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Preferential regeneration of thioredoxin from parasitic flatworm Fasciola gigantica using glutathione system
Accepted Manuscript Title: Preferential regeneration of thioredoxin from parasitic flatworm Fasciola gigantica using glutathione system Author: Ankita Gupta Tripti Pandey Bijay Kumar Timir Tripathi PII: DOI: Reference:
S0141-8130(15)00650-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.09.035 BIOMAC 5371
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
13-7-2015 3-9-2015 21-9-2015
Please cite this article as: A. Gupta, T. Pandey, B. Kumar, T. Tripathi, Preferential regeneration of thioredoxin from parasitic flatworm Fasciola gigantica using glutathione system, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.09.035 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.
*Abstract
ABSTRACT The maintenance of cellular redox homeostasis is a crucial adaptive problem faced by parasites, and its disruption can shift the biochemical balance toward the host. The thioredoxin (Trx) system plays a key role in redox metabolism and defense against oxidative stress. In this study,
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biochemical experiments were performed on Fasciola gigantica Thioredoxin1 (FgTrx1). The recombinant FgTrx1 exists as a monomer and catalyzes the reduction of insulin. FgTrx1 is
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preferentially regenerated by the glutathione (GSH) system using glutathione reductase (GR). The regeneration of FgTrx1 by the conventional Trx system is much less as compared to the
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GSH system, suggesting that FgTrx1 could be acting as glutaredoxin (Grx). DNA nicking and hydroperoxide assay suggests that it protects the DNA from radical-induced oxidative damage.
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Thus, FgTrx1 might play a role in parasite survival as it can regenerate itself even in the absence of the canonical Trx system and also protect the cells from ROS induced damage. Further, we propose that the GR activity of FgTrx1 is not restricted to -CXXC- motif but is regulated by
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residues present in close proximity to the -CXXC- motif, through manipulation of the redox
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GRAPHICAL ABSTRACT
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potential or the pKa of the active site Cys residues.
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*Manuscript Click here to view linked References
Preferential regeneration of thioredoxin from parasitic flatworm Fasciola gigantica using glutathione system
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Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern
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Running title: Properties of Fasciola gigantica Thioredoxin1
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Hill University, Shillong -793022, India
To whom correspondence should be addressed: Dr. Timir Tripathi, Assistant Professor,
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*
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Ankita Gupta1, Tripti Pandey1, Bijay Kumar1, and Timir Tripathi1*
Department of Biochemistry, North-Eastern Hill University, Shillong- 793022, India. Email:
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[email protected], [email protected]; Tel: +91-364-2722141; Fax: +91-364-2550108.
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ABSTRACT
The maintenance of cellular redox homeostasis is a crucial adaptive problem faced by parasites,
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and its disruption can shift the biochemical balance toward the host. The thioredoxin (Trx)
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system plays a key role in redox metabolism and defense against oxidative stress. In this study, biochemical experiments were performed on Fasciola gigantica Thioredoxin1 (FgTrx1). The recombinant FgTrx1 exists as a monomer and catalyzes the reduction of insulin. FgTrx1 is preferentially regenerated by the glutathione (GSH) system using glutathione reductase (GR). The regeneration of FgTrx1 by the conventional Trx system is much less as compared to the GSH system, suggesting that FgTrx1 could be acting as glutaredoxin (Grx). DNA nicking and hydroperoxide assay suggests that it protects the DNA from radical-induced oxidative damage. Thus, FgTrx1 might play a role in parasite survival as it can regenerate itself even in the absence of the canonical Trx system and also protect the cells from ROS induced damage. Further, we propose that the GR activity of FgTrx1 is not restricted to -CXXC- motif but is regulated by residues present in close proximity to the -CXXC- motif, through manipulation of the redox potential or the pKa of the active site Cys residues.
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Keywords: Redox; antioxidant; thioredoxin; liver fluke; glutathione; free radicals; oxidative stress; parasite.
1. INTRODUCTION
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Fascioliasis, a food-borne zoonotic disease of livestock and humans, is responsible for global annual health issues and economic losses that are estimated to be at several billion USD. It is the
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most neglected tropical disease. WHO has estimated that at least 2.4 million people are infected with the disease in more than 70 countries worldwide, and several million are at risk,
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particularly, in developing countries like India. Fascioliasis is caused by the liver flukes, Fasciola hepatica and Fasciola gigantica, which are parasitic trematodes [1]. Their adaptation to
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different habitats is potentially linked to their wide geographical distribution and their ability to infect a large population intensely. F. hepatica is found in the temperate regions while F. gigantica is customary in the tropics [1-3]. Animal-based crop cultivation is still in practice
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mostly in the tropical regions of Asia and Africa for sustained economic growth. In these regions, fascioliasis is prevalent and considered to be one of the foremost sources of various
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livestock’s (especially, cattle and buffaloes) diseases. The farm animals’ feces are the main route of transmission of infection in the environment. Recent reports have suggested that, in some
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areas, the disease transmission to humans has increased and become intense [4-6]. In addition,
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reports have suggested the increasing resistance of the disease against triclabendazole, which is the only WHO-recommended drug against fascioliasis [7-9]. During the progressive life cycle between the primary and secondary hosts, flukes are exposed to free radicals and reactive species to a greater extent. Homeostatic equilibrium imbalance between the reactive oxygen species (ROS) or reactive nitrogen species (RNS) and biological antioxidant molecules causes oxidative stress, leading to the modification of essential biomolecules such as DNA, lipids, and proteins [10, 11]. Appropriate levels of ROS and RNS are necessary for the normal physiological functioning of the living organisms since the redox status of cells regulates the various transcription factors/activators via activator protein-1, NFκB, p53 etc. and thus influences gene expression and modulating cellular signaling pathway [1215]. Excessive redox-active species may damage DNA, repress the activity of cellular enzymes, and induce cell death due to the activation of kinases and caspase cascades [16, 17]. Thus, to protect themselves from oxidative damage and maintain homeostasis, aerobic organisms have 2 Page 3 of 33
developed effective and efficient defense mechanisms consisting of both enzymatic and nonenzymatic molecules. This indicates that the redox-active protein system can be a potential drug target. Trx and GSH systems are two different systems that concurrently maintain the reduced
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state of proteins and thereby govern the regulation of redox homeostasis in most organisms [1820]. The Trx system consists of Trx, thioredoxin reductase (TrxR), thioredoxin peroxidase (TPx),
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and NADPH while the GSH system consists of GSH, Grx, GR, glutathione peroxidase (GPx), and NADPH. Trx is an important redox-active enzyme whose key function involves reduction of
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disulfides linkage in the proteins. After target reduction, Trx is brought back to its reduced state by TrxR that obtains the electrons from NADPH [18, 21, 22]. In addition, Trx also performs
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several other important functions inside the cell such as protecting cells from toxicants, especially oxidants and electrophiles [20, 23]. Trxs not only act as a scavenger but also function as key regulators of ROS-induced signaling transducers [16]. It also exerts anti-inflammatory
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effects in various tissues [24]. Trx is released from cells in response to oxidative stress, and shows cytoprotective effects under oxidative and inflammatory conditions. Trx treatment could
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inhibit the synthesis of multiple pro-inflammatory cytokines and chemokines in animal models of inflammatory disorders [25, 26]. Trx also has extracellular chemotactic activity [27] and
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regulates the expression of cytokines as a potent costimulator [28]. Structurally, Trxs are smallsized (around 10–12 kDa) thiol disulfide-oxidoreductases, containing two conserved Cys
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residues (-CXXC-) in their active site motif [29-31]. They consist of four to five β-strands surrounded by four α-helices [32]. A small variation in the polarity of amino acid residues near the active site can cause functional differences in proteins having Trx-fold domains [29, 33-38]. A few studies have revealed the prominent involvement of the two internal residues in between active site Cys and their effects on redox potential [39-41]. Although these residues are important, they are not solely responsible for the differences in activity between Trx-fold containing proteins.
