Characterization of the immune roles of cathepsin L in turbot (Scophthalmus maximus L.) mucosal immunity

Journal Pre-proof Characterization of the immune roles of cathepsin L in turbot (Scophthalmus maximus L.) mucosal immunity Jinghua Chen, Lu Zhang, Ning Yang, Min Cao, Mengyu Tian, Qiang Fu, Baofeng Su, Chao Li PII:

S1050-4648(19)31133-7

DOI:

https://doi.org/10.1016/j.fsi.2019.12.005

Reference:

YFSIM 6647

To appear in:

Fish and Shellfish Immunology

Received Date: 28 August 2019 Revised Date:

25 November 2019

Accepted Date: 1 December 2019

Please cite this article as: Chen J, Zhang L, Yang N, Cao M, Tian M, Fu Q, Su B, Li C, Characterization of the immune roles of cathepsin L in turbot (Scophthalmus maximus L.) mucosal immunity, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2019.12.005. 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

Characterization of the immune roles of Cathepsin L in

2

turbot (Scophthalmus maximus L.) mucosal immunity

3 4 5 6 7

Jinghua Chena,1, Lu Zhanga,1, Ning Yanga, Min Caoa, Mengyu Tiana, Qiang Fua, Baofeng Sub,*, Chao Lia,*

8

266109, People’s Republic of China

9

b

10

a

School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao

School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn,

AL 36849, USA

11 12 13 14 15 16 17 18 19 20 21 22 23

1

24

*

25

Baofeng Su, [email protected].

All these authors contributed equally to this work.

Corresponding author: Chao Li, [email protected];

1

26 27

Abstract: Cathepsin L (CTSL) is one of the crucial enzymes in cathepsin family,

28

which has been widely known for its involvement in the innate immunity. However, it

29

still remains poorly understood how CTSL modulates the immune system of teleosts.

30

In this study, we captured three cathepsin L genes (SmCTSL, SmCTSL.1 and

31

SmCTSL1) from turbot (Scophthalmus maximus). The coding sequences of SmCTSL,

32

SmCTSL.1 and SmCTSL1 are 1,026 bp, 1,005 bp and 1,017 bp in length and encode

33

341, 334 and 338 amino acids, respectively. In details, transcripts of CTSL genes share

34

same domains as other CTSL genes, one signal peptide, one propeptide and one

35

papain family cysteine protease domain. Protein interaction network analysis

36

indicated that turbot CTSL genes may play important roles in apoptotic signaling and

37

involve in innate immune response. Evidence from subcellular localization

38

demonstrated that the three Cathepsin L proteins were ubiquitous in nucleus and

39

cytoplasm. The cathepsin L genes were widely expressed in all the tested tissues with

40

the highest expression level of SmCTSL in spleen, and SmCTSL.1 and SmCTSL1 in

41

intestine. Following Vibrio anguillarum, Edwardsiella tarda and Streptococcus iniae

42

challenge, these cathepsin L genes were significantly regulated in mucosal tissues in

43

all the challenges, especially significantly down-regulated rapidly in intestine in all

44

the three challenges. In addition, the three cathepsin L genes showed strong binding

45

ability to all the examined microbial ligands (LPS, PGN and LTA). Further studies

46

should be used to analyze the function of these three cathepsin L genes. Therefore, we

47

can use their function to maintain the integrity of the mucosal barrier, thereby

48

promoting the disease resistance line and family selection in turbot.

49 50

Keyword: Cathepsin L, Turbot, Vibrio anguillarum, Edwardsiella tarda,

51

Streptococcus iniae, Microbial ligand binding

52 53 54 55 56 57 2

58

3

59 60

1

Introduction

61 62

Cathepsins are a group of proteases predominantly located in lysosome [1, 2].

63

According to previous reports, they play critical roles in a variety of biological

64

processes, such as antigen processing and presentation, tumor progression and

65

metastasis, bone resorption and osteolysis, parasitic infection [3-6], tissue invasion

66

[7], food digestion and uptake [8], immune evasion [9] and molting [10]. In general,

67

cathepsins are classified into three groups on the basis of amino residues in their

68

active sites. They are cysteine proteases (cathepsins B, C, F, H, K, L, O, S, T, U, V,

69

W, and X), serine proteases (cathepsins A and G), and aspartic proteases (cathepsins

70

D and E), respectively [11].

71

Cathepsin L is an important member of the cathepsin family. Among different

72

groups of cathepsins, cathepsin L is universally expressed in the majority of immune

73

tissues and cells, whereas many other cathepsins can be only found in specific cell

74

types [12]. Cathepsin L is a lysosomal cysteine protease that exhibits strong

75

endopeptidase activity, and involves in intracellular and extracellular protein

76

degradation. In mammal mucosal surfaces, cathepsin L has been revealed to play

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vital roles in controlling normal mucosal epithelial homeostasis and supporting the

78

host immune defense against infection. In cathepsin L lacking mice, it showed the

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disorder of intestinal epithelial cells and initiation of intestinal epithelial disease [13].

80

Cathepsin

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mycoplasmal infection in mice [14]. In human lung epithelial cells, cathepsin L is

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involved in regulation of cell apoptosis [15]. Previous reports in fish have shown that

83

challenge with bacterial lipopolysaccharide (LPS) or other bacteria can induce the

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expression of the cathepsin L gene [16, 17]. Cathepsin L has been also found to be

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responsible for the lysis of pathogenic bacteria in the nonspecific immunological

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defense of the Japanese eel (Anguilla japonica) and Chinese mitten crab (Eriocheir

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sinensis) [18, 19]. In tongue soles (Cynoglossus semilaevis), the expression of

L supported

airway

lymphangiogenesis

4

and

protected

against

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cathepsin L was induced in kidney and spleen following Vibrio anguillarum and

89

megalocytivirus infections [20]. Despite its important roles in mucosal health, the

90

studies on immune roles of cathepsin L in fish mucosal tissues are still lacking.

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Turbot (Scophthalmus maximus L.) is one of the most extensively maricultured

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species in China. However, bacterial diseases have resulted in dramatic economic

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losses to its farming industry, such as V. anguillarum, Streptococcus iniae, V.

94

vulnificus, and Edwardsiella tarda, and others. The mucosal surfaces are the first line

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of host defense against a wide range of pathogens, and the mucosal immune

96

responses are the most critical events to prevent pathogen adhesion and invasion.

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Especially for fish species, the mucosal surfaces (skin, gill, nose and intestine) are

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constantly interacting with various pathogens in the aquatic environment [21]. So far,

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cathepsin L hasn’t yet been reported in the turbot. In addition, the knowledge on the

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mucosal immune system of turbot is important for diseases management and

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development in turbot farming. In this regard, we characterized cathepsin L genes in

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turbot, and investigated their immune roles in turbot mucosal immunity (Fig. 1).

103 104

2

Materials and methods

105 106

2.1 Sequence identification and analysis

107 108

The protein sequence of CTSL genes from other species [including zebrafish

109

(Danio rerio), medaka (Oryzias latipes), fugu (Takifugu rubripes), tongue sole (C.

