Identification and characterization of c-raf from orange-spotted grouper (Epinephelus coioides)

Journal Pre-proof Identification and characterization of c-raf from orange-spotted grouper (Epinephelus coioides) Ze-Quan Mo, Xue-Li Lai, Wan-Tao Wang, Hong-Ping Chen, Zhi-Chang He, Rui Han, Jiu-Le Wang, Xiao-Chun Luo, Yan-Wei Li, Xue-Ming Dan PII:

S1050-4648(19)31145-3

DOI:

https://doi.org/10.1016/j.fsi.2019.12.017

Reference:

YFSIM 6659

To appear in:

Fish and Shellfish Immunology

Received Date: 18 October 2019 Revised Date:

3 December 2019

Accepted Date: 9 December 2019

Please cite this article as: Mo Z-Q, Lai X-L, Wang W-T, Chen H-P, He Z-C, Han R, Wang J-L, Luo X-C, Li Y-W, Dan X-M, Identification and characterization of c-raf from orange-spotted grouper (Epinephelus coioides), Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2019.12.017. 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. © 2019 Published by Elsevier Ltd.

1

Identification and characterization of c-Raf from orange-spotted grouper

2

(Epinephelus coioides)

3

Ze-Quan Moa,b, Xue-Li Laia, Wan-Tao Wangc, Hong-Ping Chena, Zhi-Chang Hea, Rui

4

Hana, Jiu-Le Wanga, Xiao-Chun Luod, Yan-Wei Lia,*, Xue-Ming Dana,**

5 6 7 8

a

9

ource Conservation and Exploitation, College of Marine Sciences, South China

Joint Laboratory of Guangdong Province and Hong Kong Regions on Marine Biores

10

Agricultural University, Guangzhou 510642, Guangdong Province, China

11

b

12

Guangdong Province, China

13

c

14

Fujian Province, China

15

d

16

Guangzhou 510006, China

College of Animal Science, South China Agricultural University, Guangzhou 510642,

Provincial Clinical Medical College, Fujian Medical University, Fuzhou 305001,

School of Bioscience and Biotechnology, South China University of Technology,

17 18 19 20 21 22

**

23

Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou 510642,

24

China.

25

*

26

University, 483 Wushan Street, Tianhe District, Guangzhou 510642, China.

27

E-mail addresses: [email protected] (X.-M. Dan), [email protected] (Y.-L.

28

Li).

29

Correspondence to: X.-M. Dan, College of Marine Sciences, South China

Correspondence to: Y.-L. Li, College of Marine Sciences, South China Agricultural

30

Abstract

31

C-Raf proto-oncogene serine/threonine kinase is a mitogen-activated protein

32

kinase (MAP) kinase kinase, which can initiate a mitogen-activated protein kinase

33

(MAPK) cascade by phosphorylating the dual-specific MAP kinase kinases (MEK1/2),

34

and in turn activate the extracellular signal-regulated kinases (ERK1/2). To study the

35

function of c-Raf in teleost fish, a c-Raf cDNA sequence from orange-spotted grouper

36

(Epinephelus coioides) was cloned. Ecc-Raf shared 81%–99% amino acid identity

37

with other vertebrate c-Raf molecules, and shared the highest amino acid identity

38

(99%) with Lates calcarifer c-Raf. Genomic structure analysis revealed that grouper

39

c-Raf shared a conserved exon structure with other vertebrates. Tissue distribution

40

showed that Ecc-Raf was mainly transcribed in systemic immune organs. Ecc-Raf was

41

distributed throughout the cytoplasm of transfected GS cells and the overexpression

42

of Ecc-Raf only slightly enhanced the activation of Activator protein 1. The

43

phosphorylation levels of Ecc-Raf can be induced by PMA and H2O2 treatment, in

44

contrast to DMSO or untreated HKLs. Moreover, the phosphorylation level of the

45

Raf-MEK-ERK axis was downregulated after 24 h of SGIV infection. On the other

46

hand, the total level and phosphorylation level of c-Raf significantly increased post C.

47

irritans infection and showed an enhanced level post immunization. The results of

48

this study suggested that the Raf-MEK-ERK cascade was involved in the response to

49

viral or parasitic infections.

50 51

Keywords: Epinephelus coioides, Cryptocaryon irritans, SGIV, c-Raf

52

1. Introduction

53

Mitogen-activated protein kinase (MAPK) signaling pathways play an important

54

role in the regulation of cell growth and differentiation, and respond to a variety of

55

extracellular stimuli [1-3]. Four distinct groups of MAPK signaling cascades are

56

found in mammals: extracellular signal-regulated kinase (ERK) 1/2, p38 MAP kinase,

57

c-Jun N-terminal kinases (JNK), and ERK5 [4-7]. These groups are activated by

58

specific MAPKKs: MEK1/2 for ERK1/2, MKK3/6 for p38, MKK4/7 for JNKs, and

59

MEK5 for ERK5. MAPKKs can be activated by more than one MAP kinase kinase

60

kinase (MAPKKK), depending on the type of stimulation [8].

