A novel hepatic lectin of zebrafish Danio rerio is involved in innate immune defense

Journal Pre-proof A novel hepatic lectin of zebrafish Danio rerio is involved in innate immune defense Qingyun Yang, Peng Wang, Shuaiqi Yang, Xianpeng Li, Xiangmin Zhang, Guangdong Ji, Shicui Zhang, Su Wang, Hongyan Li PII:

S1050-4648(19)31036-8

DOI:

https://doi.org/10.1016/j.fsi.2019.10.068

Reference:

YFSIM 6562

To appear in:

Fish and Shellfish Immunology

Received Date: 27 June 2019 Revised Date:

17 October 2019

Accepted Date: 30 October 2019

Please cite this article as: Yang Q, Wang P, Yang S, Li X, Zhang X, Ji G, Zhang S, Wang S, Li H, A novel hepatic lectin of zebrafish Danio rerio is involved in innate immune defense, Fish and Shellfish Immunology (2019), doi: https://doi.org/10.1016/j.fsi.2019.10.068. 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

A novel hepatic lectin of zebrafish Danio rerio is involved in innate

2

immune defense

3

Qingyun Yanga,b#, Peng Wanga,b#, Shuaiqi Yanga,b, Xianpeng Lia,b,

4

Xiangmin Zhanga,b, Guangdong Ji a,b, Shicui Zhang a,b, Su Wang a,c*,

5

Hongyan Li a,b*

6 7

a

Laboratory for Evolution & Development, Institute of Evolution & Marine

8

Biodiversity and b Department of Marine Biology, Ocean University of China,

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Qingdao 266003, China

10

c

Marine Science and Engineering College, Qingdao Agricultural University, Qingdao

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266109, China.

12 13

#

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*Correspondence author

15

Dr. Hongyan Li

16

Room 301, Darwin Building, 5 Yushan Road, Ocean University of China,

17

Qingdao 266003, China

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Tel.: +86 532 82032092

19

E-mail: [email protected]

20

Dr. Su Wang

21

Marine Science and Engineering College, Qingdao Agricultural

22

University, Qingdao 266109, China.

23

Tel.: +86 532 86550511

24

E-mail: [email protected]

These authors contributed equally.

25

Declarations of interest:

26

None.

27

Abstract

28

ASGPR (asialoglycoprotein receptor, also known as hepatic lectin)

29

was the first identified animal lectin, which participated in a variety of

30

physiological processes. Yet its detailed immune functions are not well

31

studied in lower vertebrates. After reporting a zebrafish hepatic lectin

32

(Zhl), we identified a novel hepatic lectin（zebrafish hepatic lectin-like,

33

Zhl-l） in zebrafish. The zhl-l was mainly expressed in liver in a tissue

34

specific manner. And challenge with LPS/LTA induced a significant

35

change of zhl-l expression. What’s more, recombinant C-type lectin

36

domain (rCTLD) of Zhl-l had the activity of agglutinating and binding to

37

both Gram-negative and Gram-positive bacteria. It promoted the

38

phagocytosis of bacteria by carp macrophages. Moreover, rCTLD could

39

bind to insoluble lipopolysaccharide (LPS), lipoteichoic acid (LTA) and

40

peptidoglycan (PGN) independent of Ca2+, which was inhibited by

41

galactose. Interestingly, Zhl-l was located in the membrane, and its

42

overexpression could upregulate the production of pre-inflammatory

43

cytokines. Taken together, these results indicated that Zhl-l played a role

44

in immune defense, and would provide further information to understand

45

functions of C-type lectin family and the innate immunity in vertebrates.

46

Keywords: Zebrafish; Danio rerio; C-type lectin; hepatic lectin; innate

47

immunity

48

1. Introduction

49

Teleost has a relatively immature acquired immune system and

50

depends heavily on their innate mechanisms, especially the progress that

51

PRRs

52

(pathogen-associated molecular patterns), to protect them from invading

53

pathogens [1-3]. C-type lectin receptors (CLRs), a major class of PPRs,

54

are the largest and most diverse family of animal lectins [4]. CLRs may

55

impact immunity at several levels, ranging from phagocytosis to the

56

production of effector cytokines and chemokines [5]. Moreover, various

57

CLRs act as endocytic receptors on antigen-presenting cells (APCs), thus

58

are involved in the uptake of pathogens for antigen processing and

59

presentation, and subsequent T cell activation [6,7].

(pattern

recognition

receptors)

recognize

PAMPs

60

CLRs comprise 17 groups based on phylogeny, structure, and

61

functional properties [4]. Among these groups, Groups II (oligomeric

62

type II transmembrane receptors), IV (selectins), V [natural killer (NK)

63

cell

64

immunologically relevant cell surface receptors [8]. Group II CLRs are

65

authentic

66

carbohydrate-recognition domains for pathogen recognition and cell-cell

67

interactions. It is further subcategorized into some subfamilies, including

68

asialoglycoprotein receptor (ASGPR) subgroup, dendritic cell-specific

receptors],

and

C-type

VI

(mannose

lectins

that

receptors),

utilize

are

the

most

Ca2+-dependent

69

intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)

70

subgroup, macrophage receptors subgroup, Langerin and Kuppfer cell

71

receptors, and scavenger receptors [4]. The ASGPR are multifunctional

72

membrane receptors expressed by hepatic parenchymal cells, which has

73

been linked to many biological processes like clearing circulating

74

desialylated glycoproteins, lysosomal degradation [9-12], the removal of

75

apoptotic cells [13], the disposal of cellular fibronectin [14], the clearance

76

of IgA from circulation [15-18], the removals of low density lipoprotein

77

(LDL) and chylomicron remnants, [19, 20]. Moreover, ASGPR may act

78

as an entrance site of hepatotropic viruses [12-13, 21-22], and has

79

immunomodulatory properties like facilitating trapping and elimination

80

of activated lymphocytes [15,16].

81

The study of ASGPR has thus far been focused nearly and

82

exclusively on a variety of vertebrates, specifically humans, mouse and

83

chicken. To date, the knowledge regarding ASGPR in fishes remains rare.

