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Molecular cloning, expression and antioxidative activity of 2-cys-peroxiredoxin from freshwater mussel Cristaria plicata
Accepted Manuscript Molecular cloning, expression and antioxidative activity of 2-cys-peroxiredoxin from freshwater mussel Cristaria plicata Xiaobo Wang, Baoqing Hu, Chungen Wen, Ming Zhang, Shaoqing Jian, Gang Yang PII:
S1050-4648(17)30266-8
DOI:
10.1016/j.fsi.2017.05.026
Reference:
YFSIM 4579
To appear in:
Fish and Shellfish Immunology
Received Date: 11 January 2017 Revised Date:
25 March 2017
Accepted Date: 8 May 2017
Please cite this article as: Wang X, Hu B, Wen C, Zhang M, Jian S, Yang G, Molecular cloning, expression and antioxidative activity of 2-cys-peroxiredoxin from freshwater mussel Cristaria plicata, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.05.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Molecular cloning, expression and antioxidative activity of 2-cysperoxiredoxin from freshwater mussel Cristaria plicata Xiaobo Wang1, Baoqing, Hu1, Chungen Wen1*, Ming Zhang 2*, Shaoqing Jian1, Gang Yang1 1. School of Life Sciences, Nanchang University, Nanchang 330031, China
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2. College of Jiangxi Biotech Vocational, Nanchang 330200, China
*Corresponding author:
[email protected] (CG. Wen), Tel. /fax: +86-0791-83969530
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[email protected] (M. Zhang), Tel./fax: +86-0791-87877381
Running Title: A 2-Cys peroxiredoxin gene from Cristaria plicata
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ABSTRACT: Peroxiredoxins (Prxs) play an important role against various oxidative stresses by catalyzing the reduction of hydrogen 20
peroxide (H2O2) and organic hydroperoxides to less harmful form. A 2-
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cys peroxiredoxin, designated as CpPrx, was cloned from hemocytes of of freshwater mussel Cristaria plicata. The full length cDNA of CpPrx is 1247 bp, which includes an open reading frame (ORF) of 591bp,
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encoding 196 amino acids. CpPrx possesses two conserved cysteine residues (Cys49, Cys170). The deduced amino acid sequence of CpPrx
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showed a high level (67-74%) of sequence similarity to 2-Cys Prxs from other species. The results of real-time quantitative PCR revealed that CpPrx mRNA was constitutively expressed in tissues, and the highest expression levels were in hepatopancreas and gills. After peptidoglycan 30
(PGN) and Aeromonas hydrophila challenge, the expression levels of
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CpPrx mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpPrx was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3).
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Comparison with DE3-pET-32 and DE3 strain, the cells of DE3-pET-32CpPrx exhibited resistance to the concentration of 0.4, 0.8 and 1.2
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mmoL/L H2O2 in vivo.
Key words: Cristaria plicata; Peroxiredoxin; Molecular clone; Recombinant protein; Antioxidant activity
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1. Introduction Peroxiredoxins (Prxs), known as thioredoxin peroxidases, are also cysteine-dependent peroxidases proposed to function as antioxidant 45
enzymes [1]. These enzymatic antioxidants are characterized by one or
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two conserved cysteines (Cys) that reduce hydroperoxides in the presence of thiol [2]. The feature of Prxs possess the peroxidatic Cys residue (CysSRH) which is oxidized by the peroxide substrate to Cys sulfenic acid
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(Cys-SPOH) during the reaction cycle [3]. The main function of Prxs is to eliminate H2O2, and adjust signal transduction and immune reaction
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mediated by H2O2 [4]. Prxs emerge as important factors linking reactive oxygen species (ROS) metabolism to redox-dependent signaling events [2, 5, 6]. Together with ROS, nitric oxide (NO) is also a free radical product of the cell metabolism that is essential in the signal transduction. The S55
nitrosylation of PrxII in vivo probably serve as a protective mechanism
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under oxidative and nitrosative stress [7]. In addition, Prxs can enhance cell toxicity mediated by natural killer (NK) cell [8], regulate calciumdependent potassium transport across the plasma membrane [9], block the
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activation of nuclear transcription factor-κB (NF-κB) and tumor necrosis factorα (TNFα) as well as inhibit human immunodeficiency virus type 1
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(HIV-1) replication [9, 10]. Prxs of mammalian are divided into 1-Cys and 2-Cys Prx that depend on whether they have only the peroxidatic Cys or a resolving Cys (Cys-SRH). 2-Cys Prxs are further splitted into typical (Prx1-4) and atypical Prx (Prx5) according to catalytic mechanisms [3, 5, 65
11]. All Prxs have in common an overall fold and catalytic mechanism involving a conserved, fully folded active site and an unfolding event [12]. The enzymatic mechanism relies on a conserved cysteine residue, the peroxidatic cysteine, which reduces various peroxide substrates, and the second free thiol then forms a disulfide with peroxidatic cysteine [13].
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Prxs have received a great deal of attention owing to their role in regulating levels of hydrogen peroxide, which is common to many cytokine induced signal-transduction pathways as an intracellular signaling molecule [2, 14]. Many evidences implicate that Prxs are
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important and widespread signaling molecule that are served as indicator of oxidative stress or as a part of normal cellular development [15-17].
Prxs have been isolated from archeobacteria, protozoon, fungus, parasite and mammal [18], and have been extensively investigated in
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yeast (Saccharomyces cerevisiae), mammals (Homo sapiens), plants (Arabidopsis thaliana), bacteria (Salmonella typhimurium) and parasitic protists (Plasmodium falciparum and Trypanosoma sp) [2, 19-28].
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Meanwhile, the genes of Prxs have recently been reported to be present in molluscan, Saccostrea glomerata Prx6 [29], Haliotis discus discus Prx1, Prx2, Prx6 [30, 31], Crassostrea gigas Prx6 [32], Chlamys farreri Prx5
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[35].
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[33], Argopecten irradians Prx5 [34] and Laternula elliptica Prx5, Prx6
The antioxidant function of Prxs is detected in the lung, cartilage, tendon and umbilical cord blood monocytes (UCBMC) in human [36-41].
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The overexpression of Prx V offer significant protection against ocular anomalies caused by oxidative stress in Xenopus laevis [42]. Prxs are also involved in the immune response. The expression of Prx V is up-
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regulated to be associated with acute inflammation induced by lipopolysaccharide in rat [33, 41]. The expression of Prxs is examined in the tissues of aquatic organisms [33, 35, 43, 44]. The expression level of 2-Cys Prx is enhanced by exposure to hypo-osmotic stress in gill tissue of 95
Eurypanopeus. depressus [45]. The mRNA transcripts of Prx V in hemocytes of Argopecten irradians are up-regulated after Vibrio anguillarum challenge [34]. The oxidative stress of Prxs is investigated in aquatic organisms, but the antioxidant function of that has not been
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described in freshwater mussel. 100
The freshwater mussel Cristaria plicata, which is of great economical importance, is well known as one of “freshwater pearl bivalve” in the aquaculture industry of China. However, the farming of
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freshwater pearl has been suffering serious problems due to the outbreak of mussel diseases in the cultivation process [46]. Thus, it is crucial for 105
diseases management and development of sustainable mussel culture and pearl production to research the immunity of freshwater mussel.
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The cDNA sequence of a 2-Cys Prx from the C. plicata, designated as CpPrx, was isolated and was identified in this study. Next, tissue-
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specific expression of CpPrx was detected in the tissues of normal mussels. The expression patterns of CpPrx mRNA transcripts in hemocytes and hepatopancreas were examined by quantitative real-time PCR after challenge with Aeromonas hydrophila, peptidoglycan (PGN) and PBS. The CpPrx cDNA was subcloned into the pET-32 vector and
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was transformed into Escherichia coli BL21 (DE3). The antioxidant function of pET-32-CpPrx was evaluated in vitro.
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2. Materials and methods
2.1 Collection and maintenance of mussels
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The freshwater mussel C. plicata, with shell length 18-25 cm,
collected from Poyang Lake in Jiangxi province, China, was maintaned at 25±2 ℃ in freshwater tanks with continuous oxygenation, changing the water every day for one week before processing.
2.2 RNA extraction and cDNA synthesis 125
Total RNA was extracted from haemocytes using Trizol Reagent (Invitrogen, Carlsbad, CA) following manufacturer’s instruction. The
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extracted RNA was then treated with RQ1 Rnasefree DNase I (Promega, Madison, WI) to remove any possible contaminating DNA. Smart cDNA was synthesized from total RNA by using SMARTTM cDNA synthesis kit 130
(Clontech Laboratories, Palo Alto, CA). The synthesis reactions were for 15 min, and subsequently stored at -80 ℃. 2.3 Cloning the full-length cDNA of CpPrx
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performed at 65 ℃ for 5 min, 42 ℃ for 1 h, terminated by heating at 70 ℃
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Based on the highly conserved sequences from Chlamys farreri (EF634307.1) and Crassostrea gigas (XM011443230.1), two degenerated
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primers CpPrx-F1 and CpPrx-R1 (Table 1) were designed to obtain the mid-fragment of CpPrx from C. plicata. The amplification program consisted of 5 min at 94 ℃, followed by 35 cycles of 94 ℃ for 30 s, 54 ℃ for 30 s, 72 ℃ for 1 min and an additional extension at 72 ℃ for 10 min. 140
The PCR product was then cloned into the pMD18-T vector (Promega)
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and sequenced in both directions with primers T7 and SP6. To obtained full-length cDNA of CpPrx, four gene-specific primers CpPrx-F2, CpPrx-F3, CpPrx-R2 and CpPrx-R3 were designed from the
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partial sequence of CpPrx cDNA (Table 1). PCR reactions were performed using SMART-RACE and nest-PCR. PCR amplicons were
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sequenced and the full-length cDNA of CpPrx was assembled from 5' and 3' sequences.