61

C-Raf proto-oncogene, serine/threonine kinase (c-Raf), also known as Raf-1

62

proto-oncogene, serine/threonine kinase (Raf-1), is a MAPKKK which can bind to the

63

membrane-associated Ras GTPases and be recruited to the cell membrane for

64

activation. c-Raf activation initiates a MAPK cascade that the cellular c-Raf protein

65

can phosphorylate to activate the dual-specific MAPK kinases (MEK1 and MEK2),

66

which in turn phosphorylates to activate the extracellular signal-regulated kinases

67

(ERK1 and ERK2) [9]. Activated ERK1/2 plays an important role in the regulation of

68

gene expression involved in the cell division cycle, apoptosis, cell differentiation, and

69

cell migration [10-12].

70

In mammals, c-Raf contains a Ras-binding domain (for upstream Ras GTPases

71

binding), a protein kinase C conserved region 1 domain, and a serine/threonine

72

protein kinase catalytic domain (for downstream dual-specific MAPK kinase

73

activation). Mutations in the human c-Raf gene are associated with Noonan syndrome

74

5 (characterized by unusual facial features, short stature, heart defects, and eye

75

conditions) [13, 14], and LEOPARD syndrome 2 (characterized by cardiac

76

abnormalities, short stature, and facial dysmorphia) [15]. Growth retardation was

77

observed in transgenic chimeric c-Raf-deficient mice [16]. Therefore, it has been

78

suggested that c-Raf is essential for growth and development [17].

79

Although the function of c-Raf has been extensively studied in mammals, little is

80

known about this molecule in teleosts, and just a few homolog sequences have been

81

released in public network databases. The function analysis of c-Raf has mainly

82

focused on zebrafish, which is a model organism and suitable for development studies.

83

Zebrafish embryos that were injected with mutant c-Raf mRNA showed defects in

84

posterior axis formation and tail structure [18]. Knockdown of c-Raf using

85

morpholino in zebrafish showed several developmental defects including curly tail,

86

cardiac malformation, enlarged pericardium, and craniofacial hypoplasia [14].

87

However, the activation of c-Raf during pathogen infection is poorly illustrated in

88

lower vertebrates.

89

Singapore grouper iridovirus (SGIV) and Cryptocaryon irritans are two important

90

pathogens which cause serious economic losses in the marine aquaculture industry

91

[19, 20]. Recent studies have shown that both SGIV and C. irritans infection can

92

activate the MEK-ERK signaling pathway [21, 22]. However, as the upstream kinase

93

of MEK-ERK signaling transduction, whether c-Raf is involved in the activation of

94

the MEK-ERK cascade during SGIV and C. irritans infection remains unclear. To

95

further investigate this, the cDNA sequences of c-Raf from orange-spotted grouper

96

(Epinephelus coioides) were cloned, and the expression pattern of Ecc-Raf in various

97

tissues was detected. The localization and luciferase reporter assays of Ecc-Raf were

98

determined. The specific rabbit anti-rEcc-Raf antibody which is suitable for grouper

99

c-Raf detection using the western blot technique was also generated. Grouper HKLs

100

treated with phorbol 12-myristate 13-acetate (PMA) or H2O2 showed an increased

101

phosphorylation level of Ecc-Raf. The phosphorylation level of Ecc-Raf significantly

102

decreased with SGIV infection. An increased phosphorylation level of Ecc-Raf was

103

found in C. irritans infected or immune skin. The results of this study suggested that

104

grouper Raf-MEK-ERK signal pathways were involved in the response to viral or

105

parasitic infection.

106 107

2. Materials and methods

108

2.1 Fish

109

Thirty healthy orange-spotted groupers (18.4 ± 2.2 g) were purchased from the

110

Marine Fisheries Development Center of Guangdong Province, Guangdong, China,

111

and maintained at 24°C in a flow-through water system (300 L) as previously

112

described [22]. Samples of the thymus, gill, head kidney, skin, liver, spleen, and

113

intestine tissues were taken from groupers for tissue distribution analysis. All samples

114

were immediately frozen in liquid nitrogen and stored at -80°C.

115 116

2.2 Cryptocaryon irritans infection and grouper immunization

117

C. irritans was isolated from infected Trachinotus ovatus obtained from a local

118

farm in Daya Bay, Guangdong Province, China, and propagated using T. ovatus as a

119

host [23]. Grouper surface exposure immunization was conducted as described

120

previously [22]. Briefly, C. irritans theronts were used to infect groupers every 2

121

weeks at a dose of 4,000 theronts per fish in dark conditions for 2 hours. The fish

122

were transferred into a clean tank every 3 days to avoid secondary infection.

123

Uninfected fish were also transferred every 3 days. The surface exposure

124

immunization was repeated two more times. Following this, both the uninfected

125

groupers and the immune groupers were challenged with living C. irritans theronts at

126

a dose of 12,000 theronts per fish. Three days post-challenge, groupers were

127

anaesthetized with MS-222, and skin near the dorsal fin of each group was collected.

