Molecular and functional explication of thioredoxin mitochondrial-like protein (Trx-2) from big-belly seahorse (Hippocampus abdominalis) and expression upon immune provocation

Journal Pre-proof Molecular and functional explication of thioredoxin mitochondrial-like protein (Trx-2) from big-belly seahorse (Hippocampus abdominalis) and expression upon immune provocation Kishanthini Nadarajapillai, Sarithaa Sellaththurai, D.S. Liyanage, Hyerim Yang, Jehee Lee PII:

S1050-4648(20)30120-0

DOI:

https://doi.org/10.1016/j.fsi.2020.02.034

Reference:

YFSIM 6833

To appear in:

Fish and Shellfish Immunology

Received Date: 12 December 2019 Revised Date:

11 February 2020

Accepted Date: 16 February 2020

Please cite this article as: Nadarajapillai K, Sellaththurai S, Liyanage DS, Yang H, Lee J, Molecular and functional explication of thioredoxin mitochondrial-like protein (Trx-2) from big-belly seahorse (Hippocampus abdominalis) and expression upon immune provocation, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2020.02.034. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.

CRediT author statement Each author had been sufficiently involved in the work. Their personal contributions in this work following: Kishanthini Nadarajapillai: Conceptualization, Methodology, Investigation, Formal analysis Writing - Original Draft. Sarithaa Sellaththurai: Methodology, Investigation. D. S. Liyanage: Methodology, Writing - Review & Editing. Hyerim Yang: Methodology, Investigation. Jehee Lee: Resources, Supervision, Project administration, Funding acquisition.

1

Molecular and functional explication of thioredoxin mitochondrial-like protein (Trx-2)

2

from big-belly seahorse (Hippocampus abdominalis) and expression upon immune

3

provocation

4 5

Kishanthini Nadarajapillai1,2, Sarithaa Sellaththurai1,2, D. S. Liyanage1,2, Hyerim Yang1,2 and

6

Jehee Lee1,2*

7 8 9

1

10 11

2

Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea

12 13 14 15 16 17 18

*

19

Jehee Lee, Marine Molecular Genetics Lab, Department of Marine Life Sciences, Jeju National

20

University, 102 Jejudaehakno, Jeju 63243, Republic of Korea.

21

Email: [email protected]

22

Corresponding author

23

Abstract

24

Thioredoxin (Trx) is a small ubiquitous multifunctional protein with a characteristic WCGPC

25

thiol-disulfide active site that is conserved through evolution. Trx plays a crucial role in the

26

antioxidant defense system. Further, it is involved in a variety of biological functions including

27

gene expression, apoptosis, and growth regulation. Trx exists in several forms, with the cytosolic

28

(Trx-1) and mitochondrial (Trx-2) forms being the most predominant. In this study, the

29

mitochondrial Trx protein (HaTrx-2), from the big-belly seahorse (Hippocampus abdominalis)

30

was characterized, and its molecular features and functional properties were investigated. The

31

cDNA sequence of HaTrx-2 consists of a 519 bp ORF, and it encodes a polypeptide of 172

32

amino acids. This protein has a calculated molecular mass of 18.8 kDa and a calculated

33

isoelectric point (pI) of 7.80. The highest values of identity (78.7%) and similarity (86.2%) were

34

observed with Fundulus heteroclitus Trx-2 from the pairwise alignment results. The

35

phylogenetic analysis revealed that HaTrx-2 is closely clustered with teleost fishes. The qPCR

36

results showed that HaTrx-2 was prevalently expressed at various levels in all the tissues

37

examined. The ovary showed the highest expression, followed by the brain and kidney. HaTrx-2

38

showed varying mRNA expression levels during the immune challenge experiment, depending

39

on the type of tissue and the time interval. Our results confirmed the antioxidant property of

40

HaTrx-2 by performing the MCO assay, DPPH radical scavenging activity, and cell viability

41

assays. Further, an insulin disulfide reduction assay revealed the dithiol reducing the enzymatic

42

activity of HaTrx-2. Altogether these results indicate that HaTrx-2 plays indispensable roles in

43

the regulation of oxidative stress and immune response in the seahorse.

44

45

Keywords: Hippocampus abdominalis, Immune response, Mitochondrial, Oxidative stress,

46

Thioredoxin-2

47 48

Introduction

49

Oxidative stress in aerobic animals results in a disturbance of the equilibrium between pro-

50

oxidants and anti-oxidants [1]. Organisms have a cellular antioxidant protection mechanism to

51

maintain the homeostasis of antioxidant levels. Reactive oxygen species (ROS) have essential

52

roles in many cellular functions, including immunity, cell signaling involved in gene expression

53

regulation [2], cell proliferation, and cell death [3]. Nevertheless, overproduction of ROS may

54

result in enhanced lipid peroxidation, protein oxidation, and DNA damage [4,5]. ROS encompass

55

several reactive molecules, including hydroxyl radicals (.OH), hydrogen peroxide (H2O2), singlet

56

oxygen (1O2), and superoxide anion (O2-) [6]. They are generated through various biological

57

pathways of aerobic metabolism, including ATP synthesis and electron transport chains in the

58

mitochondria [5,7]. Further, ROS can be generated upon exposure to metals and other toxic

59

compounds, light, and UV radiation [8]. The regulation of antioxidant defenses is necessary to

60

maintain a stable low concentration of ROS and thereby avoid adverse effects. The antioxidant

61

defense system consists of a broad range of small antioxidant molecules including vitamin E, C,

62

and A; biliverdin; reduced glutathione and antioxidant enzymes such as thioredoxin (Trx),

63

glutathione peroxidase, Trx peroxidase, superoxide dismutase, and catalase [3].