2.4 Sequence analysis of CpPrx The CpPrx gene sequence was analyzed by using the BLAST
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algorithm at the NCBI web site (http://www.ncbi.nlm.nih. gov/blast ), and the deduced amino acid sequence, the cellular localization prediction, signal peptide, Structure domain analysis and open reading frame (ORF) were analyzed with the Expert Protein Analysis System (http://www.expasy.org/).
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The protein sequences were aligned by the ClustalW multiple sequence 155
alignment program (version 1.8). The molecular mass was calculated, and the theoretical isoelectric points were predicted by Protein MolWt & AA Composition
Calculator The
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(http://www.proteomics.com.cn/proteomics/pi_tool.asp).
phylogenetic tree was constructed from the deduced amino acid sequences using the Neighbour-Joining (NJ) algorithm within MEGA version 4.1.
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2.5 Tissue distribution and temporal expression of CpPrx after A.
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hydrophila, PGN and PBS challenge
The tissue-specific expression of CpPrx was detected in the tissues of normal mussels, including hemocytes, mantle, hepatopancreas, gill and
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muscle, from five individuals. Sixty mussels were selected for the temporal expression of CpPrx, and were randomly divided into three groups in the same tank, and each group included 20 mussels per tank. The mussel adductor muscle of individual animals in the control or
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challenged groups were injected with 0.1 mL PBS (pH 7.0, 0.2 M Na2 HPO4, 0.2 M NaH2PO4), or 0.1 mL live bacterial suspension (A. hydrophila, dissolved in PBS, 109 cell/mL), or 0.1 mL PGN (Sigma, 0.5 mg/mL), respectively. The hemocytes and hepatopancreas were obtained 175
separately from five individual mussels at 0, 3, 6, 12, 24 and 48 h postinjection of each group and were immediately stored in liquid nitrogen until used. Total RNA samples isolated using the TRIzol reagent
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(Invitrogen) were used to synthesize the first strand cDNAs, which were used as the template of real-time quantitative-PCR (RT-qPCR). 180
2.6 Real-time quantitative PCR
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The transcripts of CpPrx in different tissues were determined by RTqPCR. The total RNAs were extracted from the tissues of mussel and were utilized to synthesize the first strand cDNAs, which were used as the
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templates of RT-qPCR amplification. The primers of CpPrx-F4 and CpPrx-R4, and of β-actin-F and β-actin-R (Table 1) were designed to
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amplify specifically partial cDNA sequence of the CpPrx and the internal reference gene Cp-β-actin, respectively. RT-qPCR was performed in a total volume of 25 µL containing 10 µL of 2 × SYBR Green Real-time PCR Master Mix (TaKaRa, DRR041A), 1 µL of cDNA, 1 µL of each 190
primer and 12 µL of PCR-grade water, conducted on an Eppendorf
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Mastercycler ep Real-plex2 PCR system. Triplicate reactions were performed for each sample. The synthesis reaction was performed at 94 ℃ denaturation for 5 min, 40 cycles for 94 ℃ 30 s, 57 ℃ for 30 s,
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72 ℃ for 15 s, and finally 72 ℃ elongation for 3 min. Fluorescence readings were performed at the end of each cycle. The CpCpx mRNA
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expression level could be calculated by 2_△△CT.
2.7 PCR amplification of CpPrx The ORF sequence of CpPrx was amplified by a pair specific primer
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CpPrx-F5 and CpPrx-R5 with the corresponding restriction enzyme sites of KpnI and BanHI (Table 1). The PCR program was performed at 95 ℃ for 5 min, 35 cycles of 94 ℃ for 30 s, 57 ℃ for 30 s, and 72 ℃ for 60s, with an additional extension step at 72 ℃ for 10 min. The amplicon was
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firstly cloned into pMD18-T vector, and the sequence was verified by 205
DNA sequencing. The prokaryotic expression plasmid pET-32 (Novagen, Madison, WI) was digested with KpnI and BanHI and then ligated with the same digested and recovered CpPrx gene from recombinant T vector.
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The resulting construct was the CpPrx recombinant expression vector, which was designated to pET-32-CpPrx. 210
2.8 Prokaryotic expression of recombinant pET-32-CpPrx
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The recombinant expression plasmid pET-32-CpPrx was transformed into E. coli BL21 (DE3) competent cells. The transformed cells were
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cultivated at 37 ℃, 200 r/min in 10 mL LB medium containing 100 mg/mL ampicillin until OD600 reached about 0.4. Final concentrations of 1 mM isopropy β-D-1-thiogalactopyranoside (IPTG) were added to the culture and the culture was induced at 20 ℃, 200 r/min. 1 ml culture
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was removed at 0, 2, 4, 6, 8 h respectively. A blank group was cultured at 0 h, induced groups were cultured in other time buckets. The cells 220
were harvested by centrifugation at 12000 g for 5 min at 4 ℃. The recombinant expression of target protein was determined by 15% SDS-
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PAGE assay.
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2.9 Antioxidation function of CpPrx in vitro The antioxidant activity of CpPrx was determined by measuring the
sensitivity of E. coli cells against H2O2 toxicity. The positive DE3-pET32-CpPrx strain was selected as challenge group, and DE3-pET-32 and DE3 strain as control groups. Both challenge and control groups were induced with 1 mM IPTG for 24 hours at 37 ℃, 200 r/min. The bacterium 230
concentration, which was determined by using spectrophotometer (at 600 nm), was diluted with sterile water into OD600=0.2, continuous dilution (1:
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3). 5 µL bacterium was extracted from each sample plated on LuriaBertani (LB) agar plates with different concentrations of H2O2 (0, 0.4, 0.8, 1.6 mmoL/L). All plates were cultivated at 37 ℃ for 2 or 3 days, and the 235
diameters of the cell clusters were compared to evaluate the H2O2
2.10 Data processing and statistical analysis
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tolerance activity of CpPrx.
All assays were done in triplicate, and were repeated at least twice. The
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statistical analysis was carried out with SPSS Statistics 13.0 software, and the data were calculated as the mean ± S.D. Differences were considered
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to be statistically significant when P values were lower than 0.05.
3. Result
3.1 The full-length cDNA of CpPrx
The nucleotide and the deduced amino acid sequences of CpPrx
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were shown in Fig. 1. The cDNA sequence was deposited in the GenBank database as accession No. HQ166838. The full-length CpPrx cDNA was comprised of 1247 bp, and contained a 5’ untranslated region (UTR) of
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90 bp, an open reading frame (ORF) of 591 bp, and a long 3’ UTR of 566 bp with a canonical polyadenylation signal sequence AATAAA and a
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polyA tail. The putative CpPrx polypeptide was predicted to contain 196 amino acid residues, with a calculated molecular mass of 21.87 kDa and a theoretical isoelectric point of 5.95. Signal IP 3.0 analysis showed that no signal peptide was found. The deduced amino acid sequence of CpPrx 255
contained two highly conserved motifs (F39YPLDFTFVCPTEI53, G167EVCPA172) and two conserved Cys residue (Cys49, Cys170) in the N-and C-terminal. The mature peptide included three typical structural domains, peroxide reductase (Leu4-Gln189), thioredoxin-2 (Leu4-Phe162)
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and Alkyl hydroperoxide reductase-Thiol specific antioxidant domain 260
(Leu6-Ile139).
3.2 Homology and phylogenetic analysis of CpPrx
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The results of multiple alignment indicated that the amino acid sequence of CpPrx had high sequence similarities with the putative Prxs 265
of Sinanodonta woodiana (95% identity), Pinctada fucata (85% identity) and Haliotis discus (80% identity). The sequence of CpPrx also displayed
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similarities with Prx1 (74-67% identity) from Litopenaeus vannamei, Danio rerio, Homo sapiens, Mus musculus and Xenopus laevis, Prx2 and
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Prx4 (75-67% identity), Prx3 (52% identity), Prx5 (17% identity) and Prx6 (25% identity) from other species (Fig. 2). Pairwise comparison of the sequences revealed that the CpPrx sequence was more similar to Prx 2 sequences than Prx 1 sequences (Table 2).
The molecular phylogenetic tree based on amino acid sequences of
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Prxs was constructed by Neighbor-joining method as shown in Fig. 3. Prxs from invertebrate and vertebrate were separated to three sub-clusters.
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The CpPrx was located in the branch of typical 2-Cys Prx.
3.3 The tissue expression patterns of CpPrx mRNA
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expression
of
CpPrx
mRNA
in
hemocytes,
muscle,
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The
hepatopancreas, mantle, and gill was examined by real-time quantitative RT-qPCR (Fig. 4). The results showed that the CpPrx mRNA was constitutively expressed in detected tissues. The highest level was expressed in hepatopancreas, followed by gill and muscle, and the lowest level was in hemocytes and mantle.
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3.4 Temporal expression of CpPrx after A. hydrophila, PGN and PBS challenge
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The expression of the CpPrx mRNA was up-regulated in hemocytes and hepatopancreas. The expression of the CpPrx mRNA in hemocytes was inceased at 6, 12, 24 and 48 h, and the highest expression was 290
observed at 48 h (Fig. 5A). The expression of CpPrx mRNA in
5B).
3.5 Expression of the recombinant protein
Comparison with non-induced culture, the DE3-pET32-CpPrx was
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hepatopancreas enhanced at 3 h and resumed to normal levels at 48 h (Fig.
induced by IPTG that expressed a fusion protein of approximately 34 kDa
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(including His-tag). The result was detected by SDS-PAGE (Fig. 6). 3.6 Antioxidation function detection of DE3-pET-32-CpPrx in vitro 300
DE3-pET-32-CpPrx, DE3-pET-32 and DE3 strain were cultured in LB media containing different concentrations of H2O2 were showed in Fig.
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7. The growth of DE3 strain was inhibited in each concentration of H2O2 and while the concentration of H2O2 was raised to 1.6 mmoL/L, the growth of bacteria was completely inhibited. With the increase of the concentration of H2O2, the survival rate of DE3-pET-32 strain decreased.