In contrast to other organisms, helminth parasites have unique, linked Trx-GSH system, where a single flavoenzyme thioredoxin glutathione reductase (TGR) harbors the functions of both TrxR and GR [42]. A Trx-transmembrane-related protein has been identified in Chlonochis sinensis (CsTMX), which play an important role in the host–parasite interaction, and is probably involved in immunoregulation of host by inducing Th1-type dominated immune response in rats 3 Page 4 of 33
[43]. Recombinant Ophisthorchis viverrini Trx was able to reduce insulin and supported the enzymatic function of O. viverrini TPx [44]. Trx was detected in the human skin proteome after the invasion of the S. mansoni larvae [45] and also in thier egg secretions [46]. Proteomic and transcriptomic analysis of the secretome of F. hepatica revealed the presence of Trx in both the
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juvenile and adult form [47, 48]. Furthermore, recent study showed that O. viverrini Trx was able to prevent the apoptosis of bile duct epithelium cells induced by hydrogen peroxide [49]. A
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Trx was detected in the tegument and in the excretory–secretory products of both juvenile and adult from of F. hepatica (FhTrx) and was therefore exposed to the immune response [47, 50,
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51]. The crystal structure of FhTrx has been solved at 1.45 Å in both its oxidized and reduced forms [52]. Recently, Changklungmoa et al. cloned F. gigantica Trx and studied its expression
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pattern during different propagation cycles. They proposed that it could be considered as a novel vaccine or drug target for antihelminthic therapy [53]. To understand and characterize the redox metabolism of parasitic trematodes, we have investigated Thioredoxin1 of the liver fluke F.
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gigantica (FgTrx1) and reported its structure-function properties. Our results suggest interesting
2. MATERIAL AND METHODS
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attributes of Trx in platyhelmenthic parasites.
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The molecular biology kits and nickel-nitrilotriacetic acid (Ni-NTA) agarose matrix were
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purchased from Qiagen, CA, USA. The deoxynucleotide triphosphates (dNTPs) and enzymes were purchased from New England Biolabs, MA, USA. The TrxR enzyme was purchased from Sigma-Aldrich Chemical Company, St. Louis, MO, USA, while the GR was purchased from MP Biomedicals, CA, USA. All other reagents and chemicals were purchased either from the SigmaAldrich Chemical Company, USA or Sisco Research Laboratories, Mumbai, India and were of the highest purity available. Bacterial culture media were purchased from Himedia Laboratories, Mumbai, India.
2.1 In silico analysis Subcellular localization prediction analysis was performed using TargetP1.1 server and Sherloc2 [54, 55]. For multiple sequence alignment, the FhTrx was aligned with FgTrx1 using the ESPript 3.0 software that utilizes the ClustalW algorithm [56, 57]. The molecular model of FgTrx1 was created with Swiss-model based on the template of FhTrx (PDB ID- 2VIM). The images were 4 Page 5 of 33
visualized using UCSF Chimera 1.9.
2.2 Fluke collection, RNA isolation, and cDNA synthesis Adult F. gigantica samples were collected from the naturally-infected liver of a pig from Bada
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Bazaar slaughterhouse, Shillong, India and washed thoroughly with chilled PBS buffer (pH 7.4). The total RNA isolation was carried out using the RNAse easy mini kit (Qiagen, USA), and the
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first strand cDNA synthesis was performed using QuantiTect reverse transcription kit (Qiagen, USA). The ethical guidelines with the recommendations from the Institutional Ethics Committee,
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North Eastern Hill University, were strictly followed.
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2.3 Construction of recombinant plasmid and cloning of trx1 gene
The trx1 gene was amplified using specific primers bearing different restriction sites. The sequences of the forward and reverse primers were 5 ′-CGGGATCCATGCGGCTCTTG-3′ and
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5′-CCCAAGCTTTTATTTGTGCCTAG-3′ having BamHI and HindIII restriction sites (underlined), respectively. The PCR consisted of a denaturation step at 98°C for 30 sec, an
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annealing step at 52°C for 10 s, and an extension step at 72°C for 10 s, which was performed for 30 cycles. The amplified 315 bp product was inserted into the pSK+ vector, and the recombinant
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plasmid (pSK+-trx1) was transformed into Escherichia coli DH5α cells. The inserts from the
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obtained clones were sequenced using an automatic DNA sequencer, and the gene, without any mutation, was inserted into the pQE30 vector at the defined restriction sites. The recombinant plasmid was then introduced into E. coli M15 cells by transformation and was selected on LuriaBertini (LB) agar plates supplemented with ampicillin (100 μg/ml) and kanamycin (25 μg/ml).
2.4 Soluble expression and purification of the recombinant enzyme A single colony of E. coli M15 transformed with the pQE30-trx1 plasmid was selected for the study. Overnight cultures were diluted 1/200 in fresh LB broth supplemented with 100 μg/ml ampicillin and 25 μg/ml kanamycin and grown at 37°C with continuous shaking at 180 rpm until an OD600 of 0.6 was achieved. The expression of recombinant protein was induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), followed by incubation at 37°C with continuous shaking. The cells were harvested after 4 h of induction. The induced E. coli M15 cells were suspended in 30 ml of lysis buffer (50 mM NaH2PO4, pH 8.0 and 300 mM NaCl) supplemented 5 Page 6 of 33
with protease inhibitors. The suspended cells were lysed by sonication with pulse‒rest cycle (45 cycles; 30 s pulse at 50% amplitude with 30 s interval after each pulse). The lysate was centrifuged at 20,000 g for 30 min at 4°C. The supernatant of the cell lysate was passed through the Ni-NTA matrix. The unbound supernatant was washed with buffer A (50 mM NaH2PO4, pH
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8.0; 300 mM NaCl; and 50 mM imidazole) until the OD reached the baseline. Elution of the bound His-tagged protein was carried out with 10 ml buffer B (50 mM NaH2PO4, pH 8.0; 300
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mM NaCl; and 200 mM imidazole). The samples obtained were then analyzed by 15% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE), and the fractions containing FgTrx1 were
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pooled and dialyzed overnight against 20 mM NaH2PO4 (pH 7.5) and 150 mM NaCl at 4°C.
2.5 Determination of the molecular mass by size exclusion chromatography (SEC)
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To determine the native molecular mass of FgTrx1, SEC was performed on a SuperdexTM S-75 column (GE Healthcare Biosciences, USA). The calibration curve was prepared using the
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correlation of the molecular mass of standard proteins vs. the elution volume for the following proteins: conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease
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A (13.7 kDa), and aprotinin (6.5 kDa) (Gel Filtration Calibration Kit, GE Healthcare
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Biosciences).
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2.6 Determination of enzyme kinetics
All the enzymatic reactions were carried in a 1 ml quartz cuvette of path-length 1 cm, with a total volume of 1 ml. The data was recorded using a Varian Cary 50 Bio UV-Visible spectrophotometer. Kinetic calculations were performed using the GraphPad Prism software. Three replications were conducted, and background data were subtracted for all the experiments. The error bars represent the mean of triplicate samples.
2.7 Insulin-based Trx activity
The insulin-based reduction assay was carried out to determine the Trx activity. In the presence of recombinant FgTrx1, an increase in OD was observed at 650 nm [58]. The 1 ml of assay mixture contained 0.17 mM porcine insulin, 20 mM NaH2PO4 (pH 7.5), 150 mM NaCl, and 0.33 mM DTT (Dithiothreitol). The reaction was initiated by adding 2.5 μM FgTrx1 at 25°C. Blank reaction (non-enzymatic reactions) involves insulin reduction in the presence of DTT without 6 Page 7 of 33
FgTrx1. The pH optimum was determined for insulin activity using citrate/glycine/hepes (CGH) buffer of various pH values. Purified FgTrx1 was incubated at 30°C for 30 min in CGH buffer of pH values ranging from 6.0 to 8.5.
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2.8 TrxR and GR assays
The TrxR activity was measured by carrying out insulin reduction [59]. The final reaction
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volume of 1 ml, contained 20 mM NaH2PO4 (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2.5 μM FgTrx1, 0.13 mM insulin, and 200 μM NADPH. The enzymatic assay was started by adding rat
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liver TrxR (50 nM) at 25°C. The insulin reduction assay was also used to measure the GR activity. The only difference was, instead of using TrxR, the enzymatic reaction was initiated by adding yeast GR (50 nM) at 25°C [60]. The decrease in the absorbance at 340 nm as a result of
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NADPH (ε340nm= 6.22 mM-1cm-1) oxidation was monitored.
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2.9 Glutathione: bis-(2-hydroxy ethyl) disulfide transhydrogenase assay (HED assay) The 1 ml of assay mixture contained 20 mM NaH2PO4 (pH 7.5), 150 mM NaCl, 2.5 μM FgTrx1,
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200 μM NADPH, 50 nM GR or 50 nM TrxR, and 750 μM HED. After 5 min of incubation, GSH was added, and the NADPH consumption was monitored spectrophotometrically at 340 nm at
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25°C [60]. In this coupled assay system, high concentrations of GSH were constantly maintained
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by the NADPH/(GR or TrxR) system. The activities were corrected for the rate of the spontaneous chemical reaction of HED with GSH by using a reference sample without FgTrx1.