64

Trx is a redox protein and has a low molecular weight (12 kDa) with a highly conserved

65

sequence in its active site (WCXXC). It is present in all living organisms, including bacteria. A

66

distinctive Trx folding motif composed of four-stranded β-sheets encompassed by three α-helices

67

and an extra α-helix and β-sheet at the N-terminus [6][9]. Trx , nicotinamide adenine

68

dinucleotide phosphate hydrogen (NADPH), and Trx reductase are primary elements of the Trx

69

system. They are involved in the response to oxidative stress. Trx is reduced by transferring

70

electrons to the oxidized protein targets via Trx reductase and Trx itself from NADPH. Two

71

conserved cysteine residues in the Trx active site are important in cleavage of the oxidized target

72

protein disulfide bond. Once the catalytic cycle has been completed, two cysteine residues are

73

oxidized to form a disulfide bond (Trx-S2). The dithiol group is reduced by NADPH (Trx-(SH)2)

74

through the catalytic function of the flavoenzyme Trx reductase. Trx reductase is involved in the

75

transfer of reducing equivalents from NADPH to other target proteins via flavin adenine

76

dinucleotide, which is vital for maintaining cell redox regulation [9]. Various Trx isoforms have

77

been characterized in different organisms; among them, Trx1 is predominantly found in the

78

cytosol and Trx2 is found in the mitochondria [9][10]. The cytosolic Trx system is involved in

79

several cellular functions such as transcription factor regulation (NF-κB or the Ref-1-dependent

80

AP1), protein repair, oxidative stress defense, and apoptosis regulation [11]. Similarly, the

81

mitochondrial Trx system is engaged in the maintenance of the inner mitochondrial membrane

82

structure. This system serves as an electron donor for peroxiredoxin-3, and for the regulation of

83

mitochondria-driven cell death [12].

84

The big-belly seahorse is considered to be the largest species of seahorse in the world. Its

85

maximum length is approximately 35 cm. The deeper trunk in adults distinguishes this species

86

from other seahorses. It is extensively spread in New Zealand and in the Australian temperate

87

waters of the South-east marine region. Hippocampus spp. are listed as threatened species on the

88

CITES Appendix-II [13] and are generally threatened by overexploitation for traditional

89

medicines and aquarium trade. Seahorses have medicinal properties and hence are used for the

90

treatment of several conditions such as wheezing, arthritis, struma, kidney disorders, impotence,

91

and various skin diseases [14]. However, environmental factors of stress in aquatic animals may

92

result in loss of immunity in the seahorse making it extremely susceptible to pathogen attacks.

93

Thus, the identification of molecular features engaged in the mechanisms of immunity in the big-

94

belly seahorse can assist in the management of diseases, while assisting in the preservation of

95

sustainable seahorse aquaculture. Therefore, to advance understanding of the immune

96

mechanisms of the seahorse, the current report focuses on the identification of big-belly seahorse

97

Trx-2 with particular attention to its molecular system, the oxidative stress response of its

98

recombinant protein, spatial expression of mRNA, and mRNA expression in response to

99

pathogenic stress.

100 101

2. Materials and methodology

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2.1. Identification and molecular characterization of HaTrx-2

103

The cDNA sequence of the mitochondrial Trx of the big-belly seahorse, designated as HaTrx-2,

104

was identified from an already created big-belly seahorse transcriptome database using the NCBI

105

BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) [15]. The Unipro UGENE software

106

was used to obtain the amino acid sequence with respect to the HaTrx-2 open reading frame

107

(ORF) [16]. Functional domains and other characteristic features of the protein were predicted

108

using the SMART online server (http://smart.embl-heidelberg.de/) [17] and ExPASy Prosite

109

(http://prosite.expasy.org/) [18]. The physical characteristics of HaTrx-2 were analyzed using the

110

ExPASy ProtParam tool (https://web.expasy.org/protparam). A mitochondrial localization signal

111

was predicted by the TargetP-2.0 Server (http://www.cbs.dtu.dk/services/TargetP). To analyze

112

the HaTrx-2 protein identity and its similarity with orthologous sequences, pairwise and multiple

113

sequence

alignments

were

generated

using

the

EMBOSS

needle

114

(http://www.ebi.ac.uk/Tools/emboss/align)

115

(http://www.Ebi.ac.uk/Tools/clustalw2) [20], respectively. A phylogenetic tree was constructed

116

using the MEGA7.0.26 software with the neighbor-joining method [21] with 5000 bootstrap

117

replications.

118

2.2. Seahorse tissue collection

119

Healthy big-belly seahorses with an average body weight of 8 g were bought from the Center of

120

Marine Ornamental Fish Breeding Center (Jeju Island, Republic of Korea). Seahorses were

121

grown in the seawater tanks of laboratory aquarium (300 L) at a constant salinity of 34 ± 0.6 g/L,

122

and a temperature of 18 ± 2°C for a week prior to the experiments. Throughout the

123

acclimatization period, the seahorses were fed twice a day on frozen mysis shrimp until the

124

moment of the experiments.

125

Six unchallenged seahorses were used for the tissue distribution analysis. Blood was collected by

126

tail-cutting, and peripheral blood cells were isolated by immediate centrifugation at 3000 × g and

127

4°C for 10 min. The thirteen tissue samples, including the kidney, spleen, gills, stomach, skin,

128

liver, testis, intestine, brain, ovary, pouch, heart, and muscle were dissected from the seahorses.

129

All tissues were snapped frozen in liquid nitrogen and stored at −80°C for the downstream

130

process.

131

2.3. In vivo challenge with immune stimulants and bacterial pathogens

132

For the immune challenges, pre-acclimatized seahorses were separated into 5 groups, each

133

containing 30 individuals. Next, 100 µL suspensions of live pathogens, including Edwardsiella

134

tarda (E. tarda; 5 × 103 CFU/µL) and Streptococcus iniae (S. iniae; 105 CFU/µL), were injected

135

separately intraperitoneally (i.p.). Further immune stimulants such as polyinosinic:polycytidylic

136

acid (poly I:C; 1.5 µg/µL) and lipopolysaccharide (LPS; 1.25 µg/µL) were diluted in sterile

[19]

and

the

ClustalW2

programs

137

phosphate-buffered saline (PBS) and injected. A volume of 100 µL of PBS was injected into the

138

control group. Seahorse kidney and peripheral blood cells from five individuals per group were

139

collected at 0, 3, 6, 12, 24, 48, and 72 h post-injection. Subsequently, the collected samples were

140

frozen in liquid nitrogen and stored at −80°C.

141

2.4. Total RNA extraction and cDNA synthesis

142

Total RNA was extracted from the collected samples using RNAiso Plus reagent (TaKaRa,

143

Japan). The RNeasy spin column (Qiagen, USA) was used to purify the total extracted RNAs.