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The growth of DE3-pET-32-CpPrx strain had no significantly change in
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0.4 and 0.8 mmoL/L H2O2. While the concentration of H2O2 was raised to 1.6 mmoL/L, the growth of DE3-Pet32-CpPrx was markedly inhibited.
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4. Discussion
The typical 2-Cys Prxs are identified by two highly conserved redox
active cysteines named as the peroxidatic cysteine generally near residue 50, and the resolving cysteine near residue 170 [30]. The former is embedded in the highly conserved F-motif (FTFVCPTEI), and the latter 315
is adjacent to a highly conserved hydrophobic region (VCPAGW). These
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features are found in all typical 2-Cys Prxs from plants, mammals, fungi, bacteria and fish [2, 47, 48]. In this study, CpPrx also contained two highly
conserved
signal
motifs
(F39YPLDFTFVCPTEI53
and
G167EVCPA172) and two highly conserved Cys residue (Cys49, Cys170) in the N-and C-terminal. Phylogenetic analysis showed that CpPrx
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clustered together with other invertebrate animals in the typeical 2-Cys subfamily. Therefore, we inferred that CpPrx belonged to the typical 2Cys Prx.
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The expression of Prx1 mRNA can be detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill tissues of
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Fenneropenaeus chinensis [43]. Prx1 and Prx2 from Haliotis discus is constitutive expression in muscle，mantle，gill and digestive tract [30]. Furthermore, Prx 4 is expressed in tissues of muscle, stomach and brain, especially high expression in genital gland, hepatopancreas and heart 330
from Penaeus monodon [44]. Although the expressions of six isoforms
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Prxs have tissue specificity, they can be detected in all tested tissues from Sparus aurata [49]. CpPrx also was constitutively expressed in the examined tissues at the transcriptional level, and the highest expression
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was in hepatopancreas and gill in C. plicata. Therefore, it was suggested that CpPrx was a ubiquitously expressed gene in C. plicata. The
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hepatopancreas seems to be the primary site for the production of immune recognition molecules and act as an accessory to the gut in digestion and absorption of nutrients [50]. Meanwhile, high expression level in gill may be due to frequent exposure and high consumption of oxygen, which 340
continuously leads to inducing the production of ROS [30]. The expression level of Prx mRNA transcripts in F. chinensis increase and reach the highest at 3 h, and then decrease at 5 h after Vibrio anguillarum infection [43]. After 8 h post-injection of lipopolysaccharide, Prx4 mRNA expression in hepatopancreas from Penaeus monodon
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increase 49.2 times comparison with control group [44]. The expression level of Prx4 from B. mori dramatically up-regulate in the fat body tissues by H2O2 and baculovirus infection [51]. Additionally, Prx1 and Prx2 mRNA transcriptional in gill and digestive tract tissues from H. discus
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discus are up-regulation after intramuscular injection of H2O2 [30]. After challenged with the bacteria V. anguillarum, the level of PrxV transcripts in hemocytes of Argopecten irradians is up-regulate and reach the highest point at 15 h post-challenge [34]. In Eurypanopeus depressus, a low
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expression level of Prx1 gene is detected in gill, hypodermis and hepatopancreas tissue in non-stressed control crabs, the expression of Prx1 increase in the gill tissue upon exposure of the crabs to hypo-
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osmotic stress, and no change in the transcript level is observed in the hypodermis and hepatopancreas tissues [45]. In this study, the expression level in hemocytes from CpPrx gradually increased at 12, 24, 48 h after intramuscular injection of PGN, and initially increased at 6 h after injection of A. hydrophila. The expression level in hepatopancreas from
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CpPrx markedly increased at 3, 12, 24 h post-injection of PGN and A. hydrophila, achieved the highest expression level at 3 h. Therefore, we
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suggested that CpPrx was transcriptionally activated in response to microbial infection.
The ability of Prxs to scavenge ROS and thereby protect bio
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molecules from oxidative damage in eukaryotes has been reported [21-23, 52]. When the concentration of H2O2 is ranged from 0 to 0.4 mM, the recombinant protein of pMALAb Prx2 from H. discus has antioxidant function. The cell growth is reduced in cells without plasmid or plasmid 370
containing only pMAL-c2X with the increases of H2O2 concentration from 0.4 to 0.8 mM [30]. Here, we attempted to study antioxidant function of recombinant pET-32-CpPrx in vitro. The result showed the
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growth of DE3-pET-32-CpPrx was not affected under the condition of 0.4 and 0.8 mmoL/L H2O2. In the control groups, the cells survival 375
decreased along with the H2O2 concentration was increased. These was suggested that DE3-pET-32-CpPrx exhibited significant antioxygenation
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function comparison with DE3-pET-32 and DE3 strain. When the concentration of H2O2 was raised to 1.6 mmoL/L, the growth of DE3pET-32-CpPrx was inhibited. These indicated that the cellular respiration was blocked.
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Acknowledgements
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This research was financially supported by grants (No. 31472305, 21467015, 31460697) from National Natural Science Foundation of China,
the Project of the Scientific and Technological (GJJ10378, GJJ12024), Key Lab of Aquatic Resources and Utilization and Nanchang University
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Seed Grant for Biomedicine of Jiangxi Province, China.
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study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis. Fish & Shellfish Immunology, 2008, 24(3): 294–307. [31] Nikapitiya C, Zoysa M, Whang I, Kim CG, Lee YH, Kim SJ, Lee J. Molecular cloning, characterization and expression analysis of peroxiredoxin6 from disk abalone Haliotis discus discus and the antioxidant activity of its recombinant protein. Fish & Shellfish Immunology, 2009,
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and mRNA expression of peroxiredoxin in Zhikong scallop Chlamys farreri. Molecular Biology Reports, 2009, 36(6): 1451–1459.
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ACCEPTED MANUSCRIPT Table 1 The primers used in this study Usage
Sequence(5'→3')
CpPrx-F1
Initial PCR
CTTCTACCCACTGGATTTCACAttygtntgycc
CpPrx-R1
Initial PCR
CATCCGGCTGGACAGacytcnccrtg
CpPrx-F2
3′- RACE
CTGTGAGATCTCCAGCGCTTATGGA
CpPrx-R2
5′- RACE
TCCAATTAGCTCCACAGACTTCTCCA
CpPrx-F3
3′- RACE
GAAAGGGAAACCTGCGACAGATCAC
CpPrx-R3
5′- RACE
ATTCGTCAACTGATCGACCCACGGGCA
CpPrx-F4
RT-qPCR
ATCAATACGCCGAGAAAGCA
CpPrx-R4
RT-qPCR
AAGTCCGAAACCTCAAGTGT
β-actin-F
RT-qPCR
CTTGACTTGGCAGGTAGAGA
β-actin-R
RT-qPCR
CAGACAGCACAGTGTTAGCA
CpPrx-F5
Expression
AAGGGTACCATGTCTCAGCTGAAACTGACCA
CpPrx-R5
Expression
CGC GGATCCTCAGTGATGGGCCTTGAAATAGCT
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SC
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Prime
Table 2 Genbank accession number of Prx family members for multiple sequence
Species
proteins
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comparison and phylogenetic tree construction proteins
Accession number
NP_859048A
Prx1
AAH86648
AAH03022
Prx2
AAH86783
Prx3
EDL01849
AAH16770
Prx4
AAH03349
Prx5
AAI10984
Prx5
AAH08174
Prx6
AAH53550
Prx6
AAH13489
Prx1
AAH88118
Prx1
NP_001085485
Prx2
AAH58481
Prx2
NP_001085414
Prx3
EDL94585
Prx3
NP_001086130
Prx1
EP
Prx2 Prx3
H. sapiens