84

We previously reported a hepatic lectin (also known as ASGPR in

85

mammalian) from zebrafish (Zhl) that bound to a wide range of bacteria

86

and participated in immune defense [23]. In the present study, we

87

identified another hepatic lectin in zebrafish，designated as Zhl-l. In brief,

88

we analyzed the pattern of expression and explored its agglutinate and

89

binding activity to bacteria as well as the effect on the expression of

90

pre-inflammatory cytokines.

91

2. Materials and methods

92

2.1. RNA extraction and cDNAs synthesis

93

Total RNA was extracted with Trizol (Takara, Dalian, China) from

94

the whole zebrafish according to the manufacturer's instructions. The

95

cDNA was synthesized with reverse transcription system (Promega)

96

using oligo (dT) primer after digestion with recombinant DNase I (RNase

97

free) (Takara) to eliminate the genomic contamination. The reaction was

98

carried out at 42 °C for 50 min and inactivated at 75 °C for 15min. The

99

cDNAs synthesized were stored at −20 °C until use.

100 101

2.2. Sequence analyses We

found

one

sequence

under

the

accession

numbers

102

XM_005170599.4 shared high identity with the zebrafish hepatic lectin

103

(zhl). The protein domains were analyzed by the SMART program

104

(http://smart.embl-heidelberg.de/)

105

structure

106

(https://zhanglab.ccmb.med.umich.edu/I-TASSER/).

107

molecular mass and isoelectric point were predicted using Edit sequence

108

editing software in the DNASTAR software package (DNASTAR, Inc.,

109

Madison, WI, USA).

110

2.3. Fish maintenance

was

predicted

and by

the

three-dimensional

I-TASSER

online The

(3D)

software theoretical

111

The AB strain zebrafish (Danio rerio) were reared in zebrafish

112

farming system of ESEN and fed twice daily under a 14 h/10 h light/dark

113

photoperiod at temperature (28.0 ± 1

114

placed together in the late evening at a female to male ratio of 2:1, and

115

the embryos were collected early in the next morning and transferred to

116

Holtfreter solution (MgSO4·7H2O 0.163 mg/ml, KCl 0.03 mg/ml, NaCl 1

117

mg/ml, CaCl2 0.04 mg/ml). For WISH, 0.0045% 1-phenyl-2-thiourea was

118

added into E3 medium to prevent embryos from pigmentation started 24

119

h post-fertilization (hpf). Different stages of embryos were sorted and

120

fixed following the guide of Kimmel et al. [24].

121

2.4. Whole-mount in situ hybridization (WISH)

). Sexually mature D. rerio were

122

Whole-mount in situ hybridization was performed as described by

123

Thisse and Thisse [25]. Briefly, fragments of zhl-l were amplified with

124

specific primers P3 and P4 (Supplementary Table 1). The purified PCR

125

products were sub-cloned into vector pGEM-T, which was sequenced to

126

verify inserts orientation. Digoxigenin (DIG)-labeled zhl-l antisense

127

riboprobes were synthesized with linearized vectors (digested by Nde

128

restriction enzyme) and T7 RNA polymerase through in vitro

129

transcription. And embryos/larvae were observed and photographed

130

under a stereomicroscope (Nikon, Japan).

131

2.5. Real-time quantitative PCR (qRT-PCR)

132

Total RNAs were extracted from 13 different tissues (skin, liver,

133

spleen, intestines, heart, muscle, eye, brain, testis, ovary, gill, gall bladder,

134

and kidney) and embryos/larvae (0, 2, 4, 6,10,14, 24, 48, 72, 96, and 120

135

hours post fertilization, hpf). After digestion with RNase-free DNase

136

(Takara) to eliminate the genomic contamination, the cDNA was

137

synthesized with reverse transcription system using random primer and

138

used for qRT-PCR. The PCR primers specific for zhl-l (P5 and P6),

139

β-actin (P7 and P8) were designed to study the expression level of zhl-l

140

(Supplementary Table1). The qRT-PCR was performed using SYBR

141

Green PCR master mix (Applied Biosystems) on the Real-time PCR

142

system (Applied Biosystems 7500 Real-Time PCR System). The

143

expression levels of zhl-l were normalized to that of β-actin in cDNA

144

samples. Three independent experiments were repeated in the same

145

condition.

146

2.6. Challenge experiments

147

The challenge experiments of adult zebrafish with LPS and LTA

148

were performed as described by Yang et al. [23]. In brief, D. rerio was

149

divided randomly into three groups (30/group). Fish of two experimental

150

groups were injected intraperitoneally with 20 µl of 80 µg/ml LPS or

151

LTA. For control, fish were similarly injected with saline. Of each group,

152

three fish were anaesthetized on ice, liver and spleen tissues were

153

dissected at 0, 2, 4, 8, 12, 24, 48, and 72 hours post injection (hpi). All

154

samples were homogenized in Trizol Reagent and stored at -80 °C. RNA

155

extraction, cDNAs synthesis and qRT-PCR were carried out as described

156

above in the section 2.5.

157

2.7. Expression and purification of recombinant C-type lectin domain

158

(rCTLD) of Zhl-l and TRX-His-tag peptide (rTRX)

159

The cDNA region encoding CTLD of Zhl-l was amplified by PCR

160

from D. rerio using the primer pairs P9 and P10 (Supplementary Table1).

161

The PCR products were digested with EcoRI and HindⅢ and sub-cloned

162

into the plasmid expression vector pET32a (Novagen, Darmstadt,

163

Germany). The recombinant plasmid was verified by sequencing and

164

named pET32a/CTLD. The cells of E. coli Transetta (DE3) were

165

transformed with the recombinant plasmids pET32a/CTLD and the

166

transformed E. coli cells were cultured 3 h in LB broth containing

167

ampicillin (100 µg/ml). When OD600 reached about 1.0, Isopropyl

168

β-D-1-thiogalactopyranoside (IPTG) was added to the cultures at a final

169

concentration of 0.5 mM, and the cultures were allowed to rock at 28°C

170

for 12 h. The inclusion bodies were prepared as described previously by

171

Liu et al. with slight modification [26]. And rCTLD was purified by

172

chromatography on a Ni-NTA resin column (GE Healthcare), and then

173

refolded by dialysis according to the methods of Xu et al [27].