2.10 DNA protection assay
The pUC19 plasmid DNA was used as a substrate for the detection of DNA damage mediated by the metal catalyzed oxidation (MCO) or mixed function oxidase (MFO) system, which generates hydroxyl and thiol radicals capable of nicking the DNA template [60-62]. The MCO system consists of 1 mM FeCl3 and 1 mM DTT in 25 mM HEPES buffer (pH 7.5). 800 ng of plasmid DNA was incubated in the MCO system without or with the recombinant FgTrx1 at 37°C for 30 min. The degree of DNA damage was studied by monitoring the change in the intensity of a plasmid when it converts from supercoiled to nicked form. The extent of MCO-mediated DNA nicking was observed through ethidium bromide-staining in 1% agarose gel.
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2.11 Hydroperoxide assay The hydroperoxide reaction assay contained 2.5 µM FgTrx1, 50 nM TrxR or GR, 1 mM GSH, and 250 µM H2O2. Measurement was taken after the addition of 200 µM NADPH. The decrease in the absorbance at 340 nm as a result of NADPH (ε340nm= 6.22 mM-1cm-1) oxidation was
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monitored [60, 63].
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3. RESULTS 3.1 In silico analysis
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The screening of the transcriptome data with different known Trx sequences led to the identification of two Trxs in the liver fluke, F. gigantica —one with an active site motif -
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WCGPC- and the other with -WCTPC- —; the proteins are designated as FgTrx1 and FgTrx2, respectively. Prediction analyses for the cellular localization through TargetP1.1 server and Sherloc2 predominantly hypothesized the localization of FgTrx1 to cytoplasm and FgTrx2 to
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mitochondria. Outside the active site (Cys32 and Cys35), FgTrx1 did not contain any additional Cys residues that are present in many Trx fold-containing proteins (in higher eukaryotes) and
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have been proposed to have potential regulatory roles [31-33].
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3.2 FgTrx1 exists as a monomer under native conditions
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We observed that the N-terminus His6-FgTrx1 protein was over-expressed and more than 90% of protein was in the soluble fraction of the culture. The presence of the His-tag enabled the single-step purification of the protein using Ni-NTA matrix. Initially, the protein purity and homogeneity was confirmed by SDS-PAGE (Figure 1A), and the presence of the His-tagged recombinant FgTrx1 was validated by immunoblot analysis (Figure not shown). The molecular mass of the protein was found to be approximately 12 kDa. Using SuperdexTM S-75 gel filtration column, the oligomeric status of the protein was determined to include a retention volume of 14.3 ml corresponding to a protein of 12 kDa when compared to molecular weight standards (Figure 1B). These results indicate that FgTrx1 is stabilized in a monomeric state under native conditions.
3.3 Catalyzed reduction of insulin by FgTrx1 The active site of Trxs containing Cys residues are involved in the reversible oxidation and 8 Page 9 of 33
reduction of disulfide bonds in proteins that is necessary to maintain in the intracellular milieu. The DTT-mediated reduction of the disulfide bonds of insulin showed no activity in the reaction mixture containing only DTT even after 60 min. However, the reaction mixture containing both DTT and FgTrx1 showed precipitation of the insulin within 5 min from the starting of the
highest activity was at pH 7.5 (Figure 2B).
3.4 Preferential regeneration of FgTrx1 by the GSH system
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reaction (Figure 2A). The insulin assay carried out at different pH from 6 to 8.5 showed that the
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Grxs are the oxidoreductases that catalyze the reduction of disulfide or GSH-mixed disulfide in a coupled reaction with GSH, NADPH, and GR. The examination of the activity of FgTrx1 with
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both the Trx and GSH system using TrxR and GR based insulin reduction assays showed that FgTrx1 was unable to reduce insulin using GR in the absence of GSH. However, in the presence of GSH, FgTrx1 was regenerated by GR, thereby catalyzing insulin reduction. Figure 3 shows
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the GSH-linked activity of FgTrx1. HED was the classical substrate used to study the thioltransferase activity. It was found that the coupling of FgTrx1 with GSH and GR reduced the
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HED. In contrast, there was negligible HED activity with TrxR even in the presence of GSH
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(Figure 4). This confirms the preferential regeneration of FgTrx1 by GSH system.
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3.5 Protection of DNA from oxidative damage by FgTrx1 Few reports suggest that Trx can play a direct role as an antioxidant [17, 19]. In order to examine the antioxidant role of FgTrx1, a DNA nicking assay was performed to understand the ability of FgTrx1 as a DNA protector. The assay used metal catalyzed oxidation that generated free radicals that damaged DNA. It was found that when the supercoiled plasmid was exposed to the MCO assay, it was almost completely converted to nicked form within 30 min; however, when FgTrx1 was added to the assay, it resulted in protection from nicking. It was found that 5 µM of FgTrx1 protected at least 30% of the plasmid DNA from nicking (Figure 5A), suggesting that the FgTrx1 can protect DNA from free radical damage. The radical scavenging activity of FgTrx1 was further confirmed using hydroperoxide assay; FgTrx1 was found to be highly active in the peroxide assay. Figure 5B shows the summary of the assay. The Km of FgTrx1 for H2O2 (73.4 ± 13.0 μM) was found to be low, suggesting its high affinity towards H2O2. In comparison to FgTrx1, the Km of S. mansoni Trx with H2O2 is much lower (~5 μM) [64], indicating that its 9 Page 10 of 33
affinity is higher than that of FgTrx1. Though, the Km of FgTrx1 is less than that of SmTRx, it lies in the micromolar range, suggesting its high affinity towards H2O2. The Vmax of FgTrx1 was observed to be around 1.28 ± 0.08 µmol.min-1, which is comparable to that of SmTrx (1.5
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µmol.min-1)[64].
4. DISCUSSION
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Members of the Trx superfamily share two common features: (a) they contain an active site motif -CXXC-, and (b) the overall structure containing this motif corresponds to a Trx-like fold.
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The amino acid sequence of F. hepatica and F. gigantica shows more than 96% identity (Figure 6A), suggesting that both the proteins have a similar structure and biochemical properties. The molecular modeling of FgTrx1 based on the template of FhTrx shows that the overall
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architecture of FgTrx1 consists of a typical Trx-topology with four stranded β-sheets surrounded by four α-helices (Figure 6B). The analysis of the Ramachandran plot using PROCHECK
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indicates that 92.6% of the amino acid residues are within the most favored region while 7.4% residues are in the additionally allowed region. These two structures generate an RMSD of 0.07
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Å over 104 alpha carbon pairs, suggesting negligible spatial variation between them. Similar to FhTrx, the active site of FgTrx1 contains a redox-sensitive Cys pair -31CGPC34- that can be
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either oxidized in disulfide state or reduced in dithiol state. The active site also contains a
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conserved Trp30 residue preceding the -31CGPC34- motif. In FgTrx1, Trp30 is found on the surface of the protein and forms a flat surface close to the active site, which is similar to that of FhTrx (Figure 6C).