110

semilaevis) and olive flounder (Paralichthys olivaceus)] were collected as queries to

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BLAST against turbot genome and transcriptome database with a cut off E-value of

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1e-10 to capture ctsl gene in turbot (CTSL) [22, 23]. The primers were designed

113

based on the transcript sequence for sequencing using Primer Premier 5.0 software

114

(Premier Biosoft Company, Palo Alto, CA) (Table 1). Then, the verified sequences

115

were translated using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The

116

predicted

ORF

sequences

were

further 5

verified

by

BLASTP

117

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) against NCBI non-redundant (nr) protein

118

sequence database. The conserved domains were further identified using the simple

119

modular

120

(http://www.cbs.dtu.dk/services/SignalP) was used to determine signal peptides of

121

these CTSL genes. The theoretical pI, molecular mass and N-glycosylation sites were

122

characterized by ExPASy server [24]. The Splign program was used to predict their

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intron and exon structures [25]. The identity and similarity among the turbot and

124

other species of CTSL genes were calculated using MatGAT program [26]. The

125

online program PHYRE2 (Protein Homology/analogY Recognition Server, V2.0)

126

(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) was used to establish

127

the presumed 3D protein structural model.

architecture

research

tool

(SMART).

The

SignalP

5.0

Server

128

To determine the protein-protein interaction network of expressed proteins,

129

amino acid sequences of CTSL were blasted against C. semilaevis by using STRING

130

software 11.0. Representation of the protein-protein network was analyzed at

131

confidence score 0.40 in the Textmining, Experiments, Databases, Co-expression,

132

and Neighborhood sources.

133 134

2.2 Phylogenetic analysis

135 136

The amino acid sequences of CTSL from turbot, together with those from other

137

organisms were retrieved from NCBI databases to construct the phylogenetic tree,

138

including human (Homo sapiens), chicken (Gallus gallus), tongue sole (C.

139

semilaevis), Japanese flounder (Paralichthys olivaceus), channel catfish (Ictalurus

140

punctatus), zebrafish (D. rerio), Chinese softshell turtle (Pelodiscus sinensis),

141

barramundi (Lates calcarifer), greater amberjack (Seriola dumerili). Multiple protein

142

sequence alignments were performed using the Clustal Omega program [27].

143

Molecular

144

neighbor-joining method was used to proceed multiple protein sequence alignments.

145

Bootstrapping with 1,000 replications was conducted to evaluate the phylogenetic

Evolutionary

Genetics

Analysis

6

package

(MEGA6)

with

the

146

tree [28].

147 148

2.3 Syntenic analysis

149 150

In order to further verify the identified CTSL, the syntenic analysis was performed

151

across several species. The FGENESH Program was used to predict the protein

152

sequences of neighboring genes of the CTSL from the turbot scaffolds. The BLASTP

153

program against nr database was used to annotate the identified protein sequences.

154

The conserved syntenic patterns of CTSL genes in other species were identified in

155

Ensemble database and Genomicus.

156 157

2.4 Bacteria challenge and sample collection

158 159

In order to characterize the immune roles of CTSL genes and investigate their

160

expression patterns, turbot was exposed to Gram-negative bacteria V. anguillarum, E.

161

tarda and Gram-positive bacteria S. iniae, respectively. The turbot fingerlings

162

(average body weight: ~15.6 g and average body length: ~5.5 cm) were obtained from

163

the turbot hatchery (Haiyang, Shandong, China). The turbot fingerlings were reared in

164

the laboratory in a flow-through system. After a pre-challenge, the bacteria were

165

re-isolated from symptomatic fish and biochemically confirmed before cultured.

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Mucosal tissues (skin, gill and intestine samples) were collected following challenge

167

with three biological replications at each timepoint.

168

For bacteria challenge, the V. anguillarum, E. tarda and S. iniae were inoculated

169

in Luria-Bertain (LB) broth and incubated in a shaker (180 rpm/min) at 28

170

overnight, respectively. The fish were equally divided into four experimental groups

171

and one control group with 30 fish in each group. And then the fish in experimental

172

groups were immersed 2 h in V. anguillarum，E. tarda and S. iniae with a final

173

concentration of 5×107 CFU/mL [29], 1×107 CFU/mL [30] and 5×106 CFU/mL [31],

174

respectively. In contrast, the fish in control groups were immersed in sterilized media.

175

After immersion, the fish were transferred back in the flow-through system. Samples

176

from control group and V. anguillarum, E. tarda and S. iniae infected groups were

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separately collected at 2 h, 4 h, 8 h and 12 h post challenge. All the samples from both 7

178

experiments were flash-frozen in liquid nitrogen and then stored in a -80

179

freezer.

ultra-low

180 181

2.5 Total RNA extraction and cDNA synthesis

182 183

Prior to RNA extraction, the mortar and pestle were used to homogenize tissue

184

samples under liquid nitrogen. Total RNA was extracted using Trizol® Reagent

185

(Invitrogen, USA) following the manufacturer’s protocol. RNA concentration and

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integrity of each sample was measured on a Nanodrop 2000 (Thermo Electron North

187

America LLC, FL). All the RNA samples had an A260/280 ratio greater than 1.8.

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The ration of A260/280 of these samples was ranged from 1.8 to 2.1. The iScriptTM

189

cDNA Synthesis Kit (Bio-Rad) according to manufacturer’s protocol was used to

190

synthesize the first strand cDNA. The iScript chemistry uses a blend of oligo-dT and

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random hexamer primers. All the RNA from different time points was diluted to 500

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ng/µl for cDNA synthesis, and the first strand cDNA was synthesized by Prime

193

Script RT reagent Kit (TaKaRa) according to manufacturer's protocol (500 ng RNA

194

per 10µl reaction), and utilized for the quantitative real-time PCR (qPCR) reaction.

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The ddH2O was used as negative control for the cDNA synthesis.

196 197

2.6 Quantitative real-time PCR analysis

198 199

Primer Premier 5 was used to design the CTSL gene specific primers based on

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the isolated cathepsin L sequences in turbot [32]. And 18S rRNA gene was used as a

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reference gene (Table 1). The quantitative real-time PCR (qPCR) was performed on

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a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA)

203

using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China). The real-time qPCR

204

reaction system was as follows: The PCR reaction mixture was denatured at 95°C for

205

30s and then subjected to 40 cycles of 95°C for 5s, 58°C for 5s and followed by the

206

dissociation curve analysis, 5 s at 65°C, then up to 95°C at a rate of 0.1 °C/s 8

207

increment, to verify the specificity of the amplicons. The mRNA expression levels of

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all samples were normalized to the levels of 18S rRNA in the same samples. The

209

expression patterns were confirmed by repeating in triplicates (technical replicates)

210

for the qPCR analysis.

211 212

2.7 Plasmid construction expression, purification of recombinant SmCTSL

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Three different cDNA and putative amino acid sequences (SmCTSL, SmCTSL.1

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and SmCTSL1) were captured in turbot with the specific primers (Table 1) following

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cDNA synthetization to construct the expression plasmids of SmCTSL, SmCTSL.1

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and SmCTSL1, respectively. The PCR was used for amplifying the coding sequence

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of CTSL and then electrophoretically analyzed. The pEASY-Blunt-E1 vector was

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used for ligating the PCR product following the gel extraction, after that, the plasmid

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was transformed into competent Trans-T1 cells. The constructed plasmid was

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reproduced on ampicillin-containing LB plates, and cultivated overnight at 37 .

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Following blue-white spotting selection, the positive clone was selected and

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sequenced. The verified recombinant plasmid was extracted and marked as

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pEASY-E1-CTSL. In the same way, the plasmids pEASY-E1-CTSL.1 and

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pEASY-E1-CTSL1 were constructed by inserting the mature SmCTSL.1 and

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SmCTSL1 nucleotide sequences into the pEASY vector, respectively.

