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Research article
Peroxiredoxin System of Aspergillus nidulans Resists Inactivation by High Concentration of Hydrogen Peroxide-Mediated Oxidative Stress
1State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R. China, 2School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China, 3Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan
Correspondence to:J. Microbiol. Biotechnol. 2018; 28(1): 145-156
Published January 28, 2018 https://doi.org/10.4014/jmb.1707.07024
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
Peroxiredoxins (Prxs) are a family of thiol-specific peroxidases that catalyze the reduction of peroxides, including H2O2 and organic peroxides. These antioxidant enzymes are found across all living organisms, and multiple Prxs are commonly found in one species (three in
Reduction of the disulfide (CP-CR) formed in oxidized 2-Cys Prxs requires Trx systems consisting of Trx combined with Trx reductase (TrxR) [13, 14]. Trxs are small proteins with a molecular mass of 12-13 kDa, and ubiquitously distributed in most eukaryotes and prokaryotes. Trxs are characterized at the level of their sequence by the presence of two redox-active cysteines in the active motif (WCGPC). The two vicinal cysteines are key for reducing the disulfides in other proteins by cysteine thiol-disulfide exchange [15]. On the other hand, TrxR is a homodimeric protein containing an active disulfide and FAD and NADPH binding sites in each subunit. During the reduction of Prx, Trx directly reduces (CP-CR) in the oxidized 2-Cys Prx by disulfide exchange. Subsequently, TrxR catalyzes the reduction of oxidized Trx (Trx-S2) by using NADPH as the electron donor and its own redox-active cysteine pair with FAD as the cofactor [13, 16].
Some 2-Cys Prxs are readily inactivated by a high concentration of peroxide during catalysis. Such inactivation has been detected during in vitro characterizations of 2-Cys Prxs from eukaryotic cells [5, 17-19]. At high levels of peroxide, the CP-SOH of the sensitive 2-Cys Prxs can be further oxidized by another peroxide molecule to form cysteine-sulfinic acid (Cys-SO2H) in a process termed hyperoxidation, causing Prx inactivation [20-23]. The two sequence motifs “GGLG” and “YF” are well conserved in most eukaryotic typical 2-Cys Prxs, and these sequence features are common to sensitive 2-Cys Prxs [24]. The inactivation of 2-Cys Prxs has been suggested to be an evolutionarily adapted mechanism for eukaryotic cells, for the allowance of H2O2 accumulation to substantial levels for signaling purposes under certain circumstances [24].
Peroxide dose-dependent inactivation of 2-Cys Prxs is generally considered to be caused by irreversibly over-oxidizing the reduced CP to cysteine-sulfinic acid (CP-SO2H) in Prxs. However, no concern was paid to the effects of the other two key components of the Prx systems, Trx and TrxR, on the inactivation during the oxidation process. In fact, at least TrxR has been reported to be inactivated by oxidants such as H2O2, which was proposed to give the enzyme the status of a cellular “redox sensor” [25]. Whether the activity of Trx is also sensitive to peroxide requires experimental investigation.
Many studies have clearly indicated that the substrates of Prx, peroxides, mediate a variety of physiological processes, such as life span, spore germination, cell survival, biomass production, and pathogen attack [26-30]. Therefore, systematic investigations on of the enzymatic properties of Prxs are necessary. To date, the biochemical properties and physiological functions of Prxs from most species, including bacterial, yeast, plant, and mammalian cells, have been investigated and described in detail, whereas the corresponding information on industrially and medically important filamentous fungi is very limited and fragmentary. Until now, only two Prxs from filamentous fungi,
In the present study, we biochemically characterized
Materials and Methods
Fungus, Media, and Culture Conditions
Gene Cloning and Expression Vector Construction
Trizol reagent (Invitrogen, USA) was used to extract total RNA from
Expression and Purification of the Recombinant Proteins
The above-mentioned expression plasmids and the previously constructed An.TrxA and An.TrxR expression plasmids [35] were introduced into
Molecular Mass Determination
The purified protein was applied to a Superdex 200 increase 10/300 column (GE Healthcare) and then eluted at a flow rate of 0.5 ml/min with 20 mM sodium phosphate buffer containing 150 mM NaCl and 5 mM dithiothreitol (DTT). The molecular mass of the enzyme was determined on the basis of the mobilities of the standard proteins containing ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa) and thyroglobulin (660 kDa).
Construction of AN8692 Disruption Strain
The primers used for
Southern Blot Analysis of AN8692 Disruption Strain
Genomic DNAs from the parent strain ABPU1 and the candidate disruption strain An.PrxA were digested using NdeI and HindIII, respectively, separated by gel electrophoresis, and then transferred to Hybond N+ membranes (Amersham Pharmacia Biotech, UK). Probes for hybridization were prepared by PCR using primers (Table S1) and then labeled using a digoxigenin (DIG) PCR labeling kit (Roche Diagnostics, Switzerland). Hybridization and signal detection were operated according to the manual supplied by the manufacturer.
Enzymatic Assays and Kinetic Parameter Determination
Peroxidase activity was measured by spectrophotometric assay monitoring the consumption of NADPH at 340 nm [13]. In the pretreatment operation, H2O2 and DTT were removed by the P6 column (Bio-Rad Laboratories, USA) prior to the activity assays. To determine the kinetic parameter, the reaction was carried out in a final volume of 1 ml of reaction mixture containing 50 mM phosphate buffer (pH 7.0), 200 μM NADPH, 1.13 μM Trx, 0.38 μM TrxR, 0.3 μM PrxA, and various concentrations of H2O2 or t-butyl hydroperoxide (
Results
Molecular Characterization of Recombinant An.PrxA
In 2007, Thon
cDNA sequence analysis revealed that
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Fig. 1. Sequence alignment of deduced CR ahead of CP type of 2-Cys peroxiredoxin (Prx) family members. The deduced Prx sequences of
Aspergillus fumigatus (XP_747849.1),Aspergillus niger (XP_001395908.1),Neurospora crassa (XP_964200.2),Penicillium nordicum (KOS40786.1),Saccharomyces cerevisiae (NP_013210.1),Candida albicans (XP_720512.1),Candida glabrata (XP_449850.1),Pichia pastoris (XP_002490288.1),Polynucleobacter wuianus (WP_068947794.1),Ralstonia syzygii (CCA854164.1),Burkholderia vietnamiensis (YP_001118388.1), andHerminiimonas arsenicoxydans (WP_011872175.1) were aligned using the programs Clustal and ESPrint. The conserved Cys 31, Cys 61, and Arg 134 of An.PrxA are indicated by the closed triangle, opened triangle, and closed circle, respectively. The putative PXXXS/TXXC motifs are indicated by the bar under the sequences.