Accession number
AC C
Prx4
Species
AAH02685
R .norvegicus
M. musculus
X. laevis Prx4
AAH59122
Prx4
NP_001085918
Prx5
AAH78771
Prx5
NP_001085580
Prx6
NP_446028
Prx6
NP_001084316
ACCEPTED MANUSCRIPT Prx1
AAH84184
C. plicata
Prx
ADM88874
Prx2
AAH61276
H. discus discus
Prx
ABO26635
Prx3
NP_001025608
P. fucata
Prx
ADC35419
Prx4
AAH76692
B.ignitus
Prx
ACP44066
Prx5
NP_001106525
L.vannamei
Prx
ACX53642
Prx6
NP_989102
AC C
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M AN U
SC
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X .tropicalis
ACCEPTED MANUSCRIPT
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Table 3 Amino acid sequence identity matrix comparison CpPrx with other members of Prx family from representative species
C.plicata H.sapiens X. laevis H.sapiens X. laevis H.sapiens X. laevis H.sapiens X. laevis H.sapiens X. laevis H.sapiens X. laevis C.plicata Prx Prx1 Prx1 Prx 2 Prx2 Prx3 Prx3 Prx4 Prx4 Prx5 Prx5 Prx6 Prx6 Prx6 63.9 69.5 67.5 73.3 100
52.7 48.6 44.7 49.6 46.9 100
51.4 50.4 46.4 49.8 48.2 71.9 100
51.7 49.6 48.9 50.2 48.3 43.6 50.7 100
SC
69.2 77.4 72.4 100
M AN U
66.3 83.4 100
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69.3 100
EP
100
AC C
C.plicata Prx H.sapiens Prx1 X. laevis Prx1 H.sapiens Prx2 X. laevis Prx2 H.sapiens Prx3 X. laevis Prx3 H.sapiens Prx4 X. laevis Prx4 H.sapiens Prx5 X. laevis Prx5 H.sapiens Prx6 X. laevis Prx6 C.plicata Prx6
53.6 51.1 50.7 51.3 50.2 54.1 50.7 81.0 100
17.4 15.4 13.6 17.4 15.5 21.4 19.3 17.5 19.9 100
16.8 16.0 15.5 16.7 16.8 16.9 16.1 16.6 15.5 55.8 100
26.8 27.9 25.7 27.5 27.7 18.3 21.6 18.0 17.9 11.6 15.5 100
25.4 27.3 25.5 27.0 27.8 18.5 18.6 18.8 19.4 13.0 15.1 78.7 100
24.9 29.8 27.2 27.8 27.2 18.4 19.0 18.4 17.3 13.7 16.8 61.2 61.2 100
ACCEPTED MANUSCRIPT TAA CGA CAA CCT AAC CAT ACT GCC CTT GAC GGA GTA CGA GTG AAG TTA TAT AAC TTG TGC TTT TTT GTT TGT TGA AAA CAA TCT TCC ATA 90 ATG TCT CAG CTG AAA CTG ACC AAA CCA GCC CCA GAG TGG AGT GGA ACT GCC GTT GTC AAT 150 M
S
Q
L
K
L
T
K
P
A
P
E
W
S
G
T
A
V
V
N
20
G
E
F
K
D
I
S
L
A
D
Y
R
G
K
Y
L
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GGA GAA TTT AAA GAT ATT TCA TTG GCA GAT TAT AGG GGC AAA TAC CTC GTC CTG TTT TTC 210 V
L
F
F
40
TAT CCA TTG GAT TTC ACT TTT GTT TGC CCA ACA GAG ATC ATA GCC TTC AGT GAC AGG GTG 270 Y
P
L
D
F
T
F
V
*
C
P
T
E
I
I
A
F
S
D
R
V
60
GAA GAA TTC CGA GCC ATC AAC TGT GAA GTT GTA GCC TGC TCC ACA GAT AGC CAT TTC TCT 330 E
F
R
A
I
N
C
E
V
V
A
C
S
T
D
S
H
SC
E
F
S
80
CAC TTG GCA TGG ATC AAT ACG CCG AGA AAG CAG GGT GGC TTG GGC AGC ATG AAT ATA CCT 390 L
A
W
I
N
T
P
R
K
Q
G
G
L
G
S
M AN U
H
M
N
I
P
100
CTT CTG GCC GAC AAA ACC TGT GAG ATC TCC AGC GCT TAT GGA GTT CTT AAG GAA GAT GAG 450 L
L
A
D
K
T
C
E
I
S
S
A
Y
G
V
L
K
E
D
E
120
GGA GTG GCA TTC AGA GGA CTG TTT ATA ATT GAT GGA AAG GGA AAC CTG CGA CAG ATC ACA 510 G
V
A
F
R
G
L
F
I
I
D
G
K
G
N
L
R
Q
I
T
140
GTG AAT GAT ATG CCC GTG GGT CGA TCA GTT GAC GAA ACC TTG AGA CTA GTT CAG GCT TTC 570 N
D
M
P
V
G
R
S
V
D
TE D
V
E
T
L
R
L
V
Q
A
F
160
CAG TTC ACA GAT AAG CAT GGA GAA GTC TGT CCA GCT AAT TGG AAG CCT GGT TCC GAC ACG 630 Q
F
T
D
K
H
G
E
V
*
C
P
A
N
W
K
P
G
S
D
T
180
ATG AAG CCC AGC CCT AAA GAA AGC CAG AGC TAT TTC AAG GCC CAT CAC TAA TTA ATT ATA 690 K
P
S
P
K
E
S
EP
M
Q
S
Y
F
K
A
H
H
ATC AAA TGT CGT TTA CAG GAT AAG CTT ATG TCC CAT GTG CAT CTC TCA CTA CCA TGT GCT 750
AC C
ACT AGG TCT TAT TTT CTC TGG GTA TTC AAA TGG TTT TCA CAA ATT TAG GTA CTT TAC ATG 810 CTG TCT CTC AGA TAC TTC AGT TAG AGA TTT GAG AAC ATA TTC TAA TTT AAG TGA TAC ATG 870 TGT GCA TTA TTA TTG CAC AAG AAT TTT TCA CTT GTT TTC AGC AGT AAT TGT GTA AGC CAC 930 CAG CAC AGA TGA GGG AAT AAT CTC GTT AAA ATA ATT TCC ATC AGG TAT ATT AGC TTT CAG 990 GTC TGT TAC AAT TTT CGT CTG TTG CAA TAA TTT GGA TAT CTT TTT ACA TAC TCT TCA CCT 1050 TCG GTG AAG TCT TTA TCT CGT ATT TTG CCG TAT TTT GTT CAT GAT ATT ACC CTT TTG TTC 1110 ACT TTA TTG TTA GAT TTT GCA TTA A AT ATA AAT AA AGG TAG TTG CTA CCC AGT TTT TTT TGT GAG 1170 TAC TGT AAA ATC AGG CTG TGA TGT TTG TCT TGT GTT TGA TAT TGT GCC ATA AAA AAA AAA 1230 AAA AAA AAA AAA AAA A
1246
Fig. 1 Nucleotide and deduced amino acid sequence of Prx cDNA gene from Cristaria plicata
ACCEPTED MANUSCRIPT Note: Underline shows the start codon (ATG), the stop codon (TGA) and putative polyadenylation
signals
(AATAAA).
Conserved
signature
sequences
“FYPDFTFVCPTEI” and “GEVCPA” are shaded by gray. The asterisk showed N-
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terminal conserved cysteine.
M AN U
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ACCEPTED MANUSCRIPT
Fig. 2 ClustalW multiple alignment analysis of CpPrx with known Prxs Note: Amino acid residues shared by all the sequence are denoted by an asterisk. Similar amino acids residues are denoted by (* or :). Solid boxes indicate predicted the Prx signature motif 1 and 2 sequences. Dashed box indicates that the conservative
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motifs are associated with sensitive of typical 2-Cys Prxs for H2O2 concentration. Two conserved cysteines in each motif are highlighted with star (★) on top of each residue. GenBank accession numbers for the protein sequences are as follows. H. discus Prx2: ABO26635.1. C. plicata TPx: ADM88874.1. H. sapiens Prx1-4: AAH03022,
EP
NP_859048A,
AAH02685,
AAH16770.
M.
musculus
Prx1-4:
AAH86648, AAH86783, EDL01849, AAH03349. X. laevis Prx1-4: NP_001085485,
AC C
NP_001085414, NP_001086130, NP_001085918. D. rerio Prx1-4: NP_001013489, AAH76347, AAH92846, NP_001082894.
ACCEPTED MANUSCRIPT 52 M. musculus Prx1 100 R. norvegicus Prx1 H. sapiens Prx1 97
Prx1
X. laevis Prx1
100
X. tropicalis Prx1
48
X. laevis Prx2
100 50
H. sapiens Prx2
100
26
RI PT
X. tropicalis Prx2
100
Prx2
M. musculus Prx2
R. norvegicus Prx2 B. ignites Prx
58 61 52
▲ C. plicata Prx
M AN U
99
Prx
H. discus Prx
91 88
Typical 2-Cys
SC
L. vannamei Prx
P. fucata Prx
99 X. laevis Prx4 100 X. tropicalis Prx4 Prx4
H. sapiens Prx4
EP
TE D
91 M. musculus Prx4 99 R. norvegicus Prx4
AC C
1-Cys Prx
atypical 2-Cys Prx
97 X. laevis Prx3 X. tropicalis Prx3 100 H. sapiens Prx3
Prx3
97 M. musculus Prx3 93 R. norvegicus Prx3 100 X. laevis Prx6 X. tropicalis Prx6
100 99
H. sapiens Prx6 M. musculus Prx6
92 100
R. norvegicus Prx6 X. laevis Prx5 X. tropicalis Prx5
100 99
H. sapiens Prx5 M. musculus Prx5
67
0.2
Prx6
R. norvegicus Prx5
Prx5
ACCEPTED MANUSCRIPT Fig. 3 Neighbor-joining phylogenetic tree of Prx amino acid sequences from ten species animals Note: GenBank accession numbers for the protein sequences are as follows. M. musculus Prx5-6: AAH08174, AAH13489. R. norvegicus Prx1-6: AAH88118, AAH58481, EDL94585, AAH59122, AAH78771, NP_446028. H. sapiens Prx5-6:
tropicalis
Prx1-6:
AAH84184,
AAH61276,
RI PT
AAI10984, AAH53550. X. laevis Prx5-6: NP_001085580, NP_001084316. X. NP_001025608,
AAH76692,
NP_001106525, NP_989102. P. fucata Prx, ADC35419. B. ignitus, ACP44066. L. vannamei, ACX53642. Other abbreviations and accession numbers are the same as in
AC C
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M AN U
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Fig. 2.
Fig. 4 Tissueexpression of Prx gene in different tissues of Cristaria plicata
AC C
EP
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M AN U
SC
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ACCEPTED MANUSCRIPT
Fig. 5 The expression of Prx mRNA in hemocytes (A) and hepatopancreas (B) from Cristaria plicata after challenge by Real-time quantitative PCR Note: All values represent the mean ± S.D. (n=4). CpPrx transcript levels were normalized by injecting 0.1 mL PBS at 0 hour. Asterisk (*) are significantly different (* and **represent p < 0.05 and p < 0.01; respectively, t-test).
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
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Fig. 6 Expression of recombinant plasmid in Escherichia. coli by SDS-PAGE Note: M: molecular weight marker, 1. Uninduced plasmid, 2. Plasmid with IPTG induced 2 h, 3. Plasmid with IPTG induced 4 hr, 4. Plasmid with IPTG induced 6 h, 5.
AC C
EP
Plasmid with IPTG induced 8 h.
2
ACCEPTED MANUSCRIPT
1 2
A
B
3 4
A
SC
B
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C
M AN U
C
Fig. 7 The cell tolerance of DE3 (A), DE3-pET-32 (B) and DE3-pET-32-CpPrx (C) to H2O2
Note: 1. Culture medium without H2O2, 2, 3, 4. Culture medium with 0.4, 0.8 and 1.6
AC C
EP
TE D
mmoL/L H2O2.