128

Skin samples from uninfected groupers were collected as negative controls.

129 130

2.3. RNA extraction and cDNA synthesis

131

Total RNA was extracted from all samples with TRIzol reagent (Invitrogen)

132

following the manufacturer’s protocol and stored at -80°C. The cDNA for obtaining

133

full-length grouper c-Raf was synthesized from the total RNA of healthy grouper

134

spleen using the SMARTerTM RACE cDNA Amplification Kit (Clontech, Palo Alto,

135

CA, USA). The cDNA for tissue distribution analysis was synthesized from total RNA

136

from all collected samples using a ReverTra Ace-a-Kit (Toyobo, Katata, Ohtsu,

137

Japan).

138 139

2.4. Cloning of gene sequences

140

An expressed sequence tag (EST) sequence of c-Raf was identified from grouper

141

transcriptome data using the BLASTx program [19]. However, it was noted that this

142

c-Raf EST sequence was missing 3′ regions. To obtain the 3′ unknown regions of

143

c-Raf, c-Raf 3′GSP1/UPM, and c-Raf 3′GSP2/NUP primers (Table 1) were designed

144

for primary and nested PCR to obtain the full-length sequence of c-Raf from groupers.

145

The amplification protocol was performed as follows: (98°C, 10 s; 63°C, 15 s; 72°C,

146

2 min) for 35 cycles, and one cycle of 72°C for 5 min. The amplification products

147

were purified and sequenced. The open reading frame (ORF) of c-Raf was amplified

148

using the primers c-Raf ORF F/R (Table 1). The amplification protocol was

149

performed as follows: (98°C, 10 s; 58°C, 15 s; 72°C, 2 min) for 35 cycles, and one

150

cycle of 72°C for 5 min. The amplification products were purified and ligated into

151

pEASY-Blunt Cloning Vector (TRANS, Beijing, China) for sequencing.

152 153

2.5. Gene structure and phylogenetic analysis

154

The

ORF

of

Ecc-Raf

was

predicted

using

ORF

finder

155

(https://www.ncbi.nlm.nih.gov/orffinder/). The theoretical isoelectric point (pI) and

156

molecular weight (Mw) of Ecc-Raf were predicted using the Compute pI/Mw tool

157

(http://web.expasy.org/compute_pi/). Conserved domains were searched for in the

158

CDD tool (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Gene information

159

of c-Raf from other vertebrates was downloaded from NCBI, and the gene sequences

160

of grouper c-Raf were kindly provided by Dr. Zhao Mi (unpublished genome data).

161

Alignment and phylogenic analysis of c-Raf were performed using the MEGA 5.04

162

program. All GeneBank accession numbers used in this study are listed in

163

Supplementary Table 1.

164 165

2.6. Tissue distribution analysis

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The expression level of Ecc-Raf in various tissues was determined using the

167

SYBR Green Realtime PCR Master Mix (Toyobo) as previously described [24].

168

Gene-specific primers of Ecc-Raf RTF/R (Table 1) were used for real-time PCR.

169

β-actin primers (β-actin F/β-actin R) were used to amplify the reference gene. The

170

cycling protocol was: 94°C for 2 min and 94°C, 15 s; 58°C, 15 s; 72°C, 20 s for 40

171

cycles. Melting curve analysis and sequencing were used to detect the specificity of

172

PCR products. The PCR products were verified by sequencing. All samples were

173

analyzed in triplicate. The expression of the target gene was normalized to the β-actin

174

gene and calculated using the 2−∆∆Ct method [25].

175 176

2.7. Expression, purification, and polyclonal antibody preparation of Ecc-Raf

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An N-terminal 336-amino acid fragment of Ecc-Raf was amplified with rc-Raf

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F/R primers (Table 1) and restriction enzymes KpnI or EcoRI at the 5′-termini. The

179

fragment was then cloned into a pET32a vector and transformed into E. coli BL21

180

(DE3). The recombinant Ecc-Raf protein (rEcc-Raf) was induced as previously

181

described [24]. Briefly, positive E. coli were cultured and induced by 1 mM

182

isopropyl-β-d-thiogalactoside (IPTG) for 4 hours. rEcc-Raf protein was purified using

183

a nickel-nitrilotriacetic acid column (Ni-NTA; Qiagen, Germany), according to the

184

manufacturer’s instructions. The purified rEcc-Raf was emulsified with Freund’s

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Complete Adjuvant (Sigma-Aldrich, St. Louis, MO, USA) and 1 mg of protein was

186

injected into rabbits. Animals were then boosted with 0.5 mg of purified antigen in

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Freund's incomplete adjuvant (Sigma-Aldrich) on two separate occasions. A rEcc-Raf

188

affinity column was prepared according to the manufacturer’s instructions

189

(Smart-Lifesciences, Changzhou, Jiangsu, China). Rabbit anti-rEcc-Raf IgG was

190

purified using rEcc-Raf affinity column. The specificity of the polyclonal antibody

191

was detected by western blotting as described in section 2.11 below.