174

To express the TRX-His-tag peptide as control, DE3 cells were also

175

transformed by plasmid pET-32a (+) (Novagen) and induced with IPTG

176

at a final concentration of 0.5 mM at 28 ℃ for 12 h. The peptide was

177

purified as rCTLD with slight modifications. The protein concentrations

178

were determined with BCA protein assay kit (CWBIO) according to the

179

manufacturer’s instruction.

180

2.8. Western blotting

181

The purified proteins rCTLD as well as the extracts of E. coli

182

transetta (DE3) containing pet-32a/CTLD before and after IPTG

183

induction were examined on a 12% SDS-PAGE gel. The proteins on the

184

gels were electroblotted onto PVDF membrane (Amersham) by a

185

semi-dry technique (Bio-Rad). After blocking with 4% bovine serum

186

albumin (BSA) in PBS, pH 7.4 at room temperature for 2 h, the

187

membranes were incubated with anti-His-tag mouse monoclonal antibody

188

(CWBIO) diluted 1:4000 with 4% BSA in PBS at 4 °C overnight. After

189

washing five times with PBS containing 0.1% Tween-20 (PBST), the

190

membranes were incubated with horseradish peroxidase conjugated goat

191

anti-mouse IgG Ab (CWBIO) diluted 1:8000 with 4% BSA in PBS at

192

room temperature for 40 min. The bands were visualized using DAB kit

193

(CWBIO) according to the manufacturer's instruction.

194

2.9. Bacterial agglutination assay

195

To test the agglutination activity of rCTLD, a representative

196

Gram-negative bacteria Escherichia coli (ATCC 25922) and a

197

representative Gram-positive bacteria Staphylococcus aureus (ATCC

198

25923) were cultured to mid-logarithmic phase and harvested by

199

centrifugation at 6000 g for 5 min. The bacteria were washed three times

200

with PBS and re-suspended in PBS yielding a density of 2 × 108 cells/ml.

201

Aliquots of 25 µl bacterial suspensions were mixed with 25 µl of 5 µg

202

rCTLD or rTRX (control) in PBS, incubated at 37 °C for 1 h in the

203

presence or absence of 5 mM CaCl2, and observed under a microscope.

204

2.10. Bacterial binding assay

205

To test the bacterial binding activity of rCTLD, two Gram-negative

206

bacterium E. coli and Aeromonas hydrophila (ATCC 35654) and two

207

Gram-positive bacterium Bacillus subtilis (ATCC 6633) and S. aureus

208

were cultured to logarithmic phase, and collected by centrifugation at

209

6000 g for 5 min. After washing three times with PBS, the bacteria were

210

re-suspended in PBS giving a density of 2×108 cells/ml. Aliquots of 150

211

µl of bacterial suspensions were mixed with 150 µl of 5 µg rCTLD or

212

rTRX (control) in PBS. The mixtures were incubated at 25 °C for 1 h in

213

the presence or absence of 5 mM CaCl2 and centrifuged at 6000 g for

214

5min. The bacterial pellets were washed three times with PBS and

215

re-suspended in 200 µl PBS. The bacterial suspensions were subjected to

216

12% SDS-PAGE and the binding activity was determined by Western

217

blotting.

218

2.11. Ligand binding assay

219

An assay for binding of rCTLD to LPS, LTA and PGN was

220

conducted as described by Wang et al. [28]. Aliquots of 150 µl LTA (100

221

µg/ml), LPS (100 µg/ml), PGN (100 µg/ml) or PBS (pH 7.4) were mixed

222

with10 µg of rCTLD, respectively, and incubated for 1 h at 25 °C. In

223

order to absorb the residual free recombinant proteins, S. aureus cells

224

(2×108 cells) were introduced in the LTA, PGN and PBS groups, and E.

225

coli cells (2×108cells) in the LPS, PGN and PBS groups. After incubation

226

at 25 °C for 1 h, the mixtures were centrifuged at 6000 rpm at 4 °C for 5

227

min to collect the bacterial cells. After washing three times with PBS (pH

228

7.4), the bacterial pellets were re-suspended in 200 µl of PBS. An aliquot

229

of 20 µl of each bacterial sample was then electrophoresed on a 12%

230

SDS-PAGE gel and immunostained as Western blotting.

231

To quantify the binding of rCTLD to LTA, LPS and PGN, an

232

enzyme-linked immunosorbent assay (ELISA) was performed as

233

described previously by Qu et al. and Sun et al. with several

234

modifications [29-30]. Aliquots of 50 µl of 40 µg/ml LPS, LTA and PGN

235

were applied to each well of a 96-well microplate and air-dried at 25 °C

236

overnight. The plates were incubated at 60 °C for 30 min to fix the

237

ligands, and then each well was blocked with 100 µl of 1 mg/ml BSA in

238

PBS at 37 °C for 2 h. After washing five times with PBST, a total of 100

239

µl PBS containing 0.1 mg/ml BSA and different concentrations (0, 1, 5,

240

10, 15 ,20, 40, 60, 80, and 100 µg/ml) of rCTLD or rTRX (control) was

241

added into each well and incubated at 25 °C for 3 h in the presence or

242

absence of 5 mM CaCl2. The wells were rinsed five times with PBST and

243

incubated with 100 µl of mouse anti-His-tag antibody (CWBIO) diluted

244

1:4000 with 4% BSA in PBS at 37 °C for 1 h. After washing five times

245

with PBST, the wells were then incubated with 100 µl of HRP-labeled

246

goat anti-mouse IgG Ab (CWBIO) diluted 1:8000 with 4% BSA in PBS

247

at room temperature for 1 h. Subsequently, the wells were washed five

248

times with PBST, added with 75 µl of 0.4 mg/ml O-phenylenediamine

249

(Amresco) in the buffer consisting of 51.4 mM Na2HPO4, 24.3 mM citric

250

acid and 0.045% H2O2 (ph5.0), and reacted at 37 °C for 10 min. Finally,

251

25 µl of 2 M H2SO4 was added into each well to terminate the reaction,

252

and absorbance at 492 nm was monitored by a microplate reader (GENios

253

Plus; Tecan).