The purified recombinant An.PrxA gave a single band with a molecular mass of approximately 20 kDa on SDS-PAGE under reducing conditions (Fig. 2A, lane 1). FPLC of An.PrxA on a gel filtration column in the presence of a buffer containing DTT yielded a peak at position corresponding to 40 kDa (Fig. 2B), suggesting that the native An.PrxA exists as a homodimer independent of the disulfide bond. When DTT was removed from the reduced mixture, the An.PrxA monomer band was oxidized gradually to a dimer band by oxygen in air under nonreducing conditions (Fig. 2A, lines 2 and 3). Under the same conditions, two major bands of the monomers with slightly different electrophoretic mobility were detected on SDS-PAGE (Fig. 2A, lines 2 and 3). The bands above and beneath might represent the originally reduced and the intramolecular disulfide bond formed forms of An.PrxA, respectively, because the ring-shaped protein should migrate slightly faster than the linear protein owing to the compactness of the protein conformation. To further confirm the redox state of two monomer forms, we treated the DTT-free An.PrxA with increasing concentrations of DTT. As a result, a gradual shift in the protein band from the position of higher mobility to that of lower mobility was observed (Fig. 2C), which supported our abovementioned supposition about the redox states of the two monomers. These observations suggested that oxidation of An.PrxA might result in the formation of both intramolecular and intermolecular linkages between Cys 31 and Cys 61, which seems to disturb the classification rule of An.PrxA between typical 2-Cys and atypical 2-Cys Prxs. However, it has been previously reported that in mammalian 1-Cys Prx, the CP oxidation product, cysteine sulfenic acid (Cys-SOH), readily undergoes condensation with another noncatalytic cysteine to form disulfides in the denatured conformation of the protein during heating, which does not occur in its native state [38]. To investigate this possibility, we incubated the air-oxidized An.PrxA in the absence or presence of a thiol blocking reagent N-ethylmaleimide (NEM) to prevent the Cys-SOH group from forming disulfide after denaturation, before heating and followed by nonreducing SDS-PAGE analysis. As shown in Fig. 2D, the higher mobility monomer band was occluded by the thiol block reagent, while the dimer band still existed. These results suggested the higher mobility monomer contains a disulfide bond formed by heating during the denaturation process of SDS-PAGE, whereas the oxidized nondenatured An.PrxA contains a disulfide bond between intermolecular linkages but not intramolecular linkages. These structure features clearly indicate that An.PrxA belongs to the subfamily of typical 2-Cys Prxs.
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Fig. 2. SDS-PAGE and gel filtration analysis of recombinant
Aspergillus nidulans peroxiredoxin (An.PrxA). (A) Nonreducing SDS-PAGE analysis (15% polyacrylamide gel) of An.PrxA. Line 1, An.PrxA (4 μg) with 10 mM dithiothreitol (DTT); line 2, An.PrxA (4 μg) first treated with 10 mM DTT followed by DTT removal by P6 column; line 3, DTT-free reduced An.PrxA (4 μg) exposed to air for 12h. (B) Estimation of the molecular mass of An.PrxA by gelfiltration. Inset: correlation between the molecular mass and elution volume of proteins. (C) Nonreducing SDS-PAGE analysis (15% polyacrylamide gel) of the DTT-free An.PrxA (4 μg) incubated with the indicated concentrations of DTT (lanes 1-7: 0, 1, 2, 4, 6, 8, and 10 mM DTT). (D) Effect of N-ethylmaleimide (NEM) treatment on the electrophoretic mobility (disulfide formation) of air-oxidized An.PrxA. The air-oxidized An.PrxA (4 μg) was incubated in the absence (Lane 1) or presence (Lane 2) of 10 mM NEM for 30 min before nonreducing SDS-PAGE analysis.
Catalytic Properties of An.PrxA
The oxidation of An.PrxA by peroxide was directly observed by non-reducing SDS-PAGE. Both H2O2 and
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Fig. 3. Peroxidase activity analyses of
Aspergillus nidulans peroxiredoxin (An.PrxA) variants. (A and B) NADPH oxidation was monitored as the decrease in A340 in a 1 ml reaction mixture containing 50 mM phosphate buffer (pH 7.0), 1.2 μM An.TrxR, 10 μM An.TrxA, 150 μM NADPH, and 2 μM An.PrxA-C31S or An.PrxA-C61S or the mixture of 1 μM An.PrxA-C31S and 1 μM An.PrxAC61S, or 2 μM An.PrxA, with 0.5 mM H2O2 (A) or t-butyl hydroperoxide (t -BOOH) (B) as substrates, respectively. All the experiment were performed three times. (C) Growth ofA. nidulans strains on agar plates containing 0 mM and 1 mM H2O2 or 0.5 mMt -BOOH. WT, wild type.
