characterization and expression of the schistosoma japonicum ...

J. Parasitol., 99(1), 2013, pp. 68–76 Ó American Society of Parasitologists 2013

CHARACTERIZATION AND EXPRESSION OF THE SCHISTOSOMA JAPONICUM THIOREDOXIN PEROXIDASE-2 GENE Yang Hong, Yanhui Han, Zhiqiang Fu, Hongxiao Han, Chunhui Qiu, Min Zhang, Jianmei Yang, Yaojun Shi, Xiangrui Li*, and Jiaojiao Lin† Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai, 200241, P.R. China. e-mail: [email protected] ABSTRACT:

We analyzed proteins that were differentially expressed by 10-day-old schistosomula from 3 different hosts and determined that a functional thioredoxin peroxidase-2 gene has an important antioxidant role in Schistosoma japonicum, which we investigated further. A full-length cDNA encoding the S. japonicum thioredoxin peroxidase-2 (SjTPx-2) had an open reading frame of 681 bp that encoded 226 amino acids with a signal peptide of 24 amino acids. A cDNA encoding SjTPx-2 without the signal peptide sequence was isolated from 42-day-old schistosome cDNAs. Real-time quantitative RT-PCR analysis revealed that SjTPx-2 was upregulated in 7- and 13-day-old schistosomes, while the expression level in females was around 2-fold higher than that in male worms at 42 days. SjTPx was subcloned into pET28a(þ) and expressed as both inclusion bodies and supernatant in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjTPx-2 (rSjTPx-2) was immunogenic. The purified recombinant protein could form disulfide-bonded dimers and it had peroxidase activity in vitro. An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjTPx-2 could induce 31.2% and 34.0% reductions in the numbers of worms and eggs in the liver, respectively. This study suggests that SjTPx-2 may be an important antioxidative enzyme in scavenging ROS, and it may be a potential vaccine candidate or new drug target for schistosomiasis.

balance systems to protect themselves from the ROS generated by host responses and their own oxygen metabolism (Wang et al., 2006; Suttiprapa et al., 2008). Cysteine-based peroxidases, known as peroxiredoxins (Prx) or, more commonly, thioredoxin peroxidase (TPx), are important thiol-specific antioxidant proteins that prevent oxidative damage by a variety of peroxides and alkyl hydroperoxides in cells (Chae et al., 1994; Rhee et al., 1999; Pushpamali et al., 2008). TPx was first reported in Saccharomyces cerevisiae, but it was later found to be ubiquitous in prokaryotes and eukaryotes (Hofmann et al., 2002; Wood et al., 2003). Superoxide dismutase (SOD) and glutathione peroxidase (GPx) have been detected in a number of parasites, in the absence of catalase or GPx activity (McGonigle et al., 1998). TPx was considered to be the principal enzyme for hydrogen peroxide removal in these helminth parasites. Molecular cloning and characterization of SmTPx-1 (Kwatia et al., 2000); SmTPx-2 and SmTPx-3 (Sayed and Williams, 2004); and SjTPx-1, SjTPx-2, and SjTPx-3 (Kumagai et al., 2006) have recently been identified. TPx-1 and TPx-2 gene expressions in S. japonicum eggs, cercariae, and adult worms were analyzed by RT-PCR. Gene expression was distributed in different schistosome tissues and organs, where they protect the parasite against ROS from a different source (Kumagai et al., 2006). However, the biochemical properties of these S. japonicum TPx types were not characterized in these reports. In the present study, we cloned and expressed the SjTPx-2 gene that was differentially expressed in 10-day-old schistosomula from schistosome-susceptible and -resistant hosts (Hong et al., 2011) before characterizing its biochemical properties in vitro. We also analyzed SjTPx-2 expression at different life stages using real-time quantitative RT-PCR and evaluated the potential efficacy of recombinant SjTPx-2 as a vaccine candidate against schistosome challenge.

Schistosomiasis is one of the world’s most prevalent tropical parasitic diseases and remains one of the major public health problems, with an estimated 200 million infected individuals in 74 endemic countries. Schistosomiasis is caused by blooddwelling trematodes. Schistosoma japonicum is 1 of the 3 major species of blood fluke and is distributed in the central lakes and the Yangtze River in China, as well as other southeast Asian regions and islands (Xianyi et al., 2005; Utzinger et al., 2009). The current strategy for schistosomiasis control is aimed at reducing morbidity, which involves treatment with praziquantel, the only drug that is widely used for clinical treatment and chemotherapy (WHO, 2002). However, a prophylactic vaccine that is used alone or in combination with anthelmintic drugs may be more effective for the long-term sustainable control of schistosomiasis (Bergquist et al., 2005). The identification of target proteins for use in the development of vaccines or new drugs would contribute enormously to the control of this disease. Reactive oxygen species (ROS) generally exist in normal metabolic living organisms where they are the products of a variety of physiological processes, or they may result from exposure to natural sources of oxidative stress caused by environmental stimuli, toxic heavy metals, ionizing irradiation, heat shock, immune responses, and inflammation. ROS mass accumulation can exert a toxic effect on biomolecules, leading to DNA base modifications, DNA strand breaks, protein oxidation, lipid peroxidation, and subsequent serious oxidative damage of cells (Yu, 1994; Nikapitiya et al., 2009). ROS are mainly produced in 2 conditions in schistosomes, i.e., erythrocyte ingestion by worms produces ROS and host effector cell adhesion to worms also releases ROS. The latter are also known to be highly toxic to schistosomes, and the hydrogen peroxide generated by cells can kill schistosomula of Schistosoma mansoni in vitro (Kazura et al., 1981). Parasites have developed antioxidant defenses and redox