The results of the biochemical studies demonstrated that the most important feature of FgTrx1 is its preferential regeneration with the GSH system. In addition, FgTrx1, a disulfide oxidoreductase, is unique in its enzymatic behavior with various substrates such as HED, insulin, GR, and TrxR. The HED activity is considered to be specific for Grxs; however, a few reports have suggested that Trx can also be active against HED [65]. Recent reports showed that Haemonchus contortous [60] and some plant Trxs may be reduced by the GSH system [65]. It has been proposed that the presence of unique Arg and Ser residues in the active site -CRSCmotif is responsible for the GR-like activity in HcTrx5 [60]. FgTrx1 has also been shown to be active with GSH and GR; however, in contrast to HcTrx5, it does not contain these residues. Hence, we have proposed that, apart from the active site residues, some other residues could be 10 Page 11 of 33
involved in the activity of FgTrx1 with GR. Molecular modeling of FgTrx1 shows the presence of three charged amino acid residues, Gln29, Arg35, and Asn36, near the active site region that can influence the reduction potential and pKa values and can be responsible for Grx-like activity of FgTrx1 (Figure 6D). The effect of neighboring residues on Trx like activity and specificity for
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different substrate should be further studied. The active Cys residues of -CXXC- motif lies in the N-terminal region of α2-helix [66]. In E. coli Trx, the substitution of Lys for Gly at the first X
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does not affect the redox potential but affects the interaction of EcTrx with other proteins, which in turn decreases the catalytic efficiency of the enzyme [67]. In case of Grx, the presence of basic
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amino acid environment around the active site has been considered to be responsible for interaction with insignificant changes in pKa at N-terminal Cys [68]. In HcTrx5, the Arg residue,
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present between the active site -CRSC-, has been suggested to mediate critical proton transfer during the catalytic cycles, which is similar to Grx containing the conserved Arg residue in its active site [69]. E. coli -CVWC- Trx mutation has shown to have an increased reduction
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potential but a decreased pKa compared to the wild type -CGPC- Trx. The addition of Val and Trp residues in the active site has been found to alter the catalytic properties without varying the
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structure. The comparative analysis of DsbD transmembrane thiol:disulfide oxidoreductase clearly shows the catalytic activity of Trx-fold containing protein is not solely driven by their -
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CXXC- motif [70]. The functionality of -CXXC- motif depends on the balance between the
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reduction potential (dithiol-disulfide equilibrium) and pKa value (protonation state). Such equilibrium involves a proton moment that in turn regulates the reduction potential. It has been proposed that an extended active site motif of the Trx superfamily proteins and residues forming the α1 helices and β1, β2 strands could predominantly influence the pKa value of the reactive Cys [70]. The evolutionary change in Trx superfamily proteins has resulted in a family of enzymes (Trx, PDI, DsbA, etc) that have a similar structure but different specificity for their substrate with distinguished efficiency and catalytic mechanism. These enzymes have evolved in such a way that they can act in different environments, such as the cytoplasm, periplasm or endoplasmic reticulum, under different environmental conditions [71-73]. Free radicals can oxidize supercoiled DNA, which result in DNA nicking. We have utilized the MCO/MFO assay to carry out this experiment. MCO system is composed of Fe 3+, O2, and DTT as an electron donor. DTT oxidation produces H2O2 in a reaction catalyzed by Fe3+, which then reacts in a metal-catalyzed Fenton reaction to produce the ultimate toxic species, 11 Page 12 of 33
OH. It is well established that a DTT/Fe3+/O2 system induces strand breaks in plasmid and mammalian DNA [74-76]. The results of DNA nicking assay showed the ability of FgTrx1 to protect DNA against the free radical species during oxidative stress thereby suggesting the antioxidant role of FgTrx1. This type of DNA protection has previously been reported in some
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plant and parasite Trxs [61, 77]. The data suggest that FgTrx1 concentration dependently protects plasmid DNA from oxidative damage. To endorse the specificity of the antioxidant
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activity of FgTrx1, similar experiments were performed in the presence of bovine serum albumin (BSA) replacing FgTrx1, but BSA did not protect DNA from oxidative damage by free radical
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damage (data not shown). We further confirmed the free radical scavenging property of FgTrx1 by the hydroperoxide assay, and FgTrx1 was found to be highly active, thereby, establishing its
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antioxidant functions.
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5. CONCLUSIONS
Growing evidence suggests that redox-active proteins are not only involved in target protein reduction, but also linked with the detoxification of ROS. Trx is a highly conserved
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oxidoreductase, which regulates the reduction of many important proteins. The present
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biochemical observations suggested that Trx1 of F. gigantica might play a valuable role in parasitic survival within the host as it participates not only in linking the Trx and GSH system
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but also in protecting cells from ROS-induced oxidative damage. Thus, their inhibition could be tested for the development of novel therapeutic compounds against fascioliasis. Data mining of helminthic parasites has provided a clue for the presence of several redox-active proteins. Complete elucidation of the redox-sensitive protein system in Fasciola can suggest the mechanism and evolutionary conservation of Trx and GSH system in helminths, as well as help in evaluating their drug target potential. We plan to characterize these proteins in our future studies.
ACKNOWLEDGEMENTS The study was supported by a research grant from Department of Biotechnology, Govt of India, New Delhi, India to TT (Grant no. BT/28/NE/TBP/2010 dated 28.02.2011). AG thanks DBT for providing fellowship. Authors thank A. Sharma and M. Yogavel, ICGEB, New Delhi, India and Banchob Sripa, Khon Kaen University, Thailand for constructive suggestions and valuable 12 Page 13 of 33
scientific discussions. Thanks also go to N. D. Young, The University of Melbourne, Australia for generously providing the transcriptome data of F. gigantica.
FOOTNOTES
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The coordinates for the model of FgTrx1 has been submitted to the Protein Model Database with
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the following id: PM0080291.
ABBREVIATIONS
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Trx, thioredoxin; Grx, glutaredoxin; TrxR, thioredoxin reductase; GR, glutathione reductase,
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GSH, reduced glutathione; HED, 2-hydroxyethyldisulfide; DTT, dithiothreitol.
CONFLICT OF INTEREST
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The authors declare that there are no conflicts of interests.
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FIGURE LEGENDS
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Figure 1. Purification and oligomeric status of FgTrx1. (A) SDS-PAGE profile of recombinant FgTrx1. Lane 1: Molecular weight marker; Lane 2: un-induced culture of E. coli extract; Lane 3: induced culture of E. coli extract; Lane 4: purified FgTrx1. (B) Size exclusion
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profile of FgTrx1. The peak shows a monomeric structure of proteins in Superdex S-75TM
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column. The inset shows the column calibration curve.
Figure 2. Insulin assay. (A) Dithiothreitol mediated reduction of insulin by FgTrx1. The
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increase in OD at 650 nm was measured and plotted against time. The reaction mixture contains 0.33 mM DTT, 0.17 mM insulin, and 2 mM EDTA in 20 mM NaH2PO4 (pH 7.5) with 2.5 µM of
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FgTrx1 (solid line). Dotted line represents the control reaction in the absence of FgTrx1. (B)
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Effect of pH on the insulin activity of purified FgTrx1. Purified FgTrx1 was incubated at 30°C for 30 min in CGH buffer of pH values ranging from 6.0 to 8.5, and then readings were taken as above. Three replications were conducted, and background data were subtracted for all the experiments.
Figure 3. GSH-linked activity of FgTrx1. FgTrx1 activity with GSH and Trx system was determined by the insulin disulfide reduction assay. The reaction mixture contained 2.5 µM of FgTrx1, 200 µM of NADPH, 0.17 mM of insulin, 1 mM of GSH, 50 nM of GR or TrxR and 2 mM EDTA in 20 mM NaH2PO4 (pH 7.5). Absorbance was monitored at 340 nm for NADPH consumption.
Figure 4. HED assay using TrxR and GR. (A) Reduction of the disulfide bond in HED by FgTrx1 using TrxR; (B) Reduction of the disulfide bond in HED by FgTrx1 using GR. In both 17 Page 18 of 33
the experiments, the reaction mixture contains 2.5 µM FgTrx1, 200 µM of NADPH, 750 µM of HED, 50 nM of TrxR or GR and 2 mM EDTA in 20 mM NaH2PO4 (pH 7.5). After 5 min of incubation, GSH was added, and the reaction was monitored at 340 nm. Solid blocks represent the reaction in the presence of GSH while the hollow box represents reaction in the absence of
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GSH.
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Figure 5. Antioxidant property of FgTrx1. (A) The protection of DNA from nicking was studied using MCO assay. The system consists of 25 mM HEPES (pH 7.5), 1 mM DTT, 1 mM
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FeCl3, and 800 ng of supercoiled pUC19 plasmid DNA in each lane. Lane 1: pUC19 plasmid alone with no incubation with MCO, Lane 2: pUC19 plasmid incubated in MCO for 30 min
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without FgTrx1, Lane 3-7: pUC19 plasmid incubated in MCO with 1-5 µM of FgTrx1 for 30 min. (B) Hydroperoxide assay of FgTrx1. The reaction mixture contains 2.5 µM of FgTrx1, 50 nM of TrxR or GR, 1 mM GSH, 200 µM H2O2, and 250 µM NADPH in 20 mM NaH2PO4 buffer
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pH 7.5.
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Figure 6. In silico analysis of FgTrx1. (A) Multiple sequence alignment of FgTrx1 with FhTrx is shown. Identical residues are shown in red boxes, similar residues in yellow boxes while
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amino acid residue having different properties does not have any box. (B) Cartoon diagram of
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the FgTrx1 monomer structure. Secondary structural elements are labeled along with the protein termini. (C) Zoom view of the FgTrx1 showing the active site Cys residues (C31 and C34) in dithiol state in red. The surface Trp30 is shown in pink. (D) Close-up view of the active site region of FgTrx1 showing the Gln29, Arg35, and Asn36 residues in sticks along with the Cys31 and Cys34 arranged in ball and stick model. The 3D model was made using Swiss-model and visualized with UCSF Chimera 1.9.
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Highlights (for review)
HIGHLIGHTS Structure-function studies with FgTrx1 were performed.
FgTrx1 is preferentially regenerated by the GSH system.