Protection of An.PrxA against H2O2 and t -BOOH
The in vitro catalytic properties strongly suggested that An.PrxA could provide protection against oxidative stress in vivo. To examine the effect of the An.PrxA gene on oxidative stress tolerance, we constructed a null mutant of the AnPrxA gene (Fig. S3). The unimpaired viability of the resulting
Extremely High Resistance of An.PrxA to H2O2
In other peroxidases, the sequence motifs “GGLG” and “YF” promote hyperoxidation by hindering the local unfolding necessary for disulfide formation with the resolving cysteine. By sequence analysis, we noticed that these motifs are not conserved in the An.PrxA sequence, thus suggesting An.PrxA may be a robust and resistant peroxidase to high oxidative stress. To prove this point, we investigated the resistance of An.PrxA to inactivation by high concentrations of H2O2. In comparison, we have also studied yeast Sc.Ahp1, another “GGLG” and “YF” absent Prx, which has been revealed to be more resilient to hyperoxidation than other eukaryotic Prxs [5, 39]. The time course of NADPH oxidation towards various concentrations of H2O2 showed that both peroxidase activities of the two Prx isoforms exhibited high resistance to high H2O2- mediated inactivation, but with different degrees (Figs. 4A and 4B). The A340 decrease rates summarized in Fig. 4C indicated that even under high oxidative stress conditions, the peroxidase activity of An.PrxA increased or remained stable with the concentration of substrates increasing until to an extreme concentration of 600 mM. In comparison, 100 mM of H2O2 was the threshold concentration for Sc.Ahp1 to resist inactivation, while above 100 mM, the activities decreased in a dose-dependent manner. These results clearly indicated that both “GGLG” and “YF” motifabsent 2-Cys Prxs exhibited high abilities to resist inactivation by extremely high concentrations of H2O2, but An.PrxA system showed stronger tolerability.
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Fig. 4. Comparison of peroxidase activities of
Aspergillus nidulans peroxiredoxin (An.PrxA) andSaccharomyces cerevisiae alkyl hydroperoxide reductase 1 (Sc.Ahp1) towards various concentrations of H2O2. (A and B) Peroxidase activities were measured in a 1 ml reaction mixture containing 5 μM An.TrxA, 0.6 μM An.TrxR, 150 μM NADPH, and 1 μM An.PrxA (A) or 5 μM Sc.Trx, 0.6 μM Sc.TrxR, 150 μM NADPH, and 1 μM Sc.Ahp1 (B) in the presence of 10-600 mM H2O2. (C) Quantitative comparison of peroxidase activities of An.PrxA and Sc.Ahp1 measured in (A) and (B). Peroxidase activities were reflected by A340 decrease values at 1 min after the beginning of reaction. Results are the means ± SD of three independent experiments. *,p < 0.05; **,p < 0.01; ***,p < 0.001.
Molecular Basis for the Extremely High Resistance of An.PrxA to H2O2
An.PrxA and Sc.Ahp1 possess similar sequence characteristics, such as the absence of H2O2-sensitive feature motifs and the same order arrangements of CP and CR (Fig. 1). Therefore, we assumed that the great degree of different H2O2-dependent inactivation of the two peroxidases may be due to the Trx/TrxR systems rather than considering the H2O2-susceptibility distinction of the two Prxs. To determine the contribution of Trx/TrxR systems to the robust resistance, we set up a new assay by interchanging the Trx and TrxR pairs between the two Prx systems. The assay was carried out with 10 mM H2O2 and monitored by A340 decrease. Unexpectedly, the catalytic activities of both recombination sets were even higher than those of their individual native forms, indicating that the Trx/TrxR combinations can functionally substitute in peroxidase assays of An.PrxA and Sc.Ahp1 (Fig. 5A). Interestingly, the enzyme recombination substantially altered the H2O2 tolerance of the two Prxs. As shown in Fig. 5B, equivalently changing An.TrxA/TrxR to Sc.Trx/TrxR critically promoted the susceptibility of An.PrxA to 600 mM H2O2, whereas An.TrxA/TrxR greatly increased the H2O2 tolerance of Sc.Ahp1, even slightly exceeding the level of the native An.PrxA combination, suggesting that the extremely high H2O2 tolerance of Prx requires robust An.TrxA/TrxR.
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Fig. 5. TrxR determined the H2O2-sensitivity of the peroxiredoxin (Prx) systems towards high conditions of H2O2. (A) An.PrxA combined with An.TrxA/TrxR or Sc.Trx/TrxR, and Sc.Ahp1 combined with Sc.Trx/TrxR or An.TrxA/TrxR were constituted to measure their peroxidase activities in the presence of 10 mM H2O2. (B) The peroxidase activities of the four Prx combination systems were measured in the presence of 600 mM H2O2. (C and D) An.TrxA/TrxR (C) and Sc.Trx/TrxR (D) were treated with indicated concentrations of H2O2 for 5 min before starting the reactions. (E) An.TrxA, An.TrxR, Sc.Trx, and Sc.TrxR was individually pretreated with 600 mM H2O2. (F) Sc.TrxR was pretreated with the indicated concentrations of H2O2. After pretreatment, the excess H2O2 was removed by P6 column and the pretreated enzymes were used for the peroxidase reaction systems in the presence of 0.5 mM H2O2. The reaction mixture contained the same amount of individual enzymes and NADPH as described in Figs. 4A and 4B. The results are representative of three independent experiments.
Further experiments were performed to compare the H2O2 tolerance of the two Trx/TrxR systems from the two separate sources. Individual Trx/TrxR systems were preexposed to a range of H2O2 concentrations for 5 min, and the activities were assayed by measuring NADPH oxidation coupled to 0.5 mM H2O2 reduction catalyzed by the two Prxs. As shown in Figs. 5C and 5D, for each pretreated concentration of H2O2, An.TrxA/TrxR displayed robust reduction activities, whereas concentrations higher than 200 mM H2O2 seriously inactivated Sc.Trx/TrxR in a dose-dependent manner, which is consistent with the data shown in Fig. 4. These results confirmed that it is Trx/TrxR dominating the H2O2 sensitivity of Prx systems under extreme conditions.