MATERIALS AND METHODS

Received 16 January 2012; revised 22 June 2012; accepted 27 June 2012. * College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, P.R. China. † To whom correspondence should be addressed. DOI: 10.1645/GE-3096.1

Parasites and animals Schistosomes of the Anhui strain of S. japonicum at 7, 13, 21, 28, 35, and 42 days were obtained by perfusion of artificially infected New Zealand rabbits. We manually separated 42-day-old males and females. 68

HONG ET AL.—EXPRESSION OF S. JAPONICUM PEROXIDASE GENE

Cercariae were collected, and eggs were isolated from the livers of the infected mice using previously described methods (Cheng et al., 2005; Yang et al., 2009). BALB/c mice (male, ~25 g each) and New Zealand rabbits (male, ~2.5 kg each) were purchased from Shanghai Laboratory Animal Center at the Chinese Academy of Sciences (Shanghai, China). All animal care procedures were conducted according to the principles of the Shanghai Veterinary Research Institute for the Care and Use of Laboratory Animals. Sequence characterization and phylogenetic analysis of SjTPx-2 The S. japonicum EST corresponding to TPx (accession number AY813893) was identified in previous proteomic studies of S. japonicum that were conducted in our laboratory (data not shown). Sequence identities of SjTPx-2 with known sequences were analyzed using the DNAStar program. Multiple amino acid sequence alignments were generated using the ClustalX 1.83 program. In the phylogenetic analysis, the deduced SjTPx-2 amino acid sequence was determined using the Neighbor-Joining method in the MEGA 5.05 program. The signal peptide was predicted using the SignalP worldwide server (http:// www.cbs.dtu.dk/services/SignalP/). The domains were evaluated using a motif scan program (http://myhits.isb-sib.ch/cgi-bin/motif_scan). The subcellular location prediction was performed using the WoLF PSORT worldwide server (http://wolfpsort.org/). Preparation of mRNA and real-time quantitative RT-PCR Total RNAs were extracted from schistosomes and all RNA samples were initially treated with RNase-free DNase I (TaKaRa, Shiga, Japan) to remove genomic DNA contaminants. mRNAs were purified using an RNeasy mini Kit (Qiagen, Dusseldorf, Germany) and quantified by spectrophotometry (Eppendorf, Hamburg, Germany). Reverse transcription was performed with 5 lg of mRNA from each stage, random hexamer primers, and Superscript III reverse transcriptase (Invitrogen, Carlsbad, California), according to standard protocols. Primers for real-time PCR were designed using the primer design tool Beacon Designer 7.0, which generated the recommended amplicons measuring 100–200 bp. The primers of SjTPx (forward 5 0 -GCT GGT GGA TTA GGA CAA ATG-3 0 ; and reverse 5 0 -AGA AGA CGA ATC GCC TCA TC-3 0 ) amplified a product of 200 bp. NADH-ubiquinone reductase of S. japonicum (forward 5 0 -CGA GGA CCT AAC AGC AGA GG-3 0 ; and reverse 5 0 -TCC GAA CGA ACT TTG AAT CC-3 0 , product size 174 bp) was selected as the housekeeping gene that was used as an internal standard (Gobert et al., 2009; Hong et al., 2010). Real-time quantitative PCR was conducted on triplicate samples using SYBR Green tag (TaKaRa) in a Rotor-Gene 3000A Dual Channel Multiplexing System (Corbett Research, Sydney, Australia). Negative controls with no template were included in each PCR run. Expression and purification of recombinant SjTPx-2 Primers of SjTPx-2 were designed based on its nucleotide sequence using the corresponding restriction enzyme sites of BamH I and Sal I (underlined) at the N-terminus and C-terminus, respectively. A cDNA fragment encoding SjTPx-2 was amplified by PCR using the forward primer 5 0 -GAG GAT CCT CAG AGT CAG TTA ATC GG-3 0 and the reverse primer 5 0 -GGG TCG ACT CAG TTT ACA GAG GAA AAG3 0 . The SjTPx-2 cDNA fragment was then ligated into the expression vector pET28a(þ) (Novagen, Darmstadt, Germany). The ligation products were transformed into Escherichia coli BL21 (DE3) cells (Invitrogen), and recombinant clones were obtained by antibiotic selection. The recombinant proteins were overexpressed in the presence of isopropyl-b-d-thiogalactopyranoside (IPTG). Transformed cells were grown in Luria broth plus kanamycin (1 mg ml1) at 30 C until OD600 ¼ 0.6, and IPTG was added to the culture at a final concentration of 1 mM. After 5–6 hr of induction, cells were harvested and the expression of recombinant protein was analyzed by SDS-PAGE. The histidine-tagged fusion recombinant protein was then purified from E. coli lysates by metal affinity chromatography using HisBindtResin Chromatography (Novagen). Purified SjTPx-2 was dialyzed against phosphate-buffered saline (PBS). The concentrations of the purified proteins were determined by the Bradford method using bovine serum albumin (BSA) as the standard (Bradford 1976), and recombinant SjTPx-2 was stored at 80 C until use. The purified rSjTPx-2 was sent to Shanghai GeneCore BioTechnologies Co. Ltd.