ACCEPTED MANUSCRIPT
Highlights The full cDNA sequences of CpPrx were cloned. The transcripts of CpPrx were
AC C
EP
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M AN U
SC
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up-regulated after stimulation. The recombinant had antioxidant activity.
S1050-4648(17)30266-8
DOI:
10.1016/j.fsi.2017.05.026
Reference:
YFSIM 4579
To appear in:
Fish and Shellfish Immunology
Received Date: 11 January 2017 Revised Date:
25 March 2017
Accepted Date: 8 May 2017
Please cite this article as: Wang X, Hu B, Wen C, Zhang M, Jian S, Yang G, Molecular cloning, expression and antioxidative activity of 2-cys-peroxiredoxin from freshwater mussel Cristaria plicata, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.05.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Molecular cloning, expression and antioxidative activity of 2-cysperoxiredoxin from freshwater mussel Cristaria plicata Xiaobo Wang1, Baoqing, Hu1, Chungen Wen1*, Ming Zhang 2*, Shaoqing Jian1, Gang Yang1 1. School of Life Sciences, Nanchang University, Nanchang 330031, China
RI PT
5
SC
2. College of Jiangxi Biotech Vocational, Nanchang 330200, China
*Corresponding author:
[email protected] (CG. Wen), Tel. /fax: +86-0791-83969530
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[email protected] (M. Zhang), Tel./fax: +86-0791-87877381
Running Title: A 2-Cys peroxiredoxin gene from Cristaria plicata
AC C
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15
ACCEPTED MANUSCRIPT
ABSTRACT: Peroxiredoxins (Prxs) play an important role against various oxidative stresses by catalyzing the reduction of hydrogen 20
peroxide (H2O2) and organic hydroperoxides to less harmful form. A 2-
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cys peroxiredoxin, designated as CpPrx, was cloned from hemocytes of of freshwater mussel Cristaria plicata. The full length cDNA of CpPrx is 1247 bp, which includes an open reading frame (ORF) of 591bp,
25
SC
encoding 196 amino acids. CpPrx possesses two conserved cysteine residues (Cys49, Cys170). The deduced amino acid sequence of CpPrx
M AN U
showed a high level (67-74%) of sequence similarity to 2-Cys Prxs from other species. The results of real-time quantitative PCR revealed that CpPrx mRNA was constitutively expressed in tissues, and the highest expression levels were in hepatopancreas and gills. After peptidoglycan 30
(PGN) and Aeromonas hydrophila challenge, the expression levels of
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CpPrx mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpPrx was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3).
35
EP
Comparison with DE3-pET-32 and DE3 strain, the cells of DE3-pET-32CpPrx exhibited resistance to the concentration of 0.4, 0.8 and 1.2
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mmoL/L H2O2 in vivo.
Key words: Cristaria plicata; Peroxiredoxin; Molecular clone; Recombinant protein; Antioxidant activity
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1. Introduction Peroxiredoxins (Prxs), known as thioredoxin peroxidases, are also cysteine-dependent peroxidases proposed to function as antioxidant 45
enzymes [1]. These enzymatic antioxidants are characterized by one or
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two conserved cysteines (Cys) that reduce hydroperoxides in the presence of thiol [2]. The feature of Prxs possess the peroxidatic Cys residue (CysSRH) which is oxidized by the peroxide substrate to Cys sulfenic acid
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(Cys-SPOH) during the reaction cycle [3]. The main function of Prxs is to eliminate H2O2, and adjust signal transduction and immune reaction
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mediated by H2O2 [4]. Prxs emerge as important factors linking reactive oxygen species (ROS) metabolism to redox-dependent signaling events [2, 5, 6]. Together with ROS, nitric oxide (NO) is also a free radical product of the cell metabolism that is essential in the signal transduction. The S55
nitrosylation of PrxII in vivo probably serve as a protective mechanism
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under oxidative and nitrosative stress [7]. In addition, Prxs can enhance cell toxicity mediated by natural killer (NK) cell [8], regulate calciumdependent potassium transport across the plasma membrane [9], block the
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activation of nuclear transcription factor-κB (NF-κB) and tumor necrosis factorα (TNFα) as well as inhibit human immunodeficiency virus type 1
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(HIV-1) replication [9, 10]. Prxs of mammalian are divided into 1-Cys and 2-Cys Prx that depend on whether they have only the peroxidatic Cys or a resolving Cys (Cys-SRH). 2-Cys Prxs are further splitted into typical (Prx1-4) and atypical Prx (Prx5) according to catalytic mechanisms [3, 5, 65
11]. All Prxs have in common an overall fold and catalytic mechanism involving a conserved, fully folded active site and an unfolding event [12]. The enzymatic mechanism relies on a conserved cysteine residue, the peroxidatic cysteine, which reduces various peroxide substrates, and the second free thiol then forms a disulfide with peroxidatic cysteine [13].
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Prxs have received a great deal of attention owing to their role in regulating levels of hydrogen peroxide, which is common to many cytokine induced signal-transduction pathways as an intracellular signaling molecule [2, 14]. Many evidences implicate that Prxs are
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important and widespread signaling molecule that are served as indicator of oxidative stress or as a part of normal cellular development [15-17].
Prxs have been isolated from archeobacteria, protozoon, fungus, parasite and mammal [18], and have been extensively investigated in
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yeast (Saccharomyces cerevisiae), mammals (Homo sapiens), plants (Arabidopsis thaliana), bacteria (Salmonella typhimurium) and parasitic protists (Plasmodium falciparum and Trypanosoma sp) [2, 19-28].
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Meanwhile, the genes of Prxs have recently been reported to be present in molluscan, Saccostrea glomerata Prx6 [29], Haliotis discus discus Prx1, Prx2, Prx6 [30, 31], Crassostrea gigas Prx6 [32], Chlamys farreri Prx5
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[35].
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[33], Argopecten irradians Prx5 [34] and Laternula elliptica Prx5, Prx6
The antioxidant function of Prxs is detected in the lung, cartilage, tendon and umbilical cord blood monocytes (UCBMC) in human [36-41].
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The overexpression of Prx V offer significant protection against ocular anomalies caused by oxidative stress in Xenopus laevis [42]. Prxs are also involved in the immune response. The expression of Prx V is up-
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regulated to be associated with acute inflammation induced by lipopolysaccharide in rat [33, 41]. The expression of Prxs is examined in the tissues of aquatic organisms [33, 35, 43, 44]. The expression level of 2-Cys Prx is enhanced by exposure to hypo-osmotic stress in gill tissue of 95
Eurypanopeus. depressus [45]. The mRNA transcripts of Prx V in hemocytes of Argopecten irradians are up-regulated after Vibrio anguillarum challenge [34]. The oxidative stress of Prxs is investigated in aquatic organisms, but the antioxidant function of that has not been
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described in freshwater mussel. 100
The freshwater mussel Cristaria plicata, which is of great economical importance, is well known as one of “freshwater pearl bivalve” in the aquaculture industry of China. However, the farming of
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freshwater pearl has been suffering serious problems due to the outbreak of mussel diseases in the cultivation process [46]. Thus, it is crucial for 105
diseases management and development of sustainable mussel culture and pearl production to research the immunity of freshwater mussel.
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The cDNA sequence of a 2-Cys Prx from the C. plicata, designated as CpPrx, was isolated and was identified in this study. Next, tissue-
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specific expression of CpPrx was detected in the tissues of normal mussels. The expression patterns of CpPrx mRNA transcripts in hemocytes and hepatopancreas were examined by quantitative real-time PCR after challenge with Aeromonas hydrophila, peptidoglycan (PGN) and PBS. The CpPrx cDNA was subcloned into the pET-32 vector and
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was transformed into Escherichia coli BL21 (DE3). The antioxidant function of pET-32-CpPrx was evaluated in vitro.
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2. Materials and methods
2.1 Collection and maintenance of mussels
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The freshwater mussel C. plicata, with shell length 18-25 cm,
collected from Poyang Lake in Jiangxi province, China, was maintaned at 25±2 ℃ in freshwater tanks with continuous oxygenation, changing the water every day for one week before processing.
2.2 RNA extraction and cDNA synthesis 125
Total RNA was extracted from haemocytes using Trizol Reagent (Invitrogen, Carlsbad, CA) following manufacturer’s instruction. The
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extracted RNA was then treated with RQ1 Rnasefree DNase I (Promega, Madison, WI) to remove any possible contaminating DNA. Smart cDNA was synthesized from total RNA by using SMARTTM cDNA synthesis kit 130
(Clontech Laboratories, Palo Alto, CA). The synthesis reactions were for 15 min, and subsequently stored at -80 ℃. 2.3 Cloning the full-length cDNA of CpPrx
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performed at 65 ℃ for 5 min, 42 ℃ for 1 h, terminated by heating at 70 ℃
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Based on the highly conserved sequences from Chlamys farreri (EF634307.1) and Crassostrea gigas (XM011443230.1), two degenerated
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primers CpPrx-F1 and CpPrx-R1 (Table 1) were designed to obtain the mid-fragment of CpPrx from C. plicata. The amplification program consisted of 5 min at 94 ℃, followed by 35 cycles of 94 ℃ for 30 s, 54 ℃ for 30 s, 72 ℃ for 1 min and an additional extension at 72 ℃ for 10 min. 140
The PCR product was then cloned into the pMD18-T vector (Promega)
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and sequenced in both directions with primers T7 and SP6. To obtained full-length cDNA of CpPrx, four gene-specific primers CpPrx-F2, CpPrx-F3, CpPrx-R2 and CpPrx-R3 were designed from the
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partial sequence of CpPrx cDNA (Table 1). PCR reactions were performed using SMART-RACE and nest-PCR. PCR amplicons were
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sequenced and the full-length cDNA of CpPrx was assembled from 5' and 3' sequences.