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The three recombinant plasmids pEASY-E1-CTSL, pEASY-E1-CTSL.1 and

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pEASY-E1-CTSL1 were subsequently transformed into E. coli BL21 (DE3)

229

(Novagen, USA), respectively. A single colony of the above E. coli BL21 was

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inoculated in 5 ml of LB broth at 37 °C, and shaken at 180 rpm/min overnight. After

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that, the cultures were diluted at 1:20 with fresh LB medium and cultured until

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mid-logarithmic phase, and then respectively induced by adding (0.05, 0.1 and 0.5

233

mM) IPTG (isopropyl-β-D-thiogalactopyranoside). The nickel-nitrilotriacetic acid

234

chromatography were used for purifying the expressed proteins under denaturing

235

conditions following the manufacturer's instructions. The protein was analyzed in 12% 9

236

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained

237

with Coomassie Brilliant Blue R-250. The accuracy of the recombinant protein was

238

confirmed using Western blot. Monoclonal mouse anti-His-tag antibody and

239

HRP-labeled Goat Anti-Mouse IgG (Solarbio, China; 1:1000 dilution in 5% BSA)

240

were applied as primary and secondary antibodies, respectively. The specific

241

antigen-bound antibody was visualized with DAB (Diaminobenzidine) reagent

242

(Sigma, USA). Bradford's method was performed to determine the concentration of

243

the purified protein.

244 245

2.8 Solid-phase enzyme-linked immunosorbent assay (ELISA)

246 247

In order to evaluate the binding ability of recombinant proteins (SmCTSL

248

SmCTSL.1 and SmCTSL1 with lipopolysaccharide (LPS), lipoteichoic acid (LTA)

249

and peptidoglycan (PGN), the 96 microtiter plates (Corning, NY, USA) were

250

respectively coated with 100 µl of 5 µg/ml LPS, LTA and PGN at 4°C overnight.

251

After discarding the liquid in the plate, the wells were washed by 300 µl PBST (0.05%

252

Tween-20 in PBS) three times, then blocked with 100 µl 5% BSA at 4

253

After that, each ligand-coated well was added a series of 100µl increased

254

concentration of purified recombinant SmCTSL, SmCTSL.1 and SmCTSL1 (0.5, 1, 2,

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4, 8 and 16 µg/mL). Each concentration had four replicates, and the plate was

256

incubated at 37°C for 1.5 h. Subsequently, the wells were washed with 300 µl PBST

257

three times, then they were incubated at 37°C for 1 h with 100 µl mouse anti-His

258

antibody (Solarbio, Beijing, China) (diluted 1:1000 in 5% BSA). Followed by three

259

times washing with 300 µl PBST, the wells were incubated with addition of 100 µl

260

horseradish peroxidase conjugated goat anti-mouse IgG (Solarbio, Beijing, China)

261

(diluted 1:1000 in 5% BSA) at 37°C for 40 min. According to the manufacturer's

262

instructions, after washing three times with PBS, 0.01% of TMB (3, 3′, 5, 5’

263

-Tetramethylbenzidine) was used to visualize ligand-binding results. Finally, the

264

reaction was terminated by adding 0.5 M sulfate, and the plate was then read at 450 10

for 1 h.

265

nm wavelength with an ELISA reader. The pEASY-E1 vector protein was employed

266

as negative control in our experiment.

267 268

2.9 Subcellular localization of CTSL genes

269 270

The DNA fragments for subcellular localization, SmCTSL, SmCTSL.1 and

271

SmCTSL1 ORFs were amplified with the primers in Table 1. Subsequently, the

272

amplified ORF were cloned into enzyme-cut pEGFP-N2 vector with endonuclease

273

Nhe I and Sac I, respectively for construct pEGFP-N2-CTSL, pEGFP-N2-CTSL.1,

274

and pEGFP-N2-CTSL1 which was then sequencing confirmed. For the subcellular

275

localization analysis, the human embryonic kidney 293T cells (HEK293T cells) were

276

cultured overnight in DMEM medium (Hyclone, USA) containing 10% fetal bovine

277

serum (FBS) (Hyclone, USA), and incubated at 37 °C with 5% CO2. Coverslips were

278

placed into the wells of a 6-well plate, and the HEK293T cells were subsequently

279

seeded and cultured. Empty plasmid pEGFP-N2 group (vector control) and

280

recombinant

281

pEGFP-N2-CTSL1 were transfected into HEK293T cells by Xfect™ transfection

282

reagent (Clontech, USA) according to the manufacturer’s instructions, respectively.

283

The cells were washed with PBS and fixed with immunostaining fixative (4%

284

paraformaldehyde). The cells were then washed with PBS and stained with l µg/mL

285

4,6-diamidino-2-phenylindole (DAPI) for 5 min. After washing, anti-fluorescence

286

quenching sealing solution was added to the coverslips. The subcellular localizations

287

of SmCTSL, SmCTSL.1 and SmCTSL1 were observed under a fluorescence

288

microscope (Leica, German).

plasmids,

pEGFP-N2-CTSL,

pEGFP-N2-CTSL.1,

and

289 290

2.10 Statistical analysis

291 292

All the experiments were triplicated in our study. For the data analysis, statistical

293

analysis was performed utilizing SPSS 21.0 software package (Chicago, IL, USA).

294

The data were presented as the mean ± standard error. The differences between 11

295

control and experimental groups were considered statistically significant when the p

296

value less than 0.05.

297 298

3. Result

299 300

3.1 Identification of turbot CTSL genes

301 302

Three different cDNA sequences, SmCTSL (Genbank: ARR29131.1),

303

SmCTSL.1 (Genbank: MK110652) and SmCTSL1 (Genbank: MK110653) were

304

captured in turbot. In detail, the full-length transcripts of SmCTSL, SmCTSL.1 and

305

SmCTSL1 contain 1,026 bp, 1,005 bp and 1,017 bp open reading frame (ORF)

306

encoding 341, 334 and 338 amino acids, respectively. The deduced SmCTSL,

307

SmCTSL.1 and SmCTSL1 proteins were predicted to have a molecular mass of 37.59,

308

36.6 and 37.97 kDa and a theoretical pI of 8.64, 4.86 and 5.82, respectively. The

309

deduced (SmCTSL, SmCTSL.1 and SmCTSL1) proteins were predicted to have (7,2,4)

310

protein kinase C phosphorylation sites, (2,7,1) casein kinase II phosphorylation sites,

311

and (4,8,1) N-glycosylation sites, respectively. In addition, they had (31, 46 and 44)

312

negatively charged residues (Asp + Glu), (36, 30 and 35) positively charged residues

313

(Arg + Lys), and with an instability index of (32.53, 37.64 and 27.53) and aliphatic

314

index of (78.59, 72.46 and 66.36) (supplementary Table 1).

315

Comparative domain organization was analyzed by aligning turbot SmCTSL,

316

SmCTSL.1 and SmCTSL1 predicted peptide sequences with those of human,

317

zebrafish, channel catfish, tongue sole and others (supplementary Fig.1). Multiple

318

sequence alignment demonstrated that the three turbot CTSL genes were highly

319

homologues to that of other species (42-88% identities and 59-95% similarity,

320

supplementary table 2). The multiple sequence alignment demonstrated that high

321

sequence conservation existed in signal peptide, inhibitor_I29, and Pept_C1 among

322

the three turbot cathepsin genes (supplementary Fig.2). Highly conserved four active

323

residues, Gln, Cys, His and Asn, existed among the aligned sequences 12

324

(supplementary Fig. 2). Other features, propeptide region and four loops were also

325

predicted from the multiple sequence alignment (Fig. 2 and supplementary Fig. 2).