To further explore which element in Trx/TrxR is responsible for the extreme H2O2 tolerance of the An.PrxA system, we separately pretreated Trx and TrxR from the Prx assay set with 600 mM H2O2 and compared the inactivation levels reflected by the rate of NADPH oxidation catalyzed by Prx with 0.5 mM H2O2. As shown in Fig. 5E, pretreatment of Sc.TrxR seriously impaired the peroxidase activities of its corresponding Sc.Ahp1, whereas An.TrxR exhibited robust resistance to inactivation by 600 mM H2O2. It was easy to ascertain that neither of the pretreated Trxs determined the H2O2 sensitivity of their corresponding Prx systems because they showed identically high resistant abilities to extreme H2O2. To further confirm the H2O2 sensitivity of Sc.TrxR, we investigated the effect of the H2O2 concentration on the inactivation of Sc.TrxR using the pretreatment method. We found that the H2O2-induced decrease of H2O2 reduction activities was evident and that the inactivation rates were dose-dependent (Fig. 5F). To exclude the possibility that the high resistant abilities to H2O2 is Trx specific, we also tested the effects of the remaining Trx and TrxR combinations (An.Trx paired with Sc.TrxR and Sc.Trx paired with An.TrxR) on H2O2 sensitivity of the Prx systems (Figs. S5A and S5B). As was expected, possessing An.TrxR in the assay combinations would be beneficial to H2O2 tolerance, regardless of which Trx was combined. These data clearly indicated that it is the outstanding H2O2 tolerence of An.TrxR rather than Trx that ensures the extremely high resistance of An.PrxA to H2O2.
Activity Intolerance of the An.PrxA System to Excess t -BOOH
We subsequently investigated whether the same oxidative stress tolerance mechanism is present in the reduction process of
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Fig. 6. Comparison of t-butyl hydroperoxide (
t -BOOH)-tolerant abilities of theAspergillus nidulans peroxiredoxin (An.PrxA) andSaccharomyces cerevisiae alkyl hydroperoxide reductase 1 (Sc.Ahp1) systems. Peroxidase activity was measured in a 1 ml reaction mixture containing 150 μM NADPH, 1-8 mMt -BOOH, and the following different enzyme combinations: (A) 2 μM An.PrxA, 10 μM An.Trx, and 1.2 μM An.TrxR. (B) 2 μM Sc.Ahp1, 10 μM Sc.Trx, and 1.2 μM Sc.TrxR. (C) 2 μM An.PrxA, 10 μM Sc.Trx, and 1.2 μM Sc.TrxR. (D) 2 μM Sc.Ahp1, 10 μM An.Trx, and 1.2 μM An.TrxR. The results are representative of three independent experiments.
Discussion
Filamentous fungi have industrial, pharmaceutical, and medical importance, and they frequently encounter high oxidative stress caused by host pathogen defense and fermentation conditions [40, 41]. Catalases, glutathione peroxidases, and Prxs have been considered as the central enzymes for protecting filamentous fungi from peroxide injury [30, 42-44]. The first two antioxidant enzymes have been intensively studied in filamentous fungi, but the information of filamentous fungal Prxs is very limited. Therefore, we molecularly characterized a recombinant filamentous fungal Prx cloned from
Although the bioinformatics analysis indicated that at least six Prx-like genes exist in the
Our data indicated that the peroxidase tolerance to the extremely high concentrations of H2O2 is likely completely dependent on the oxidative stabilities of the reaction partner TrxR. In fact, TrxR sensitivity to peroxides has also been confirmed by other evidence. Incubation of mammalian TrxR (TR1) with H2O2 led to TR1 inactivation due to the oxidation of the enzyme in its reactive selenocysteine residue at the C-terminal end, which is required for catalytic activity [25]. The inactivation of TR1 will result in ROS accumulation, which will then affect cellular signaling components, including transcription factors, phosphatases, antioxidant enzymes, and kinases. Thus, the H2O2 sensitivity of TR1 was proposed to be involved in the redox-regulated cell signaling. Another possible reason for the sensitivity differences of the Prx systems to H2O2 is correlated to ROS-promoting protein denaturation or degradation. Many studies have shown that oxidation of proteins may lead to hydroxylation of aliphatic amino-acid side chains and aromatic groups, sulfoxidation of methionine residues, and conversion of some amino acids to carbonyl derivatives [47]. Such modifications directly lead to the loss of the enzymatic activities and were observed in some physiological processes such as aging, disease, and apoptosis. The modification efficiencies of the target proteins are dependent on their amino acid sequences and structural differences. Although the TrxR from
TrxRs from different organisms show a great diversity in their chemical mechanism of thioredoxin reduction [48]. However, the TrxR/Trx system-mediated Prx-dependent peroxide reduction processes are similar in most organisms. The Trx/TrxR system from
In spite of the outstanding resistance of An.PrxA to H2O2, the An.PrxA system was not resistant to excess
Supplemental Materials
Acknowledgments
This work was sponsored by the National Natural Science Foundation of China (21672065, 21636003, and 31471659), the Shanghai Pujiang Program (16PJ1402500), and the National Special Fund for State Key Laboratory of Bioreactor Engineering (Grant No. 2060204).
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2018; 28(1): 145-156
Published online January 28, 2018 https://doi.org/10.4014/jmb.1707.07024
Copyright © The Korean Society for Microbiology and Biotechnology.