192 193 194

2.8. Subcellular localization and luciferase reporter assay Two

plasmids

pEGFP-Ecc-Raf-SL

(for

subcellular

localization)

195

pEGFP-Ecc-Raf-LR (for luciferase reporter assay) were constructed by amplification

196

with primers c-Raf EGFP F/R1 and c-Raf EGFP F/R2 (Table 1), which contained

197

20-bp end sequences identical to pEGFP-N1 at the 5′-termini, respectively. Linearized

198

pEGFP-N1 were generated by using EcoR I and Kpn I double-digestion. The PCR

199

product was mixed with linearized pEGFP-N1 and ligated using ClonExpress Ultra

200

One Step Cloning Kit (Vazyme, China) following the manufacturer's protocol. Both

201

plasmids were extracted using E.Z.N.A. Endo-free Plasmid Mini Kit (Promega,

202

Madison, WI, USA) for subsequent transformation.

203

Culture and transfection of GS cells was performed as previously described [26,

204

27] with some modifications. Briefly, GS cells were cultured in Leibovitz’s L-15

205

medium containing 10% fetal bovine serum at 27°C. At the 90% confluence, the

206

pEGFP-Ecc-Raf-SL and pEGFP-N1 plasmids Endo-free plasmid (1 µg for each

207

plasmid) were transfected into GS cells using Lipofectamine™ 2000 Reagent

208

(Invitrogen). Twenty-four hours after transfection, the cells were fixed with 4%

209

paraformaldehyde for 15 min and then 1 mg/mL of 4′, 6-diamidino-2-phenylindole

210

(DAPI) was added followed by a further 5 min incubation. The intracellular

211

localization was observed and photographed using a NIH-Elements System (Nikon,

212

Tokyo, Japan). In addition, transfected GS cells were harvested and the positive

213

transfected ratio was detected using flow cytometry (CytoFLEX, Beckman Coulter).

214

The luciferase reporter assay was performed as described previously [27]. In

215

brief, pEGFP-Ecc-Raf-LR (150 ng) and pEGFP-N1 (150 ng) were co-transfected with

216

40 ng of AP-1 dependent firefly luciferase reporter vector and 10 ng of Renilla

217

luciferase vector. Twenty-four hours after transfection, cells were collected and lysed

218

for dual-luciferase reporter assay analysis according to the manual. The relative

219

luciferase activity was measured by the ratio between firefly luciferase activity and

220

Renilla luciferase activity.

221

222

2.9. In vitro stimulation

223

Grouper head kidney leukocytes (HKLs) were prepared as described in [22]

224

using Ficoll-Paque PLUS (1.077 g/ml; GE Healthcare). The leukocyte layer was

225

collected and washed twice with PBS then cultured in L-15 (Gibco) at 28 °C. 1 × 106

226

HKLs were treated with DMSO, 100 nM or 200 nM PMA (Selleck) for 15 min, or 1

227

mM and 10 mM H2O2 for 5 min at 28°C and washed with PBS. Cell lysates were

228

prepared and western blotting analysis was performed as described in section 2.11

229

below.

230 231

2.10. SGIV infection

232

SGIV infection was performed as described in [21]. Briefly, GS cells were

233

cultured at the 90% confluence and added to a medium containing SGIV for infection.

234

24 hours post-infection, mock- and SGIV-infected GS cells were harvested. Cell

235

lysates were prepared and western blotting analysis was performed as described in the

236

following section.

237 238

2.11. Western blotting

239

Total proteins of grouper skin, head kidney, spleen, GS cells, and HKLs were

240

extracted using RIPA lysis buffer (Beyotime) by adding an extra 1 mM

241

Phenylmethanesulfonyl fluoride (Beyotime) and 1 mM pervanadate (Sigma),

242

respectively. The supernatant was mixed with SDS sample buffer and boiled for 10

243

min. Protein samples were electrophoresed and transferred to polyvinylidene fluoride

244

(PVDF) membranes as described in [24]. The PVDF membranes were blocked in 5%

245

dried milk (diluted with PBST) for 1 hour at room temperature, followed by

246

incubation with rabbit anti-rEcc-Raf IgG (1:1,000 dilution), rabbit anti-pc-Raf IgG

247

(1:1000 dilution, CST), rabbit anti-MEK1/2 IgG (1:1000 dilution, CST), rabbit

248

anti-pMEK1/2 IgG (1:1,000 dilution, CST), rabbit anti-ERK1/2 IgG (1:1,000 dilution,

249

CST), rabbit anti-pERK1/2 IgG (1:1,000 dilution, CST), or mouse anti-actin IgG

250

(1:1,000 dilution, Proteintech) overnight at 4°C with gentle shaking. Membranes were

251

washed with PBST three times and incubated with goat anti-rabbit IgG antibodies

252

conjugated to horseradish peroxidase (HRP), or goat anti-mouse IgG antibodies

253

conjugated to HRP, for 1 hour at room temperature. Membranes were washed in

254

PBST three times and incubated with SuperSignal West Pico Chemiluminescent

255

Substrate (Thermo), then exposed and

analyzed using the Tanon 5200

256

chemiluminescence imaging analysis system (Tanon).