254

2.12. Assay for effects of sugars on binding of rCTLD to ligands

255

The effects of sugars on the binding of rCTLD to ligands were

256

detected with the method as described by Qu et al. [29]. Aliquots of 50 µl

257

of 40 µg/ml LPS, LTA and PGN were applied to each well of a

258

96-wellmicroplate and air-dried at 25 °C overnight. Aliquots of 10 µg

259

rCTLD in 50 µl PBS were mixed with 50 µl of galactose or fucose or

260

mannose or glucose solutions in the presence of 5 mM CaCl2 and

261

0.1mg/ml BSA and incubated at 4 °C overnight. The mixtures were then

262

added into each well and processed as described above in ELISA.

263

2.13. Assay for the effects of rCTLD on phagocytosis

264

As it was difficult to isolate the macrophages from D. rerio, we thus

265

used the macrophages of carp (Cyprinus carpio) to test the effects of

266

rCTLD on phagocytosis. The head kidney-derived macrophages of

267

common carp were isolated by the method of Yang et al [23]. And

268

concentration of the macrophages was determined and adjusted to 2×107

269

cells/ml with a Burker cell counter. In addition, the viability of the

270

macrophages isolated was determined by trypan blue exclusion assay.

271

And the resulting cell suspension was stored at 4

272

following experiments within 2 h. Labeling the cells of S. aureus and E.

273

coli with fluorescein isothiocyanate (FITC; Sigma) was performed

274

according to method of Li et al. [31].

, and used for the

275

Flow cytometric analysis was carried out following the method of Li

276

et al. with slight modifications [32]. For each sample, 10,000 individual

277

cells were analyzed. The phagocytic activity (PA) was defined as

278

percentage of the macrophages which had ingested one or more bacteria

279

within the total macrophage population, and the phagocytic index (PI)

280

defined as the mean fluorescence intensity of the cells.

281

2.14. Assay for subcellular localization

282

The complete coding region of zhl-l was amplified by PCR using the

283

primer P11 and P12 (Supplemental Table 1), and the PCR products were

284

digested with Hind Ⅲ and EcoR Ⅰ and ligated into the eukaryotic

285

expression vector pcDNA3.1/V5/eGFP (which was cut with the same

286

restriction enzymes) upstream, to construct the recombinant eukaryotic

287

expression vector, pcDNA3.1/V5/zhl-l/eGFP. To examine the subcellular

288

localization of Zhl-l, HEK 293T cells were seeded in 6-well plates and

289

cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium

290

(DMEM) containing 10% fetal bovine serum (FBS), 10 U/m penicillin

291

and

292

pcDNA3.1/V5/eGFP and pcDNA3.1/V5/zhl-l/eGFP were transfected into

293

cells using Lipofectamine 2000 Reagent (Invitrogen) according to the

294

instructions of the manufacturer. At 30 h after transfection, the cells were

295

washed with PBS, fixed with 4% paraformaldehyde, and stained with 10

296

µg/mL DAPI as described previously. The samples were observed under

297

Leica fluorescence microscopy (DMI 300B, Germany).

298

2.15. Assay for effect of overexpression of zhl-l on cytokines

299

expression in RAW264.7 cells

100

mg/ml

streptomycin.

Subsequently,

the

plasmids

300

The zhl-l was subcloned into the eukaryotic expression vector

301

pcDNA3.1/V5-His A vector (Invitrogen) with the primers P13 and P14

302

(Supplemental Table 1) by the method mentioned in assay for subcellular

303

localization. The plasmid designated pcDNA3.1/V5/zhl-l. Subsequently,

304

murine RAW264.7cells were seeded in 24-well plates and cultured in

305

DMEM at 37

306

control vector or zhl-l expression vector was carried out using

307

Lipofectamine2000 reagent according to the manufacturer’s protocol. At

308

12 h, 18 h, and 24 h after transfection, the transfected murine RAW264.7

309

cells were scraped off, suspended, centrifuged at 1000g for 2 min at 4 °C

310

and washed with PBS three times, lastly homogenized in Trizol Reagent

under 5% CO2. Transfection of cells with pcDNA3.1

311

and stored at -80

312

cDNA were carried out as described above in section 2.5. The qRT-PCR

313

was used to test the expression level of pro-inflammatory cytokines in

314

macrophages with the PCR primers specific for β-actin (P15 and P16),

315

TNF-α (P17 and P18), IL-1β (P19 and P20) and IL-6 (P21 and P22).

316

(Supplementary Table1)

317

2.16. Statistical analysis

. Preparation of total RNA from cells and synthesis of

318

All the experiments were conducted 3 times. Statistical analyses

319

were performed using the Graphpad Prism 5. The significance of

320

difference was determined by two-way ANOVA or unpaired Student's

321

t-test. Difference at p < 0.05 was considered as significant.

322

3. Results

323

3.1. Structures and characteristics of Zhl-l

324

The cDNA sequence of zhl-l was 1291 bp containing a 5’-UTR of

325

110 bp, a 3’-UTR of 81 bp，and an ORF of 900 bp coding 299 amino

326

acids (Fig. 1A). The deduced protein contained an N-terminal

327

cytoplasmic tail, a trans-membrane domain of 23 aa, a neck region, and a

328

C-terminal extracellular canonical CTLD of 126-amino acids, (Fig. 1A).

329

Within the CTLD, a conserved QPD motif essential for determining the

330

carbohydrate binding specificity was identified (Fig. 1A). Molecular 3D

331

structure modeling revealed that the Zhl-l consists of 5 α-helices and 4

332

β-sheets, which was closely similar to that of human ASGPR containing

333

5 α-helices and 3 β-sheets, and that of zebrafish Zhl containing 3

334

α-helices and 5 β-sheets (Fig. 1B). And they had 6 highly conserved

335

cysteine residues to form the internal disulfide bridges (Fig. 1A and B).

336

3.2. Expression analysis of zhl-l.

337

WISH was performed to explore zhl-l expression patterns in early

338

development. zhl-l was widely expressed in the embryo before

339

segmentation stages (Fig. 2A-2F), then zhl-l mRNA was predominantly

340

detected in the head region (Fig. 2G). The expression of zhl-l was

341

restricted to liver from 48 hpf and the signals gradually expanded

342

coinciding with liver growth during larva development (Fig. 2H-2K). The

343

qRT-PCR also showed that zhl-l mRNAs were abundant in zygotes and

344

decreased with development until 48 hpf. The expression increased

345

between 72 and 120 hpf (Fig. 2L). We also performed qRT-PCR to

346

determine the expression profile of zhl-l in different tissues of adult

347

zebrafish. The expression in gill was used to normalize the expression

348

level in thirteen uninfected tissues. The dissociation curve of

349

amplification products showed a single peak, indicating that the

350

amplification was specific (Data not shown). The expression of zhl-l was

351

mainly detected in liver and low expression level in spleen, intestine,

352

testis, ovary and gallbladder was observed (Fig. 2M).