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(Shanghai, China) for MALDI-TOF-MS peptide mass fingerprint (PMF) analysis. PMF data were submitted to Mascot for identification against the NCBInr database using default parameters. Antioxidant activity and peroxidase assays To measure the antioxidant activity of SjTPx-2, a DNA cleavage (nicking) assay was performed to assess the protection from DNA damage by a metal-catalyzed oxidation (MCO) system containing metals (Fe3þ), O2, and dithiothreitol (DTT) as the electron donor. Assays were performed with previously described modifications (Kim et al., 1988; Li et al., 2004). Briefly, a reaction mixture (50 ll) containing 33 lM FeCl3, 3.3 mM DTT, and concentrations of purified SjTPx-2 fusion proteins ranging from 6.25 to 400 lg ml1 was incubated at 37 C for 2 hr. Then 300 ng of pUC19 supercoiled plasmid DNA was added to each reaction mixture before incubating at 37 C for 2.5 hr. Finally, the mixtures were examined by agarose gel electrophoresis containing ethidium bromide to detect evidence of DNA nicking. Purified recombinant S. japonicum proteasome subunit alpha type 5 (rSjPSMA5) was included as a control protein. The ability of the recombinant SjTPx-2 to remove hydrogen peroxide was measured essentially according to a previously described method (Lim et al., 1993). A reaction mixture (100 ll) containing different concentrations of purified recombinant SjTPx-2 and 50 mM Tris-HCl (pH 8.0) was incubated at 37 C for 30 min. After incubation, H2O2 was added to a final concentration of 50 lM before incubating for an additional 30 min. Then, 0.9 ml (8% v/v) trichloroacetic acid was added to stop the reaction, and the protein was removed by centrifugation. Finally, 0.2 ml ferrous ammonium sulfate (10 mM) and 0.1 ml potassium thiocyanate (2.5 M) were added and the absorbance of the mixture was measured at 480 nm. The amount of H2O2 was determined based on a known amount of H2O2 that was used as the standard. Vaccination and immune response assays The 206 adjuvant (Seppic, Paris, France) was used according to the manufacturer’s instructions. The BALB/c mice were randomly allocated into 3 groups with 10 mice per group. The 3 groups were injected subcutaneously (SC) 3 times at 2-wk intervals with rSjTPx-2 emulsified with 206 adjuvant (20 lg 100 ll1 mouse1), 206 adjuvant in PBS (100 ll mouse1), or PBS only (100 ll mouse1). Before the first vaccination and 10 days after each vaccination, sera were collected from the mice in each group by retro-orbital bleeding. Specific IgG antibodies against SjTPx-2 were detected by ELISA in the serum of individual mice. We coated 96-well microtiter plates (Costar, Tewksbury, Massachusetts) overnight at 4 C with 100 ll soluble rSjTPx-2 (10 lg ml1) diluted in carbonate–bicarbonate buffer (pH 9.6). The plates were washed 3 times using PBST (0.05% Tween 20 in PBS), then blocked with 3% BSA in PBST for 1 hr at 37 C. After washing 3 times, all test sera were diluted 1:100 with PBST and incubated at 100 ll well1 for 2 hr at 37 C. The plates were then washed 3 times, and goat anti-mouse IgG conjugated to horseradish peroxidase (Sigma, St. Louis, Missouri) diluted 1:2,500 in PBST was added to the wells (100 ll well1). After 1 hr incubation at 37 C, the plates were washed 3 times and 100 ll 3,3 0 5,5 0 -tetramethyl benzidine dihydrochloride was added to each well. The reaction was incubated for 10 min at 37 C in the dark and stopped using 2 M sulfuric acid (50 ll well1). Optical densities were read at 450 nm using a microplate reader (BioTek, Winooski, Vermont). Mice were killed 2 wk after the last vaccination, and their spleens were harvested aseptically. The splenocytes were enriched by passage through a nylon wool column before being washed twice and adjusted to a concentration of 1 3 107 ml1. Each cell suspension was incubated with PE-conjugated rat anti-mouse CD4 antibody, APC-conjugated rat anti-mouse CD8, and FITC-conjugated rat anti-mouse CD3 antibody (eBioscience, San Diego, California) for 40 min at room temperature in the dark. After 2 washes with PBS, the cell suspensions were resuspended and analyzed by flow cytometry (FCM). Western blotting Purified rSjTPx-2 or protein extracts from S. japonicum worms at 42 days were subjected to 12% SDS-PAGE and then transferred electrophoretically onto a nitrocellulose membrane (GE Healthcare,