257 258

3.

Results

259

3.1. Characteristics of Ecc-Raf and phylogenetic analysis

260

The ORF of Ecc-Raf (GenBank no. MN577973) was 1,914 bp, which encoded

261

sequences of 637 amino acids with a theoretical pI of 9.3, and a molecular mass of

262

72.0 kDa. The amino acid sequence of Ecc-Raf contains a Ras-binding domain, a

263

Protein kinase C conserved region 1 domain and a serine/threonine protein kinase

264

catalytic domain which is well conserved in both teleosts and mammals (Fig. 1).

265

There were 11 strand parallel β-shells and 10 α-helices in Ecc-Raf, which matched the

266

structure of human c-Raf (PDB id: 3omv.1.A, QMEAN = -3.44) (Fig. 2). Homology

267

analysis showed that Ecc-Raf shared 81%–99% overall amino acid identity with c-Raf

268

molecules of other teleosts and mammals, and shares the highest amino acid identity

269

(99%) with Lates calcarifer c-Raf. The Ras-binding domain, protein kinase C

270

conserved region 1 domain and serine/threonine protein kinases catalytic domain of

271

Ecc-Raf shared 73%–99%, 94%–100%, and 91%–99% amino acid identity with those

272

in teleosts and mammals, respectively (Table 2). Similar to other vertebrates’ c-Raf

273

genes, the grouper c-Raf gene showed a conserved exon structure, containing 16

274

exons (Fig. 3). The exon sizes of the grouper c-Raf gene were 201, 113, 103, 159, 89,

275

154, 25, 116, 118, 85, 177, 47, 119, 132, 135, and 141 bp. Four clades were formed in

276

the phylogenetic tree, which represented mammalian c-Raf, avian c-Raf, reptilian

277

c-Raf, and teleostein c-Raf, of which Ecc-Raf shared the closest homology with the L.

278

calcarifer (Fig. 4).

279 280

3.2. Tissue distribution of Ecc-Raf in healthy grouper

281

The mRNA expression level of Ecc-Raf was detected in seven types of grouper

282

tissue. The results showed that Ecc-Raf was transcribed in all tissues and was mainly

283

expressed in the immune organs. The highest transcript levels of Ecc-Raf were found

284

in head kidney tissue followed by spleen, skin, intestine, thymus, liver, and gill (Fig.

285

5).

286 287

3.3. Expression and purification of rEcc-Raf and polyclonal antibodies

288

preparation

289

A ~40 kDa rEcc-Raf protein, which contained a N-terminal 336-amino acid

290

fragment of Ecc-Raf, was successfully induced from a positive clone of E. coli (Fig.

291

6A). The rEcc-Raf protein was purified using a nickel-nitrilotriacetic acid column,

292

and was injected into rabbits. A rEcc-Raf affinity column was used to purify the

293

specific rabbit anti-rEcc-Raf IgG from immune rabbit serum. Total protein from

294

grouper head kidney and spleen tissues were extracted and western blot analysis

295

showed a ~72 kDa band, corresponding to the predicted size of Ecc-Raf. This

296

suggested that grouper c-Raf protein could be detected using rabbit anti-rEcc-Raf IgG

297

(Fig. 6B), but not with pre-immune rabbit serum (data not shown).

298 299

3.4. Subcellular localization and luciferase reporter assay

300

Although a specific rabbit anti-rEcc-Raf polyclonal antibody was generated, it

301

failed to label the c-Raf protein from grouper cells by immunofluorescence. Therefore,

302

a plasmid pEGFP-Ecc-Raf-SL was constructed, which contained a fusion Green

303

fluorescent protein (GFP) followed by the grouper c-Raf, for subcellular localization

304

analysis. The positive transfected ratio was detected using flow cytometry and showed

305

that about 25% of transfected GS cells are able to express GFP (Fig. 7A). Under

306

fluorescence microscope observation, GS cells transfected with pEGFP-Ecc-Raf-SL

307

showed that Ecc-Raf were distributed throughout the cytoplasm (Fig. 7B). To study

308

the function of Ecc-Raf in the activation of AP1, a plasmid pEGFP-Ecc-Raf-LR was

309

constructed, which only expresses grouper c-Raf, for dual-luciferase reporter assays.

310

The results showed that there was an increase of 37.3% in pEGFP-Ecc-Raf-LR

311

transfected GS cells compared with mock transfected cells, but this was not

312

statistically significant (P > 0.05) (Fig. 7C).