144

Next, RNA purity and concentration were determined spectrophotometrically in a µDrop Plate

145

(Thermo Scientific, USA) at 260 nm and then verified with 1.5% agarose gel electrophoresis.

146

The first-stranded cDNA was synthesized from purified RNA samples (2.5 µg) using the

147

PrimeScript™ II 1st strand cDNA Synthesis Kit (TaKaRa, Japan). The cDNA was stored at

148

−20°C.

149

2.5. Analysis of spatial and temporal expression of HaTrx-2 by quantitative real-time PCR

150

(qPCR)

151

The spatial and temporal expression pattern of HaTrx-2 mRNA in all tissues was studied by

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qPCR using prepared cDNA samples. As the invariant control gene, seahorse 40S ribosomal

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protein S7 (Accession number KP780177) was used. All qPCR primers (Table 1) were designed

154

using the IDT Primer Quest Tool [22]. The qPCR reaction was carried out in a Thermal Cycler

155

Dice™ Real-Time System III (TaKaRa). The final reaction volume (10 µL) contained 1.2 µL of

156

nuclease-free water, 0.4 µL of each forward and reverse primer (10 pmol/µL), 5 µL of TaKaRa

157

Ex Taq™ SYBR premix (2×), and 3 µL of cDNA template from each tissue. The following

158

conditions were used for the PCR reaction: initial denaturation at 95°C for 10 s, followed by 45

159

cycles at 95°C for 10 s, 58°C for 10 s, and 72°C for 20 s. Finally, a melting cycle at 95°C for 15

160

s, 60°C for 30 s, and 95°C for 15 s were performed. To increase the reliability of the results,

161

qPCR was performed in triplicate for each sample, and the relative expression of HaTrx-2

162

mRNA was calculated according to the 2-∆∆CT method [23]. The expression level of HaTRx-2 in

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immune-challenged seahorses has been normalized to PBS-injected controls to exclude errors.

164

2.6. Preparation of recombinant HaTrx-2 plasmid constructs

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The ORF of HaTrx-2 was amplified by PCR using the primers HaTrx-2-cF and HaTrx-2-cR

166

(Table 1) containing EcoRV and EcoRI restriction enzyme sites, respectively (Table 1), and

167

cloned into the pMAL-c5X vector (BioLabs Inc.). The gene HaTrx-2 was amplified using 50 µL

168

reaction mixture containing 10 pmol of each primer (Table 1), 5 µL of 10× ExTaq buffer, 4 µL of

169

2.5 mM dNTPs, 0.2 µL of ExTaq polymerase, and 50 ng of cDNA obtained from the ovary tissue

170

as a template. The following parameters were used to perform the PCR: initial denaturation at

171

94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 1 min, with a

172

final extension of 72°C for 7 min. The amplification of PCR products was confirmed by

173

electrophoresis on a 1% agarose gel. The desired size band was cut and purified using the

174

Accuprep® gel purification kit (Bioneer Co., Korea). The purified HaTrx-2 gene and the cloning

175

vector of pMAL-c5X (BioLabs Inc.) were digested with EcoRV and EcoRI restriction enzymes

176

in buffer H. The ligation was performed using the DNA Ligation Mighty Mix (5.0 µL; TaKaRa

177

Bio Inc.) for 30 min at 16°C followed by overnight incubation at 4°C. The heat-shock method

178

was used to transform the ligated product into competent cells of Escherichia coli (E. coli) DH5α,

179

and successful clones were verified by sequencing.

180

2.7. Recombinant HaTrx-2 fusion protein (rHaTrx-2-MBP) expression and purification

181

The recombinant plasmid was confirmed by sequencing and was then transformed into E. coli

182

BL21 (DE3) competent cells for overexpression. Transformed cells were grown until the

183

absorbance (OD600) reached 0.6 in 500 mL Luria-Bertani rich medium supplemented with 0.2%

184

glucose and 100 µg/mL ampicillin at 37°C/200 rpm. Isopropyl-β-D-1-thiogalactopyranoside

185

(IPTG 0.5 mM) was added into the medium to induce the protein production, followed by

186

incubation for 3 h at 37°C/200 rpm. Afterward, cells were collected by centrifugation at 1,200 ×

187

g for 20 min at 4°C. A volume of 25 mL of column buffer containing 20 mM Tris-HCl and

188

200 mM sodium chloride (NaCl) at pH 7.4 was used to resuspend the pellet, centrifuged using

189

the above-mentioned parameters, and incubated overnight at -20°C. The pellet was thawed,

190

subjected to cold sonication, and centrifuged at 9,000 × g and 4°C for 20 min. Next, 1 mL

191

amylose resin was mixed with the supernatant and left on ice for 20 min to facilitate better

192

binding. The mixture was then poured into a column and washed with 60 mL of column buffer.

193

The 10 mM maltose buffer was used to elute the rHaTrx-2-MBP fusion protein, and the

194

concentration was calculated using the Thermo Scientific NanoDrop™ 2000/2000c

195

Spectrophotometer machine. The protein band size was analyzed by 12% sodium dodecyl

196

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

197

2.8. Insulin disulfide reduction assay

198

This assay was carried out using the method described in a previous study with slight

199

modifications [24] to analyze the enzymatic activity of rHaTrx-2. Briefly, a 200 µL reaction

200

mixture consisting of purified rHaTrx-2, 0.6 mM dithiothreitol (DTT), 130 µM bovine insulin

201

(Sigma, USA), 4 mM EDTA, and 100 mM potassium phosphate buffer (pH 7.0) was prepared.

202

The addition of DTT to the wells started the reaction, which was kept for 10 min at 25°C. The

203

precipitation was measured at 650 nm every 5 min. The control experiments were conducted

204

with recombinant MBP (rMBP) and without DTT separately. The assay was conducted in

205

triplicate. The half-maximal inhibitory concentration (IC50) value and specific activity of HaTrx-

206

2 were calculated.