353 354

The expression profiles of zebrafish zhl-l in response to LPS/LTA challenge,

which

mimics

infection

by

pathogenic bacteria,

were

355

investigated. In liver, the zhl-l expression upon challenge with LPS

356

exhibited a two-peak pattern. It was significantly up-regulated at 4 hpi.

357

Subsequently, it dropped under the basal level until 24h. The second peak

358

was observed at 48 hpi, eventually, it dropped to under the basal level

359

again at 72 hpi (Fig. 3A). The zhl-l gene expression pattern upon

360

challenge with LTA was similar to that response to LPS, except the first

361

peak came up early at 2 hpi (Fig. 3B). By contrast, the zhl-l expression

362

upon challenge with LPS/LTA matched one-peak pattern in spleen. It was

363

significantly down-regulated till 24 hpi, then it was up-regulated

364

significantly at 48 and72 hpi (Fig. 3C and D). These results indicated that

365

the expression of zhl-l was regulated by LPS and LTA.

366

3.3. Agglutinating activity of rCTLD

367

Expression of recombinant proteins, rCTLD and rTRX were induced

368

by IPTG, and they were purified by chromatography on a Ni-NTA resin

369

column. The purified rCTLD and rTRX all yielded a single band of

370

approximately 33 and 21 kDa, respectively, well matching the expected

371

sizes (Fig. 4A). Western blotting showed that they reacted with

372

anti-His-tag antibody, indicating that they were correctly expressed. We

373

then tested if rCTLD could induce agglutination of bacteria. As shown in

374

Fig. 4B, rCTLD showed a conspicuous agglutinating activity towards

375

Gram-negative bacterium E. coli and Gram-positive bacterium S. aureus

376

in the presence of Ca2+, while they did not in the absence of Ca2+. By

377

contrast, rTRX (control) showed little agglutinating activity towards E.

378

coli and S. aureus in the presence of Ca2+. These indicated that the

379

bacterial agglutinating activity of rCTLD depended on Ca2+ (Fig. 4B).

380

We also examined if rCTLD possessed any antibacterial activity by a

381

colony formation assay. The results showed that it did not display

382

antibacterial activity against E. coli and S. aureus (data not shown).

383

3.4. Bacterial binding activity of rCTLD

384

The bacterial binding activity assay revealed that rCTLD showed an

385

intense affinity to the Gram-negative bacteria E. coli and A. hydrophila

386

and the Gram-positive bacteria S. aureus and B. subtilis in the presence or

387

absence of Ca2+ (Fig. 5A and 5B). By contrast, rTRX displayed no

388

positive signal to interact with the examined microbes (Fig. 5A and 5B).

389

These indicated that rCTLD was able to interact specifically with the

390

Gram-negative and Gram-positive bacteria, and the bacterial binding

391

activity of rCTLD did not depend upon the presence of Ca2+.

392

3.5. Ligand binding activity of rCTLD

393

To better understand the mechanisms of microbial-binding activity,

394

Western blotting was carried out to examine the effects of the signature

395

components of bacteria, LPS, LTA and PGN, on the binding of rCTLD to

396

microbes. As shown in Fig. 6A, the binding of rCTLD to E. coli was

397

inhibited by pre-incubation with LPS and PGN, and to S. aureus was

398

inhibited by pre-incubation with LTA and PGN. These results suggested

399

that rCTLD bound to the Gram-positive and Gram-negative bacteria via

400

interaction with the LTA, LPS and PGN on microbial surfaces.

401

Furthermore, an ELISA was performed to verify the interaction of

402

rCTLD (rTRX was used as control) to LTA, LPS and PGN. Although

403

rTRX slightly bound to LTA, LPS and PGN, rCTLD had a significantly

404

stronger affinity to the immobilized ligands LTA, LPS and PGN (Fig.

405

6B-D). These indicated that the microbial signature molecules of bacteria

406

were specifically recognized by rCTLD.

407

3.6. Inhibition of bindings of rCTLD to ligands by sugars

408

As shown in Fig. 7, the bindings of rCTLD to LPS, LTA and PGN

409

were all significantly inhibited by galactose. Similarly, the bindings to

410

LPS and PGN were markedly inhibited by glucose and mannose. The

411

binding to LTA were also considerably inhibited by mannose and fucose.

412

These suggested that among the four sugars examined, galactose was a

413

potent inhibitor capable of suppressing the binding of rCTLD to the

414

ligands.

415

3.7. Enhancement of macrophage phagocytosis by rCTLD

416

Flow cytometric assay was used to assess the effects of recombinant

417

proteins on the phagocytosis of microbes by macrophages. According to

418

the dot plots of the microbes (E. coli and S. aureus), the macrophages and

419

the macrophages challenged with E. coli and S. aureus, we defined a

420

region of macrophages cluster as Gate A (Fig. 8A). The fluorescence data

421

below were all limited in Gate A, which ensured the accuracy of analysis.

422

Based on the part of the macrophages without any phagocytosis C (Fig.

423

8A), we marked the other part as phagocytosis part B (Fig. 8A). The PA

424

(Gate%) and PI (X-Mean) values of the macrophages phagocytosing

425

microbes pre-incubated with PBS, rTRX or rCTLD were shown in

426

histograms. Statistical analyses revealed that the PA and PI values of the

427

macrophages engulfing E. coli and S. aureus pre-incubated with rCTLD

428

were significantly increased compared with those of the macrophages

429

engulfing the same microbes that had been pre-incubated with PBS,

430

rTRX (Fig. 8B). All these data showed that rCTLD was able to promote

431

the phagocytosis of the microbes by the macrophages.