Peroxiredoxin System of Aspergillus nidulans Resists Inactivation by High Concentration of Hydrogen Peroxide-Mediated Oxidative Stress
Yang Xia 1, Haijun Yu 1, Zhemin zhou 2, Naoki Takaya 3, Shengmin Zhou 1* and Ping Wang 1
1State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R. China, 2School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China, 3Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan
Correspondence to:Shengmin Zhou
zhoushengmin@ecust.edu.cn
Abstract
Most eukaryotic peroxiredoxins (Prxs) are readily inactivated by a high concentration of hydrogen peroxide (H2O2) during catalysis owing to their “GGLG” and “YF” motifs. However, such oxidative stress sensitive motifs were not found in the previously identified filamentous fungal Prxs. Additionally, the information on filamentous fungal Prxs is limited and fragmentary. Herein, we cloned and gained insight into Aspergillus nidulans Prx (An.PrxA) in the aspects of protein properties, catalysis characteristics, and especially H2O2 tolerability. Our results indicated that An.PrxA belongs to the newly defined family of typical 2-Cys Prxs with a marked characteristic that the “resolving” cysteine (CR) is invertedly located preceding the “peroxidatic” cysteine (CP) in amino acid sequences. The inverted arrangement of CR and CP can only be found among some yeast, bacterial, and filamentous fungal deduced Prxs. The most surprising characteristic of An.PrxA is its extraordinary ability to resist inactivation by extremely high concentrations of H2O2, even that approaching 600 mM. By screening the H2O2- inactivation effects on the components of Prx systems, including Trx, Trx reductase (TrxR), and Prx, we ultimately determined that it is the robust filamentous fungal TrxR rather than Trx and Prx that is responsible for the extreme H2O2 tolerence of the An.PrxA system. This is the first investigation on the effect of the electron donor partner in the H2O2 tolerability of the Prx system.
Keywords: Peroxiredoxin, filamentous fungus, thioredoxin, thioredoxin reductase, oxidative stress, hydrogen peroxide
Introduction
Peroxiredoxins (Prxs) are a family of thiol-specific peroxidases that catalyze the reduction of peroxides, including H2O2 and organic peroxides. These antioxidant enzymes are found across all living organisms, and multiple Prxs are commonly found in one species (three in
Reduction of the disulfide (CP-CR) formed in oxidized 2-Cys Prxs requires Trx systems consisting of Trx combined with Trx reductase (TrxR) [13, 14]. Trxs are small proteins with a molecular mass of 12-13 kDa, and ubiquitously distributed in most eukaryotes and prokaryotes. Trxs are characterized at the level of their sequence by the presence of two redox-active cysteines in the active motif (WCGPC). The two vicinal cysteines are key for reducing the disulfides in other proteins by cysteine thiol-disulfide exchange [15]. On the other hand, TrxR is a homodimeric protein containing an active disulfide and FAD and NADPH binding sites in each subunit. During the reduction of Prx, Trx directly reduces (CP-CR) in the oxidized 2-Cys Prx by disulfide exchange. Subsequently, TrxR catalyzes the reduction of oxidized Trx (Trx-S2) by using NADPH as the electron donor and its own redox-active cysteine pair with FAD as the cofactor [13, 16].
Some 2-Cys Prxs are readily inactivated by a high concentration of peroxide during catalysis. Such inactivation has been detected during in vitro characterizations of 2-Cys Prxs from eukaryotic cells [5, 17-19]. At high levels of peroxide, the CP-SOH of the sensitive 2-Cys Prxs can be further oxidized by another peroxide molecule to form cysteine-sulfinic acid (Cys-SO2H) in a process termed hyperoxidation, causing Prx inactivation [20-23]. The two sequence motifs “GGLG” and “YF” are well conserved in most eukaryotic typical 2-Cys Prxs, and these sequence features are common to sensitive 2-Cys Prxs [24]. The inactivation of 2-Cys Prxs has been suggested to be an evolutionarily adapted mechanism for eukaryotic cells, for the allowance of H2O2 accumulation to substantial levels for signaling purposes under certain circumstances [24].
Peroxide dose-dependent inactivation of 2-Cys Prxs is generally considered to be caused by irreversibly over-oxidizing the reduced CP to cysteine-sulfinic acid (CP-SO2H) in Prxs. However, no concern was paid to the effects of the other two key components of the Prx systems, Trx and TrxR, on the inactivation during the oxidation process. In fact, at least TrxR has been reported to be inactivated by oxidants such as H2O2, which was proposed to give the enzyme the status of a cellular “redox sensor” [25]. Whether the activity of Trx is also sensitive to peroxide requires experimental investigation.
Many studies have clearly indicated that the substrates of Prx, peroxides, mediate a variety of physiological processes, such as life span, spore germination, cell survival, biomass production, and pathogen attack [26-30]. Therefore, systematic investigations on of the enzymatic properties of Prxs are necessary. To date, the biochemical properties and physiological functions of Prxs from most species, including bacterial, yeast, plant, and mammalian cells, have been investigated and described in detail, whereas the corresponding information on industrially and medically important filamentous fungi is very limited and fragmentary. Until now, only two Prxs from filamentous fungi,
In the present study, we biochemically characterized
Materials and Methods
Fungus, Media, and Culture Conditions
Gene Cloning and Expression Vector Construction
Trizol reagent (Invitrogen, USA) was used to extract total RNA from
Expression and Purification of the Recombinant Proteins
The above-mentioned expression plasmids and the previously constructed An.TrxA and An.TrxR expression plasmids [35] were introduced into
Molecular Mass Determination
The purified protein was applied to a Superdex 200 increase 10/300 column (GE Healthcare) and then eluted at a flow rate of 0.5 ml/min with 20 mM sodium phosphate buffer containing 150 mM NaCl and 5 mM dithiothreitol (DTT). The molecular mass of the enzyme was determined on the basis of the mobilities of the standard proteins containing ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa) and thyroglobulin (660 kDa).
Construction of AN8692 Disruption Strain
The primers used for
Southern Blot Analysis of AN8692 Disruption Strain
Genomic DNAs from the parent strain ABPU1 and the candidate disruption strain An.PrxA were digested using NdeI and HindIII, respectively, separated by gel electrophoresis, and then transferred to Hybond N+ membranes (Amersham Pharmacia Biotech, UK). Probes for hybridization were prepared by PCR using primers (Table S1) and then labeled using a digoxigenin (DIG) PCR labeling kit (Roche Diagnostics, Switzerland). Hybridization and signal detection were operated according to the manual supplied by the manufacturer.