207

2.9. DPPH radical-scavenging assay

208

To evaluate the radical-scavenging ability of rHaTrx-2, the DPPH (α, α-diphenyl-β-

209

picrylhydrazyl) assay was conducted in a 96-well plate according to a previously described

210

method [25]. Briefly, 100 µL of the rHaTrx-2 sample with different concentrations (15, 30, 45,

211

60, 90, and 120 µg/mL) and 120 µg/mL of rMBP were mixed with 100 µL of 0.4 mM DPPH

212

solution dissolved in dimethyl sulfoxide. Ascorbic acid solutions were prepared at various

213

concentrations (15, 30, 45, 60, 90, and 120 µg/mL) and used as a reference. The mixture was

214

kept for 30 min at room temperature. The optical density of the mixtures was then measured at

215

517 nm. The following formula was used to calculate the radical-scavenging activity percentage:

216

([Acontrol − Asample]/Acontrol × 100). The IC50 value of HaTrx-2 was calculated with respect to

217

ascorbic acid as reference DPPH radical scavenger.

218

2.10. Metal-catalyzed oxidation (MCO) protection assay

219

The ability of rHaTrx-2 to protect supercoiled DNA from oxidative stress was evaluated by

220

conducting an MCO assay based on a previously described method [26]. Briefly, the 50 µL

221

reaction mixture consisting of 3.3 mM DTT, 33 µM FeCl3, and various concentrations of

222

purified rHaTrx-2 protein (0.05, 0.1, 0.2, 0.4, and 0.8 µg/µL) were incubated for 2 h at 37°C.

223

After adding 1 µg of pUC19 supercoiled DNA, the mixtures were again kept for 2 h at 37°C.

224

Thereafter, each reaction mixture was purified using a PCR purification kit (Bioneer Co., Korea),

225

and the degradation of super-coiled DNA was confirmed by electrophoresis in agarose gel (1%).

226

The purified MBP was used as a control experiment under similar reaction conditions.

227

2.11. Construction of pcDNA3.1(+) vector and transfection of HaTrx-2

228

The coding sequence of HaTrx2 with EcoRI and XhoI restriction sites (Table 1) was cloned into

229

the pcDNA3.1(+) vector. The sequence of the cloned plasmid was confirmed by sequencing

230

(Macrogen, Korea), and the QIAfilter™ Plasmid Midi Kit (Qiagen, Germany) was used to purify

231

the confirmed plasmid sequence. The 5 × 105 cell/mL concentration of FHM cells were seeded in

232

6-well plates and incubated at 25°C for 24 h in L-15 Leibovitz's medium supplemented with 1%

233

penicillin and streptomycin and fetal bovine serum (2%). The X-tremeGENE™ 9 reagent was

234

then used to transfect the empty pcDNA3.1(+) vector (1 µg) and the recombinant pcDNA3.1(+)

235

plasmid containing the HaTrx-2 gene into cultured FHM cells according to the manufacturer's

236

protocol.

237

2.12. Cell viability analysis via MTT assay

238

The empty pcDNA3.1(+) and HaTrx-2-inserted pcDNA3.1(+) transfected cells were treated with

239

various concentrations of H2O2 (0, 250, and 500 µM) for 24 h. A volume of 200 µL of a solution

240

with 2 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was then

241

added to all wells and the plates were incubated for 3 h. The purple MTT formazan crystals were

242

dissolved in 150 µL of dimethyl sulfoxide. The absorbance was then measured at 570 nm using

243

the Multiskan Sky microplate reader (Thermo Fisher Scientific, USA). All assays were

244

performed in triplicate. Cell viability percentage was calculated using the following formula:

245

Cell viability = Optical density of the sample/Optical density of the control × 100%.

246

2.13. Statistical analysis

247

All qPCR data, insulin disulfide reduction and DPPH radical-scavenging assays were performed

248

in triplicate, and the results are presented as the mean ± standard deviation (SD). A p-value

249

<0.05 was considered to be statistically significant. Spatial and temporal expression data were

250

evaluated by one-way ANOVA and Student’s t-test, respectively.

251

3. Results

252

3.1. Identification and molecular characterization of HaTrx-2

253

The amino acid sequence of HaTrx-2 was identified from the cDNA library of the Hippocampus

254

abdominalis (accession number: MN812710). It contains a 519 bp ORF encoding 172 amino

255

acids with a predicted molecular weight of 18.8 kDa and an estimated isoelectric point of 7.8.

256

The Trx family domain was identified from 73–166 amino acid residues with a conserved redox-

257

active site motif C95-C98 using the ExPASy Prosite program. HaTrx-2 contains the N-terminal

258

mitochondrial localization signal peptide with a cleavage site between amino acids 64 and 65.

259

3.2. Phylogenetic and homology analysis of HaTrx-2

260

Pairwise sequence analysis (Table 2) of HaTrx-2 revealed that HaTrx-2 shared the highest

261

identity (78.7%) and similarity (86.2%) with Fundulus heteroclitus Trx-2 and shared 50–80%

262

identity with the other Trx-2 sequences. The multiple sequence alignment of HaTrx-2 with other

263

Trx-2 amino acid sequences (Fig. 1) showed sequences that were fully conserved, strongly

264

conserved, and weakly conserved and have been indicated by asterisks (*), semicolons (:), and

265

periods (.), respectively. Moreover, it was shown that all aligned Trx-2 sequences contained the

266

CGPC active site. The phylogenetic tree, generated by the neighbor-joining method using

267

bacterial Trx-2 (Streptomyces malaysiensis) as outgroup (Fig. 2), revealed the evolutionary

268

relationship between HaTrx-2 and orthologs from other species. The phylogenetic tree was

269

divided into 2 main clusters, namely vertebrates and invertebrates. Further, Hippocampus

270

abdominalis was positioned in the vertebrate cluster and sub clustered with other fish orthologs.

271

3.3. Spatial expression of HaTrx-2

272

qPCR was performed to understand the tissue-specific mRNA expression by using gene-specific

273

primers. The gene encoding the 40S ribosomal protein S7 was used as the internal control gene.

274

The HaTrx-2 expression fold in each tissue was calculated relative to the spleen, which had a

275

basal expression level. The highest mRNA expression was detected in the ovary, followed by the

276

brain and kidney among the fourteen tissues examined (Fig. 3).