2.4 Sequence analysis of CpPrx The CpPrx gene sequence was analyzed by using the BLAST
150
algorithm at the NCBI web site (http://www.ncbi.nlm.nih. gov/blast ), and the deduced amino acid sequence, the cellular localization prediction, signal peptide, Structure domain analysis and open reading frame (ORF) were analyzed with the Expert Protein Analysis System (http://www.expasy.org/).
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The protein sequences were aligned by the ClustalW multiple sequence 155
alignment program (version 1.8). The molecular mass was calculated, and the theoretical isoelectric points were predicted by Protein MolWt & AA Composition
Calculator The
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(http://www.proteomics.com.cn/proteomics/pi_tool.asp).
phylogenetic tree was constructed from the deduced amino acid sequences using the Neighbour-Joining (NJ) algorithm within MEGA version 4.1.
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2.5 Tissue distribution and temporal expression of CpPrx after A.
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hydrophila, PGN and PBS challenge
The tissue-specific expression of CpPrx was detected in the tissues of normal mussels, including hemocytes, mantle, hepatopancreas, gill and
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muscle, from five individuals. Sixty mussels were selected for the temporal expression of CpPrx, and were randomly divided into three groups in the same tank, and each group included 20 mussels per tank. The mussel adductor muscle of individual animals in the control or
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challenged groups were injected with 0.1 mL PBS (pH 7.0, 0.2 M Na2 HPO4, 0.2 M NaH2PO4), or 0.1 mL live bacterial suspension (A. hydrophila, dissolved in PBS, 109 cell/mL), or 0.1 mL PGN (Sigma, 0.5 mg/mL), respectively. The hemocytes and hepatopancreas were obtained 175
separately from five individual mussels at 0, 3, 6, 12, 24 and 48 h postinjection of each group and were immediately stored in liquid nitrogen until used. Total RNA samples isolated using the TRIzol reagent
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(Invitrogen) were used to synthesize the first strand cDNAs, which were used as the template of real-time quantitative-PCR (RT-qPCR). 180
2.6 Real-time quantitative PCR
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The transcripts of CpPrx in different tissues were determined by RTqPCR. The total RNAs were extracted from the tissues of mussel and were utilized to synthesize the first strand cDNAs, which were used as the
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templates of RT-qPCR amplification. The primers of CpPrx-F4 and CpPrx-R4, and of β-actin-F and β-actin-R (Table 1) were designed to
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amplify specifically partial cDNA sequence of the CpPrx and the internal reference gene Cp-β-actin, respectively. RT-qPCR was performed in a total volume of 25 µL containing 10 µL of 2 × SYBR Green Real-time PCR Master Mix (TaKaRa, DRR041A), 1 µL of cDNA, 1 µL of each 190
primer and 12 µL of PCR-grade water, conducted on an Eppendorf
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Mastercycler ep Real-plex2 PCR system. Triplicate reactions were performed for each sample. The synthesis reaction was performed at 94 ℃ denaturation for 5 min, 40 cycles for 94 ℃ 30 s, 57 ℃ for 30 s,
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72 ℃ for 15 s, and finally 72 ℃ elongation for 3 min. Fluorescence readings were performed at the end of each cycle. The CpCpx mRNA
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expression level could be calculated by 2_△△CT.
2.7 PCR amplification of CpPrx The ORF sequence of CpPrx was amplified by a pair specific primer
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CpPrx-F5 and CpPrx-R5 with the corresponding restriction enzyme sites of KpnI and BanHI (Table 1). The PCR program was performed at 95 ℃ for 5 min, 35 cycles of 94 ℃ for 30 s, 57 ℃ for 30 s, and 72 ℃ for 60s, with an additional extension step at 72 ℃ for 10 min. The amplicon was
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firstly cloned into pMD18-T vector, and the sequence was verified by 205
DNA sequencing. The prokaryotic expression plasmid pET-32 (Novagen, Madison, WI) was digested with KpnI and BanHI and then ligated with the same digested and recovered CpPrx gene from recombinant T vector.
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The resulting construct was the CpPrx recombinant expression vector, which was designated to pET-32-CpPrx. 210
2.8 Prokaryotic expression of recombinant pET-32-CpPrx
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The recombinant expression plasmid pET-32-CpPrx was transformed into E. coli BL21 (DE3) competent cells. The transformed cells were
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cultivated at 37 ℃, 200 r/min in 10 mL LB medium containing 100 mg/mL ampicillin until OD600 reached about 0.4. Final concentrations of 1 mM isopropy β-D-1-thiogalactopyranoside (IPTG) were added to the culture and the culture was induced at 20 ℃, 200 r/min. 1 ml culture
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was removed at 0, 2, 4, 6, 8 h respectively. A blank group was cultured at 0 h, induced groups were cultured in other time buckets. The cells 220
were harvested by centrifugation at 12000 g for 5 min at 4 ℃. The recombinant expression of target protein was determined by 15% SDS-
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PAGE assay.
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2.9 Antioxidation function of CpPrx in vitro The antioxidant activity of CpPrx was determined by measuring the
sensitivity of E. coli cells against H2O2 toxicity. The positive DE3-pET32-CpPrx strain was selected as challenge group, and DE3-pET-32 and DE3 strain as control groups. Both challenge and control groups were induced with 1 mM IPTG for 24 hours at 37 ℃, 200 r/min. The bacterium 230
concentration, which was determined by using spectrophotometer (at 600 nm), was diluted with sterile water into OD600=0.2, continuous dilution (1:
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3). 5 µL bacterium was extracted from each sample plated on LuriaBertani (LB) agar plates with different concentrations of H2O2 (0, 0.4, 0.8, 1.6 mmoL/L). All plates were cultivated at 37 ℃ for 2 or 3 days, and the 235
diameters of the cell clusters were compared to evaluate the H2O2
2.10 Data processing and statistical analysis
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tolerance activity of CpPrx.
All assays were done in triplicate, and were repeated at least twice. The
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statistical analysis was carried out with SPSS Statistics 13.0 software, and the data were calculated as the mean ± S.D. Differences were considered
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to be statistically significant when P values were lower than 0.05.
3. Result
3.1 The full-length cDNA of CpPrx
The nucleotide and the deduced amino acid sequences of CpPrx
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were shown in Fig. 1. The cDNA sequence was deposited in the GenBank database as accession No. HQ166838. The full-length CpPrx cDNA was comprised of 1247 bp, and contained a 5’ untranslated region (UTR) of
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90 bp, an open reading frame (ORF) of 591 bp, and a long 3’ UTR of 566 bp with a canonical polyadenylation signal sequence AATAAA and a
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polyA tail. The putative CpPrx polypeptide was predicted to contain 196 amino acid residues, with a calculated molecular mass of 21.87 kDa and a theoretical isoelectric point of 5.95. Signal IP 3.0 analysis showed that no signal peptide was found. The deduced amino acid sequence of CpPrx 255
contained two highly conserved motifs (F39YPLDFTFVCPTEI53, G167EVCPA172) and two conserved Cys residue (Cys49, Cys170) in the N-and C-terminal. The mature peptide included three typical structural domains, peroxide reductase (Leu4-Gln189), thioredoxin-2 (Leu4-Phe162)
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and Alkyl hydroperoxide reductase-Thiol specific antioxidant domain 260
(Leu6-Ile139).
3.2 Homology and phylogenetic analysis of CpPrx
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The results of multiple alignment indicated that the amino acid sequence of CpPrx had high sequence similarities with the putative Prxs 265
of Sinanodonta woodiana (95% identity), Pinctada fucata (85% identity) and Haliotis discus (80% identity). The sequence of CpPrx also displayed
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similarities with Prx1 (74-67% identity) from Litopenaeus vannamei, Danio rerio, Homo sapiens, Mus musculus and Xenopus laevis, Prx2 and
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Prx4 (75-67% identity), Prx3 (52% identity), Prx5 (17% identity) and Prx6 (25% identity) from other species (Fig. 2). Pairwise comparison of the sequences revealed that the CpPrx sequence was more similar to Prx 2 sequences than Prx 1 sequences (Table 2).
The molecular phylogenetic tree based on amino acid sequences of
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Prxs was constructed by Neighbor-joining method as shown in Fig. 3. Prxs from invertebrate and vertebrate were separated to three sub-clusters.
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The CpPrx was located in the branch of typical 2-Cys Prx.
3.3 The tissue expression patterns of CpPrx mRNA
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expression
of
CpPrx
mRNA
in
hemocytes,
muscle,
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The
hepatopancreas, mantle, and gill was examined by real-time quantitative RT-qPCR (Fig. 4). The results showed that the CpPrx mRNA was constitutively expressed in detected tissues. The highest level was expressed in hepatopancreas, followed by gill and muscle, and the lowest level was in hemocytes and mantle.
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3.4 Temporal expression of CpPrx after A. hydrophila, PGN and PBS challenge
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The expression of the CpPrx mRNA was up-regulated in hemocytes and hepatopancreas. The expression of the CpPrx mRNA in hemocytes was inceased at 6, 12, 24 and 48 h, and the highest expression was 290
observed at 48 h (Fig. 5A). The expression of CpPrx mRNA in
5B).
3.5 Expression of the recombinant protein
Comparison with non-induced culture, the DE3-pET32-CpPrx was
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hepatopancreas enhanced at 3 h and resumed to normal levels at 48 h (Fig.
induced by IPTG that expressed a fusion protein of approximately 34 kDa
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(including His-tag). The result was detected by SDS-PAGE (Fig. 6). 3.6 Antioxidation function detection of DE3-pET-32-CpPrx in vitro 300
DE3-pET-32-CpPrx, DE3-pET-32 and DE3 strain were cultured in LB media containing different concentrations of H2O2 were showed in Fig.