326

The tertiary structures of turbot SmCTSL1 (residues 22-338), SmCTSL.1 (residues

327

16-334) and SmCTSL1 (residues 17-341) were constructed using the Phyre2 server,

328

which were 55%, 49% and 49% identical to the same model, d7pcka, with 93-95%

329

confidence, and images were colored by rainbow from N to C terminus (Fig 2A, 2B,

330

and 2C). A relatively high conserved structures, including 5 helixes, 2 β-turns and 5

331

β-sheets and other motifs were found in the tertiary structures (Fig.2). Collectively,

332

these structures might indicate the functional similarity of cathepsin genes among

333

turbot and other species.

334

Using protein-protein interaction (PPI) network analysis, we found that these

335

proteins showing high connectivity to each other (Fig. 3, supplementary table 3.).

336

The results showed that all three CTSL genes might participate in some signal

337

transductions associated with immunity and share same interacting genes, including

338

survivin, XP_008313347.1, birc2, and bcl2l1 (supplementary table 3). Both survivin

339

(Baculoviral IAP repeat-containing protein 5.1-A-like) and XP_008313347.1

340

(Baculoviral IAP repeat-containing protein 5-like) have a baculoviral inhibition of

341

apoptosis protein repeat (BIR) domain and act as a direct inhibitor of caspase

342

enzymes [33]. Both birc2 and XP_008325831.1 have three BIR domains and one

343

RING (ring finger) domain which has E3 ubiquitin-protein ligase activity, while the

344

former gene has one CARD domain which is the caspase recruitment domain and

345

involved in apoptotic signaling [34,35]. Three proteins, bcl2l1, bcl2 and

346

XP_008316516.1 have a BCL domain and a BH4 domain, which belong to the

347

BCL-2 family. They are central regulators of caspase activation and regulates the

348

apoptotic process [36]. Proteins atg4c (cysteine protease ATG4C isoform X1), atg4b

349

(cysteine protease ATG4B isoform X6) and XP_008316516.1 (cysteine protease

350

ATG4D-like) are a group of cysteine peptidases and have a peptidase_C54 domain

351

that constitute MEROPS peptidase family C54 [37]. Both XP_008320464.1

352

(containing cystatin-like domain, CY) and XP_008306187.1(CY domain) have the 13

353

cysteine protease activity [38]. Cystatin-like may interact with cathepsin L, for

354

example, Cystatin-A1-like and Nile tilapia cathepsin L (NtCatL) might interact to

355

form a stable complex through hydrogen bond and hydrophobic function, which

356

inhibits the protease activity of NtCatL [39]. CD74 is responsible for the activity of

357

MHC class II protein binding [40].

358 359

3.2 Genomic structure analysis of CTSL genes

360 361

The Splign was used to compare the exon/intron architecture of CTSL genes in

362

turbot with other species in vertebrates (Fig.4). In general, a comparison of the

363

structures of CTSL genes, i.e., SmCTSL, SmCTSL.1 and SmCTSL1, revealed a highly

364

conserved exon size, except chicken and zebrafish SmCTSL1 and sole CTSL. In

365

CTSL, only the tongue sole has 8 exons, turbot has the same number of exons as

366

flounder and silver seaperch. All of the four species also show the same exon size in

367

the second, third and the last three exons (123 bp, 153 bp, 166 bp, 106 bp and 100 bp)

368

(Fig. 4A). In CTSL.1, six of seven exons (143 bp, 198 bp, 192 bp, 166 bp, 106 bp

369

and 100 bp) are identical across all the species (Fig. 3B). In CTSL1, the same

370

number of exon and intron of CTSL1 gene was observed in human, mouse and turbot

371

(Fig. 4C). The exon sizes of CTSL1of all examined species showed strong similarity

372

and high conservation. However, wide variations were observed in the intron size in

373

all compared species (Fig. 4C).

374 375

3.3 Phylogenetic analysis

376 377

The neighbor-joining method was used to perform the phylogenetic analysis for

378

amino acid sequences of cathepsin L (Fig.5). In our results, the turbot SmCTSL1

379

gene was first clustered with tongue sole SmCTSL1, then clustered with the subclade

380

of zebrafish and channel catfish, and then clustered with the other single clade

381

formed by tetrapod species chicken and Chinese softshell turtle. The turbot 14

382

SmCTSL.1 gene was first clustered with Asian sea bass (Barramundi), and then

383

clustered with zebrafish and channel catfish, which was then clustered with tongue

384

sole. The turbot SmCTSL gene first clustered with Greater amberjack and

385

Barramundi, and then clustered with Oliver flounder. This subclade was further

386

clustered with tongue sole. All branching nodes were supported by high bootstrap

387

values (bootstrap value >90%).

388 389

3.4 Syntenic analysis

390 391

In order to further validate the identified turbot ctsl gene, the syntenic analysis

392

was performed. In general, a conserved synteny was detected among the selected

393

species (Fig. 6). For ctsl gene, with the exception of 14-3-3 protein beta/alpha

394

(YWHAB) and Ninja-family protein AFP4 (AFP4) in the tongue sole and turbot, the

395

genes

396

Polyadenylate-binding protein 1 (PABP1), Synaptic vesicle glycoprotein 2A (SV2A),

397

Cathepsin

398

domain-containing nogo receptor-interacting protein 1 (LINGO1) and Vacuolar

399

protein sorting-associated protein 72 homolog (VPS72). However, turbot ctsl had

400

similar neighboring genes as tongue sole (Fig. 6A). The CTSL.1 homologous genes

401

were highly consistent across the five species as the same neighboring genes for

402

SmCTSL.1 were observed in all compared species. Of the eight genes listed, only

403

Lysine-specific histone demethylase 1A (KDM1A) was missing in channel catfish

404

(Fig. 6B). In case of ctsl1 gene, a well conserved synteny was identified among all

405

the examined species under analysis, which included KN motif and ankyrin repeat

406

domain-containing protein 1 (KANK1), Fructose-1,6-bisphosphatase 1 (FBP1),

407

Doublesex- and mab-3-related transcription factor 1 (DMRT1), Doublesex- and

408

mab-3-related transcription factor 3 (DMRT3). In addition, Doublesex- and

409

mab-3-related transcription factor 2 (DMRT2) was also observed in the genomic

410

neighborhood of all species except channel catfish (Fig. 6C).

remained

S

the

(CTSS),

same

in

all

Leucine-rich

15

four

repeat

selected

and

species,

including

immunoglobulin-like

411 412

3.5 Tissue distribution of CTSL genes

413 414

In order to characterize the tissue distribution patterns of the cathepsin L genes

415

in turbot, the expression analysis was conducted in eight healthy turbot tissues,

416

including blood, liver, spleen, gill, skin, intestine, head kidney and brain by real-time

417

PCR. Before detection of expression levels of these cathepsin L genes, their

418

amplicons were verified by Sanger sequencing (Supplementary Table 4). Overall, all

419

three cathepsin L genes were widely expressed in all the examined tissues, but with

420

distinct expression patterns (Fig. 7). In detail, SmCTSL showed the highest

421

expression level in spleen, followed by head kidney, and the lowest expression level

422

in liver. The modest expression lever of SmCTSL was observed in intestine and skin

423

(Fig. 7). In case of SmCTSL.1, the highest expression was revealed in intestine, but

424

the lowest expression level was detected in blood and liver. Moderate expression

425

was detected in brain, followed by brain, gill, head kidney, spleen and skin. In case

426

of SmCTSL1, the highest expression level was observed in intestine and blood,

427

followed by gill and head kidney (Fig. 7). In general, the expression of SmCTSL1

428

was ubiquitously expressed in all the tested tissues but not as strong expressed as

429

that of SmCTSL and SmCTSL.1. (Fig. 7).