Our data suggests that it protects cells from ROS induced damage.
Residues present in close proximity to the -CXXC- motif may regulate the GR activity of
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S0141-8130(15)00650-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.09.035 BIOMAC 5371
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
13-7-2015 3-9-2015 21-9-2015
Please cite this article as: A. Gupta, T. Pandey, B. Kumar, T. Tripathi, Preferential regeneration of thioredoxin from parasitic flatworm Fasciola gigantica using glutathione system, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.09.035 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.
*Abstract
ABSTRACT The maintenance of cellular redox homeostasis is a crucial adaptive problem faced by parasites, and its disruption can shift the biochemical balance toward the host. The thioredoxin (Trx) system plays a key role in redox metabolism and defense against oxidative stress. In this study,
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biochemical experiments were performed on Fasciola gigantica Thioredoxin1 (FgTrx1). The recombinant FgTrx1 exists as a monomer and catalyzes the reduction of insulin. FgTrx1 is
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preferentially regenerated by the glutathione (GSH) system using glutathione reductase (GR). The regeneration of FgTrx1 by the conventional Trx system is much less as compared to the
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GSH system, suggesting that FgTrx1 could be acting as glutaredoxin (Grx). DNA nicking and hydroperoxide assay suggests that it protects the DNA from radical-induced oxidative damage.
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Thus, FgTrx1 might play a role in parasite survival as it can regenerate itself even in the absence of the canonical Trx system and also protect the cells from ROS induced damage. Further, we propose that the GR activity of FgTrx1 is not restricted to -CXXC- motif but is regulated by
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residues present in close proximity to the -CXXC- motif, through manipulation of the redox
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GRAPHICAL ABSTRACT
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potential or the pKa of the active site Cys residues.
1 Page 1 of 33
*Manuscript Click here to view linked References
Preferential regeneration of thioredoxin from parasitic flatworm Fasciola gigantica using glutathione system
1
Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern
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Running title: Properties of Fasciola gigantica Thioredoxin1
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Hill University, Shillong -793022, India
To whom correspondence should be addressed: Dr. Timir Tripathi, Assistant Professor,
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*
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Ankita Gupta1, Tripti Pandey1, Bijay Kumar1, and Timir Tripathi1*
Department of Biochemistry, North-Eastern Hill University, Shillong- 793022, India. Email:
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[email protected], [email protected]; Tel: +91-364-2722141; Fax: +91-364-2550108.
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ABSTRACT
The maintenance of cellular redox homeostasis is a crucial adaptive problem faced by parasites,
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and its disruption can shift the biochemical balance toward the host. The thioredoxin (Trx)
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system plays a key role in redox metabolism and defense against oxidative stress. In this study, biochemical experiments were performed on Fasciola gigantica Thioredoxin1 (FgTrx1). The recombinant FgTrx1 exists as a monomer and catalyzes the reduction of insulin. FgTrx1 is preferentially regenerated by the glutathione (GSH) system using glutathione reductase (GR). The regeneration of FgTrx1 by the conventional Trx system is much less as compared to the GSH system, suggesting that FgTrx1 could be acting as glutaredoxin (Grx). DNA nicking and hydroperoxide assay suggests that it protects the DNA from radical-induced oxidative damage. Thus, FgTrx1 might play a role in parasite survival as it can regenerate itself even in the absence of the canonical Trx system and also protect the cells from ROS induced damage. Further, we propose that the GR activity of FgTrx1 is not restricted to -CXXC- motif but is regulated by residues present in close proximity to the -CXXC- motif, through manipulation of the redox potential or the pKa of the active site Cys residues.
1 Page 2 of 33
Keywords: Redox; antioxidant; thioredoxin; liver fluke; glutathione; free radicals; oxidative stress; parasite.
1. INTRODUCTION
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Fascioliasis, a food-borne zoonotic disease of livestock and humans, is responsible for global annual health issues and economic losses that are estimated to be at several billion USD. It is the
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most neglected tropical disease. WHO has estimated that at least 2.4 million people are infected with the disease in more than 70 countries worldwide, and several million are at risk,
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particularly, in developing countries like India. Fascioliasis is caused by the liver flukes, Fasciola hepatica and Fasciola gigantica, which are parasitic trematodes [1]. Their adaptation to
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different habitats is potentially linked to their wide geographical distribution and their ability to infect a large population intensely. F. hepatica is found in the temperate regions while F. gigantica is customary in the tropics [1-3]. Animal-based crop cultivation is still in practice
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mostly in the tropical regions of Asia and Africa for sustained economic growth. In these regions, fascioliasis is prevalent and considered to be one of the foremost sources of various
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livestock’s (especially, cattle and buffaloes) diseases. The farm animals’ feces are the main route of transmission of infection in the environment. Recent reports have suggested that, in some
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areas, the disease transmission to humans has increased and become intense [4-6]. In addition,
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reports have suggested the increasing resistance of the disease against triclabendazole, which is the only WHO-recommended drug against fascioliasis [7-9]. During the progressive life cycle between the primary and secondary hosts, flukes are exposed to free radicals and reactive species to a greater extent. Homeostatic equilibrium imbalance between the reactive oxygen species (ROS) or reactive nitrogen species (RNS) and biological antioxidant molecules causes oxidative stress, leading to the modification of essential biomolecules such as DNA, lipids, and proteins [10, 11]. Appropriate levels of ROS and RNS are necessary for the normal physiological functioning of the living organisms since the redox status of cells regulates the various transcription factors/activators via activator protein-1, NFκB, p53 etc. and thus influences gene expression and modulating cellular signaling pathway [1215]. Excessive redox-active species may damage DNA, repress the activity of cellular enzymes, and induce cell death due to the activation of kinases and caspase cascades [16, 17]. Thus, to protect themselves from oxidative damage and maintain homeostasis, aerobic organisms have 2 Page 3 of 33
developed effective and efficient defense mechanisms consisting of both enzymatic and nonenzymatic molecules. This indicates that the redox-active protein system can be a potential drug target. Trx and GSH systems are two different systems that concurrently maintain the reduced
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state of proteins and thereby govern the regulation of redox homeostasis in most organisms [1820]. The Trx system consists of Trx, thioredoxin reductase (TrxR), thioredoxin peroxidase (TPx),
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and NADPH while the GSH system consists of GSH, Grx, GR, glutathione peroxidase (GPx), and NADPH. Trx is an important redox-active enzyme whose key function involves reduction of
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disulfides linkage in the proteins. After target reduction, Trx is brought back to its reduced state by TrxR that obtains the electrons from NADPH [18, 21, 22]. In addition, Trx also performs
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several other important functions inside the cell such as protecting cells from toxicants, especially oxidants and electrophiles [20, 23]. Trxs not only act as a scavenger but also function as key regulators of ROS-induced signaling transducers [16]. It also exerts anti-inflammatory
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effects in various tissues [24]. Trx is released from cells in response to oxidative stress, and shows cytoprotective effects under oxidative and inflammatory conditions. Trx treatment could
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inhibit the synthesis of multiple pro-inflammatory cytokines and chemokines in animal models of inflammatory disorders [25, 26]. Trx also has extracellular chemotactic activity [27] and
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regulates the expression of cytokines as a potent costimulator [28]. Structurally, Trxs are smallsized (around 10–12 kDa) thiol disulfide-oxidoreductases, containing two conserved Cys
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residues (-CXXC-) in their active site motif [29-31]. They consist of four to five β-strands surrounded by four α-helices [32]. A small variation in the polarity of amino acid residues near the active site can cause functional differences in proteins having Trx-fold domains [29, 33-38]. A few studies have revealed the prominent involvement of the two internal residues in between active site Cys and their effects on redox potential [39-41]. Although these residues are important, they are not solely responsible for the differences in activity between Trx-fold containing proteins.
In contrast to other organisms, helminth parasites have unique, linked Trx-GSH system, where a single flavoenzyme thioredoxin glutathione reductase (TGR) harbors the functions of both TrxR and GR [42]. A Trx-transmembrane-related protein has been identified in Chlonochis sinensis (CsTMX), which play an important role in the host–parasite interaction, and is probably involved in immunoregulation of host by inducing Th1-type dominated immune response in rats 3 Page 4 of 33
[43]. Recombinant Ophisthorchis viverrini Trx was able to reduce insulin and supported the enzymatic function of O. viverrini TPx [44]. Trx was detected in the human skin proteome after the invasion of the S. mansoni larvae [45] and also in thier egg secretions [46]. Proteomic and transcriptomic analysis of the secretome of F. hepatica revealed the presence of Trx in both the
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juvenile and adult form [47, 48]. Furthermore, recent study showed that O. viverrini Trx was able to prevent the apoptosis of bile duct epithelium cells induced by hydrogen peroxide [49]. A
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Trx was detected in the tegument and in the excretory–secretory products of both juvenile and adult from of F. hepatica (FhTrx) and was therefore exposed to the immune response [47, 50,
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51]. The crystal structure of FhTrx has been solved at 1.45 Å in both its oxidized and reduced forms [52]. Recently, Changklungmoa et al. cloned F. gigantica Trx and studied its expression
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pattern during different propagation cycles. They proposed that it could be considered as a novel vaccine or drug target for antihelminthic therapy [53]. To understand and characterize the redox metabolism of parasitic trematodes, we have investigated Thioredoxin1 of the liver fluke F.