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7. The growth of DE3 strain was inhibited in each concentration of H2O2 and while the concentration of H2O2 was raised to 1.6 mmoL/L, the growth of bacteria was completely inhibited. With the increase of the concentration of H2O2, the survival rate of DE3-pET-32 strain decreased.
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The growth of DE3-pET-32-CpPrx strain had no significantly change in
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0.4 and 0.8 mmoL/L H2O2. While the concentration of H2O2 was raised to 1.6 mmoL/L, the growth of DE3-Pet32-CpPrx was markedly inhibited.
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4. Discussion
The typical 2-Cys Prxs are identified by two highly conserved redox
active cysteines named as the peroxidatic cysteine generally near residue 50, and the resolving cysteine near residue 170 [30]. The former is embedded in the highly conserved F-motif (FTFVCPTEI), and the latter 315
is adjacent to a highly conserved hydrophobic region (VCPAGW). These
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features are found in all typical 2-Cys Prxs from plants, mammals, fungi, bacteria and fish [2, 47, 48]. In this study, CpPrx also contained two highly
conserved
signal
motifs
(F39YPLDFTFVCPTEI53
and
G167EVCPA172) and two highly conserved Cys residue (Cys49, Cys170) in the N-and C-terminal. Phylogenetic analysis showed that CpPrx
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clustered together with other invertebrate animals in the typeical 2-Cys subfamily. Therefore, we inferred that CpPrx belonged to the typical 2Cys Prx.
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The expression of Prx1 mRNA can be detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill tissues of
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Fenneropenaeus chinensis [43]. Prx1 and Prx2 from Haliotis discus is constitutive expression in muscle，mantle，gill and digestive tract [30]. Furthermore, Prx 4 is expressed in tissues of muscle, stomach and brain, especially high expression in genital gland, hepatopancreas and heart 330
from Penaeus monodon [44]. Although the expressions of six isoforms
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Prxs have tissue specificity, they can be detected in all tested tissues from Sparus aurata [49]. CpPrx also was constitutively expressed in the examined tissues at the transcriptional level, and the highest expression
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was in hepatopancreas and gill in C. plicata. Therefore, it was suggested that CpPrx was a ubiquitously expressed gene in C. plicata. The
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hepatopancreas seems to be the primary site for the production of immune recognition molecules and act as an accessory to the gut in digestion and absorption of nutrients [50]. Meanwhile, high expression level in gill may be due to frequent exposure and high consumption of oxygen, which 340
continuously leads to inducing the production of ROS [30]. The expression level of Prx mRNA transcripts in F. chinensis increase and reach the highest at 3 h, and then decrease at 5 h after Vibrio anguillarum infection [43]. After 8 h post-injection of lipopolysaccharide, Prx4 mRNA expression in hepatopancreas from Penaeus monodon
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increase 49.2 times comparison with control group [44]. The expression level of Prx4 from B. mori dramatically up-regulate in the fat body tissues by H2O2 and baculovirus infection [51]. Additionally, Prx1 and Prx2 mRNA transcriptional in gill and digestive tract tissues from H. discus
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discus are up-regulation after intramuscular injection of H2O2 [30]. After challenged with the bacteria V. anguillarum, the level of PrxV transcripts in hemocytes of Argopecten irradians is up-regulate and reach the highest point at 15 h post-challenge [34]. In Eurypanopeus depressus, a low
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expression level of Prx1 gene is detected in gill, hypodermis and hepatopancreas tissue in non-stressed control crabs, the expression of Prx1 increase in the gill tissue upon exposure of the crabs to hypo-
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osmotic stress, and no change in the transcript level is observed in the hypodermis and hepatopancreas tissues [45]. In this study, the expression level in hemocytes from CpPrx gradually increased at 12, 24, 48 h after intramuscular injection of PGN, and initially increased at 6 h after injection of A. hydrophila. The expression level in hepatopancreas from
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CpPrx markedly increased at 3, 12, 24 h post-injection of PGN and A. hydrophila, achieved the highest expression level at 3 h. Therefore, we
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suggested that CpPrx was transcriptionally activated in response to microbial infection.
The ability of Prxs to scavenge ROS and thereby protect bio
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molecules from oxidative damage in eukaryotes has been reported [21-23, 52]. When the concentration of H2O2 is ranged from 0 to 0.4 mM, the recombinant protein of pMALAb Prx2 from H. discus has antioxidant function. The cell growth is reduced in cells without plasmid or plasmid 370
containing only pMAL-c2X with the increases of H2O2 concentration from 0.4 to 0.8 mM [30]. Here, we attempted to study antioxidant function of recombinant pET-32-CpPrx in vitro. The result showed the
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growth of DE3-pET-32-CpPrx was not affected under the condition of 0.4 and 0.8 mmoL/L H2O2. In the control groups, the cells survival 375
decreased along with the H2O2 concentration was increased. These was suggested that DE3-pET-32-CpPrx exhibited significant antioxygenation
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function comparison with DE3-pET-32 and DE3 strain. When the concentration of H2O2 was raised to 1.6 mmoL/L, the growth of DE3pET-32-CpPrx was inhibited. These indicated that the cellular respiration was blocked.
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Acknowledgements
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This research was financially supported by grants (No. 31472305, 21467015, 31460697) from National Natural Science Foundation of China,
the Project of the Scientific and Technological (GJJ10378, GJJ12024), Key Lab of Aquatic Resources and Utilization and Nanchang University
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Seed Grant for Biomedicine of Jiangxi Province, China.
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ACCEPTED MANUSCRIPT Table 1 The primers used in this study Usage
Sequence(5'→3')
CpPrx-F1
Initial PCR
CTTCTACCCACTGGATTTCACAttygtntgycc
CpPrx-R1
Initial PCR
CATCCGGCTGGACAGacytcnccrtg
CpPrx-F2
3′- RACE
CTGTGAGATCTCCAGCGCTTATGGA
CpPrx-R2
5′- RACE
TCCAATTAGCTCCACAGACTTCTCCA
CpPrx-F3
3′- RACE
GAAAGGGAAACCTGCGACAGATCAC
CpPrx-R3
5′- RACE
ATTCGTCAACTGATCGACCCACGGGCA
CpPrx-F4
RT-qPCR
ATCAATACGCCGAGAAAGCA
CpPrx-R4
RT-qPCR
AAGTCCGAAACCTCAAGTGT
β-actin-F
RT-qPCR
CTTGACTTGGCAGGTAGAGA
β-actin-R
RT-qPCR
CAGACAGCACAGTGTTAGCA
CpPrx-F5
Expression
AAGGGTACCATGTCTCAGCTGAAACTGACCA
CpPrx-R5
Expression
CGC GGATCCTCAGTGATGGGCCTTGAAATAGCT
M AN U
SC
RI PT
Prime
Table 2 Genbank accession number of Prx family members for multiple sequence
Species
proteins
TE D
comparison and phylogenetic tree construction proteins
Accession number
NP_859048A
Prx1
AAH86648
AAH03022
Prx2
AAH86783
Prx3
EDL01849
AAH16770
Prx4
AAH03349
Prx5
AAI10984
Prx5
AAH08174
Prx6
AAH53550
Prx6
AAH13489
Prx1
AAH88118
Prx1
NP_001085485
Prx2
AAH58481
Prx2
NP_001085414
Prx3
EDL94585
Prx3
NP_001086130
Prx1
EP
Prx2 Prx3
H. sapiens