277

3.4. Immune challenges modulated HaTrx-2 mRNA expression profile

278

To understand the immune responses of HaTrx-2 against immune stimulants, we selected kidney

279

and blood tissues. In those tissues, we investigated the transcription level of HaTrx-2 at different

280

time intervals after the immune challenges by qPCR. The transcriptional profile of HaTrx-2 in

281

the kidney (Fig. 4(A)) is different from that of blood Trx-2. Kidney HaTrx-2 showed upregulated

282

expression with LPS at 3, 12, 24, and 72 h post-injection (p.i). HaTrx-2 was found to be

283

downregulated by all stimulants at 6 and 48 h p.i. A significant upregulation of HaTrx-2 was

284

detected with poly I:C and E. tarda at 24 and 72 h p.i. with S. iniae stimulation at 12 h p.i.

285

The HaTRx-2 transcriptional level in the blood is shown in Fig. 4(B). LPS showed upregulation

286

at 3–72 h p.i. except at 12 h p.i. Significant upregulations were observed with poly I:C and E.

287

tarda at 6, 24, 48, and 72 h p.i. Following S. iniae challenge, downregulation was observed at

288

mid-phase (12–48 h p.i.).

289

3.5. Expression and purification of HaTrx-2 recombinant fusion protein (rHaTrx-2-MBP)

290

The rHaTrx-2 was overexpressed in E. coli BL21(DE3) by IPTG induction. Affinity

291

chromatography was then performed to elute the MBP tagged Trx-2 protein. After elution, the

292

purified protein was examined by SDS-PAGE. The purified fusion protein size was 61.3 kDa

293

including the 42.5 kDa of MBP according to the SDS-PAGE analysis (Fig. 5). The predicted

294

molecular weight of Trx-2 was 18.8 kDa.

295

3.6. Insulin disulfide reduction assay

296

To evaluate the ability of HaTrx-2 to reduce insulin disulfide bonds in the presence of DTT,

297

insulin disulfide reduction assay was carried out (Fig. 6). The results showed that insulin

298

precipitation started to form after 10 min and the absorbance increased with increasing time and

299

concentration of rHaTrx-2. The plateau level was reached after 75 min. The control assays were

300

carried out with MBP protein, without rHaTrx-2, and without DTT separately. The control also

301

showed some readings, but the insulin precipitation and rate were lower than the lowest

302

concentration of rHaTrx-2. The IC50 values were 43.83 ± 5.64, 37.66 ± 1.31, 48.12 ± 1.00, and

303

63.43 ± 3.77 for 32, 16, 8, and 4 µg of rHaTrx-2, respectively. The specific activity of rHaTrx-2

304

was 1.417 U/mg.

305

3.7. DPPH radical-scavenging assay

306

The plotted graph (Fig. 7) shows the antioxidant capacity of rHaTrx-2 with respect to ascorbic

307

acid as a positive control in the DPPH radical experiment. The free radical scavenging activity

308

increased with increasing concentration of rHaTrx-2, indicating a dose-dependent activity. The

309

highest inhibition concentration of DPPH radicals was 74.90% within the concentration range of

310

rHaTrx-2 protein used. rHaTrx-2 showed an IC50 value at a concentration of 43.87 ± 1.90 µg/mL.

311

Compared to 120 µg/mL rHaTrx-2, 120 µg/mL of rMBP showed the lowest radical scavenging

312

activity (26.79 %).

313

3.8. Metal-catalyzed oxidation (MCO) protection assay

314

To understand the antioxidant potential of rHaTrx-2, the MCO assay was performed. The

315

principle of the MCO system is that the DNA can be damaged by OH· radicals. Owing to the

316

oxidative stress, the supercoiled form of DNA will be converted to a nicked form. Here, the

317

untreated pUC19 sample was run with treated samples for confirmation of results. According to

318

the results (Fig. 8), when pUC19 was treated with the MCO system only, the smear was detected,

319

and there was no significant DNA protection in the MCO system with rMBP. The range of DNA

320

damage was reduced in direct proportion to the amount of rHaTrx-2 protein added into the MCO

321

system. The lowest level of nicking activity was observed at the highest concentration of

322

rHaTrx-2.

323

3.9. Cell protective role of HaTrx-2 against H2O2

324

The cell protective role of Trx-2 in H2O2-induced cell death was assessed using FHM cells with

325

transfected HaTrx-2. Our results (Fig. 9) demonstrated that overexpressed HaTrx-2 diminished

326

the cell death caused by H2O2-mediated ROS production compared with pcDNA3.1(+)-

327

transfected cells.

328 329

4. Discussion

330

Trx is a multifunctional protein that regulates redox homeostasis, controls cell growth, and

331

prevents apoptosis [27] in most living cells. Trx-(SH)2 is a constituent of T7 DNA polymerase in

332

E. coli [28]. Further, Trx-2 participates in the assembly of filamentous phage [29]. Members of

333

the Trx family have in their active site an amino acid sequence (CXXC) that has been conserved

334

throughout evolution. The mitochondrion is a major source of ROS generation by the electron

335

transport chain. Overproduction of ROS can cause oxidative damage to biopolymers (e.g.,

336

proteins, lipids and DNA) in the cell, leading to apoptosis by the release of cytochrome c and

337

other mitochondrial apoptotic factors [27,30].

338

Based on the results of in-silico analysis, HaTrx-2 was comprised of a 519 bp ORF that encodes

339

a putative protein of 172 amino acids with a molecular weight of 18.8 kDa. The same molecular

340

weight has been reported previously in Trx-2 proteins of different organisms; for instance, disk

341

abalone [26], Manila clam [24], human [31], and mouse species. Trx-1 is a 12 kDa protein,

342

whereas Trx-2 encodes a higher molecular weight protein than Trx-1, with a low number of other

343

cysteine residues, which makes it more resistant to oxidative stress compared with Trx-1 [26].

344

Trx-2 is encoded in the nucleus and localized to mitochondria by N-mitochondrial leader

345

sequence targeting signals. The cleavage at a mitochondrial peptidase cleavage site would give a

346

mature protein of 12 kDa, which is a size similar to that of Trx-1 [31]. Multiple alignments

347

revealed that the WCXXC motif was highly conserved in all analyzed Trx-2 sequences. The

348

phylogenetic tree construction showed that HaTrx-2 clustered together with fish Trx-2.