Enzymatic Assays and Kinetic Parameter Determination
Peroxidase activity was measured by spectrophotometric assay monitoring the consumption of NADPH at 340 nm [13]. In the pretreatment operation, H2O2 and DTT were removed by the P6 column (Bio-Rad Laboratories, USA) prior to the activity assays. To determine the kinetic parameter, the reaction was carried out in a final volume of 1 ml of reaction mixture containing 50 mM phosphate buffer (pH 7.0), 200 μM NADPH, 1.13 μM Trx, 0.38 μM TrxR, 0.3 μM PrxA, and various concentrations of H2O2 or t-butyl hydroperoxide (
Results
Molecular Characterization of Recombinant An.PrxA
In 2007, Thon
cDNA sequence analysis revealed that
-
Figure 1. Sequence alignment of deduced CR ahead of CP type of 2-Cys peroxiredoxin (Prx) family members. The deduced Prx sequences of
Aspergillus fumigatus (XP_747849.1),Aspergillus niger (XP_001395908.1),Neurospora crassa (XP_964200.2),Penicillium nordicum (KOS40786.1),Saccharomyces cerevisiae (NP_013210.1),Candida albicans (XP_720512.1),Candida glabrata (XP_449850.1),Pichia pastoris (XP_002490288.1),Polynucleobacter wuianus (WP_068947794.1),Ralstonia syzygii (CCA854164.1),Burkholderia vietnamiensis (YP_001118388.1), andHerminiimonas arsenicoxydans (WP_011872175.1) were aligned using the programs Clustal and ESPrint. The conserved Cys 31, Cys 61, and Arg 134 of An.PrxA are indicated by the closed triangle, opened triangle, and closed circle, respectively. The putative PXXXS/TXXC motifs are indicated by the bar under the sequences.
The purified recombinant An.PrxA gave a single band with a molecular mass of approximately 20 kDa on SDS-PAGE under reducing conditions (Fig. 2A, lane 1). FPLC of An.PrxA on a gel filtration column in the presence of a buffer containing DTT yielded a peak at position corresponding to 40 kDa (Fig. 2B), suggesting that the native An.PrxA exists as a homodimer independent of the disulfide bond. When DTT was removed from the reduced mixture, the An.PrxA monomer band was oxidized gradually to a dimer band by oxygen in air under nonreducing conditions (Fig. 2A, lines 2 and 3). Under the same conditions, two major bands of the monomers with slightly different electrophoretic mobility were detected on SDS-PAGE (Fig. 2A, lines 2 and 3). The bands above and beneath might represent the originally reduced and the intramolecular disulfide bond formed forms of An.PrxA, respectively, because the ring-shaped protein should migrate slightly faster than the linear protein owing to the compactness of the protein conformation. To further confirm the redox state of two monomer forms, we treated the DTT-free An.PrxA with increasing concentrations of DTT. As a result, a gradual shift in the protein band from the position of higher mobility to that of lower mobility was observed (Fig. 2C), which supported our abovementioned supposition about the redox states of the two monomers. These observations suggested that oxidation of An.PrxA might result in the formation of both intramolecular and intermolecular linkages between Cys 31 and Cys 61, which seems to disturb the classification rule of An.PrxA between typical 2-Cys and atypical 2-Cys Prxs. However, it has been previously reported that in mammalian 1-Cys Prx, the CP oxidation product, cysteine sulfenic acid (Cys-SOH), readily undergoes condensation with another noncatalytic cysteine to form disulfides in the denatured conformation of the protein during heating, which does not occur in its native state [38]. To investigate this possibility, we incubated the air-oxidized An.PrxA in the absence or presence of a thiol blocking reagent N-ethylmaleimide (NEM) to prevent the Cys-SOH group from forming disulfide after denaturation, before heating and followed by nonreducing SDS-PAGE analysis. As shown in Fig. 2D, the higher mobility monomer band was occluded by the thiol block reagent, while the dimer band still existed. These results suggested the higher mobility monomer contains a disulfide bond formed by heating during the denaturation process of SDS-PAGE, whereas the oxidized nondenatured An.PrxA contains a disulfide bond between intermolecular linkages but not intramolecular linkages. These structure features clearly indicate that An.PrxA belongs to the subfamily of typical 2-Cys Prxs.
-
Figure 2. SDS-PAGE and gel filtration analysis of recombinant
Aspergillus nidulans peroxiredoxin (An.PrxA). (A) Nonreducing SDS-PAGE analysis (15% polyacrylamide gel) of An.PrxA. Line 1, An.PrxA (4 μg) with 10 mM dithiothreitol (DTT); line 2, An.PrxA (4 μg) first treated with 10 mM DTT followed by DTT removal by P6 column; line 3, DTT-free reduced An.PrxA (4 μg) exposed to air for 12h. (B) Estimation of the molecular mass of An.PrxA by gelfiltration. Inset: correlation between the molecular mass and elution volume of proteins. (C) Nonreducing SDS-PAGE analysis (15% polyacrylamide gel) of the DTT-free An.PrxA (4 μg) incubated with the indicated concentrations of DTT (lanes 1-7: 0, 1, 2, 4, 6, 8, and 10 mM DTT). (D) Effect of N-ethylmaleimide (NEM) treatment on the electrophoretic mobility (disulfide formation) of air-oxidized An.PrxA. The air-oxidized An.PrxA (4 μg) was incubated in the absence (Lane 1) or presence (Lane 2) of 10 mM NEM for 30 min before nonreducing SDS-PAGE analysis.