430 431

3.6 Expression profiles of CTSL following bacterial challenge

432 433

To further reveal the immunological roles of CTSL in turbot mucosal immunity,

434

the expression profiles of three cathepsin L (SmCTSL, SmCTSL.1 and SmCTSL1)

435

were examined in mucosal tissues (gill, skin and intestine) at early time points

436

following immersion challenge with Gram-negative bacteria V. anguillarum and E.

437

tarda, and Gram-positive bacteria S. iniae, respectively.

438

Following V. anguillarum challenge, SmCTSL showed different expression

439

patterns in different tissues (Fig. 8). The expression of SmCTSL had no significant 16

440

changes observed in gill and skin in earlier time points except that the expression

441

was significant induced in gill with 2.39 fold at 24 h. However, in intestine, SmCTSL

442

was down-regulated at all the time points compared to the control and the expression

443

varied from -3.32 fold to -5.19 fold (Fig. 8A). The SmCTSL.1 was only induced for

444

2.84 fold at 2 h and 2.73 fold at 24 h in gill, and was repressed for -26.38 fold at 2 h

445

in intestine (Fig. 8B). Different from the expression pattern of SmCTSL and

446

SmCTSL.1, the SmCTSL1 had a unique expression pattern following challenge. In

447

skin, SmCTSL1 was significantly up-regulated for 2.84 fold at 2 h and 4.24 fold at 6

448

h. While in intestine, SmCTSL1 was only suppressed at 2 h with -4.43 fold.

449

Repression of SmCTSL1 in gill was detected at 12 h for -3.12 fold (Fig. 8C).

450

Following E. tarda challenge, general expression patterns with up-regulation in

451

skin, down-regulation in intestine and in gill (Fig. 9), were observed among the three

452

turbot cathepsin L genes. In detail, the SmCTSL was rapidly down-regulated in gill

453

with -25.48 fold at 2 h, but returned to basal level quickly in other tested time points.

454

For its expression in intestine, the down-regulation of SmCTSL was detected at 12 h

455

with -23.73 fold, and continued to be down-regulated with -5.76 fold at 24 h and

456

-3.33 fold at 48 h. In contrast, the SmCTSL was up-regulated in skin with 8.95 fold at

457

6 h, 3.54 fold at 12 h, and 20.81 fold at 24 h (Fig. 9A). Similarly, the SmCTSL.1 was

458

also down-regulated at 2 h with -7.33 fold in gill, but no significant expression was

459

observed at other time points. Notably, it was down-regulated in intestine at all the

460

time points after 2 h post treatment with -12.84 fold at 6 h, -68.98 fold at 12 h,

461

-40.01 fold at 24 h, and -15.71 fold at 48 h. While it was only up-regulated in skin

462

with 3.38 fold at 24 h and 10.82 fold at 48 h (Fig. 9B). Differently, the SmCTSL1

463

was up-regulated at all the time points in skin with 11.81 fold at 2 h, 20.49 fold at 6 h,

464

12.99 fold at 12 h, 38.59 fold at 24 h and 3.54 fold at 48 h. Although SmCTSL1 was

465

also up-regulated in intestine at 2 h with 3.63 fold, it was dramatically

466

down-regulated at 12 h for 60.33 fold, and returned to basal level quickly. In gill,

467

down-regulation was only observed at 2 h with -4.08 fold (Fig. 9C).

468

The expression patterns of three cathepsin L genes were also detected in turbot 17

469

after S. iniae infection (Fig. 10). In detail, SmCTSL was only down-regulated at 8 h

470

for -3.13 fold in intestine, and no significant expression changes were detected in

471

skin at other time points following challenge. In the gill, the expression of SmCTSL

472

was reduced to -4.80 fold at 2 h post treatment but up-regulated to 4.27 fold at 12 h

473

(Fig. 10A). For the expression of SmCTSL.1, almost all three tissues had significant

474

changes at all-time points after the challenge except for gill and skin at 2 h and skin

475

at 8 h post treatment. The expression of SmCTSL.1 in gill was gradually increased to

476

4.24 fold, 5.66 fold and 10.81 fold at 4 h, 8 h and 12 h after challenge. The

477

expression of SmCTSL.1 in skin was significantly up-regulated to 7.06 fold at 4 h

478

and 6.71 fold at 12 h. The expression was most pronounced in the intestine for

479

SmCTSL.1. The smallest down-regulation was -29.29 fold 2 h after treatment,

480

fluctuated at 4 h and the repression at 8 h was -19.20 fold. It restored to -6.38 fold at

481

12 h (Fig. 10B). Different to expression of SmCTSL.1, SmCTSL 1showed an early

482

down-regulation response at 2 h with -4.30 fold in gill, but was quickly returned to

483

basal level at 4 h and 8 h, and was up-regulated again at 12 h with 3.05 fold, which

484

had the similar expression pattern as that of SmCTSL. In the skin, significant gene

485

expression was only observed at 12 h post-challenge for 2.95 fold. Intestine had the

486

highest down-regulation at 2 h with -9.13 fold, followed by -5.28 fold at 8 h (Fig.

487

10C).

488 489

3.7 Microbial ligand-binging in vitro

490 491

The binding abilities of the three CTSL genes were investigated in order to

492

further characterize their immune function. Three CTSL proteins with His-tagged

493

were purified from E. coil and then were named as rSmCTSL, rSmCTSL.1 and

494

rSmCTSL1, respectively. For these three CTSL proteins, only a single band was

495

observed in SDS-PAGE analysis (Fig.11). The rSmCTSL and rSmCTSL.1 showed

496

strong binding ability to all the examined microbial ligands (Fig. 12A &12B). In

497

detail, the highest peak value for the binding ability of three microbial ligands was 18

498

LPS, followed by PGN and LTA. The rSmCTSL.1 had the strongest binding ability to

499

LPS, PGN and LTA as even the lowest amount, 0.5 µg of SmCTSL.1, showed highest

500

absorbance than that of SmCTSL and SmCTSL1 binding to the same ligand. Unlike

501

the binding ability of SmCTSL and SmCTSL.1, SmCTSL1 had generally lower

502

binding ability to LPS and PGN (Fig. 12C). The highest concentration of 16.0 µg of

503

SmCTSL1 revealed similar binding ability to 4.0 µg SmCTSL and 1.0 µg SmCTSL.1

504

to LTA, respectively (Fig. 12).