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THE JOURNAL OF PARASITOLOGY, VOL. 99, NO. 1, FEBRUARY 2013

FIGURE 1. Comparison of the protein sequence of SjTPx-2 with that found in other species. ClustalX alignment of the derived amino acid sequences of TPx-2 from Schistosoma japonicum (AAW25625), Schistosoma mansoni (AAG15508), Clonorchis sinensis (AEK86199), Onchocerca ochengi (AAC77922), Danio rerio (AAH76347), and Haliotis discus discus (ABO26635). Two conserved domains of 2-Cys Prx (FYPLDFTFVCPTE and GEVCPA) are boxed and the 2 conserved cysteines in each motif are highlighted with an asterisk (*) on top of each sequence. The predicted signal peptide of SjTPx-2 is underlined at the N-terminal.

Pittsburgh, Pennsylvania) in transfer buffer (25 mM Tris, 192 mM glycine, and 20% v/v methanol, pH 8.4) at 260 mA for 2 hr at 4 C. The membrane was incubated with 5% nonfat dry milk in PBST overnight at 4 C. Subsequently, the membrane was incubated in a 1:100 dilution of anti-SjTPx-2 mice serum, anti-SWAP (an antigen preparation from adult S. japonicum worms) rabbit serum, and normal mouse or rabbit serum as the primary antibody in a 1:2,500 dilution of secondary goat anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase in blocking buffer for 1 hr at 37 C. PBST was used to wash the membrane 3 times for 10 min each wash between the steps. Finally, diaminobenzidine was added, according to the manufacturer’s instructions. Evaluation of immune protection against S. japonicum challenge infection All mice were percutaneously infected with 40 6 1 viable cercaria via a wet-glass lid 2 wk after the last vaccination. To evaluate the efficacy of immunization, worms were obtained by perfusion of infected BALB/c mice from a hepatic portal system 42 days after challenge, and they were then counted. The worm reduction rates were calculated using the formula: percentage reduction in worm burden ¼ (mean worm burden of control group mean worm burden of vaccinated group)/mean worm burden of control group 3 100%. The number of eggs in the liver was also estimated using the following method. A sample of 1 g of liver tissue from each infected mouse was homogenized in 10 ml PBS, and 10 ml of 10% NaOH was added before each mixture was incubated at 56 C for 1 hr. An average of 3 counts per 20 ll thoroughly mixed product was used to determine the number of eggs. The count was converted to eggs per gram (EPG). The number of eggs used to determine the liver reduction rate was calculated as follows: percentage reduction in liver egg count ¼ (mean EPG from control group mean EPG from vaccinated group)/mean EPG from control group 3 100%. Statistical analysis Analysis of variance and Duncan’s multiple range tests were performed using SPSS 11.5 software. P , 0.05 was considered statistically significant.

RESULTS Cloning and molecular characterization of SjTPx-2 The full-length sequence cDNA encoding SjTPx-2 was obtained by RT-PCR amplification from the mRNA of 42day-old schistosomes using specific oligonucleotides that were designed based on the EST sequence (GenBank accession No. AY813893.1). The resulting full-length cDNA contained an open reading frame of 681 bp, which encoded a protein of 226 amino acid residues with a predicted signal peptide of 24 amino acids, a calculated molecular mass of approximately 25.1 kDa, and an isoelectric point of 6.2. Two highly conserved domains (the N-terminal FYPLDFTFVCPTE and C-terminal GEVCPA motifs) containing the VCP motifs of 2-Cys peroxiredoxin active sites were present in SjTPx-2. BlastP comparisons of the SjTPx with GenBank sequences showed that the TPx-2 of S. mansoni shared the highest identity (90% identity). TPx-2 from the related trematode, Clonorchis sinensis, shared 76% identity with SjTPx-2. TPx-2 from other species shared approximately 60% identity with SjTPx-2 (Fig. 1). The phylogenetic analyses of TPx-2 are provided in Figure 2, showing that SjTPx-2 was most closely related to SmTPx-2, while the next closest relation was the TPx-2 of C. sinensis. Real-time quantitative RT-PCR assay The expression of SjTPx-2 at the transcript level was evaluated in S. japonicum for eggs and cercariae, and at 7, 13, 21, 28, 35, and 42 days using real-time quantitative RTPCR analysis with NADH-ubiquinone reductase as the housekeeping gene (Fig. 3). SjTPx-2 mRNA was found in all investigated stages. The level was approximately 2–3 times