Catalytic Properties of An.PrxA
The oxidation of An.PrxA by peroxide was directly observed by non-reducing SDS-PAGE. Both H2O2 and
-
Figure 3. Peroxidase activity analyses of
Aspergillus nidulans peroxiredoxin (An.PrxA) variants. (A and B) NADPH oxidation was monitored as the decrease in A340 in a 1 ml reaction mixture containing 50 mM phosphate buffer (pH 7.0), 1.2 μM An.TrxR, 10 μM An.TrxA, 150 μM NADPH, and 2 μM An.PrxA-C31S or An.PrxA-C61S or the mixture of 1 μM An.PrxA-C31S and 1 μM An.PrxAC61S, or 2 μM An.PrxA, with 0.5 mM H2O2 (A) or t-butyl hydroperoxide (t -BOOH) (B) as substrates, respectively. All the experiment were performed three times. (C) Growth ofA. nidulans strains on agar plates containing 0 mM and 1 mM H2O2 or 0.5 mMt -BOOH. WT, wild type.
Protection of An.PrxA against H2O2 and t -BOOH
The in vitro catalytic properties strongly suggested that An.PrxA could provide protection against oxidative stress in vivo. To examine the effect of the An.PrxA gene on oxidative stress tolerance, we constructed a null mutant of the AnPrxA gene (Fig. S3). The unimpaired viability of the resulting
Extremely High Resistance of An.PrxA to H2O2
In other peroxidases, the sequence motifs “GGLG” and “YF” promote hyperoxidation by hindering the local unfolding necessary for disulfide formation with the resolving cysteine. By sequence analysis, we noticed that these motifs are not conserved in the An.PrxA sequence, thus suggesting An.PrxA may be a robust and resistant peroxidase to high oxidative stress. To prove this point, we investigated the resistance of An.PrxA to inactivation by high concentrations of H2O2. In comparison, we have also studied yeast Sc.Ahp1, another “GGLG” and “YF” absent Prx, which has been revealed to be more resilient to hyperoxidation than other eukaryotic Prxs [5, 39]. The time course of NADPH oxidation towards various concentrations of H2O2 showed that both peroxidase activities of the two Prx isoforms exhibited high resistance to high H2O2- mediated inactivation, but with different degrees (Figs. 4A and 4B). The A340 decrease rates summarized in Fig. 4C indicated that even under high oxidative stress conditions, the peroxidase activity of An.PrxA increased or remained stable with the concentration of substrates increasing until to an extreme concentration of 600 mM. In comparison, 100 mM of H2O2 was the threshold concentration for Sc.Ahp1 to resist inactivation, while above 100 mM, the activities decreased in a dose-dependent manner. These results clearly indicated that both “GGLG” and “YF” motifabsent 2-Cys Prxs exhibited high abilities to resist inactivation by extremely high concentrations of H2O2, but An.PrxA system showed stronger tolerability.
-
Figure 4. Comparison of peroxidase activities of
Aspergillus nidulans peroxiredoxin (An.PrxA) andSaccharomyces cerevisiae alkyl hydroperoxide reductase 1 (Sc.Ahp1) towards various concentrations of H2O2. (A and B) Peroxidase activities were measured in a 1 ml reaction mixture containing 5 μM An.TrxA, 0.6 μM An.TrxR, 150 μM NADPH, and 1 μM An.PrxA (A) or 5 μM Sc.Trx, 0.6 μM Sc.TrxR, 150 μM NADPH, and 1 μM Sc.Ahp1 (B) in the presence of 10-600 mM H2O2. (C) Quantitative comparison of peroxidase activities of An.PrxA and Sc.Ahp1 measured in (A) and (B). Peroxidase activities were reflected by A340 decrease values at 1 min after the beginning of reaction. Results are the means ± SD of three independent experiments. *,p < 0.05; **,p < 0.01; ***,p < 0.001.
Molecular Basis for the Extremely High Resistance of An.PrxA to H2O2
An.PrxA and Sc.Ahp1 possess similar sequence characteristics, such as the absence of H2O2-sensitive feature motifs and the same order arrangements of CP and CR (Fig. 1). Therefore, we assumed that the great degree of different H2O2-dependent inactivation of the two peroxidases may be due to the Trx/TrxR systems rather than considering the H2O2-susceptibility distinction of the two Prxs. To determine the contribution of Trx/TrxR systems to the robust resistance, we set up a new assay by interchanging the Trx and TrxR pairs between the two Prx systems. The assay was carried out with 10 mM H2O2 and monitored by A340 decrease. Unexpectedly, the catalytic activities of both recombination sets were even higher than those of their individual native forms, indicating that the Trx/TrxR combinations can functionally substitute in peroxidase assays of An.PrxA and Sc.Ahp1 (Fig. 5A). Interestingly, the enzyme recombination substantially altered the H2O2 tolerance of the two Prxs. As shown in Fig. 5B, equivalently changing An.TrxA/TrxR to Sc.Trx/TrxR critically promoted the susceptibility of An.PrxA to 600 mM H2O2, whereas An.TrxA/TrxR greatly increased the H2O2 tolerance of Sc.Ahp1, even slightly exceeding the level of the native An.PrxA combination, suggesting that the extremely high H2O2 tolerance of Prx requires robust An.TrxA/TrxR.
-
Figure 5. TrxR determined the H2O2-sensitivity of the peroxiredoxin (Prx) systems towards high conditions of H2O2. (A) An.PrxA combined with An.TrxA/TrxR or Sc.Trx/TrxR, and Sc.Ahp1 combined with Sc.Trx/TrxR or An.TrxA/TrxR were constituted to measure their peroxidase activities in the presence of 10 mM H2O2. (B) The peroxidase activities of the four Prx combination systems were measured in the presence of 600 mM H2O2. (C and D) An.TrxA/TrxR (C) and Sc.Trx/TrxR (D) were treated with indicated concentrations of H2O2 for 5 min before starting the reactions. (E) An.TrxA, An.TrxR, Sc.Trx, and Sc.TrxR was individually pretreated with 600 mM H2O2. (F) Sc.TrxR was pretreated with the indicated concentrations of H2O2. After pretreatment, the excess H2O2 was removed by P6 column and the pretreated enzymes were used for the peroxidase reaction systems in the presence of 0.5 mM H2O2. The reaction mixture contained the same amount of individual enzymes and NADPH as described in Figs. 4A and 4B. The results are representative of three independent experiments.