505 506

3.8 Subcellular localization of CTSL in HEK293T cells

507 508

In order to investigate the subcellular localization of turbot ctsl proteins, the

509

recombinant plasmids pEGFP-N2-CTSL, pEGFP-N2-CTSL.1, pEGFP-N2-CTSL1

510

were constructed and transfected into HEK293T cells. As shown in Fig. 13, after

511

transfection, in general, all the plasmids expressed green fluorescence and the

512

nucleus expressed blue fluorescence following DAPI staining. In details, SmCTSL

513

and SmCTSL1 were distributed both in the cytoplasm and nucleus, the SmCTSL.1

514

was uniformly distributed in the nucleus, but not in the cytoplasm (Fig. 13).

515 516

4. Discussion

517

Cathepsin L and K are both implicating in the functional specificity of antigen

518

processing in immune system. The cathepsin L function has been reported to be

519

associated with antigen processing and presentation, tumor progression and

520

metastasis, bone resorption and osteolysis, a variety of parasitic infection [3-6],

521

tissue invasion [7] and immune evasion [9]. In mammals, cathepsin L is involved in

522

MHC II-associated Ag presentation and regulation of CD4+ T lymphocyte selection

523

[41]. Mice defective in cathepsin L exhibited reduced number of CD4+ T cells and

524

were affected in the ability to degrade invariant chain, a critical step in MHC

525

II-restricted antigen presentation, in cortical thymic epithelial cells, which are

526

antigen presenting cells for positive selection of T lymphocytes in the thymus 19

527

[41,42]. Currently, the Cathepsin L has also been identified from a few of fishes,

528

which showed broad functional roles. For example, it was found that Cathepsin L

529

was involved in the regulation of growth in rainbow trout (Oncorhynchus mykiss)

530

[43]. The ctsl was proposed to hydrolyze the main protein in carp (Cyprinus carpio)

531

surimi, and participated in the modori phenomenon in carp surimi gel [44]. In olive

532

flounder (P. olivaceus), ctsl was down-regulated after starvation [45]. In zebrafish,

533

ctsl involved in the yolk formation and processing [46]. Furthermore, it has been

534

proved that cathepsin L plays key roles in host immune defense. For example, it has

535

been proved that CTSL plays an important role in degradation of the extracellular

536

matrix in [47]. In Nile tilapia, osteoclasts do not use matrix metalloproteinases

537

(MMPs) for resorption without cathepsin L [48]. Besides, previous study showed

538

that the upregulation of CTSL genes was observed in response to bacterial challenge

539

in catfish [49]. The cathepsin L knockout Japanese flounder were more susceptible to

540

the invading pathogens [50]. However, previous study focused on characterizing the

541

gene expression levels of cathepsin L in liver, spleen and kidney rather than mucosal

542

tissues. The involvement of cathepsin L in mucosal immunity has been poorly

543

studied. To explore their immune responses to bacterial infection, we have identified

544

and characterized the transcripts of three cathepsin L genes (SmCTSL, SmCTSL.1

545

and SmCTSL1) from turbot for the first time.

546

The deduced amino acid sequences of three cathepsin L genes showed similar

547

molecular properties to other fish species. The conserved three domains

548

(supplementary Fig. 1) in turbot protect the active sites from substrate binding and

549

maintains the peptidase in an inactive state. In addition, it also stabilizes the enzyme

550

against denaturing at neutral to alkaline pH conditions. Experimental studies

551

demonstrated that removal of this region by proteolytic cleavage results in activation

552

of the enzyme [51,52]. The residuals for Pept_C1 domain of CTSL1, CTSL.1 and

553

CTSL are 117-337, 118-333, 114-340, respectively. Such domain belongs to the

554

papain family cysteine protease (clan CA), which has a wide variety of activities,

555

including broad-range (papain) and narrow-range endo-peptidases, aminopeptidases, 20

556

carboxypeptidases, dipeptidyl peptidases and enzymes with both exo- and

557

endo-peptidase activity [53]. Similar to other cathepsin belonging to Clan CA, turbot

558

ctsl genes also possess papain-like fold, which consists two subdomains, referred to

559

as L- (left) and R- (right). The two subdomains enclose the active sites between them.

560

The L-domain consists of a bundle of helices, with the catalytic Cys at the end of

561

one of them, and the other R-domain is a beta-barrel motif with the active sites, His

562

and Asn (or Asp) [54]. A catalytic triad occurs in the order: Cys/His/Asn (or Asp) as

563

revealed in the multiple amino sequence alignment (supplementary Fig. 2). Gln,

564

usually preceding the active site Cys, is important for stabilizing the acyl

565

intermediate that forms during catalysis and helps form the 'oxyanion hole', and Asn

566

orientates the imidazole ring of His. Potential N-glycosylation sites were predicted in

567

the turbot ctsl genes, and variation in N-glycosylation sites, 4,8,1 were found in

568

SmCTSL, SmCTSL.1 and SmCTSL1, suggesting a different role in the transportation

569

of cathepsin into lysosomes [35, 55].

570

Because most of turbot protein data has not been certified experimentally, the

571

protein-protein interaction predicted were therefore based on C. semilaevis database,

572

where the protein identity ranged from 43% to 94% (supplementary table 3). The

573

PPI network of the three turbot ctsl genes share some interacting functional genes,

574

including survivin, XP_008313347.1, birc2, and bcl2l1, which are all related to

575

apoptosis pathways. The similar protein interaction network may suggest similar

576

regulatory functions. Therefore, we speculated that these genes were predicted using

577

protein-protein interaction in turbot might be responsible for apoptosis process and

578

immune response. Other Cystatin-like gene domain/motif and turbot CTSL may form

579

a stable complex through hydrogen bond which can inhibit the protease activity. It is

580

intriguing that the turbot PPI network pathway will become more reliable with the

581

increasing high-throughput lab experiments, experimentally certified fish proteins

582

and the availability of turbot database form PPI databases. Thus, it is worthwhile to

583

putting more effort on the development of turbot PPI database and investigating the

584

interactions when available. 21

585

Little is known about the subcellular localization of the three CTSL proteins in

586

turbot. Our current results showed that the three GFP fused CTSL proteins mainly

587

distributed in the cytoplasm of HEK-293T cells. SmCTSL.1 is uniformly distributed

588

in the nucleus while both SmCTSL and SmCTSL1 are ubiquitously detected in the

589

cytoplasm and nucleus. The different localizations may be indication of their

590

different function in cell signaling transduction. Previous studies demonstrated that

591

isoforms of cathepsin L in mouse are distributed in the nucleus and regulating cell

592

cycle by cutting the histone H3’s N-terminus end [56]. A CTSL variant (Ctsla) in

593

zebrafish is a putative yolk processing enzyme, involved in yolk absorption during

594

embryogenesis [57]. Ctsl genes which are ubiquitously expressed as endopeptidases

595

in cysteine cathepsins family, play significant roles in signaling pathway and are

596

recognized as vital regulators in many physiological events.