349

According to the tissue-specific expression results, HaTrx-2 mRNA was ubiquitously and

350

differentially expressed in all analyzed tissues. The highest-level expression of HaTrx-2 was

351

observed in ovary followed by brain, kidney, heart, and muscle, while the lowest expression was

352

observed in the spleen, suggesting that Trx-2 is essential for metabolically active and

353

energetically demanding tissues and highlighting its important role against ROS generated in

354

mitochondria. Previous studies reported that the mouse Trx-2 has its highest expression in the

355

heart and muscle, followed by the kidney [31], which is similar to our results. Based on the tissue

356

distribution analysis of Manila clam (Ruditapes philippinarum), the highest expression was

357

detected in hemocytes and gills [24].

358

The proper functioning of the ovaries is critical for maintaining fertility and overall health. The

359

high level of ROS has been associated with persistently poor oocyte quality, embryo

360

development, aging, and ovarian dysfunction [32]. Several mechanisms have already been

361

identified to be in charge of maintaining the homeostasis of ROS. Cells have several enzymatic

362

and nonenzymatic antioxidants that include vitamin C, vitamin E, catalase, Trx glutathione, and

363

superoxide dismutase [33]. The expression and functions of human Trxs have been reported in

364

several female reproductive organs, including the uterine endometrium and the ovary [32]. The

365

upregulation and induction of Trx are influenced by several factors, such as UV exposure, viral

366

infection, and H2O2. In addition to this, estrogen is one of the strongest inducers of Trx [34,35].

367

Previous studies in the rat show that Trx-2 is highly expressed in the neurons in most brain

368

regions exhibiting severe oxidative stress, including the olfactory bulb, cerebellum, and frontal

369

cortex. Trx-2 is induced not only by oxidative stress but also by ischemia/reperfusion and

370

cerebral infarction [36]. The pathophysiology of ischemia resulted in the overproduction of ROS

371

and an imbalance in redox homeostasis [37]. Further, this study showed that single

372

dexamethasone treatment upregulated Trx-2 mRNA expression in the thalamic reticular nucleus

373

and the paraventricular hypothalamic nucleus [36]. Another study revealed that an increase in the

374

level of Trx-2 and Prx3 in the hippocampus and spinal cord of aged dogs might be associated

375

with a reduction in oxidative stress-related neuronal damage throughout normal aging [38].

376

Further investigation has shown that the medullary thick ascending limb in the kidney is most

377

susceptible to ischemia and reperfusion because of its high demand for ATP-dependent

378

reabsorption. These findings were further clarified using transgenic human Trx-overexpressing

379

mice that were resistant to the injury and functional deterioration of the medullary thick

380

ascending limb caused by ischemia/reperfusion [39].

381

Red blood cells (RBC) transfer oxygen to all cells continuously exposed to oxidative stress. Free

382

radicals can deteriorate RBC products by lipid and protein oxidation. Further, the RBC

383

membrane is affected by oxidative damage [40]. Therefore, blood has a more powerful

384

antioxidant system than other tissues. Phagocytic leukocytes may also trigger oxidative stress by

385

producing ROS in response to certain stimuli [41]. Altogether, the tissue distribution analysis

386

revealed the HaTrx-2 activity in response to the oxidative stress of tissues.

387

To examine the temporal expression profile of HaTrx-2, the kidney and blood of the immune

388

challenged seahorse were collected. To evaluate the response to an immune challenge, S. iniae, E.

389

tarda, poly I:C and LPS were used. E. tarda is a gram-negative, rod-shaped bacterium whose

390

infection leads to severe economic losses in the aquaculture of teleost fishes. S. iniae is a gram-

391

positive bacterium associated with acute and chronic fish diseases [36]. LPS is a gram-negative

392

bacteria cell wall component and endotoxin. Poly I:C is a viral mimic used to stimulate viral-like

393

infections. The HaTrx-2 mRNA expression in the blood upon immune challenges at later hours

394

is higher than that in the kidney. Since the blood is constantly exposed to oxidative stress,

395

erythrocytes are continuously damaged by free radicals. Further, macrophages and neutrophils

396

are involved in phagocytosis. During phagocytosis, the NADPH oxidase activation process

397

produces O2− (superoxide) by reducing oxygen via phagocyte NADPH oxidase 2. The process is

398

known as “oxidative burst.” Consecutive dismutation occurs to form H2O2 and as a consequence,

399

some reactive microbicidal oxidants are produced by myeloperoxidase-catalyzed oxidation of

400

Cl− and reduction of H2O2, including hypochlorous acid (HOCl), OH· radicals, and peroxynitrite

401

(ONOO−) [42]. This might be one of the reasons for the upregulation of HaTrx-2 upon immune

402

challenges. The expression of the HaTrx-2 transcript showed upregulation only upon LPS

403

stimulation at 3 h p.i. in the kidney. In contrast, HaTrx-2 mRNA was upregulated with four

404

stimulants at 3 h p.i., and upregulation was significant upon LPS and S. iniae treatments in blood.

405

The reason may be that the blood exhibited higher levels of oxidative stress than the kidney, and

406

basal level expression may not be enough to sustain oxidative stress. Moreover, LPS can directly

407

interact with complement receptor 3 (CR3) present in phagocytic cells to induce an inflammation

408

that results in phagocytosis [43]. Consequently, respiratory burst occurs and ROS are formed

409

[42]. The overproduction of ROS stimulates the antioxidant system.

410

Both kidney and blood mRNA expressions revealed a significant upregulation of HaTrx-2 with

411

the presence of poly I: C. In both tissues, peak values were observed for poly I:C at 24 h p.i. Fish

412

infected by viruses generally produce interferons [44]. Poly I:C is considered as a potent inducer

413

of interferons (IFNs) [45]. The previous study demonstrated that human Trx is induced by IFN-γ,

414

and both protein and mRNA levels of Trx were increased by 2~3 fold within 4 to 24 h after IFN-

415

γ treatment [46]. HaTrx-2 mRNA expression level reached its peak value gradually in both

416

tissues at 72 h p.i. upon E. tarda challenge, but no upregulation was observed in blood at the

417

early phases (3, 6 and 12 h p.i.). The reason is that when gram-negative bacteria enter the

418

bloodstream, LPS interacts directly with leukocytes to trigger an inflammatory cascade [43]. In

419

blood, HaTrx-2 expression was downregulated in the long mid-secretory phase (12, 24, and 48 h)

420

upon S. iniae stimulation. A previous study showed that S. iniae has adapted to survive in

421

phagocytes and induces their apoptosis [47]. It causes a decrease in other functions relying on

422

phagocytosis. The mRNA turnover [48] might be the reason for the fluctuation in Trx-2

423

expression in both tissues with all stimulants, owing to the maintenance of ROS levels in cells, as

424

ROS are involved in the elimination of various pathogens [49] and signal transduction. Our

425

results suggest that HaTrx-2 might be involved in pathogen attacks and its expression can be

426

varied according to time and tissue type.