432

3.8. Subcellular localization

433

Protein subcellular localization is tightly linked to its function. The

434

green fluorescence of Zhl-l-eGFP fusion protein was visualized at the rim

435

of the cells, indicating Zhl-l-eGFP was localized on the cell membrane

436

(Fig.9). In contrast, the eGFP alone was evenly distributed throughout the

437

cell (Fig.9). The subcellular localization of Zhl-l was consistent with the

438

predicted structure with a transmembrane region. This result indicated a

439

possible function of Zhl-l as a receptor of hepatocyte.

440

3.9. Effects of ectopic overexpression of zhl-l on production of

441

pro-inflammatory cytokines in macrophages

442

MTT assay was carried out and concluded that Zhl-l didn’t show any

443

cytotoxicity to murine RAW264.7 cells at the tested concentrations

444

(Supplementary Table 3). Expression of pro-inflammatory cytokines,

445

such TNF-α, IL-1β, and IL-6, is a hallmark of macrophage activation [33].

446

We evaluated whether overexpression of zhl-l was capable of affecting

447

expression of pro-inflammatory cytokines in macrophages. As shown in

448

Fig.10, overexpression of zhl-l significantly increased the expression of

449

TNF-α, IL-1β, and IL-6 mRNAs. These results suggested that

450

overexpression of zhl-l may stimulate production of pro-inflammatory

451

mediators in macrophages.

452

4. Discussion

453

The zebrafish has unique advantages for understanding the evolution

454

of vertebrate immunity and for modeling human diseases [34-35]. Up to

455

date, as a major component of the immune system, several CLRs have

456

been identified in zebrafish. For example, Mannose/mannan binding

457

lectin/protein was a Ca2+-dependent collagenous (C-type) lectin that plays

458

an important role in the innate immune system [36,37]. CLEC14A

459

induced filopodia and facilitated endothelial migration, tube formation

460

and vascular development in zebrafish [38]. And Ai-Fu Lin described the

461

identification and biological characterization of the DC-SIGN from

462

zebrafish and its involvement in adaptive immunity [39]. In this study, we

463

identified a novel liver specific expressed CLR, which contained a

464

canonical CTLD and located on the cell membrane. Its bindings to

465

ligands were inhibited significantly by galactose according with ASGPR

466

(hepatic lectin in mammals) specifically recognizing galactose and

467

GlcNAc [9]. Therefore, we concluded that this CLR was a putative

468

hepatic lectin. To distinguish our previously identified hepatic lectin Zhl,

469

it was named as hepatic lectin-like in zebrafish (Zhl-l) [23]. It is well

470

known that three rounds (3R) whole genome duplications (WGD)

471

occurred in bony fish [40], so zhl-l may generate from the duplication of

472

zhl. But the syntenic analysis cannot absolutely confirm this speculation

473

(data not shown), therefore, they were named as zhl and zhl-l, rather than

474

zhla and zhlb.

475

In fish, several CLRs were prominently expressed in spleen, kidney,

476

liver, or intestine tissues, such as Zhl, zfMR, DC-SIGN in zebrafish,

477

DC-SIGN and L-SIGN in Miichthys miiuy, and OppCTL from

478

Oplegnathus punctatus [23, 39, 41-43]. In this study, zhl-l was mainly

479

observed in liver, low expression level in spleen, testis, and gallbladder.

480

Of which, liver and spleen are immune-related organs that participate in

481

humoral immune and inflammatory responses [44, 45]. Challenge with

482

LPS/LTA both resulted upregulation of zhl-l, while the expression of zhl-l

483

exhibited two-peak pattern in liver but one-peak profile in spleen. The

484

temporal expression of zhl-l mRNA was all significantly up-regulated at

485

certain time points post infection. The similar expression pattern was

486

found for zfMR of zebrafish in response to A. sobria challenge [41]. The

487

reasons for the variation of the temporal expression patterns of zhl-l in

488

different tissues were enigmatic. However, the mRNA expression of zhl-l

489

was indeed induced by the infection of LPS/LTA, indicating that Zhl-l

490

may involve in innate defenses. Zhl exhibited different expression

491

profiles with Zhl-l in response to LPS/LTA challenge [23]. This might

492

suggest that two hepatic lectins might participate the immune responses

493

in different stages. Similarly, FcLec3 and Fc-hsL, as members of CLRs

494

family, were all specifically found in hepatopancreas, they also displayed

495

different expression patterns to V. anguillarum challenge [46, 47].

496

Agglutinating and binding bacteria activity are basic and important

497

characteristics of authentic C-type lectins [4, 46]. The EsLecB from

498

Eriocheir sinensis exhibited agglutinating and bacteria-binding activity in

499

Ca2+-independent

500

previously shown that rCTLD of Zhl was capable of agglutinating and

501

binding bacteria in the presence of Ca2+ [23]. And we also demonstrated

502

that rCTLD of Zhl-l had these activities. While its bacterial agglutinating

503

activity depended on the presence of Ca2+, its bindings to bacteria even

504

LPS, LTA and PGN were independent of Ca2+. Similarly, a novel C-type

505

lectin Fc-Lec2 from Fenneropenaeus chinensis and its two CRDs bound

506

to microorganisms in the absence of Ca2+, but agglutinated some bacteria

507

in a Ca2+ -dependent manner [49]. And CaNTC from Carassius auratus

508

agglutinated and bound to tested bacteria in the same manner with Zhl-l

and

carbohydrate-dependent

manner

[48].

We

509

and Fc-Lec2 [50]. The relationship between conserved Ca2+ binding motif

510

and Ca2+-dependent activity is unclear. The C-type lectins mentioned

511

above all have a Ca2+ binding motif which known as an EPN/WND motif,

512

but their activity depending on Ca2+ are different. Ca2+ requirements for

513

C-type lectins may be affected by interactions with side chains of amino

514

acids in other regions in addition to the CTLD, or by formation of

515

oligomeric structures [49, 51]. Furthermore, proteins of Groups II and V

516

share overall structural similarities. And Group V CLRs, which typically

517

recognize protein ligands independent of Ca2+, might not be present in

518

bony fish [4, 52]. Therefore, Zhl-l, a member of group II CLRs, may

519

possess some properties of Group V CLRs so that it bound to bacteria in

520

Ca2+ -independent manner.