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gigantica (FgTrx1) and reported its structure-function properties. Our results suggest interesting
2. MATERIAL AND METHODS
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attributes of Trx in platyhelmenthic parasites.
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The molecular biology kits and nickel-nitrilotriacetic acid (Ni-NTA) agarose matrix were
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purchased from Qiagen, CA, USA. The deoxynucleotide triphosphates (dNTPs) and enzymes were purchased from New England Biolabs, MA, USA. The TrxR enzyme was purchased from Sigma-Aldrich Chemical Company, St. Louis, MO, USA, while the GR was purchased from MP Biomedicals, CA, USA. All other reagents and chemicals were purchased either from the SigmaAldrich Chemical Company, USA or Sisco Research Laboratories, Mumbai, India and were of the highest purity available. Bacterial culture media were purchased from Himedia Laboratories, Mumbai, India.
2.1 In silico analysis Subcellular localization prediction analysis was performed using TargetP1.1 server and Sherloc2 [54, 55]. For multiple sequence alignment, the FhTrx was aligned with FgTrx1 using the ESPript 3.0 software that utilizes the ClustalW algorithm [56, 57]. The molecular model of FgTrx1 was created with Swiss-model based on the template of FhTrx (PDB ID- 2VIM). The images were 4 Page 5 of 33
visualized using UCSF Chimera 1.9.
2.2 Fluke collection, RNA isolation, and cDNA synthesis Adult F. gigantica samples were collected from the naturally-infected liver of a pig from Bada
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Bazaar slaughterhouse, Shillong, India and washed thoroughly with chilled PBS buffer (pH 7.4). The total RNA isolation was carried out using the RNAse easy mini kit (Qiagen, USA), and the
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first strand cDNA synthesis was performed using QuantiTect reverse transcription kit (Qiagen, USA). The ethical guidelines with the recommendations from the Institutional Ethics Committee,
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North Eastern Hill University, were strictly followed.
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2.3 Construction of recombinant plasmid and cloning of trx1 gene
The trx1 gene was amplified using specific primers bearing different restriction sites. The sequences of the forward and reverse primers were 5 ′-CGGGATCCATGCGGCTCTTG-3′ and
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5′-CCCAAGCTTTTATTTGTGCCTAG-3′ having BamHI and HindIII restriction sites (underlined), respectively. The PCR consisted of a denaturation step at 98°C for 30 sec, an
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annealing step at 52°C for 10 s, and an extension step at 72°C for 10 s, which was performed for 30 cycles. The amplified 315 bp product was inserted into the pSK+ vector, and the recombinant
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plasmid (pSK+-trx1) was transformed into Escherichia coli DH5α cells. The inserts from the
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obtained clones were sequenced using an automatic DNA sequencer, and the gene, without any mutation, was inserted into the pQE30 vector at the defined restriction sites. The recombinant plasmid was then introduced into E. coli M15 cells by transformation and was selected on LuriaBertini (LB) agar plates supplemented with ampicillin (100 μg/ml) and kanamycin (25 μg/ml).
2.4 Soluble expression and purification of the recombinant enzyme A single colony of E. coli M15 transformed with the pQE30-trx1 plasmid was selected for the study. Overnight cultures were diluted 1/200 in fresh LB broth supplemented with 100 μg/ml ampicillin and 25 μg/ml kanamycin and grown at 37°C with continuous shaking at 180 rpm until an OD600 of 0.6 was achieved. The expression of recombinant protein was induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), followed by incubation at 37°C with continuous shaking. The cells were harvested after 4 h of induction. The induced E. coli M15 cells were suspended in 30 ml of lysis buffer (50 mM NaH2PO4, pH 8.0 and 300 mM NaCl) supplemented 5 Page 6 of 33
with protease inhibitors. The suspended cells were lysed by sonication with pulse‒rest cycle (45 cycles; 30 s pulse at 50% amplitude with 30 s interval after each pulse). The lysate was centrifuged at 20,000 g for 30 min at 4°C. The supernatant of the cell lysate was passed through the Ni-NTA matrix. The unbound supernatant was washed with buffer A (50 mM NaH2PO4, pH
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8.0; 300 mM NaCl; and 50 mM imidazole) until the OD reached the baseline. Elution of the bound His-tagged protein was carried out with 10 ml buffer B (50 mM NaH2PO4, pH 8.0; 300
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mM NaCl; and 200 mM imidazole). The samples obtained were then analyzed by 15% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE), and the fractions containing FgTrx1 were
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pooled and dialyzed overnight against 20 mM NaH2PO4 (pH 7.5) and 150 mM NaCl at 4°C.
2.5 Determination of the molecular mass by size exclusion chromatography (SEC)
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To determine the native molecular mass of FgTrx1, SEC was performed on a SuperdexTM S-75 column (GE Healthcare Biosciences, USA). The calibration curve was prepared using the
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correlation of the molecular mass of standard proteins vs. the elution volume for the following proteins: conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease
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A (13.7 kDa), and aprotinin (6.5 kDa) (Gel Filtration Calibration Kit, GE Healthcare
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Biosciences).
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2.6 Determination of enzyme kinetics
All the enzymatic reactions were carried in a 1 ml quartz cuvette of path-length 1 cm, with a total volume of 1 ml. The data was recorded using a Varian Cary 50 Bio UV-Visible spectrophotometer. Kinetic calculations were performed using the GraphPad Prism software. Three replications were conducted, and background data were subtracted for all the experiments. The error bars represent the mean of triplicate samples.
2.7 Insulin-based Trx activity
The insulin-based reduction assay was carried out to determine the Trx activity. In the presence of recombinant FgTrx1, an increase in OD was observed at 650 nm [58]. The 1 ml of assay mixture contained 0.17 mM porcine insulin, 20 mM NaH2PO4 (pH 7.5), 150 mM NaCl, and 0.33 mM DTT (Dithiothreitol). The reaction was initiated by adding 2.5 μM FgTrx1 at 25°C. Blank reaction (non-enzymatic reactions) involves insulin reduction in the presence of DTT without 6 Page 7 of 33
FgTrx1. The pH optimum was determined for insulin activity using citrate/glycine/hepes (CGH) buffer of various pH values. Purified FgTrx1 was incubated at 30°C for 30 min in CGH buffer of pH values ranging from 6.0 to 8.5.
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2.8 TrxR and GR assays
The TrxR activity was measured by carrying out insulin reduction [59]. The final reaction
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volume of 1 ml, contained 20 mM NaH2PO4 (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2.5 μM FgTrx1, 0.13 mM insulin, and 200 μM NADPH. The enzymatic assay was started by adding rat
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liver TrxR (50 nM) at 25°C. The insulin reduction assay was also used to measure the GR activity. The only difference was, instead of using TrxR, the enzymatic reaction was initiated by adding yeast GR (50 nM) at 25°C [60]. The decrease in the absorbance at 340 nm as a result of
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NADPH (ε340nm= 6.22 mM-1cm-1) oxidation was monitored.
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2.9 Glutathione: bis-(2-hydroxy ethyl) disulfide transhydrogenase assay (HED assay) The 1 ml of assay mixture contained 20 mM NaH2PO4 (pH 7.5), 150 mM NaCl, 2.5 μM FgTrx1,
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200 μM NADPH, 50 nM GR or 50 nM TrxR, and 750 μM HED. After 5 min of incubation, GSH was added, and the NADPH consumption was monitored spectrophotometrically at 340 nm at
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25°C [60]. In this coupled assay system, high concentrations of GSH were constantly maintained
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by the NADPH/(GR or TrxR) system. The activities were corrected for the rate of the spontaneous chemical reaction of HED with GSH by using a reference sample without FgTrx1.