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FIGURE 2. Phylogenetic tree analysis of SjTPx-2 and its homologues (the accession numbers of the other members are cited in the legend for Fig. 1).

higher in female worms compared with males and higher at 7, 13, and 42 days compared with other stages. Expression, purification, MALDI-TOF-MS identification and antigenicity analysis of rSjTPx-2 The gene was cloned into the pET28a(þ) expression vector and expressed in E. coli BL21 (DE3). A recombinant protein containing a His tag was successfully expressed with the expected molecular weight of 27 kDa (Fig. 4). The bacteria induced with IPTG were sonicated briefly, and the lysate was separated into soluble and insoluble fractions. The supernatant contained the majority of the recombinant protein. The protein was purified by affinity chromatography using His-binding columns under native conditions, with dialysis against PBS. Purified rSjTPx-2 was analyzed by SDS-PAGE, which detected the expected band and another band with an apparent molecular mass of 54 kDa. The larger band was cut from the gel and confirmed by mass spectrometry (Fig. 5; Table I). rSjTPx-2 and SjTPx-2 in native worms was analyzed by Western blotting. Two positive bands with molecular weights approximately 27 kDa and 54 kDa were detected in the rSjTPx2 lane, while a positive band around 23 kDa was detected in the adult worm antigen lane, as shown in Figure 6. Normal mouse and rabbit sera were used as the negative control, and no bands were observed (data not shown).

Supercoiled DNA protection activity of rSjTPx-2 and peroxidase activity The DNA protection activity of rSjTPx-2 was determined using a DNA nicking assay. Hydroxyl radicals produced by the MCO system cause nicking of pUC19 supercoiled plasmid DNA. The pUC19 supercoiled plasmid DNA in the MCO system was measured based on the shift in gel mobility after the treatment. The results showed that separately incubated MCO components did not cause any damage to the pUC19 supercoiled plasmid. Nicking of the plasmid in the MCO system was prevented in the presence of rSjTPx-2, but not with the control rSjPSMA5 under the same conditions. The protective activity of rSjTPx-2 was dosedependent because reducing the level of this protein in the reaction decreased the plasmid protection from oxidative cleavage. The addition of purified rSjTPx-2 initially protected the nicking of pUC19 at a concentration of 12.5 lg ml1. In general, it was found that almost 100% of the supercoiled form of pUC19 DNA was protected by rSjTPx-2 at a concentration of 200 lg ml1 in the MCO assay (Fig. 7). To determine the antioxidant properties of purified rSjTPx-2, peroxidase activity was assayed using H2O2 as the substrate. The reduction of peroxide was monitored based on the remaining H2O2 in the ferri-thiocyanate system. In the H2O2 reduction assay, rSjTPx-2 showed the ability to remove H2O2. When the concentration of rSjTPx-2 was 10 lg ml1, it removed almost 20%

FIGURE 3. Stage and gender differential expression of SjTPx-2 in Schistosoma japonicum using real-time RTPCR. e and c represent eggs and cercariae, respectively; 7d, 13d, 21d, 28d, and 35d represent worms at 7 days, 13 days, 21 days, 28 days, and 35 days, respectively, whereas 42d (f) is for adult female worms at 42 days, and 42d (m) for adult male worms at 42 days. Expression of the gene encoding NADH-ubiquinone reductase was used as a control.

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Detection of SjTPx-2 specific IgG antibody and evaluation of cell-mediated immunity The level of specific anti-rSjTPx-2 IgG antibody in the sera of rSjTPx-2 immunized and control mice was detected by ELISA (Fig. 9). After the first immunization with rSjTPx-2, the amount of specific IgG antibody was significantly increased in the vaccination group. This antibody remained at a high level until the mice were killed. No significant differences were shown in specific antibody levels compared with mice that received 206 adjuvant or PBS only. This showed that rSjTPx-2 could stimulate a strong antibody response (P , 0.01). The results of the assay of the cell-mediated immune responses in all mice in each group are shown in Table III. The proportions of different subsets of splenocytes in immunized mice were detected by FCM 2 wk after the last immunization, and no þ significant changes in CDþ 4 and CD8 cells were observed among the 3 groups of mice. FIGURE 4. SDS-PAGE (12%) analysis of the expression of recombinant protein SjTPx-2. Lanes 1 and 2, total extracts from a clone after and before induction with 1 mM IPTG. Lanes 3 and 4, total extracts of pET28a(þ) after and before induction with 1 mM IPTG. Lanes 5 and 6, supernatant and inclusion bodies of pET28a(þ)-SjTPx-2 after lysis, respectively. Lane 7, rSjTPx-2 purified using Ni2þ-charged column chromatography and after dialysis.

of the H2O2 in the assay system. As the concentration of rSjTPx-2 was increased, more H2O2 was removed from the system. When the concentration of rSjTPx-2 reached 50 lg ml1, it removed almost 40% of the H2O2 (Fig. 8). However, there was no H2O2 removal activity with rSjPSMA5 in the control reaction (data not shown). This indicated that rSjTPx-2 possessed peroxidase activity. Protective immune efficacy induced by rSjTPx-2 To evaluate the protective effect induced by rSjTPx-2, the worm burden and egg counts in the livers of each group were calculated, as shown in Table II. Mice immunized with rSjTPx-2 exhibited a 31.2% reduction in the worm burden (P , 0.05) and a 34.0% reduction in the egg count (P , 0.05) compared with the blank control. There was no significant difference (P . 0.05) in the worm burden and liver EPG between the blank and the adjuvant control group. Thus, rSjTPx-2 induced partial protection against challenge with S. japonicum in mice.

