiefer, Biasini, Bordoli, & Schwede  with some modification. Briefly, the EXPASY database located at https://swissmodel.expasy.org was used to simulate target protein tertiary structures to examine the resulting structural models. Another web site (http://bioinf.cs.ucl.ac.uk/psipred/) was used to predict target protein secondary structure to analyze the relationship between the peptide structure and its antioxidant activity. Theoretical molecular weight, amino acid compositions, isoelectric point, estimated half-life, instability index, hydrophobicity and hydrophilicity scales were obtained for further functional expression and preparation from https://web.expasy.org/protparam/and https://web.expasy.org/protscale/. Moreover, the molecular interaction between PFAOP and porcine elastase (PDB ID: 1ELB) was examined by AutoDock Vina 4.0 according to the reference of Trott & Olson  with some modification for virtual screening complex interactions between the receptor 1ELB and antioxidant peptides and to assist with analyzing the potential activity mechanism. A docking grid box (with dimensions of 40×40×40 Å and a certain grid spacing of 0.375 Å) was made to cover the entire binding pocket including the active site. Vina scores (kcal/mol) were calculated with the predicted affinity of peptides difference conformation for binding to the porcine elastase, and the 2D diagram and 3D structure diagram were produced by Discovery Studio 4.5 Visualizer (USA).
Preparation of Antioxidant Peptide
DNA sequence of PFAOP was codon-optimized for further expression in Escherichia coli system using Primer Premier 5.0  and chemically synthesized by SANGON (China), ligated into the pET30a vector by the Bam HI and Xho I restriction enzymes. Code-optimized sequence of PFAOP was 5’-
PFAOP was prepared using a slightly modified method for protein expression and purification reported by Dagar, Adivitiya, & Khasa . For this purpose, DE3-pET-PFAOP was pre-cultured at 37°C in LB medium using a rotary shaker operated at 220 rpm, after which it was grown to a log phase with OD600 in the 0.6-0.8 range. It was then induced by 0.5 mM IPTG at 37°C and also cultured for another 12, 6, and 4 h at three different temperatures (20, 30, and 37°C). Cells were centrifuged for 5 min at 5,000 ×
Antioxidant Activity Assay for Antioxidant Peptide
Cellular Antioxidant Activity Assay for Antioxidant Peptide
Cellular antioxidant activity of PFAOP was analyzed using slightly modified method reported by Wolfe & Liu . HepG2 cells used as a cell model were pre-cultured in a cell culture flask at 37°C under atmosphere containing 5% CO2. Then the cells were placed into 96-well black plate (2 × 105 cells per well) and incubated again at the same conditions for 24 h. Old culture medium was absorbed and cells were rinsed once with PBS. One hundred microliters of antioxidant peptide dissolved in medium containing 25 mM 2, 7-dichlorodihydrofluorescein diacetate was added and the whole mixture was further incubated for 60 min. Next, the microplate added with 0.6 mM 2,20-azobis(2-amidinopropane) dihydrochloride solution was immediately placed into a Filter Max F5 Multi-Mode Microplate Reader for analysis performed at 535 nm after the solutions in wells were excited at 485 nm every 5 min for 60 min at 37°C. Glutathione was used as a positive control. Cellular antioxidant activity was calculated as 1−∫SA/∫CA, where ∫SA and∫CA are integral areas under the absorbance curves of the test and blank samples, respectively.
Cytotoxicity Assay and Anti-Proliferation Activity of Antioxidant Peptide
PFAOP cytotoxicity activity and anti-proliferation activity were analyzed by a slightly modified method described in the literature [22-24]. Briefly, the cell culturing method was similar to the method used for the cellular antioxidant activity assay. However, HepG2, Caco-2 and MCF-7 cells pre-cultured as described above were used as model cells. All further incubation and treatment steps were same for all cells. First, cells were seeded at 2 × 105 or 5×105 cells per well in a 96-well microplate for further analysis of anti-proliferation and cytotoxicity activity, respectively. The cells were incubated for 4 h at 37°C under atmosphere containing 5% CO2, after which the old culture medium was absorbed and washed with PBS once. Next, 100 μl of peptide solution dissolved in the medium was added to each well and incubated for 24 h. Quantification of viable cells was performed using a Cell Counting Kit-8 (Sangon Biotech Co.). Absorbance was measured at 450 nm.