313 314

3.4. PMA or H2O2 treatment increases the phosphorylation level of Ecc-Raf.

315

To ensure the anti-pc-Raf antibody was suitable for Ecc-Raf phosphorylation

316

detection, grouper HKLs were isolated and treated with PMA (a potent activator of

317

protein kinase C) [28, 29] or H2O2 (a potent inhibitor of protein tyrosine phosphatases)

318

[30]. Cell lysates from each group were probed with rabbit anti-rEcc-Raf IgG (1:1,000

319

dilution) or rabbit anti-pc-Raf IgG (1:1,000 dilution, CST). The phosphorylation level

320

of Ecc-Raf was increased in HKLs at a concentration of 100 nM or 200 nM PMA, as

321

well as 1 mM or 10 mM H2O2 compared to the vehicle (Fig. 8). This suggested that

322

the phosphorylation level of Ecc-Raf could be induced by PMA or H2O2 treatment.

323

324

3.5. Downregulation of Raf-MEK-ERK phosphorylation level post SGIV

325

infection

326

Previous studies have determined that MEK-ERK signaling is involved in SGIV

327

infection [21]. To examine whether c-Raf was also activated during MAPK signal

328

transduction, the phosphorylation level of the Raf-MEK-ERK signal pathway post

329

SGIV infection was detected. Western blot analysis showed that grouper pc-Raf was

330

significantly downregulated after SGIV infection for 24 h (Fig. 9). Similar to the

331

findings of previous studies, the phosphorylation level of MEK-ERK was also

332

downregulated. Together, these results indicated that the Raf-MEK-ERK signaling

333

pathway was activated during SGIV infection.

334 335

3.6. Activation of Raf-MEK-ERK post-C. irritans infection

336

Our previous study showed that MEK-ERK was activated during C. irritans

337

infection [22]. However, whether c-Raf was activated during infection was not

338

determined. In order to examine this, immune groupers were prepared as described in

339

a previous study [31]. Together with the infected groupers and negative control

340

groupers, skin samples from the three groups were collected and used to detect the

341

total protein level and phosphorylation level of grouper c-Raf. Both the total level and

342

phosphorylation level of c-Raf were significantly upregulated in the infected grouper

343

skin and enhanced in immune grouper skin (Fig. 10). This suggested that the grouper

344

Raf-MEK-ERK signal pathway was activated during C. irritans infection.

345 346

4. Discussion

347

Although c-Raf has been extensively study in mammals, functional studies of

348

these molecules in lower vertebrates are limited. In this study, the cDNA sequence of

349

c-Raf was cloned from orange-spotted grouper. Genomic structure analysis revealed

350

that grouper c-Raf shared a conserved exon structure with other vertebrates including

351

zebrafish, turtles, chickens and humans. Sequence analysis showed that Ecc-Raf

352

shared a very high amino acid identity with those molecules in other vertebrates, and

353

had the highest amino acid identity (99%) with L. calcarifer c-Raf. In addition, like

354

mammals, three conserved domains were found in Ecc-Raf: Ras-binding domain,

355

protein kinase C conserved region 1 domain and serine/threonine protein kinase

356

catalytic domain. In addition to the 3D modeling and phylogenetic analysis, the

357

results suggested that grouper c-Raf may had a similar function as that displayed in

358

mammals.

359

The expression pattern of mRNA in specific tissues can indicate important clues

360

about gene function. In mammals, c-Raf is mainly transcripted in leukocytes

361

including monocytes, B cells, and T cells [32]. Although some functional studies of

362

c-Raf have been carried out in zebrafish [14, 15], little is known about the expression

363

pattern of c-Raf in fish. To investigate the tissue distribution of c-Raf, gene specific

364

primers were designed to detect the expression levels of this molecule in various

365

tissues. Ecc-Raf mRNA is present in various tissues including head kidney, spleen,

366

skin, intestine, thymus, liver, and gill. The highest expression level of Ecc-Raf was

367

found in head kidney tissue followed by spleen tissue, which are two systemic

368

immune organs in teleosts. This indicated that Ecc-Raf may play a vital role in fish

369

hematopoietic organs.

370

Subcellular localization is essential for protein functional properties. In this

371

study a specific rabbit anti-rEcc-Raf polyclonal antibody was prepared. Unfortunately,

372

it failed to detect c-Raf protein from grouper cells by immunofluorescence. A plasmid

373

which contained a GFP tag for c-Raf tracking was then constructed. Under

374

fluorescence microscope observation, GS cells transfected with pEGFP-Ecc-Raf-SL

375

showed that Ecc-Raf was distributed throughout the cytoplasm, which is similar to the

376

subcellular localization of mammal c-Raf [33]. The c-Raf activation initiates a MAPK

377

by phosphorylating MEK1 and MEK2, and in turn phosphorylating to activate ERK1

378

and ERK2. This cascade activation plays an important role in transferring

379

extracellular signals and regulating downstream AP1 activation [34]. To determine

380

whether Ecc-Raf played a role in the activation of AP1, GS cells were transfected

381

with pEGFP-Ecc-Raf-LR as well as an AP1 dependent firefly luciferase reporter for

382

luciferase activity detection. Although relative luciferase activity increased by 37.3%

383

in pEGFP-Ecc-Raf-LR transfected GS cells compared with mock transfected cells,

384

this was not statistically significant (P > 0.05). The insufficiency of AP1 activation

385

may be partly due to the low transfection efficiency of GS cells (~25%). Nevertheless,

386

it suggested that the overexpression of Ecc-Raf could only slightly enhance the

387

activation of AP1 in fish cell lines.