521

CLRs could bind to CLR ligands including carbohydrate, protein

522

and lipid components of both pathogens and self, which variably trigger

523

immune

524

anti-inflammatory reactions [5]. And a number of CLRs have potential

525

anti-inflammatory or pro-inflammatory activity in fish. Sbgalectin-1 from

526

Dicentrarchus labrax can down-regulate the expressions of IL-1β, TNF-α,

527

and Mx and played a potential anti-inflammatory, protective role during

528

viral infection [53]. OppCTL from Oplegnathus punctatus had potential

529

anti-inflammatory activity [43]. A mannose receptor in zebrafish was

530

involved in synthesis of pro-inflammatory cytokines such as IL-1β and

responses

including

phagocytic,

pro-inflammatory

or

531

TNF-α by binding of natural or synthetic ligands [54]. In our study, Zhl-l

532

overexpression on significantly enhanced expression of pro-inflammatory

533

cytokines, TNF-α, IL-1β, and IL-6 at approximately 12 h, 18 h and 24 h

534

post transfection respectively. The reason for out of synchronism maybe

535

that TNF-α is originally derived from macrophages, which induce the

536

production of IL-1, IL-6, IL-8, and pro-inflammatory mediators, thereby

537

further inducing inflammatory reaction [55]. In addition, we previously

538

shown that overexpression of Zhl inhibited the production of

539

pro-inflammatory cytokines [23]. The opposite responses on modulating

540

inflammatory cytokines between Zhl and Zhl-l are interesting.

541

Inflammation is a complex biological responses of body tissues to

542

harmful stimuli, such as pathogen, is a protective response. However,

543

excessive inflammation can break the body's homeostasis when suffered

544

from pathogens infection. So, Zhl may function as a potential

545

immunosuppressive factor in anti-inflammatory reaction protecting host

546

from injury of excessive inflammatory reaction, and Zhl-l may have

547

immunopromotive activity and cooperate with Zhl to maintain body's

548

homeostasis. And the speculated antagonistic action on expression of

549

pro-inflammatory cytokines between Zhl-l and Zhl needs to be further

550

investigated.

551

In conclusion, Although Zhl-l did not display any antibacterial

552

activity against E. coli and S. aureus (Data not shown), it can agglutinate

553

and bind bacteria directly, and function as pattern recognition receptors to

554

recognize Gram-negative and Gram-positive bacteria via interacting with

555

LPS, LTA and PGN. Moreover, it can also act as opsonin to enhance the

556

phagocytosis of bacteria by macrophages. In addition, Zhl-l, as a

557

membrane receptor, its overexpression could increase the production of

558

pro-inflammatory

559

participates in the immune response to against bacterial infection. Our

560

study will enrich the study of immune function of fish lectins and provide

561

more information for future research.

562

Acknowledgements

cytokines.

These

results

indicated

that

Zhl-l

563

This work was supported by the grant (2018YFD0900502) of the

564

Ministry of Science and Technology (MOST) of China and the grants

565

(31872187，31572219) of Natural Science Foundation of China (NSFC).

566

This work was also supported by Grants 201941009 from the

567

Fundamental Research Funds for Central Universities.

568 569

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[52] P.G. Panagos, K.P. Dobrinski, X. Chen, A.W. Grant, D. Traver, J.Y.

736

Djeu, S. Wei, J.A. Yoder, Immune-related, lectin-like receptors are

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differentially expressed in the myeloid and lymphoid lineages of

738

zebrafish, Immunogenetics. 58 (2006) 31–40.

739

[53] L. Poisa-Beiro, S. Dios, H. Ahmed, G.R. Vasta, A. Martínez-López,

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A. Estepa, J. Alonso-Gutiérrez, A. Figueras, B. Novoa, Nodavirus

741

infection of sea bass (Dicentrarchus labrax) induces up-regulation of

742

galectin-1 expression with potential anti-inflammatory activity, J.

743

Immunol. 183 (10) (2009) 6600–6611.

744

[54] S.D. Tachado, J. Zhang, J. Zhu, N. Patel, M. Cushion, H. Koziel,

745

Pneumocystis-mediated

746

coexpression of mannose receptors and TLR2, J Leukoc Biol 81(1) (2007)

747

205-11.

748

[55] N.C. Riedemann, R.F. Guo, P.A. Ward, Novel strategies for the

749

treatment of sepsis, Nat Med. 9 (2003) 517–524.

750

IL-8

release

by

macrophages

requires

751

Figure legends

752

Figure 1. Nucleotide sequence, deduced amino acid sequence and

753

domain architecture of Zhl-l.

754

(A) The full-length nucleotide and deduced amino acid sequences of Zhl-l.

755

The nucleotides and amino acids are numbered on the right margin. The

756

termination codon is indicated with asterisk (*). Several motifs were in

757

red box and conserved cysteine-rich motifs were in green box. The

758

transmembrane domain (TM) and C-type lectin domain (CTLD) was

759

shaded in blue and pink, respectively. (B). 3D structure of Zhl-l. In 3D

760

structure, red: α-helix; yellow: β-sheets; green: loops. The six conserved

761

cysteines forming three disulfide bridges. C168-C179, C196/C292 and

762

C270/C284, in blue, magenta and cyan, respectively.

763 764

Figure 2. Expression patterns of zhl-l at different developmental

765

stages and in different tissues.

766

Stages of embryonic development: A, two cells; B, sixteen cells; C,

767

sphere; D, 50% epiboly; E, bud; F, 10-somites; G, 24 hpf; H, 48 hpf; I, 72

768

hpf. J, 96hpf; K, 120 hpf. hpf, hour post-fertilization. L, Expression

769

profiles of zhl-l at different developmental stages. M, Expression profiles

770

of zhl-l in different tissues.

771 772

Figure 3. LPS/LTA-induced expression of zhl-l in liver and spleen.

773

(A) Quantitative analysis of zhl-l in infected liver by intraperitoneal

774

injection LPS. (B) Quantitative analysis of zhl-l in infected liver by

775

intraperitoneal injection LTA. (C) Quantitative analysis of zhl-l in

776

infected spleen by intraperitoneal injection LPS. (D) Quantitative analysis

777

of zhl-l in infected spleen by intraperitoneal injection LTA. The results

778

shown are mean values ± SD, n = 3 replicates per tissue, and are pooled

779

from three experiments. Asterisks indicate statistically different (*p <

780

0.05, **p < 0.01, ***p< 0.001) compared to control. Data were

781

normalized to the β-actin gene as internal control.