PFAOP and glutathione activities related to their radical scavenging, cellular antioxidation, cytotoxicity and anti-proliferation were analyzed using three separate batches. Statistical analysis was conducted using SPSS 13.0 software (SPSS Inc., USA). All values reported in this work represent average values with uncertainties calculated as standard deviations.
Bioinformatics Analysis Result
PFAOP chemical formula, instability index (II), theoretical molecular weight and isoelectric point were C708H1155N275O221S4, 26.52 (stable), 17183.91 Da and 11.04, respectively. The total number of positively (
Structural representation depicting PFAOP hydrophobicity (A), secondary structure (B) and 3D spatial conformation (C).
To forecast spatial conformation and next-step preparation by bio-fermentation using engineering strains fermentation method, spatial conformation of PFAOP 3D structural representation was constructed by SWISS-MODEL using 2v53.1.B as a template. PFAOP is spatially conformed as a random coil resembled α-helix structure (Fig. 1C). Such a structure could expose active amino acids and their reactive sites toward free radical scavenging, which would result in higher antioxidant activity of the peptide as a whole. Random coil resembling an can also provide data needed to establish the relationship between peptide activity and its spatial conformation.
To investigate the interaction between antioxidant peptide PFAOP and porcine elastase 1ELB, molecular docking was carried out. The result (Fig. 2) demonstrated that PFAOP could strongly interact with 1ELB, which displayed the highest affinity to elastase (6.6 kcal/mol). Obviously, twelve hydrogen bonds (including conventional hydrogen bond and carbon hydrogen bond) were observed of PFAOP and the Cys199, Ser203, Val224, Gln200, Cys229, Ser225, Ser203, Thr100, Val224, Asn153, Gln200, and Ser222 residues with a length of 2.3 Å, 2.2 Å, 2.5 Å, 2.7 Å, 3.1 Å, 2.7 Å, 2.1 Å, 1.9 Å, 2.5 Å, 3.4 Å, 2.5 Å, and 3.5 Å, respectively (Figs. 2B-2D). It has been proved that the hydrogen bond interaction force represents a positive effect on the inhibitory activity property of antioxidants. As for GSH (Fig. S1), fewer hydrogen bonds between GSH and 1ELB residues were observed compared to PFAOP, indicating that PFAOP exhibited a higher activity than GSH.
Molecular docking for the interaction of antioxidant peptide PFAOP with porcine elastase (PDB ID: 1ELB).( A) Schematic representations of PFAOP and the selective site used for molecule docking, ( B) Putative binding mode of PFAOP in the binding cavity of 1ELB, ( C) 3D view of docking pose of PFAOP and 1ELB molecular catalytic site, ( D) 2Ddiagram of the interaction.
Validation of PFAOP Preparation
Fig. 3A summarizes the preparation using the bio-fermentation method for PFAOP. And this result suggested that this method of preparing PFAOP had some advantages such as cost, degree of automation, preparation speed or scale-up processes compared with the enzymatic hydrolysis or chemical synthesis methods. Preparation of PFAOP was validated in the IPTG absence and presence using 12% SDS–PAGE (Fig. 3B). Only after IPTG induction, a protein band corresponding to molecular weight below 25KD was observed. Comparison of sizes of PFAOP bands with the control (which was non-induced lysate) indicated that molecular weights of target proteins containing designed linker protein sequences corresponded to expected theoretical molecular weights. Thus, successful expression of PFAOP was confirmed. Typically, higher growth temperatures result in increased aggregation and reduced solubility of the heterologous proteins in
(A) Diagram of preparation using bio-fermentation method for PFAOP; (B) SDS-PAGE analysis of PFAOP at 20, 30, and 37°C. Lane M corresponds to a protein molecular weight marker. Lane 1 corresponds to non-induced lysates. Lanes 2 and 3 correspond to the supernatant induced for 12 h at 20°C, respectively. Lanes 4 and 5 correspond to lysates and to supernatant, respectively, induced for 6 h at 30°C. Lanes 6 and 7 correspond to lysates and to supernatant, respectively, induced for 4 h at 37°C; (C) SDS-PAGE analysis of PFAOP purification process. Lane M corresponds to protein molecular weight marker. Lane 1 corresponds to supernatant of PFAOP induced at 37°C. Lane 2 corresponds to the eluent at the time the sample was loaded. Lane 3 corresponds to rinsing with binding buffer. Lanes 4-5 correspond to eluent containing PFAOP.