FIGURE 5. Peptide mass fingerprinting map of recombinant SjTPx-2 dimers.

DISCUSSION In the present study, we performed molecular cloning and characterization of SjTPx-2. Bioinformatic analysis revealed that SjTPx-2 was most closely related to SmTPx-2, and it exhibited 2 conserved hallmark features of the 2-Cys Prx family. Furthermore, it was predicted that SjTPx-2 has an N-terminal signal peptide that is not found in this protein in other species we investigated. This complete sequence of SjTPx-2 was also the first reported in S. japonicum, and this finding will help us recognize it in schistosomes or other species. We also analyzed the subcellular location of SjTPx-2, and the result demonstrated that SjTPx-2 could be secreted from parasite cells and it produced a marked effect. The results of the real-time quantitative RT-PCR analysis showed that the SjTPx-2 transcript was expressed at all stages investigated. This agreed with a previous study that also demonstrated constitutive SjTPx-2 expression in eggs, miracidia, cercariae, and adult worms using the RT-PCR technique. But they did not investigate the change of mRNA level in these stages of this gene (Kumagai et al., 2006). Here, we found that the SjTPx-2 transcript was significantly upregulated in schistosomes at 7, 13, and 42 days in female worms. Comparison of the level of expression by gender in the adult worms at 42 days revealed that the mRNA level of SjTPx-2 in female worms was over 2-fold


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TABLE I. Identification of SjTPx-2 by MALDI-TOF-MS analyses and database search.

Species

Accession no.

Mascot score

Sequence coverage (%)

Theoretical Mr/pI

Total ion score

Schistosoma japonicum

gij56754885

319

42.5

25360.9/6.0

276

higher than that in male worms. This showed that the upregulation of SjTPx-2 mRNA occurred in the early stages of schistosomes infecting hosts and during female worm egg-laying. The schistosomulum is the early stage of schistosomes where the parasite encounters a new living environment in the final host; it is a critical stage for the development and maturation of schisto-

FIGURE 6. Western blotting analysis of rSjTPx-2 and protein extracts from Schistosoma japonicum. M, marker; (1) Purified rSjTPx-2 was probed with the serum anti-SWAP rabbit serum; (2) Purified rSjTPx-2 was probed with the serum from BALB/c mice immunized with rSjTPx-2; (3) Protein extracts from S. japonicum worms at 42 days were probed with the serum from BALB/c mice immunized with rSjTPx-2.

Matched peptides LLDAFIFFEK QITVNDRPVGR GMFLIDPNGVLR AYGVLDEEEGHAFR GCQVIACSTDSIYSHLAWTK TNMLLPNQPAPDFEGTAVIGTEFHPITLR

somes. They are exposed to ROS produced by host immune cells and in the metabolized erythrocytes consumed by the adult worms (Loverde, 1998). Thus, they have to express more antioxidant enzymes to resist the ROS produced by the host and themselves, so upregulation of the SjTPx-2 gene is required to meet the physiological needs of the parasite. Female schistosomes at 42 days produced many eggs, and this might explain why the mRNA level of SjTPx-2 was upregulated at this stage. The SDS-PAGE analysis and the MALDI-TOF-MS identification showed that rSjTPx-2 could be expressed in E. coli as a soluble fusion protein and that purified rSjTPx-2 could exist in the form of a monomer and a dimer at the same time. Previous structural and mechanistic studies have shown that 2-Cys Prxs are divided into 2 classes known as the ‘typical’ and ‘atypical’ 2-Cys Prxs (Wood et al., 2003). Typical 2-Cys Prxs are obligate homodimers containing 2 identical active sites, whereas atypical 2-Cys Prxs are functionally monomeric (Suttiprapa et al., 2008). Recombinant SjTPx-2 can form dimers under native conditions, which supports its classification as a typical 2-Cys Prx. Western blotting analysis revealed that rSjTPx-2 had good immunogenicity and that it could be recognized by the sera from BALB/c mice immunized with purified rSjTPx-2. Hydroxyl radicals produced by the MCO system inflict damage on proteins, lipids, and DNA, which leads to modified bases and strand breaks. The antioxidant activity of TPx proteins from other helminths has been described, and they have similar activity in protecting supercoiled plasmid DNA (Klimowski et al., 1997; Ghosh et al., 1998; Lu et al., 1998; Kumagai et al., 2006; Suttiprapa et al., 2008). In our study, rSjTPx-2 showed antioxidant activity by protecting pUC19 supercoiled DNA from oxidative nicking via hydroxyl radicals generated from the MCO system. This suggests that rSjTPx-2 could protect the schistosome nucleic acid from oxidative damage by ROS produced by host and worms. Recombinant SjTPx-2 also possessed peroxidase activity, and it could interact with H2O2 to reduce its content in the reaction system. In previous studies, Cys79 is directly responsible for the peroxidase activity of TPxs, and it accepts