388

To ascertain whether the anti-pc-Raf (against mammals) antibody was suitable

389

for Ecc-Raf phosphorylation detection, c-Raf phosphorylation in grouper HKLs were

390

detected

391

as12-O-Tetradecanoylphorbol-13-acetate (TPA), is a potent activator of protein kinase

using

two

stimulators:

PMA

and

H2O2.

PMA,

also

known

392

C and ERK pathways [28, 29]. H2O2 is a potent inhibitor of protein tyrosine

393

phosphatases which can hydrolyze phosphoester bonds in proteins [26]. The results

394

showed that both PMA and H2O2 were able to induce c-Raf phosphorylation within a

395

few minutes, in contrast to the DMSO or untreated cells. In addition, it also confirmed

396

that this anti-pc-Raf antibody was suitable for detecting grouper c-Raf

397

phosphorylation.

398

Recent studies have confirmed that the MEK-ERK cascade can be activated by

399

SGIV or C. irritans infection [21, 22]. However, whether c-Raf initiates the activation

400

of MEK-ERK cascade during SGIV and C. irritans infection remains unclear. Similar

401

to previous reports, the results of the current study showed that SGIV infection

402

induced

403

phosphorylation level of grouper c-Raf significantly downregulated after SGIV

404

infection as well. It has been demonstrated that ERK signaling is involved in SGIV

405

replication in host cells [21]. Further evidence was provided in the current study that

406

grouper had an integral Raf-MEK-ERK axis and this was activated during SGIV

407

infection.

dephosphorylation

in

MEK1/2

and

ERK1/2.

Furthermore,

the

408

Previous reports have shown that grouper inflammatory factors interleukin-1β

409

(IL-1β) and interleukin-8 (IL-8) were upregulated in C. irritans infected skin [23, 31].

410

In addition, it was found that the phosphorylation level of grouper MEK1/2 and

411

ERK1/2 were significantly increased in C. irritans infected grouper skin on day three

412

and enhanced in the immune grouper skin [22]. In this study the phosphorylation level

413

of c-Raf during C. irritans infection was further determined. The results also showed

414

that both the total level and phosphorylation level of Ecc-Raf were significantly

415

increased post-infection and in immune grouper skin. It has been demonstrated that

416

ERK can mediate the production of IL-1β and IL-8 [35]. Therefore, it is assumed that

417

the grouper Raf-MEK-ERK signaling pathway was activated and mediated the

418

production of IL-1β and IL-8 in response to C. irritans infection. In addition, C.

419

irritans infection can induce host humoral immunity responses by producing specific

420

antibodies [36]. The upstream molecules of the grouper B cell antigen receptor (BCR)

421

signaling pathway (CD79a, CD79b, LYN, SYK, BTK, BLNK), which plays a crucial

422

role in B cell development and antibody production, was thought to be involved in the

423

response against C. irritans infection [24, 27, 37]. The present study showed an

424

enhanced expression of phosphorylate c-Raf post immunization and indicated that the

425

signal for B cell activation may be transduced by the Raf-MEK-ERK pathway during

426

infection. Nevertheless, the function of c-Raf during B cell activation in fish still

427

requires further study.

428

In this study, c-Raf cDNA sequences from orange-spotted grouper and Ecc-Raf

429

were mainly transcribed in the systemic immune organs. Overexpression of Ecc-Raf

430

can only slightly enhance the activation of AP1. Both PMA and H2O2 treatment can

431

induce the phosphorylation levels of Ecc-Raf. Moreover, the Raf-MEK-ERK axis was

432

activated during SGIV or C. irritans infection, suggesting that the ERK cascade was

433

involved in the response to viral or parasitic infection.

434 435

Acknowledgements

436

This work was supported by the National Natural Science Foundation of China

437

(41876162), the Pearl River S&T Nova Program of Guangzhou (201806010181), the

438

China Postdoctoral Science Foundation (2019M662938), the Guangdong MEPP Fund

439

(No. GDME-2018C002), the Guangdong Provincial Special Fund for Modern

440

Agriculture Industry Technology Innovation Teams (2019KJ141), and the China

441

Modern Agricultural Industry Technology System (The Control of Parasites Infection

442

on Marine Fish, CARS-47-18).