Antioxidant Activity Analysis Results of Antioxidant Peptides
Antioxidant activity of purified PFAOP, C-PFAOP, E-PFAOP and glutathione antioxidants were investigated based on their scavenging activity relative to radicals of DPPH, superoxide and hydroxyl, as well as based on their cytotoxicity and cellular antioxidant activities. The half maximal cytotoxicity concentration (CC50) values of PFAOP, C-PFAOP, E-PFAOP and glutathione obtained using cytotoxicity analysis were significantly over 10 mg/ml (Table 1), which is much higher than the corresponding half maximal effect concentration (EC50) value, indicating that the inhibitory effects of PFAOP, C-PFAOP, E-PFAOP and glutathione in each case were not attributed to cytotoxic effect. EC50 values related to PFAOP antioxidant activity relative to the free radicals of DPPH, hydroxyl, superoxide and its cellular antioxidant activity were 0.018 ± 0.005, 0.126 ± 0.008, 0.168 ± 0.005, and 0.105 ± 0.005 mg/ml, respectively (Table 1). Obviously, the analogous EC50 values of antioxidant activity of glutathione (a common cosmetics ingredient also used in this study as a positive control) were lower (
Anti-Proliferation and Cytotoxicity Analysis of Antioxidant Peptides
Anti-proliferation and cytotoxicity activities of PFAOP, C-PFAOP, E-PFAOP and glutathione antioxidants against HepG2, Caco-2 and MCF-7 cells expressed as EC50 and CC50 are shown in Table 2. PFAOP EC50 values with regard to HepG2, Caco-2 and MCF-7 cells were 0.069 ± 0.008, 0.145 ± 0.012, and 0.182 ± 0.009 mg/ml, respectively. Particularly, PFAOP had the highest inhibitory effect against HepG2 cell proliferation, followed by Caco-2 and MCF-7 cells. A similar trend was observed for glutathione, C-PFAOP and E-PFAOP, but the corresponding values indicated higher anti-proliferation activity of PFAOP than the other three (
Antioxidant activity stability and content changes of purified PFAOP during its antioxidant reaction process as well as clarification of the relationship between spatial conformation structure and antioxidant activity were determined based on analysis of samples taken at different periods during total antioxidant capacity experiment performed using 12% SDS-PAGE (Fig. 4A). PFAOP content decreased as reaction time increased, and relative intensity of PFAOP was continuously decreasing (
SDS-PAGE analysis results (Lane M corresponds to the marker. Letters a through g in ( A) and relative intensity ( B) of PFAOP at 0, 6, 30, 60, 120, 240, and 360 min (Lanes 1 through 7, respectively). B) indicate significant differences ( p< 0.05).
Differences between the enzymatic hydrolysis, bio-fermentation and chemical synthesis methods occur mostly whether spatial conformation exists of antioxidant peptide from
Previous literature reports some antioxidant peptides from
In this work, antioxidant peptides from
Moreover, PFAOP activities related to free radical scavenging, cytotoxicity, cellular antioxidation and anti-proliferation were examined to evaluate its overall antioxidant properties, as well as provide test data to clarify the relationship between structure and activity. A comparison of the antioxidant activities in vivo and in vitro of PFAOP, C-PFAOP, E-PFAOP and glutathione (Table 1) illustrated that glutathione, C-PFAOP, E-PFAOP and PFAOP had bioactivity properties. PFAOP exhibited higher scavenging activity towards free radicals of DPPH, OH and superoxide, as well as higher cellular antioxidant activity than that of glutathione (
Additionally, PFAOP exhibited higher in vitro radical scavenging activity, and the same is true for cell cellular antioxidant activity and anti-proliferation activity in vivo experiments. Judging by the EC50 values of cellular antioxidant and cytotoxicity activities (Table 1), PFAOP showed better cellular antioxidant activity than glutathione, C-PFAOP and E-PFAOP with no cytotoxicity with EC50 value equal to 0.105 ± 0.005 mg/ml (
In conclusion, we prepared antioxidant peptides of PFAOP using bio-fermentation method in this work. Unlike traditionally separation and puriﬁcation techniques (
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
This work was supported by the China Agriculture Research System (CARS-47), National Key R&D Program of China (2019YFD0901900), Central Public-interest Scientific Institution Basal Research Fund, South China Sea Fisheries Research Institute, CAFS (2018ZD01), Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2020TD69), and the Dedicated Fund for Promoting High-Quality Economic Development in Guangdong Province (Marine Economic Development Project, GDOEA25).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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