FIGURE 7. Protection of supercoiled DNA cleavage by rSjTPx-2 in a MCO. (1) pUC19 plasmid; (2) pUC19 plasmid þ FeCl3; (3) pUC19 plasmid þ DTT; (4) pUC19 plasmid þ FeCl3 þ DTT; (5–11) pUC19 plasmid þ FeCl3 þ DTT þ recombinant SjTPx-2 (400, 200, 100, 50, 25, 12.5, 6.25 lg ml1, respectively); (12) pUC19 plasmid þ FeCl3 þ DTT þ 200 lg ml1 recombinant SjPSMA5. NF, nicked form of pUC19 plasmid; SF, supercoiled form of pUC19 plasmid.

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FIGURE 8. Peroxidase activity of rSjTPx-2 proteins. Peroxidase activity was assayed using the purified rSjTPx-2 fusion protein and H2O2 (50 lM) in a concentration dependent manner. The remaining H2O2 was measured spectrophotometrically and calculated as the percentage of H2O2 removal by rSjTPx-2. Values are the means 6SE from 3 similar experiments. The error bars represent the SE (n ¼ 3).

the hydrogen bond from Arg155 and donates it to the carboxylate of Glu82 (Montemartini et al., 1999). Arg155 is predicted to stabilize the ionized state of Cys79 and increase its activity (Alphey et al., 2000). Cys79 and Cys200 can form a disulfide bond, which is an intermediate product of the peroxidation reaction catalyzed by Cys79 (Hirotsu et al., 1999). These important amino acid residues found in SjTPx-2 explain the antioxidant activities of this protein. Because of its important antioxidant role in schistosomes, the rSjTPx-2 protein was used to immunize BALB/c mice to evaluate its protective efficacy against S. japonicum infection. The results showed that the number of adult worms and liver eggs was significantly lower in mice that were immunized subcutaneously with rSjTPx-2 compared with those in the other 2 control groups (P , 0.05). ELISA assay showed that the purified rSjTPx-2 could elicit a strong antibody response and that mice vaccinated with rSjTPx-2 were able to produce high levels of specific IgG antibodies. This high level of IgG antibodies could be maintained for a long time in mice challenged with cercaria. Two weeks after the last vaccination, some of the mice from the 3 groups were killed to evaluate changes in the numbers of T lymphocytes. The þ percentage of CDþ 4 and CD8 cells in mice vaccinated with rSjTPxTABLE II. Comparison of protective effectiveness against Schistosoma japonicum challenge in mice receiving SjTPx-2.

Group

Worm burden*

SjTPx-2 19.6 6 2.3A 206 27.4 6 1.6B adjuvant PBS 28.5 6 2.0B

% Reduction in worm burden

EPG

% Reduction in liver egg count

31.2 3.9

47666.7 6 8089.8A 78400.0 6 9090.5B

34.0 —

—

72200.0 6 5308.1B

—

* Data are expressed as means 6 SE. Each group contained 10 mice. Values with different superscripts in the same column differed significantly (P , 0.05). Values with the same superscripts in the same column did not differ significantly (P . 0.05).

FIGURE 9. Antibody responses specific to rSjTPx-2. Mice were injected subcutaneously with rSjTPx-2, 206 adjuvant, or PBS. Sera were collected and analyzed using ELISA. Each bar represents the mean OD (6SE, n ¼ 10), while the asterisks (*) indicate significantly increased serum antibody titers compared with the PBS control (P , 0.01).

2 were not significantly different compared with the mice in the adjuvant or blank control groups. Humoral immunity and cellular immunity are important for protection against schistosome infection, but in this study we did not investigate the role of cellular immunity. We only speculated that humoral immunity had an important immunoprotective effect against schistosome infection in BALB/c mice. Vaccination with irradiated cercariae could elicit the highest immune protection against schistosomes by now, but it has some restrictions and shortages (Siddiqui et al., 2008). Recombinant vaccine is still considered a defined and safe vaccine, although it does not reach the highest level of immunoprotection. Two laboratories have independently assessed the protective potential of the 6 WHO-designated ‘‘priority antigens’’ (paramyosin, glutathione S-transferase, fatty acid binding 14 kDa protein, IrV-5, triose phosphate isomerase, and Sm23) and reported that none of them provided the stated goal of 40% protection (Siddiqui et al., 2008). Priming with pcDNAI/Amp constructs encoding S. mansoni cytosolic superoxide dismutase (SmCTSOD) and glutathione peroxidase (SmGPX), resulted in varying degrees of protection, i.e., 44–60% and 23–55%, respectively (Shalaby et al., 2003). Recent studies using a DNA vaccination strategy with SmCT-SOD and SmGPX induced a mean of 39% protection and no protection following challenge with adult worms by surgical transfer, respectively (Cook et al., 2004). These results, taken together with our protection result, could provide þ TABLE III. Changes in CDþ 4 and CD8 T cells in splenocytes from immunized BALB/c mice.