Accession number
AC C
Prx4
Species
AAH02685
R .norvegicus
M. musculus
X. laevis Prx4
AAH59122
Prx4
NP_001085918
Prx5
AAH78771
Prx5
NP_001085580
Prx6
NP_446028
Prx6
NP_001084316
ACCEPTED MANUSCRIPT Prx1
AAH84184
C. plicata
Prx
ADM88874
Prx2
AAH61276
H. discus discus
Prx
ABO26635
Prx3
NP_001025608
P. fucata
Prx
ADC35419
Prx4
AAH76692
B.ignitus
Prx
ACP44066
Prx5
NP_001106525
L.vannamei
Prx
ACX53642
Prx6
NP_989102
AC C
EP
TE D
M AN U
SC
RI PT
X .tropicalis
ACCEPTED MANUSCRIPT
RI PT
Table 3 Amino acid sequence identity matrix comparison CpPrx with other members of Prx family from representative species
C.plicata H.sapiens X. laevis H.sapiens X. laevis H.sapiens X. laevis H.sapiens X. laevis H.sapiens X. laevis H.sapiens X. laevis C.plicata Prx Prx1 Prx1 Prx 2 Prx2 Prx3 Prx3 Prx4 Prx4 Prx5 Prx5 Prx6 Prx6 Prx6 63.9 69.5 67.5 73.3 100
52.7 48.6 44.7 49.6 46.9 100
51.4 50.4 46.4 49.8 48.2 71.9 100
51.7 49.6 48.9 50.2 48.3 43.6 50.7 100
SC
69.2 77.4 72.4 100
M AN U
66.3 83.4 100
TE D
69.3 100
EP
100
AC C
C.plicata Prx H.sapiens Prx1 X. laevis Prx1 H.sapiens Prx2 X. laevis Prx2 H.sapiens Prx3 X. laevis Prx3 H.sapiens Prx4 X. laevis Prx4 H.sapiens Prx5 X. laevis Prx5 H.sapiens Prx6 X. laevis Prx6 C.plicata Prx6
53.6 51.1 50.7 51.3 50.2 54.1 50.7 81.0 100
17.4 15.4 13.6 17.4 15.5 21.4 19.3 17.5 19.9 100
16.8 16.0 15.5 16.7 16.8 16.9 16.1 16.6 15.5 55.8 100
26.8 27.9 25.7 27.5 27.7 18.3 21.6 18.0 17.9 11.6 15.5 100
25.4 27.3 25.5 27.0 27.8 18.5 18.6 18.8 19.4 13.0 15.1 78.7 100
24.9 29.8 27.2 27.8 27.2 18.4 19.0 18.4 17.3 13.7 16.8 61.2 61.2 100
ACCEPTED MANUSCRIPT TAA CGA CAA CCT AAC CAT ACT GCC CTT GAC GGA GTA CGA GTG AAG TTA TAT AAC TTG TGC TTT TTT GTT TGT TGA AAA CAA TCT TCC ATA 90 ATG TCT CAG CTG AAA CTG ACC AAA CCA GCC CCA GAG TGG AGT GGA ACT GCC GTT GTC AAT 150 M
S
Q
L
K
L
T
K
P
A
P
E
W
S
G
T
A
V
V
N
20
G
E
F
K
D
I
S
L
A
D
Y
R
G
K
Y
L
RI PT
GGA GAA TTT AAA GAT ATT TCA TTG GCA GAT TAT AGG GGC AAA TAC CTC GTC CTG TTT TTC 210 V
L
F
F
40
TAT CCA TTG GAT TTC ACT TTT GTT TGC CCA ACA GAG ATC ATA GCC TTC AGT GAC AGG GTG 270 Y
P
L
D
F
T
F
V
*
C
P
T
E
I
I
A
F
S
D
R
V
60
GAA GAA TTC CGA GCC ATC AAC TGT GAA GTT GTA GCC TGC TCC ACA GAT AGC CAT TTC TCT 330 E
F
R
A
I
N
C
E
V
V
A
C
S
T
D
S
H
SC
E
F
S
80
CAC TTG GCA TGG ATC AAT ACG CCG AGA AAG CAG GGT GGC TTG GGC AGC ATG AAT ATA CCT 390 L
A
W
I
N
T
P
R
K
Q
G
G
L
G
S
M AN U
H
M
N
I
P
100
CTT CTG GCC GAC AAA ACC TGT GAG ATC TCC AGC GCT TAT GGA GTT CTT AAG GAA GAT GAG 450 L
L
A
D
K
T
C
E
I
S
S
A
Y
G
V
L
K
E
D
E
120
GGA GTG GCA TTC AGA GGA CTG TTT ATA ATT GAT GGA AAG GGA AAC CTG CGA CAG ATC ACA 510 G
V
A
F
R
G
L
F
I
I
D
G
K
G
N
L
R
Q
I
T
140
GTG AAT GAT ATG CCC GTG GGT CGA TCA GTT GAC GAA ACC TTG AGA CTA GTT CAG GCT TTC 570 N
D
M
P
V
G
R
S
V
D
TE D
V
E
T
L
R
L
V
Q
A
F
160
CAG TTC ACA GAT AAG CAT GGA GAA GTC TGT CCA GCT AAT TGG AAG CCT GGT TCC GAC ACG 630 Q
F
T
D
K
H
G
E
V
*
C
P
A
N
W
K
P
G
S
D
T
180
ATG AAG CCC AGC CCT AAA GAA AGC CAG AGC TAT TTC AAG GCC CAT CAC TAA TTA ATT ATA 690 K
P
S
P
K
E
S
EP
M
Q
S
Y
F
K
A
H
H
ATC AAA TGT CGT TTA CAG GAT AAG CTT ATG TCC CAT GTG CAT CTC TCA CTA CCA TGT GCT 750
AC C
ACT AGG TCT TAT TTT CTC TGG GTA TTC AAA TGG TTT TCA CAA ATT TAG GTA CTT TAC ATG 810 CTG TCT CTC AGA TAC TTC AGT TAG AGA TTT GAG AAC ATA TTC TAA TTT AAG TGA TAC ATG 870 TGT GCA TTA TTA TTG CAC AAG AAT TTT TCA CTT GTT TTC AGC AGT AAT TGT GTA AGC CAC 930 CAG CAC AGA TGA GGG AAT AAT CTC GTT AAA ATA ATT TCC ATC AGG TAT ATT AGC TTT CAG 990 GTC TGT TAC AAT TTT CGT CTG TTG CAA TAA TTT GGA TAT CTT TTT ACA TAC TCT TCA CCT 1050 TCG GTG AAG TCT TTA TCT CGT ATT TTG CCG TAT TTT GTT CAT GAT ATT ACC CTT TTG TTC 1110 ACT TTA TTG TTA GAT TTT GCA TTA A AT ATA AAT AA AGG TAG TTG CTA CCC AGT TTT TTT TGT GAG 1170 TAC TGT AAA ATC AGG CTG TGA TGT TTG TCT TGT GTT TGA TAT TGT GCC ATA AAA AAA AAA 1230 AAA AAA AAA AAA AAA A
1246
Fig. 1 Nucleotide and deduced amino acid sequence of Prx cDNA gene from Cristaria plicata
ACCEPTED MANUSCRIPT Note: Underline shows the start codon (ATG), the stop codon (TGA) and putative polyadenylation
signals
(AATAAA).
Conserved
signature
sequences
“FYPDFTFVCPTEI” and “GEVCPA” are shaded by gray. The asterisk showed N-
AC C
EP
TE D
M AN U
SC
RI PT
terminal conserved cysteine.
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 2 ClustalW multiple alignment analysis of CpPrx with known Prxs Note: Amino acid residues shared by all the sequence are denoted by an asterisk. Similar amino acids residues are denoted by (* or :). Solid boxes indicate predicted the Prx signature motif 1 and 2 sequences. Dashed box indicates that the conservative
TE D
motifs are associated with sensitive of typical 2-Cys Prxs for H2O2 concentration. Two conserved cysteines in each motif are highlighted with star (★) on top of each residue. GenBank accession numbers for the protein sequences are as follows. H. discus Prx2: ABO26635.1. C. plicata TPx: ADM88874.1. H. sapiens Prx1-4: AAH03022,
EP
NP_859048A,
AAH02685,
AAH16770.
M.
musculus
Prx1-4:
AAH86648, AAH86783, EDL01849, AAH03349. X. laevis Prx1-4: NP_001085485,
AC C
NP_001085414, NP_001086130, NP_001085918. D. rerio Prx1-4: NP_001013489, AAH76347, AAH92846, NP_001082894.
ACCEPTED MANUSCRIPT 52 M. musculus Prx1 100 R. norvegicus Prx1 H. sapiens Prx1 97
Prx1
X. laevis Prx1
100
X. tropicalis Prx1
48
X. laevis Prx2
100 50
H. sapiens Prx2
100
26
RI PT
X. tropicalis Prx2
100
Prx2
M. musculus Prx2
R. norvegicus Prx2 B. ignites Prx
58 61 52
▲ C. plicata Prx
M AN U
99
Prx
H. discus Prx
91 88
Typical 2-Cys
SC
L. vannamei Prx
P. fucata Prx
99 X. laevis Prx4 100 X. tropicalis Prx4 Prx4
H. sapiens Prx4
EP
TE D
91 M. musculus Prx4 99 R. norvegicus Prx4
AC C
1-Cys Prx
atypical 2-Cys Prx
97 X. laevis Prx3 X. tropicalis Prx3 100 H. sapiens Prx3
Prx3
97 M. musculus Prx3 93 R. norvegicus Prx3 100 X. laevis Prx6 X. tropicalis Prx6
100 99
H. sapiens Prx6 M. musculus Prx6
92 100
R. norvegicus Prx6 X. laevis Prx5 X. tropicalis Prx5
100 99
H. sapiens Prx5 M. musculus Prx5
67
0.2
Prx6
R. norvegicus Prx5
Prx5
ACCEPTED MANUSCRIPT Fig. 3 Neighbor-joining phylogenetic tree of Prx amino acid sequences from ten species animals Note: GenBank accession numbers for the protein sequences are as follows. M. musculus Prx5-6: AAH08174, AAH13489. R. norvegicus Prx1-6: AAH88118, AAH58481, EDL94585, AAH59122, AAH78771, NP_446028. H. sapiens Prx5-6:
tropicalis
Prx1-6:
AAH84184,
AAH61276,
RI PT
AAI10984, AAH53550. X. laevis Prx5-6: NP_001085580, NP_001084316. X. NP_001025608,
AAH76692,
NP_001106525, NP_989102. P. fucata Prx, ADC35419. B. ignitus, ACP44066. L. vannamei, ACX53642. Other abbreviations and accession numbers are the same as in
AC C
EP
TE D
M AN U
SC
Fig. 2.
Fig. 4 Tissueexpression of Prx gene in different tissues of Cristaria plicata
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 5 The expression of Prx mRNA in hemocytes (A) and hepatopancreas (B) from Cristaria plicata after challenge by Real-time quantitative PCR Note: All values represent the mean ± S.D. (n=4). CpPrx transcript levels were normalized by injecting 0.1 mL PBS at 0 hour. Asterisk (*) are significantly different (* and **represent p < 0.05 and p < 0.01; respectively, t-test).
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
TE D
Fig. 6 Expression of recombinant plasmid in Escherichia. coli by SDS-PAGE Note: M: molecular weight marker, 1. Uninduced plasmid, 2. Plasmid with IPTG induced 2 h, 3. Plasmid with IPTG induced 4 hr, 4. Plasmid with IPTG induced 6 h, 5.
AC C
EP
Plasmid with IPTG induced 8 h.
2
ACCEPTED MANUSCRIPT
1 2
A
B
3 4
A
SC
B
RI PT
C
M AN U
C
Fig. 7 The cell tolerance of DE3 (A), DE3-pET-32 (B) and DE3-pET-32-CpPrx (C) to H2O2
Note: 1. Culture medium without H2O2, 2, 3, 4. Culture medium with 0.4, 0.8 and 1.6
AC C
EP
TE D
mmoL/L H2O2.
ACCEPTED MANUSCRIPT
Highlights The full cDNA sequences of CpPrx were cloned. The transcripts of CpPrx were
AC C
EP
TE D
M AN U
SC
RI PT
up-regulated after stimulation. The recombinant had antioxidant activity.