782 783

Figure 4. SDS-PAGE and Western blotting of recombinant proteins

784

and bacterial agglutination activity of rCTLD.

785

(A) SDS-PAGE and Western blotting of recombinant proteins rCTLD and

786

rTRX, Lane M, marker; lane 1, total cellular extracts from E. coli

787

transetta (DE3) containing expression vector before induction; lane 2,

788

total cellular extracts from IPTG induced E. coli transetta (DE3)

789

containing expression vector; lane 3, purified recombinant proteins; lane

790

4, Western blot of purified recombinant proteins. (B) Agglutination of E.

791

coli and S. aureus by rCTLD in the presence or absence of Ca2+.

792 793

Figure 5. Bacterial binding activity of rCTLD.

794

(A) Bindings of rCTLD to two Gram-negative bacteria E. coli and A.

795

hydrophila. Lane M, molecular mass standards; lane 1, purified rCTLD

796

protein; lane 2, purified rTRX protein; lane 3, E. coli incubated with

797

purified rCTLD protein in presence of Ca2+; lane 4, E. coli incubated with

798

purified rCTLD protein in absence of Ca2+; lane 5, E. coli incubated with

799

purified rTRX protein in presence of Ca2+; lane 6, A. hydrophila

800

incubated with purified rCTLD protein in presence of Ca2+; lane 7, A.

801

hydrophila incubated with purified rCTLD protein in absence of Ca2+;

802

lane 8, A. hydrophila incubated with purified rTRX protein in presence of

803

Ca2+. (B) Binding of rCTLD to two Gram-positive bacteria B. subtilis and

804

S. aureus. Lane M, molecular mass standards; lane 1, purified rCTLD

805

protein; lane 2, purified rTRX protein; lane 3, B. subtilis incubated with

806

purified rCTLD protein in presence of Ca2+; lane 4, B. subtilis incubated

807

with purified rCTLD protein in absence of Ca2+; lane 5, B. subtilis

808

incubated with purified rTRX protein in presence of Ca2+; lane 6, S.

809

aureus incubated with purified rCTLD protein in presence of Ca2+; lane 7,

810

S. aureus incubated with purified rCTLD protein in absence of Ca2+; lane

811

8, S. aureus incubated with purified rTRX protein in presence of Ca2+.

812 813

Figure 6. Analysis of the affinity of rCTLD to the ligands.

814

(A) Binding of rCTLD to S. aureus and E. coli was inhibited by the

815

presence of LTA/PGN and LPS/PGN respectively. Lane 1, E.coli

816

incubated with recombinant proteins which were pre-incubated with PBS;

817

lane 2, E. coli incubated with recombinant proteins which were

818

pre-incubated with LPS; lane 3, E. coli incubated with recombinant

819

proteins which were pre-incubated with PGN; lane 4,S. aureus incubated

820

with recombinant proteins which were pre-incubated with PBS; lane 5, S.

821

aureus incubated with recombinant proteins which were pre-incubated

822

with LTA; lane 6, S. aureus incubated with recombinant proteins which

823

were pre-incubated with PGN.(B) the binding of rCTLD to LPS, (C) the

824

binding of rCTLD to LTA and (D) the binding of rCTLD to PGN. Data

825

are shown as mean ± SEM. The rCTLD+ means that rCTLD incubated

826

with ligands in the presence of Ca2+. The rCTLD- means that rCTLD

827

incubated with ligands in the absence of Ca2+.

828 829

Figure 7. Inhibitory effects of fucose, galactose, mannose and glucose

830

on the bindings of rCTLD to the ligands.

831

Data are shown as mean ± SEM. The symbol * indicates a significant

832

difference (p < 0.05), the symbol ** indicates an extremely significant

833

difference (p < 0.01), the symbol *** indicates an extremely significant

834

difference (p < 0.001)

835 836

Figure 8. Effects of rCTLD on the phagocytosis.

837

(A) The histograms of flow cytometric analyses of the macrophages

838

phagocytosing E. coli or S. aureus pre-incubated with PBS, rTRX or

839

rCTLD, respectively. (B) PA and PI values of rCTLD. The asterisks (*)

840

show significant difference from control (The symbol * means p < 0.05,

841

the symbol **p < 0.01 and the symbol ***p < 0.001).

842 843

Figure 9. Subcellular localization of Zhl-l in HEK293T cells.

844

The HEK293T cells was transiently transfected with pcDNA3.1/V5/eGFP

845

or pcDNA3.1/V5/zhl-l/eGFP. After 48 h, the cells were imaged by

846

fluorescence microscopy. The nucleus was stained by DAPI.One

847

representative image for each out of three independent experiments is

848

shown. Scale bar: 40µm.

849 850

Figure 10. Effects of ectopic overexpression of zhl-l on expression of

851

pro-inflammatory cytokines by macrophages.

852

RAW 264.7 cells were transfected with control vector pcDNA3.1(Control)

853

or Zhl-l expression vector. Total RNA was prepared 18h and 24h after

854

transfection. Total RNA was analyzed for mRNA expression of TNF-α,

855

IL-6 and IL-1β by qRT-PCR using specific primers. Data are shown as

856

mean ± SEM. The symbol * indicates a significant difference (p < 0.05),

857

the symbol ** indicates an extremely significant difference (p < 0.01), the

858

symbol *** indicates an extremely significant difference (p < 0.001)

► A novel zebrafish hepatic lectin (Zhl-l) was identified. ► The expression of zhl-l was up-regulated upon LPS/LTA challenge. ► Zhl-l was capable of agglutinating both Gram-negative and Gram-positive bacteria in Ca2+-dependent manner, and binding to them in Ca2+-independent manner. ► Zhl-l bound to Gram-negative and Gram-positive bacteria via interaction with LTA, LPS and PGN, which could be inhibited by galactose. ► Zhl-l could act as opsonin to enhance the phagocytosis of bacteria by macrophages. ► Overexpression of zhl-l could up-regulate the production of pre-inflammatory cytokines.