427

In the current study, Trx-2 was characterized from the big-belly seahorse, and the rHaTRx-2

428

functional properties were evaluated by insulin disulfide reductase assay, MCO assay, DPPH

429

radical scavenging activity assay, and cell viability assay. The dithiol reducing enzymatic

430

activity of HaTrx-2 was confirmed by the insulin disulfide reduction assay. Insulin consists of

431

two amino acid chains referred to as A and B chains, and they are linked together by two

432

disulfide bridges. DTT is a water-soluble reducing agent also known as Cleland’s reagent, which

433

reduces disulfide bonds to sulfhydryl groups. The two interchain disulfides of insulin are broken

434

during the reduction. The aggregation of the free B chain is the reason for the white precipitation

435

that can be observed at 650 nm by spectrophotometer [50]. Our data showed concentration-

436

dependent insulin disulfide reduction activity. Further, the specific activity of the Trx protein

437

from other species, including manila clam (3.098 U/mg) [24], disk abalone (1.825 U/mg) [26],

438

and antarctic microcrustacean (5.04 U/mg) [51] has been assessed previously using such assays.

439

These results suggest that different organisms show different Trx-2 reductase activities.

440

Collectively, these results confirm the reductase activity of HaTrx-2.

441

To examine the DNA protection activity of rHaTrx-2 from nicking, the metal-catalyzed

442

oxidation assay was performed. The supercoiled plasmid DNA disruption results in the

443

formation of OH radicals during the auto-oxidation of DTT. The MCO system containing pUC19

444

without recombinant protein showed nicked bands due to damage of supercoiled plasmid DNA.

445

A high level of DNA damage was observed when the rMBP was added to the MCO system, yet

446

this level of damage was lower than the damage observed using the MCO system without any

447

protein. In addition, the significantly higher activity of rHaTrx-2 compared with rMBP indicated

448

that rMBP did not show antioxidant activities. In the presence of an increasing concentration of

449

rHaTrx-2, the intensity of the supercoiled band increased in a concentration-dependent manner

450

due to the antioxidant activity of the recombinant protein. Similarly, previous studies have

451

demonstrated the DNA protection ability of TRx-2 in Manila clam (Ruditapes philippinarum)

452

[24] and disk abalone (Haliotis discus discus) [26].

453

The DPPH experiment is based on the reduction of α, α-diphenyl-β-picrylhydrazyl (DPPH).

454

DPPH is a persistent free radical that does not react with water, methanol, or ethanol. The

455

involvement of free radical scavenging antioxidant (Hydrogen donor) is reduced to DPPHH and

456

it loses its violet color as a consequence of absorbance decrease over time [25]. It is extensively

457

used to evaluate the antioxidant potential of hydrogen donors because it is a simple, inexpensive,

458

and rapid method. Ascorbic acid (a well-known antioxidant) was used as a positive control. The

459

IC50 value represents the concentration of recombinant protein required to inhibit 50% of DPPH

460

radicals. The IC50 value of rHaTrx-2 was 43.87 ± 1.90 µg/mL. Previous investigations in the

461

same organism but with different Trx classes showed different IC50 values; The IC50 values of

462

Trx-like protein 1 [52] and Trx domain-containing protein 17 [53] were 56.01 µg/mL and

463

23.94 µg/mL, respectively. We cannot compare the data within different Trx classes by these

464

results because they have different characteristics and localization. However, this result

465

demonstrates the antioxidant potential of rHaTrx-2 protein.

466

The MTT assay was performed to evaluate the cell viability rate. The metabolically active viable

467

cells have NAD(P)H-dependent oxidoreductase enzymes, which reduce the yellow tetrazolium

468

salt of MTT into purple formazan crystals. The main processes of apoptosis are mitochondrial

469

permeability transition activation, BCL-2 downregulation, cytochrome C liberation into the

470

cytosol, and caspase 3 activation. Trx-2 prevents apoptosis by scavenging ROS and inhibits the

471

signaling of apoptosis signal-regulating kinase 1 (ASK1). A previous study reported that the

472

overexpression of mitochondrial Trx in human osteosarcoma cells protects cells from apoptosis

473

[54]. Another study demonstrated that Trx-2 knockout mice can be recognized by depolarization

474

of the mitochondrial membrane, elevated amount of ROS in the mitochondria, lack of production

475

of ATP, and increased ASK1 signaling and cell death [55]. Taken together, these results suggest

476

that Trx-2 expression is involved in the protection of cells from ROS-related apoptosis.

477 478

Conclusion

479

In this study, HaTrx-2 was characterized by using molecular, transcriptional, and functional

480

analyses. HaTrx-2 consists of a Trx-like superfamily domain with a CXXC motif, which was

481

confirmed using in-silico tools. The spatial expression analysis revealed that HaTrx-2 was

482

pervasively expressed throughout the entire tissues examined. The temporal expression profiles

483

in the kidney and blood showed that HaTrx-2 was upregulated upon S. iniae, E. tarda, poly I:C,

484

and LPS immune stimulation. The insulin disulfide reduction assay result suggested that Trx-2 is

485

involved in keeping proteins in a reduced state. The antioxidant and free radical scavenging

486

properties of HaTrx-2 were demonstrated by cell viability assay, DPPH radical scavenging

487

activity, and MCO assays. Collectively, our study suggests that HaTrx-2 plays an imperative role

488

in ROS regulation in the protection against oxidative stress in host cells.

489 490

Acknowledgments

491

This research was supported by the Basic Science Research Program through the National

492

Research

493

(2019R1A6A1A03033553).