Group

CDþ 4 (%)*

CDþ 8 (%)

SjTPx-2 206 adjuvant PBS

62.5 6 0.5A 62.8 6 0.9A 62.8 6 1.2A

31.6 6 0.7A 30.8 6 1.0A 31.8 6 1.0A

* Data are expressed as means 6SE. Each group contained 5 mice. Values with different superscripts in the same column differed significantly (P , 0.05). Values with the same superscripts in the same column did not differ significantly (P . 0.05).


direct evidence that antioxidant enzymes are important and thus are viable candidate vaccines. TPx is an important peroxidase that can protect organisms against various oxidative stresses, and it has been extensively investigated in several helminths (Klimowski et al., 1997; McGonigle et al., 1998; Li et al., 2004). TPx proteins from other helminth parasites exhibit different expression patterns. In Onchocerca volvulus, the TPx-2 protein is predominantly localized in the hypodermis and cuticle, which may protect the parasite from being damaged by host-generated oxidative stress (Lu et al., 1998). In S. japonicum, TPx-1 was expressed in the tegument and excretory/secretory products. TPx-2 was mainly detected in the sub-tegumental tissues, parenchyma, vitelline gland, and gut epithelium of adult worms. TPx-3 has a mitochondria-targeting sequence, and it was believed to function in mitochondria. TPx-2 may have important roles in intracellular redox signaling, or the reduction of ROS generated via the hemoglobinolytic process in the digestive tract, or both (Kumagai et al., 2006). However, in recent years, SjTPx-2 was identified in our schistosome tegument proteomic study (data not shown), and a similar result was generated in Schistosoma bovis tegument proteomic research (Perez-Sanchez et al., 2008). These results were contradictory to the previous report, which showed that TPx-2 was not detected in the tegument. As mentioned above, SjTPx-2 was predicted to have an N-terminal signal peptide and could be secreted from cells. Thus, we speculate that, like SjTPx-1, SjTPx-2 protects the parasite against ROS produced by host immune cells, as well as their own oxygen metabolism, for example, hemoglobinolytic process in the digestive tract. But it might have no function in intracellular redox signaling. In recent years, RNA interference (RNAi) has been applied to study the TPx proteins. In S. mansoni, the downregulations of TPx-1 and TPx-2 by RNAi were lethal because it increased its sensitivity to hydrogen peroxide (Sayed et al., 2006). RNAi techniques were also used to suppress the specific genes of S. japonicum TPx, but neither TPx-1 nor TPx-2 was essential for the survival of schistosomula. However, TPx-1 dsRNA-treated larvae were susceptible to hydrogen peroxide, t-butylhydroperoxide, and cumene-hydroperoxide, and lethality was observed. But a similar result did not occur in TPx-2 dsRNA-treated larvae (Kumagai et al., 2009). We speculate that TPx-1 was 1 of the exposed proteins of the S. japonicum tegument and that it was at the surface of the tegument (Mulvenna et al., 2010). A similar result was observed in a study of the localization of TPx-1 in 7-day-old schistosomula by immunofluorescent staining (Kumagai et al., 2006). Thus, TPx-1 in schistosomes would initially react with peroxide when worms are exposed to oxidative stress. Another reason may be that mRNA and protein levels of TPx-1 in S. mansoni were higher than TPx-2 in S. mansoni (Sayed et al., 2006). TPx-1 may play an important role in resistance against ROS generated in the outer oxidative stress environment. Many studies of TPx proteins have been conducted in various organisms, so we expect that the essential roles of the TPx protein in the schistosome will be clarified by further investigation. In summary, we characterized a functional antioxidant enzyme SjTPx-2 from S. japonicum. It displayed good immunogenicity and it induced a high level of specific antibodies, as well as a partial protective immunity against schistosome infection in BALB/c mice. Based on these

75

findings, we conclude that SjTPx-2 may be an important antioxidative enzyme in scavenging ROS and it may be used as a potential vaccine candidate or new drug target. ACKNOWLEDGMENTS We thank Hao Li and Ke Lu from the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences for technical assistance with parasite collection. This work was supported by National Natural Science Foundation of China (No. 31172315), National Basic Research Program of China (No. 2007CB513108), Special Fund for Agroscientific Research in the Public Interest (200903036), and China Postdoctoral Science Foundation (2012M510630).

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