2.10 DNA protection assay
The pUC19 plasmid DNA was used as a substrate for the detection of DNA damage mediated by the metal catalyzed oxidation (MCO) or mixed function oxidase (MFO) system, which generates hydroxyl and thiol radicals capable of nicking the DNA template [60-62]. The MCO system consists of 1 mM FeCl3 and 1 mM DTT in 25 mM HEPES buffer (pH 7.5). 800 ng of plasmid DNA was incubated in the MCO system without or with the recombinant FgTrx1 at 37°C for 30 min. The degree of DNA damage was studied by monitoring the change in the intensity of a plasmid when it converts from supercoiled to nicked form. The extent of MCO-mediated DNA nicking was observed through ethidium bromide-staining in 1% agarose gel.
7 Page 8 of 33
2.11 Hydroperoxide assay The hydroperoxide reaction assay contained 2.5 µM FgTrx1, 50 nM TrxR or GR, 1 mM GSH, and 250 µM H2O2. Measurement was taken after the addition of 200 µM NADPH. The decrease in the absorbance at 340 nm as a result of NADPH (ε340nm= 6.22 mM-1cm-1) oxidation was
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monitored [60, 63].
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3. RESULTS 3.1 In silico analysis
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The screening of the transcriptome data with different known Trx sequences led to the identification of two Trxs in the liver fluke, F. gigantica —one with an active site motif -
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WCGPC- and the other with -WCTPC- —; the proteins are designated as FgTrx1 and FgTrx2, respectively. Prediction analyses for the cellular localization through TargetP1.1 server and Sherloc2 predominantly hypothesized the localization of FgTrx1 to cytoplasm and FgTrx2 to
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mitochondria. Outside the active site (Cys32 and Cys35), FgTrx1 did not contain any additional Cys residues that are present in many Trx fold-containing proteins (in higher eukaryotes) and
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have been proposed to have potential regulatory roles [31-33].
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3.2 FgTrx1 exists as a monomer under native conditions
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We observed that the N-terminus His6-FgTrx1 protein was over-expressed and more than 90% of protein was in the soluble fraction of the culture. The presence of the His-tag enabled the single-step purification of the protein using Ni-NTA matrix. Initially, the protein purity and homogeneity was confirmed by SDS-PAGE (Figure 1A), and the presence of the His-tagged recombinant FgTrx1 was validated by immunoblot analysis (Figure not shown). The molecular mass of the protein was found to be approximately 12 kDa. Using SuperdexTM S-75 gel filtration column, the oligomeric status of the protein was determined to include a retention volume of 14.3 ml corresponding to a protein of 12 kDa when compared to molecular weight standards (Figure 1B). These results indicate that FgTrx1 is stabilized in a monomeric state under native conditions.
3.3 Catalyzed reduction of insulin by FgTrx1 The active site of Trxs containing Cys residues are involved in the reversible oxidation and 8 Page 9 of 33
reduction of disulfide bonds in proteins that is necessary to maintain in the intracellular milieu. The DTT-mediated reduction of the disulfide bonds of insulin showed no activity in the reaction mixture containing only DTT even after 60 min. However, the reaction mixture containing both DTT and FgTrx1 showed precipitation of the insulin within 5 min from the starting of the
highest activity was at pH 7.5 (Figure 2B).
3.4 Preferential regeneration of FgTrx1 by the GSH system
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reaction (Figure 2A). The insulin assay carried out at different pH from 6 to 8.5 showed that the
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Grxs are the oxidoreductases that catalyze the reduction of disulfide or GSH-mixed disulfide in a coupled reaction with GSH, NADPH, and GR. The examination of the activity of FgTrx1 with
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both the Trx and GSH system using TrxR and GR based insulin reduction assays showed that FgTrx1 was unable to reduce insulin using GR in the absence of GSH. However, in the presence of GSH, FgTrx1 was regenerated by GR, thereby catalyzing insulin reduction. Figure 3 shows
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the GSH-linked activity of FgTrx1. HED was the classical substrate used to study the thioltransferase activity. It was found that the coupling of FgTrx1 with GSH and GR reduced the
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HED. In contrast, there was negligible HED activity with TrxR even in the presence of GSH
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(Figure 4). This confirms the preferential regeneration of FgTrx1 by GSH system.
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3.5 Protection of DNA from oxidative damage by FgTrx1 Few reports suggest that Trx can play a direct role as an antioxidant [17, 19]. In order to examine the antioxidant role of FgTrx1, a DNA nicking assay was performed to understand the ability of FgTrx1 as a DNA protector. The assay used metal catalyzed oxidation that generated free radicals that damaged DNA. It was found that when the supercoiled plasmid was exposed to the MCO assay, it was almost completely converted to nicked form within 30 min; however, when FgTrx1 was added to the assay, it resulted in protection from nicking. It was found that 5 µM of FgTrx1 protected at least 30% of the plasmid DNA from nicking (Figure 5A), suggesting that the FgTrx1 can protect DNA from free radical damage. The radical scavenging activity of FgTrx1 was further confirmed using hydroperoxide assay; FgTrx1 was found to be highly active in the peroxide assay. Figure 5B shows the summary of the assay. The Km of FgTrx1 for H2O2 (73.4 ± 13.0 μM) was found to be low, suggesting its high affinity towards H2O2. In comparison to FgTrx1, the Km of S. mansoni Trx with H2O2 is much lower (~5 μM) [64], indicating that its 9 Page 10 of 33
affinity is higher than that of FgTrx1. Though, the Km of FgTrx1 is less than that of SmTRx, it lies in the micromolar range, suggesting its high affinity towards H2O2. The Vmax of FgTrx1 was observed to be around 1.28 ± 0.08 µmol.min-1, which is comparable to that of SmTrx (1.5
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µmol.min-1)[64].
4. DISCUSSION
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Members of the Trx superfamily share two common features: (a) they contain an active site motif -CXXC-, and (b) the overall structure containing this motif corresponds to a Trx-like fold.
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The amino acid sequence of F. hepatica and F. gigantica shows more than 96% identity (Figure 6A), suggesting that both the proteins have a similar structure and biochemical properties. The molecular modeling of FgTrx1 based on the template of FhTrx shows that the overall
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architecture of FgTrx1 consists of a typical Trx-topology with four stranded β-sheets surrounded by four α-helices (Figure 6B). The analysis of the Ramachandran plot using PROCHECK
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indicates that 92.6% of the amino acid residues are within the most favored region while 7.4% residues are in the additionally allowed region. These two structures generate an RMSD of 0.07
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Å over 104 alpha carbon pairs, suggesting negligible spatial variation between them. Similar to FhTrx, the active site of FgTrx1 contains a redox-sensitive Cys pair -31CGPC34- that can be
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either oxidized in disulfide state or reduced in dithiol state. The active site also contains a
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conserved Trp30 residue preceding the -31CGPC34- motif. In FgTrx1, Trp30 is found on the surface of the protein and forms a flat surface close to the active site, which is similar to that of FhTrx (Figure 6C).