597

The distribution of basal-level tissue expression of turbot CTSL genes was

598

determined in eight tissues of healthy turbot. The results showed that the three CTSL

599

genes were expressed in all the tested tissues, which is consistent with previous

600

research that CTSL genes were ubiquitously expressed in animal tissues [2], but with

601

distinct expression patterns. In detail, SmCTSL showed the lowest expression level in

602

liver, and the highest expression level in spleen, followed by head kidney and

603

intestine, and had the modest expression level in skin and gill. Different from

604

SmCTSL, SmCTSL.1 showed the strongest expression level in intestine, followed by

605

brain, and with the lowest expression level in blood. Similar to SmCTSL, the lowest

606

expression level of SmCTSL1 was also detected, with the highest expression level in

607

intestine, followed by gill and head kidney. Previously, cathepsin L genes also

608

showed relatively high expression level in intestine and relatively low expression

609

level in liver in channel catfish [49]. In anchovy, cathepsin L mRNA was found

610

predominately in the gut, with only trace amount in liver [58]. Interestingly, in

611

orange-spotted grouper and Tongue soles the cathepsin L mRNA expression was

612

highest in liver [20, 46]. The difference may reflect the expressional difference

613

between cathepsin L of different fish species. 22

614

Turbot were immersed in different bacteria, and then the expression profiles of

615

CTSL were characterized in turbot mucosal surfaces (skin, gill and intestine). We

616

find that all three CTSL genes showed dramatic down-regulation in intestine in both

617

Gram-negative bacteria V. anguillarum and E. tarda, and Gram-positive bacteria S.

618

iniae challenge. The previous studies have suggested that intestine might serve as the

619

primary portal of entry for V. anguillarum [59], V. anguillarum cells could be

620

detected in spleens in more than 50% of orally infected fish [60]. Following bath

621

vaccination with live attenuated V. anguillarum vaccine in zebrafish, the bacteria

622

proliferated rapidly in 3 h and maintained at a high level in the intestine. Besides,

623

bacteria persisted in the intestine for a longer time whereas decreased rapidly in the

624

skin and gills [60]. In turbot, an oral challenge with V. anguillarum for turbot larvae

625

could lead to significant mortality [61]. In sea bass, after exposure to V. anguillarum,

626

intact bacteria in the gut lumen were observed in close contact with the apical brush

627

border [62]. Here, the significant down-regulation of cathepsin L in turbot intestine

628

following different bacteria challenge suggested its vital roles in intestinal immune

629

responses. Further studies are needed to characterize the exact roles of different

630

cathepsin L against infection and thus to identify the key player for disease

631

resistance selection.

632

Three representative microbial ligands (LPS, LTA and PGN) were used to

633

understand the roles of CTSL in host defense against various pathogens. The results

634

showed that the three CTSL genes had strong binding abilities to LPS, LTA and PGN.

635

Interestingly, CTSA and CTSZ in turbot also have strong binding to microbial ligands

636

[63. 64]. Previous reports in fish have shown that challenge with bacterial

637

lipopolysaccharide (LPS) or other bacteria can induce the expression of the cathepsin

638

L gene [16,17]. In crayfish, expression profiles of PcCTSL gene after LPS and Poly

639

I:C challenge showed that pathogens infection significantly influence cathepsin L

640

protein expression, and it plays a critical role in immunological reaction against

641

pathogens [65].

642

In this study, we described three CTSL genes from turbot, the first genome wide 23

643

identification of CTSL in turbot to date, profiled their gene expression levels after

644

different bacteria challenge in mucosal tissues, investigated their microbial ligand

645

binding activities, as well as their subcellular localization. Our results suggested the

646

CTSL genes might play vital roles in teleost mucosal immunity, but further studies

647

are required to uncover the specific role and mechanism of CTSL genes in teleost

648

host immune response.

649 650

Acknowledgement

651 652

This study was supported by the Scientific and Technological Innovation of Blue

653

Granary

654

(No.:31602193), the keypoint research and invention program in Shandong Province

655

(2017GHY215004), and it was financially supported also by "First class fishery

656

discipline" programme in Shandong Province, China.

(2018YFD0900503),

the

National

Science

Foundation

of

China

657 658 659

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Table 1 Primers used in this study Primer qRT-PCR Sm-CTSL F Sm-CTSL R Sm-CTSL.1 F Sm-CTSL.1 R Sm-CTSL1 F Sm-CTSL1 R 18s RNA F 18s RNA R Protein expression Sm-CTSL-pr F Sm-CTSL-pr R Sm-CTSL.1-pr F Sm-CTSL.1-pr R Sm-CTSL1-pr F Sm-CTSL1-pr R CDS clone Sm-CTSL-ORF F Sm-CTSL-ORF R Sm-CTSL.1-ORF F Sm-CTSL.1-ORF R Sm-CTSL1-ORF F Sm-CTSL1-ORF R Subcellular localization Sm-CTSL-b F Sm-CTSL-b R Sm-CTSL.1-b F Sm-CTSL.1-b R Sm-CTSL1-b F Sm-CTSL1-b R

Sequence (5’-3’) 5’ GAACCACCTGGCAGACAT 3’ 5’ GGACCCACATAACCCTTG 3’ 5’ AAATCTGGCTCAACAATCGC 3’ 5’ TGTGGCAAGAGGAGGAAGG 3’ 5’ TGGAGCCCAACTTCGTGG 3’ 5’ TCTGCTCGCTCAGGGACA 3’ 5’ ATGGCCGTTCTTAGTTGGTG 3’ 5’ CTCAATCTCGTGTGGCTGAA 3’ 5’ GCTTCAAATAAGATGTGGGAAGAG 3’ 5’ TCACAGTGTAGGATAGACTGCGAAG 3’ 5’ TCCCTGGAAGACCTGGAGTT 3’ 5’ TCAGACCAGCGGGTAGCTG 3’ 5’ GACCACTGGGGCCTGTGG 3’ 5’ TTAGACCAGAGGGTAACTGGCC 3’ 5’ ATGCCCATTTTGTGTGCGG 3’ 5’ TCACAGTGTAGGATAGACTGCGAAG 3’ 5’ ATGAAGCTTCTGTTGGTTGCTG 3’ 5’ TCAGACCAGCGGGTAGCTG 3’ 5’ ATGCTGCCGCTGCCCCTC 3’ 5’ TTAGACCAGAGGGTAACTGGCCG 3’ 5’TGAACCGTCAGATCCgctagcATGCCCATTTT GTGTGCG G 3’ 5’TGGCGACCGGCCGGTggatccCACAGTGTAG GATAGACTGCGAAG 3’ 5’TGAACCGTCAGATCCgctagcATGAAGCTTCT GTTGGTTGCTG 3’ 5’TGGCGACCGGCCGGTggatccCAGACCAGCG GGTAGCTG 3’ 5’TGAACCGTCAGATCCgctagcATGCTGCCGCT GCCCCTC 3’ 5’TGGCGACCGGCCGGTggatccAGACCAGAGG GTAACTGGCCG’ 3’

876 877 33

878 879 880 881

34

Fig.1. The schematic graph of the experiments in this study

Fig.2. The 3D structural models of turbot SmCTSL1(A), SmCTSL.1(B) and SmCTSL (C) were predicted using Phyre2. The image was colored by rainbow from N to C terminus. 95% of residues were modelled at >90% confidence. Characters A and L represented β-turn; B, C, D, F and H represented α-helix; E, G, I, J and K represented β-sheet. (For better visualization and interpretation of the references to color in the figures, the reader is referred to the web version of this article.)

Fig. 3. Protein-protein interactions which show predicted functional partners of turbot SmCTSL1(GenBank: MK110653), SmCTSL.1(GenBank: MK110652) and SmCTSL (GenBank: ARR29131.1). A: Protein-protein interactions for SmCTSL1 predicted by STRING 11.0 with the setting interaction evidence as network edges. B: Protein-protein interactions for SmCTSL.1 predicted by STRING 11.0 with the setting interaction evidence as network edges. C: Protein-protein interactions for SmCTSL predicted by STRING 11.0 with the setting interaction evidence as network edges.