Further experiments were performed to compare the H2O2 tolerance of the two Trx/TrxR systems from the two separate sources. Individual Trx/TrxR systems were preexposed to a range of H2O2 concentrations for 5 min, and the activities were assayed by measuring NADPH oxidation coupled to 0.5 mM H2O2 reduction catalyzed by the two Prxs. As shown in Figs. 5C and 5D, for each pretreated concentration of H2O2, An.TrxA/TrxR displayed robust reduction activities, whereas concentrations higher than 200 mM H2O2 seriously inactivated Sc.Trx/TrxR in a dose-dependent manner, which is consistent with the data shown in Fig. 4. These results confirmed that it is Trx/TrxR dominating the H2O2 sensitivity of Prx systems under extreme conditions.
To further explore which element in Trx/TrxR is responsible for the extreme H2O2 tolerance of the An.PrxA system, we separately pretreated Trx and TrxR from the Prx assay set with 600 mM H2O2 and compared the inactivation levels reflected by the rate of NADPH oxidation catalyzed by Prx with 0.5 mM H2O2. As shown in Fig. 5E, pretreatment of Sc.TrxR seriously impaired the peroxidase activities of its corresponding Sc.Ahp1, whereas An.TrxR exhibited robust resistance to inactivation by 600 mM H2O2. It was easy to ascertain that neither of the pretreated Trxs determined the H2O2 sensitivity of their corresponding Prx systems because they showed identically high resistant abilities to extreme H2O2. To further confirm the H2O2 sensitivity of Sc.TrxR, we investigated the effect of the H2O2 concentration on the inactivation of Sc.TrxR using the pretreatment method. We found that the H2O2-induced decrease of H2O2 reduction activities was evident and that the inactivation rates were dose-dependent (Fig. 5F). To exclude the possibility that the high resistant abilities to H2O2 is Trx specific, we also tested the effects of the remaining Trx and TrxR combinations (An.Trx paired with Sc.TrxR and Sc.Trx paired with An.TrxR) on H2O2 sensitivity of the Prx systems (Figs. S5A and S5B). As was expected, possessing An.TrxR in the assay combinations would be beneficial to H2O2 tolerance, regardless of which Trx was combined. These data clearly indicated that it is the outstanding H2O2 tolerence of An.TrxR rather than Trx that ensures the extremely high resistance of An.PrxA to H2O2.
Activity Intolerance of the An.PrxA System to Excess t -BOOH
We subsequently investigated whether the same oxidative stress tolerance mechanism is present in the reduction process of
-
Figure 6. Comparison of t-butyl hydroperoxide (
t -BOOH)-tolerant abilities of theAspergillus nidulans peroxiredoxin (An.PrxA) andSaccharomyces cerevisiae alkyl hydroperoxide reductase 1 (Sc.Ahp1) systems. Peroxidase activity was measured in a 1 ml reaction mixture containing 150 μM NADPH, 1-8 mMt -BOOH, and the following different enzyme combinations: (A) 2 μM An.PrxA, 10 μM An.Trx, and 1.2 μM An.TrxR. (B) 2 μM Sc.Ahp1, 10 μM Sc.Trx, and 1.2 μM Sc.TrxR. (C) 2 μM An.PrxA, 10 μM Sc.Trx, and 1.2 μM Sc.TrxR. (D) 2 μM Sc.Ahp1, 10 μM An.Trx, and 1.2 μM An.TrxR. The results are representative of three independent experiments.
Discussion
Filamentous fungi have industrial, pharmaceutical, and medical importance, and they frequently encounter high oxidative stress caused by host pathogen defense and fermentation conditions [40, 41]. Catalases, glutathione peroxidases, and Prxs have been considered as the central enzymes for protecting filamentous fungi from peroxide injury [30, 42-44]. The first two antioxidant enzymes have been intensively studied in filamentous fungi, but the information of filamentous fungal Prxs is very limited. Therefore, we molecularly characterized a recombinant filamentous fungal Prx cloned from
Although the bioinformatics analysis indicated that at least six Prx-like genes exist in the
Our data indicated that the peroxidase tolerance to the extremely high concentrations of H2O2 is likely completely dependent on the oxidative stabilities of the reaction partner TrxR. In fact, TrxR sensitivity to peroxides has also been confirmed by other evidence. Incubation of mammalian TrxR (TR1) with H2O2 led to TR1 inactivation due to the oxidation of the enzyme in its reactive selenocysteine residue at the C-terminal end, which is required for catalytic activity [25]. The inactivation of TR1 will result in ROS accumulation, which will then affect cellular signaling components, including transcription factors, phosphatases, antioxidant enzymes, and kinases. Thus, the H2O2 sensitivity of TR1 was proposed to be involved in the redox-regulated cell signaling. Another possible reason for the sensitivity differences of the Prx systems to H2O2 is correlated to ROS-promoting protein denaturation or degradation. Many studies have shown that oxidation of proteins may lead to hydroxylation of aliphatic amino-acid side chains and aromatic groups, sulfoxidation of methionine residues, and conversion of some amino acids to carbonyl derivatives [47]. Such modifications directly lead to the loss of the enzymatic activities and were observed in some physiological processes such as aging, disease, and apoptosis. The modification efficiencies of the target proteins are dependent on their amino acid sequences and structural differences. Although the TrxR from
TrxRs from different organisms show a great diversity in their chemical mechanism of thioredoxin reduction [48]. However, the TrxR/Trx system-mediated Prx-dependent peroxide reduction processes are similar in most organisms. The Trx/TrxR system from
In spite of the outstanding resistance of An.PrxA to H2O2, the An.PrxA system was not resistant to excess
Supplemental Materials
Acknowledgments
This work was sponsored by the National Natural Science Foundation of China (21672065, 21636003, and 31471659), the Shanghai Pujiang Program (16PJ1402500), and the National Special Fund for State Key Laboratory of Bioreactor Engineering (Grant No. 2060204).
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