The results of the biochemical studies demonstrated that the most important feature of FgTrx1 is its preferential regeneration with the GSH system. In addition, FgTrx1, a disulfide oxidoreductase, is unique in its enzymatic behavior with various substrates such as HED, insulin, GR, and TrxR. The HED activity is considered to be specific for Grxs; however, a few reports have suggested that Trx can also be active against HED [65]. Recent reports showed that Haemonchus contortous [60] and some plant Trxs may be reduced by the GSH system [65]. It has been proposed that the presence of unique Arg and Ser residues in the active site -CRSCmotif is responsible for the GR-like activity in HcTrx5 [60]. FgTrx1 has also been shown to be active with GSH and GR; however, in contrast to HcTrx5, it does not contain these residues. Hence, we have proposed that, apart from the active site residues, some other residues could be 10 Page 11 of 33
involved in the activity of FgTrx1 with GR. Molecular modeling of FgTrx1 shows the presence of three charged amino acid residues, Gln29, Arg35, and Asn36, near the active site region that can influence the reduction potential and pKa values and can be responsible for Grx-like activity of FgTrx1 (Figure 6D). The effect of neighboring residues on Trx like activity and specificity for
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different substrate should be further studied. The active Cys residues of -CXXC- motif lies in the N-terminal region of α2-helix [66]. In E. coli Trx, the substitution of Lys for Gly at the first X
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does not affect the redox potential but affects the interaction of EcTrx with other proteins, which in turn decreases the catalytic efficiency of the enzyme [67]. In case of Grx, the presence of basic
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amino acid environment around the active site has been considered to be responsible for interaction with insignificant changes in pKa at N-terminal Cys [68]. In HcTrx5, the Arg residue,
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present between the active site -CRSC-, has been suggested to mediate critical proton transfer during the catalytic cycles, which is similar to Grx containing the conserved Arg residue in its active site [69]. E. coli -CVWC- Trx mutation has shown to have an increased reduction
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potential but a decreased pKa compared to the wild type -CGPC- Trx. The addition of Val and Trp residues in the active site has been found to alter the catalytic properties without varying the
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structure. The comparative analysis of DsbD transmembrane thiol:disulfide oxidoreductase clearly shows the catalytic activity of Trx-fold containing protein is not solely driven by their -
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CXXC- motif [70]. The functionality of -CXXC- motif depends on the balance between the
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reduction potential (dithiol-disulfide equilibrium) and pKa value (protonation state). Such equilibrium involves a proton moment that in turn regulates the reduction potential. It has been proposed that an extended active site motif of the Trx superfamily proteins and residues forming the α1 helices and β1, β2 strands could predominantly influence the pKa value of the reactive Cys [70]. The evolutionary change in Trx superfamily proteins has resulted in a family of enzymes (Trx, PDI, DsbA, etc) that have a similar structure but different specificity for their substrate with distinguished efficiency and catalytic mechanism. These enzymes have evolved in such a way that they can act in different environments, such as the cytoplasm, periplasm or endoplasmic reticulum, under different environmental conditions [71-73]. Free radicals can oxidize supercoiled DNA, which result in DNA nicking. We have utilized the MCO/MFO assay to carry out this experiment. MCO system is composed of Fe 3+, O2, and DTT as an electron donor. DTT oxidation produces H2O2 in a reaction catalyzed by Fe3+, which then reacts in a metal-catalyzed Fenton reaction to produce the ultimate toxic species, 11 Page 12 of 33
OH. It is well established that a DTT/Fe3+/O2 system induces strand breaks in plasmid and mammalian DNA [74-76]. The results of DNA nicking assay showed the ability of FgTrx1 to protect DNA against the free radical species during oxidative stress thereby suggesting the antioxidant role of FgTrx1. This type of DNA protection has previously been reported in some
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plant and parasite Trxs [61, 77]. The data suggest that FgTrx1 concentration dependently protects plasmid DNA from oxidative damage. To endorse the specificity of the antioxidant
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activity of FgTrx1, similar experiments were performed in the presence of bovine serum albumin (BSA) replacing FgTrx1, but BSA did not protect DNA from oxidative damage by free radical
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damage (data not shown). We further confirmed the free radical scavenging property of FgTrx1 by the hydroperoxide assay, and FgTrx1 was found to be highly active, thereby, establishing its
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antioxidant functions.
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5. CONCLUSIONS
Growing evidence suggests that redox-active proteins are not only involved in target protein reduction, but also linked with the detoxification of ROS. Trx is a highly conserved
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oxidoreductase, which regulates the reduction of many important proteins. The present
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biochemical observations suggested that Trx1 of F. gigantica might play a valuable role in parasitic survival within the host as it participates not only in linking the Trx and GSH system
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but also in protecting cells from ROS-induced oxidative damage. Thus, their inhibition could be tested for the development of novel therapeutic compounds against fascioliasis. Data mining of helminthic parasites has provided a clue for the presence of several redox-active proteins. Complete elucidation of the redox-sensitive protein system in Fasciola can suggest the mechanism and evolutionary conservation of Trx and GSH system in helminths, as well as help in evaluating their drug target potential. We plan to characterize these proteins in our future studies.
ACKNOWLEDGEMENTS The study was supported by a research grant from Department of Biotechnology, Govt of India, New Delhi, India to TT (Grant no. BT/28/NE/TBP/2010 dated 28.02.2011). AG thanks DBT for providing fellowship. Authors thank A. Sharma and M. Yogavel, ICGEB, New Delhi, India and Banchob Sripa, Khon Kaen University, Thailand for constructive suggestions and valuable 12 Page 13 of 33
scientific discussions. Thanks also go to N. D. Young, The University of Melbourne, Australia for generously providing the transcriptome data of F. gigantica.
FOOTNOTES
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The coordinates for the model of FgTrx1 has been submitted to the Protein Model Database with
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the following id: PM0080291.
ABBREVIATIONS
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Trx, thioredoxin; Grx, glutaredoxin; TrxR, thioredoxin reductase; GR, glutathione reductase,
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GSH, reduced glutathione; HED, 2-hydroxyethyldisulfide; DTT, dithiothreitol.
CONFLICT OF INTEREST
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The authors declare that there are no conflicts of interests.
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FIGURE LEGENDS
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Figure 1. Purification and oligomeric status of FgTrx1. (A) SDS-PAGE profile of recombinant FgTrx1. Lane 1: Molecular weight marker; Lane 2: un-induced culture of E. coli extract; Lane 3: induced culture of E. coli extract; Lane 4: purified FgTrx1. (B) Size exclusion
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profile of FgTrx1. The peak shows a monomeric structure of proteins in Superdex S-75TM
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column. The inset shows the column calibration curve.
Figure 2. Insulin assay. (A) Dithiothreitol mediated reduction of insulin by FgTrx1. The
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increase in OD at 650 nm was measured and plotted against time. The reaction mixture contains 0.33 mM DTT, 0.17 mM insulin, and 2 mM EDTA in 20 mM NaH2PO4 (pH 7.5) with 2.5 µM of
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FgTrx1 (solid line). Dotted line represents the control reaction in the absence of FgTrx1. (B)
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Effect of pH on the insulin activity of purified FgTrx1. Purified FgTrx1 was incubated at 30°C for 30 min in CGH buffer of pH values ranging from 6.0 to 8.5, and then readings were taken as above. Three replications were conducted, and background data were subtracted for all the experiments.
Figure 3. GSH-linked activity of FgTrx1. FgTrx1 activity with GSH and Trx system was determined by the insulin disulfide reduction assay. The reaction mixture contained 2.5 µM of FgTrx1, 200 µM of NADPH, 0.17 mM of insulin, 1 mM of GSH, 50 nM of GR or TrxR and 2 mM EDTA in 20 mM NaH2PO4 (pH 7.5). Absorbance was monitored at 340 nm for NADPH consumption.
Figure 4. HED assay using TrxR and GR. (A) Reduction of the disulfide bond in HED by FgTrx1 using TrxR; (B) Reduction of the disulfide bond in HED by FgTrx1 using GR. In both 17 Page 18 of 33
the experiments, the reaction mixture contains 2.5 µM FgTrx1, 200 µM of NADPH, 750 µM of HED, 50 nM of TrxR or GR and 2 mM EDTA in 20 mM NaH2PO4 (pH 7.5). After 5 min of incubation, GSH was added, and the reaction was monitored at 340 nm. Solid blocks represent the reaction in the presence of GSH while the hollow box represents reaction in the absence of
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GSH.
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Figure 5. Antioxidant property of FgTrx1. (A) The protection of DNA from nicking was studied using MCO assay. The system consists of 25 mM HEPES (pH 7.5), 1 mM DTT, 1 mM
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FeCl3, and 800 ng of supercoiled pUC19 plasmid DNA in each lane. Lane 1: pUC19 plasmid alone with no incubation with MCO, Lane 2: pUC19 plasmid incubated in MCO for 30 min
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without FgTrx1, Lane 3-7: pUC19 plasmid incubated in MCO with 1-5 µM of FgTrx1 for 30 min. (B) Hydroperoxide assay of FgTrx1. The reaction mixture contains 2.5 µM of FgTrx1, 50 nM of TrxR or GR, 1 mM GSH, 200 µM H2O2, and 250 µM NADPH in 20 mM NaH2PO4 buffer
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pH 7.5.
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Figure 6. In silico analysis of FgTrx1. (A) Multiple sequence alignment of FgTrx1 with FhTrx is shown. Identical residues are shown in red boxes, similar residues in yellow boxes while
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amino acid residue having different properties does not have any box. (B) Cartoon diagram of
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the FgTrx1 monomer structure. Secondary structural elements are labeled along with the protein termini. (C) Zoom view of the FgTrx1 showing the active site Cys residues (C31 and C34) in dithiol state in red. The surface Trp30 is shown in pink. (D) Close-up view of the active site region of FgTrx1 showing the Gln29, Arg35, and Asn36 residues in sticks along with the Cys31 and Cys34 arranged in ball and stick model. The 3D model was made using Swiss-model and visualized with UCSF Chimera 1.9.
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Highlights (for review)
HIGHLIGHTS Structure-function studies with FgTrx1 were performed.
FgTrx1 is preferentially regenerated by the GSH system.
Our data suggests that it protects cells from ROS induced damage.
Residues present in close proximity to the -CXXC- motif may regulate the GR activity of
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