494 495

Foundation

of

Korea

(NRF)

funded

by

the

Ministry

of

Education

496

List of Tables

497

Table 1. Primers used for cloning and qPCR analysis of HaTrx-2

498 499 500

Name

Sequence (5′- 3′)

Amplicon Size

Tm

Application

HaTrx-2-cF

GAGAGAgatatcATGGCTCATAGGCTGCTAGCG

519 bp

61.8°C

Cloning, pMAL-c5X

HaTrx-2-cR

GAGAGAgaattcTTATTTCCCGATGATCTTGCTGACAAACGA

519 bp

60°C

Cloning, pMAL-c5X

HaTrx-2-cF

GAGAGAgaattcATGGCTCATAGGCTGCTAGCG

519 bp

61.8°C

Cloning, pcDNA3.1(+)

HaTrx-2-cR

GAGAGActcgagTTATTTCCCGATGATCTTGCTGACAAACGA

519 bp

60°C

Cloning, pcDNA3.1(+)

HaTrx-2-qF

AAGGTTGGAGAAGGCTGTTGCG

197 bp

60°C

qPCR

HaTrx-2-qR

TCTTGCTGACAAACGAGTCCAGTTCA

197 bp

60°C

qPCR

40S ribosomal S7 F

GCGGGAAGCATGTGGTCTTCATT

95 bp

60°C

qPCR internal reference

40S ribosomal S7 R

ACTCCTGGGTCGCTTCTGCTTATT

95 bp

60°C

qPCR internal reference

501

502 503

Table 2. Identity and similarity percentage of HaTrx-2 amino acid sequence with different Trx-2 orthologs Accession No

Scientific Name

Identity (%)

Similarity (%)

XP_012723347.1

Fundulus heteroclitus

78.7

86.2

XP_015800689.1

Nothobranchius furzeri

77.5

85.0

ACM09441.1

Salmo salar

75.6

86.6

XP_017337154.1

Ictalurus punctatus

69.4

81.5

NP_991204.1

Danio rerio

68.0

82.0

BAA13447.1

Bos taurus

55.3

70.4

XP_008108856.1

Anolis carolinensis

55.2

67.8

NP_036605.2

Homo sapiens

54.7

69.8

XP_007908328.1

Callorhinchus milii

54.0

72.4

NP_001230634.1

Sus scrofa

53.6

67.0

NP_001008161.1

Xenopus tropicalis

53.3

68.3

NP_001232779.1

Taeniopygia guttata

51.7

66.3

NP_445783.1

Rattus norvegicus

51.1

65.6

504

List of Figures

505 506

Fig. 1. Multiple sequence alignment of HaTrx-2 and its orthologs from different organisms. Fully conserved amino acid residues

507

are denoted by an asterisk (*). Strongly conserved and partially conserved amino acid residues are denoted by colons (:) and periods

508

(.), respectively. The WCGPC active site is enclosed in a red box. The mitochondrial localization N-terminal sequence is indicated

509

by a purple line.

510 511

Fig. 2. Phylogenetic tree of different Trx-2 amino acid sequences constructed using the neighbor-joining method (MEGA version

512

7.0.26 software). Each branch is indicated by bootstrap values.

Relative mRNA expression

7 6 5 4 3 2 1 0

Tissues 513 514

Fig. 3. Spatial mRNA expression level analysis of HaTrx-2 in different tissues. The mRNA expression fold-changes of HaTrx-2

515

were deduced by qPCR using the 2−∆∆CT method. Seahorse 40S ribosomal protein S7 was used as an internal reference. Data are

516

presented in proportion to the expression level of mRNA in the spleen. The standard deviation of triplicate samples is represented

517

by error bars.

518

519 520 521 522 523 524 525 526

527 528

Fig. 4. Expression pattern of HaTrx-2 in (A) kidney and (B) blood, after in vivo challenge with poly I:C, lipopolysaccharides (LPS),

529

Streptococcus iniae, and Edwardsiella tarda. Relative mRNA levels were determined by SYBR Green qPCR. The analysis was

530

performed using the Livak method. Fold changes at different time points during transcription are shown as normalized to the

531

mRNA level of the group injected with PBS. The results are represented as mean ± standard deviation (SD) of triplicates.

532

Statistically significant values (P < 0.05) are indicated with an asterisk (*).

533 534

Fig. 5. Analysis of purified MBP fused rHaTrx-2 protein by SDS PAGE. 1: Protein Marker, 2: Total extract of E. coli BL21 cells

535

with rHaTrx-2 prior to IPTG induction, 3: Total extract of induced E. coli BL21 cells with rHaTrx-2, 4: Supernatant after sonication,

536

5: Purified rHaTRx-2 protein, 6: MBP protein.

537 538 539

540 541

Fig. 6. Insulin disulfide reductase activity of rHaTrx-2. DTT and insulin were incubated with different concentrations of rHaTrx-2.

542

Controls were carried out with rMBP and without rHaTrx-2 separately. Negative control was carried out without DTT. Absorbance

543

measurement at 650 nm was taken in each 5 min intervals.

544 545

Fig. 7. Effect of different concentrations (15, 30, 45, 60, 90, and 120 µg/mL) of rHaTrx-2 and 120 µg/mL of rMBP on DPPH

546

radical scavenging activity. Ascorbic acid was used as a relative control in this assay. Error bars denote the standard deviations (SD)

547

of the replicates.

548 549

Fig. 8. An MCO assay revealed supercoiled DNA protection from oxidative damage by rHaTrx-2. (A) pUC19 only; (B) MCO

550

system with pUC19; (C) MCO system with pUC19 and rMBP; (D-H) MCO system with pUC19 and different concentrations of

551

rHaTRx-2 (0.05, 0.1, 0.2, 0.4, and 0.8 µg/µL) NF: Nicked form; SF: supercoiled form.

552

Cell viability percentage (%)

pcDNA 3.1(+) Trx-2

106 104 102 100 98 96 94 92 90 88

0

250 µM

500 µM

H2O2 concentration

553 554

Fig. 9. Cell viability percentage of empty pcDNA3.1(+) and HaTrx-2-inserted pcDNA3.1(+) transfected cells during H2O2

555

treatment.

556

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Highlights •

The cDNA of thioredoxin mitochondrial like gene was cloned from Hippocampus abdominalis.

•

HaTrx-2 was ubiquitously expressed in all examined tissues.

•

The mRNA expression of HaTrx-2 upon immune challenges was analyzed.

•

The rHaTrx-2 had the ability to scavenge the free radicals.

•

The rHaTrx-2 exhibited cytoprotective activity upon H2O2 stress.