443

444

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Table 1 Primers used in this study

Primer

Sequence (5’ to 3’)

c-Raf 3’GSP1 c-Raf 3’GSP2 c-Raf ORF F c-Raf ORF R c-Raf RT F c-Raf RT R rc-Raf F rc-Raf R c-Raf EGFP F c-Raf EGFP R1 c-Raf EGFP R2 UPM

GGGCTAACGGTGAAAATTGGAGAC GTGGGAAGAGGCTATTTGTCCCCA TAAAAACTTTCCCCCTCTGGCGTTATT CATCTGTGTGTAGAGATTTCAGGGCGTACT CCGACTCCAAGACTAGCAGCACTAT CGTGATCTAAAACCTCAACCAGTAGC CGGGGTACCGGTACCATGGAGCACCTCC CCGGAATTCTTACTCCCAGTAGTAACTCGAGT tcgagctcaagcttcgaattcATGGAGCACCTCCAGGGAGC ggatcccgggcccgcggtaccGTGAAAACAGGCAGTCTGGTGG ggatcccgggcccgcggtaccCTAGAAAACAGGCAGTCTGGTGG Long: CTAATACGACTCACTATAGGGCAAG CAGTGGTATCAACGCAGAGT Short: CTAATACGACTCACTATAGGGC AAGCAGTGGTATCAACGCAGAGT TGCTGTCCCTGTATGCCTCT CCTTGATGTCACGCACGAT

NUP β-actin F β-actin R

Table 2 Amino acid identities (%) between Ecc-Raf with other vertebrates

Overall

Lates calcarifer Larimichthys crocea Takifugu rubripes Danio rerio Homo sapiens Bos Taurus Rattus norvegicus

Mus musculus

99 97 95 83 81 82 81 80

Ras-binding

Protein kinase C

Serine/Threonine

domain

conserved region

protein kinases

1 domain

catalytic domain

100 100 100 94 96 96 96 96

99 99 97 94 91 92 91 90

99 95 95 91 73 73 73 73

Figure legends Fig. 1 Alignment of c-Raf. Gaps, show as dashes (----), had been introduced to maximize the alignment to the sequences. Residues identical in all the sequences were shaded by black. The Ras-binding domain, Protein kinase C conserved region 1 domain, and Serine/Threonine protein kinases catalytic domain were indicated by black letters on a grey background. All Gene Bank accession numbers used in this study are listed in Supplementary Table 1.

Fig. 2

The 3D homology model of grouper c-Raf was performed using the human

c-Raf as a template.

Fig. 3 Genomic structure of c-Raf in grouper, zebrafish and human. The boxes and lines between each box indicated exons and introns, respectively, and the numbers indicated the sizes (bp) of each exon and intron. ? represents unknown intron sizes.

Fig.4 A phylogenetic tree illustrating the relationship across vertebrate c-Raf molecules using the neighbor-joining method within the MEGA program. Node values represent percent bootstrap confidence derived from 10,000 replicates. The scale bar is 0.02.

Fig. 5 The expression pattern of Ecc-Raf in healthy grouper tissues. All data are presented as Mean ± SE, n = 3. Fig. 6 Expression, purification and western blot analysis of Ecc-Raf. SDS-PAGE of rEcc-Raf (A). Lane M: Protein marker. Lane 1: Non-induced BL21 transformed with pET-32a-c-Raf. Lane 2: Induced BL21 transformed with pET-32a-c-Raf for 4 hours. Lane 3: Purified recombinant protein rEcc-Raf using Ni column. Western blot of

recombinant protein using rabbit anti-rEcc-Raf IgG as primary antibody (B). Lane M: Protein marker. Lane 1: Grouper head kidney total protein. Lane 2: Grouper spleen total protein.

Fig. 7 Subcellular localization and AP1 activition of Ecc-Raf in GS cells. Positive transfected ratio was detected using flow cytometry (A). Fluorescence microscope observation of transfected GS cells (400 ×) (B). Detection of Ecc-Raf in AP1 activition (C). The relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase activities. Data are the fold changes relative to GS cells transfected with pEGFP-N1. All data are presented as Mean ± SE, n = 3.

Fig. 8 Western blot analysis of PMA treatment. Grouper HKLs were treated with 100 nM or 200 nM PMA for 15 min and cells lysates were probed with the indicated antibodies. All data are presented as Mean ± SE, n = 3. Significant change is indicated with different letter (P < 0.05).

Fig. 9 Western blot analysis of SGIV infection in GS cells. Detection of c-Raf phosphorylation levels in GS cells 24 h after SGIV infection. All data are presented as Mean ± SE, n = 3. Significant change is indicated with different letter (P < 0.05).

Fig. 10 Western blot analysis of C. irritans infection. Detection of pc-Raf and c-Raf in C. irritans infected or immune skin using the indicated antibodies, Con.: control skin, Inf.: infected skin; Imm.: immune skin (A). The phosphorylated and total c-Raf were measured by densitometric analysis of immunoblots and presented relative to values of β-actin (B). All data are presented as Mean ± SE, n = 3. Significant change is indicated with different letter (P < 0.05).

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Highlights

Overexpression of Ecc-Raf can only slightly enhance the activation of AP1.

Grouper pc-Raf can by inducing by the stimulation of PMA or H2O2.

Grouper Raf-MEK-ERK axis was activated in SGIV or C. irritans infction.