Fig.4. Exon/intron organizations of SmCTSL(A), SmCTSL.1(B) and SmCTSL1(C) genes were obtained by using Splign to align their cDNA sequences to the turbot genome. Boxes indicate exons and dashes indicate introns. The dark shaded boxes indicate exon sequences that encode amino acids.

Fig. 5. Phylogenetic tree for the turbot cathepsin L genes. The phylogenetic tree was constructed based on the amino acid sequences of cathepsin L from fishes and mammals, using the neighbor-joining method in MEGA 6. Gaps were removed by complete deletion and the phylogenetic tree was evaluated with 1,000 bootstrap replications. The boot strapping values were indicated by numbers at the nodes. Dark solid circle, dark filled triangle and dark filled inverted triangle indicated the newly characterized turbot SmCTSL gene, SmCTSL.1 gene and SmCTSL1 gene, respectively.

Fig. 6. Syntenic analysis of Cathepsin L genes from different species. The CTSL gene is highlighted by bottle green color filled boxes. The CTSL.1 gene is highlighted by bright red color filled boxes. The CTSL1 gene is highlighted by mazarine color filled boxes. YWHAB: 14-3-3 protein beta/alpha; PABP1: Polyadenylate-binding protein 1; AFP4: Ninja-family protein AFP4; SV2A: Synaptic vesicle glycoprotein 2A; CTSS: Cathepsin S; LINGO1: Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 1; VPS72: Vacuolar protein sorting-associated protein 72 homolog; E2F2: Transcription factor E2F2; ID3: DNA-binding protein inhibitor ID-3; YTHDF2: YTH domain-containing family protein 2; VGLL2: Transcription cofactor vestigial-like protein 2; PACRG: Parkin coregulated gene protein; PTAFR: Platelet-activating factor receptor; KDM1A: Lysine-specific histone demethylase 1A; CTSV: Cathepsin L2; FBP1: Fructose-1,6-bisphosphatase 1; DAPK1: Death-associated protein kinase 1; DMRT2: Doublesex- and mab-3-related transcription factor 2; DMRT3: Doublesex- and mab-3-related transcription factor 3; DMRT1: Doublesex- and mab-3-related transcription factor 1; KANK1: KN motif and ankyrin repeat domain-containing protein 1; HSPB8: Heat shock protein beta-8, HspB8; XBP1: X-box-binding protein 1; RUFY3: Protein RUFY3.

Fig.7. Gene expression analysis of the three cathepsin L genes in different healthy turbot tissues. Expression levels were calibrated against tissue which had the lowest expression level, and 18S rRNA was used as a reference gene. HK was the abbreviation for head kidney. The expression patterns were confirmed by repeating in triplicates runs (technical replicates) for the qPCR analysis. And the results were presented as mean ± SE of fold changes.

Fig. 8. Real-time qPCR analysis for cathepsin L expression levels following Vibrio anguillarum infection. (A) Relative gene expression level of SmCTSL following V. anguillarum infection in the mucosal tissues at different time points; (B) Relative gene expression level of SmCTSL.1 following V. anguillarum infection in the mucosal tissues at different time points; (C) Relative gene expression level of SmCTSL1 following V. anguillarum infection in the mucosal tissues at different time points. The cathepsin L expression was measured in the mucosal tissues including skin, gill, and intestine at the time points of 2 h, 6 h, 12 h, 24h, and 48 h post-infection. Fold change was calculated by the change in expression at a given time point relative to the untreated control and normalized by change in the 18S house-keeping gene. The results were presented as mean ± SE of fold changes and * indicated statistical significance at P < 0.05.

Fig. 9. Real-time qPCR analysis for cathepsin L expression levels following Edwardsiella tarda infection. (A) Relative gene expression level of SmCTSL following E. tarda infection in the mucosal tissues at different time points; (B) Relative gene expression level of SmCTSL.1 following E. tarda infection in the mucosal tissues at different time points; (C) Relative gene expression level of SmCTSL1 following E. tarda infection in the mucosal tissues at different time points. The cathepsin L expression was measured in the mucosal tissues including skin, gill, and intestine at the time points of 2 h, 6 h, 12 h, 24h, and 48 h post-infection. Fold change was calculated by the change in expression at a given time point relative to the untreated control and normalized by change in the 18S house-keeping gene. The results were presented as mean ± SE of fold changes and * indicated statistical significance at P < 0.05.

Fig. 10. Real-time qPCR analysis for cathepsin L expression following Streptococcus iniae infection. The cathepsin L expression levels were measured in the mucosal tissues including skin, gill, and intestine at the time points of 2 h, 4 h, 8 h and 12 h post- infection. Fold change was calculated by the change in expression at a given time point relative to the untreated control and normalized by changes in the 18S house-keeping gene. The results were presented as mean ± SE of fold changes and the *indicated statistical significance at P < 0.05. (A) Relative gene expression level of SmCTSL following S. iniae infection in the mucosal tissues at different time points; (B) Relative gene expression level of SmCTSL.1 following S. iniae infection in the mucosal tissues at different time points; (C) Relative gene expression level of SmCTSL1 following S. iniae infection in the mucosal tissues at different time points.

Fig. 11. SDS-PAGE analysis of the expression of the three recombinants turbot CTSL genes. (A), (B), and (C) showed the expression of rSmCTSL, rSmCTSL.1, and rSmCTSL1 using SDS-PAGE. M: protein marker. Lane 1: control; Lane 2: Induced protein; Lane 3: Purified protein; Lane 4: proteins using Western blotting analysis.

Fig. 12. Results of the in vitro binding assay of cathepsin L to microbial ligands, including lipopolysaccharide (LPS), peptidoglycan (PGN) and lipoteichoic acid (LTA). */** indicated a significant difference in the absorbance between different microbial ligands that exposed to cathepsin L and the control group; *P < 0.05; **P< 0.01. (A) Results of the in vitro binding assay of CTSL to microbial ligands; (B) Results of the in vitro binding assay of CTSL.1 to microbial ligands; (C) Results of the in vitro binding assay of CTSL1 to microbial ligands.

Fig. 13. Subcellular localization of CTSL, CTSL.1 and CTSL1 in HEK293 cells. HEK293 cells were transfected with pEGFP-N2 (panel A), pEGFP-N2-CTSL (panel B), pEGFP-N2-CTSL.1(panel C), or pEGFP-N2-CTSL1 (panel D). After 24 h, the cells were fixed and the nuclei stained with 4, 6-diamidino-2-phenylindole (DAPI). The left DAPI panels are the cell nucleus stained with DAPI, the middle GFP panels are pEGFP-N2, CTSL-, CTSL.1- and CTSL1-GFP fusion protein and GFP expression profile under fluorescence, while the right panels are the combined images of pEGFP-N2, CTSL-, CTSL.1- and CTSL1-GFP fusion proteins and GFP with cell nucleus. Bar = 100 µm.

Highlights: 1. The captured cathepsin L genes from turbot exhibited highly similarities in domains with those in other teleosts. 2. The cathepsin L proteins were ubiquitous in nucleus and cytoplasm based on subcellular localization. 3. The cathepsin L genes were all expressed in all tissues and were significantly down-regulated in the intestine following different bacterial challenge. 4. The cathepsin L genes showed strong binding ability to LPS, PGN and LTA.