AP-1 regulates the expression of IL17-4 and IL17-5 in the pacific oyster Crassostrea gigas

Journal Pre-proof AP-1 regulates the expression of IL17-4 and IL17-5 in the pacific oyster Crassostrea gigas Liyan Wang, Jiejie Sun, Zhaojun Wu, Xingye Lian, Shuo Han, Shu Huang, Chuanyan Yang, Lingling Wang, Linsheng Song PII:

S1050-4648(19)31219-7

DOI:

https://doi.org/10.1016/j.fsi.2019.12.080

Reference:

YFSIM 6722

To appear in:

Fish and Shellfish Immunology

Received Date: 27 November 2019 Revised Date:

23 December 2019

Accepted Date: 26 December 2019

Please cite this article as: Wang L, Sun J, Wu Z, Lian X, Han S, Huang S, Yang C, Wang L, Song L, AP-1 regulates the expression of IL17-4 and IL17-5 in the pacific oyster Crassostrea gigas, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2019.12.080. 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

AP-1 regulates the expression of IL17-4 and IL17-5 in the pacific

2

oyster Crassostrea gigas

3 a,c

a,c

Liyan Wang , Jiejie Sun *, Zhaojun Wua,c, Xingye Liana,c, Shuo Hana,c, Shu Huanga,c,

4

a,b,c,d

Chuanyan Yanga,c, Lingling Wang

5

a, b,c

, Linsheng Song

*

6 7 8

a

9

116023, China

Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian

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b

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Laboratory for Marine Science and Technology, Qingdao 266235, China

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c

13

University, Dalian 116023, China

14

d

15

University, Dalian 116023, China

Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National

Liaoning Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean

Dalian Key Laboratory of Aquatic Animal Disease prevention and Control, Dalian Ocean

16 17

*Corresponding to:

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Dr. Jiejie Sun, Dr. Linsheng Song

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Dalian Ocean University

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52 Heishijiao Street, Dalian 116023, China

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Tel: 86-411-84763173

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E-mail: [email protected] (J. Sun), [email protected] (L. Song)

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Abstract

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The activator protein-1 (AP-1) plays an important role in inducing the immune effector

25

production in response to cellular stress and bacterial infection. In the present study, an AP-1

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was identified from Pacific oyster Crassostrea gigas (designed as CgAP-1) and its function

27

was investigated in response against lipopolysaccharide (LPS) stimulation. CgAP-1 was

28

consisted of 290 amino acids including a Jun domain and a basic region leucine zipper (bZIP)

29

domain. CgAP-1 shared 98.6% similarities with ChAP-1 from oyster C. hongkongensis, and

30

assigned into the branch of invertebrates in the phylogenetic tree. The mRNA transcripts of

31

CgAP-1 gene were detected in all tested tissues with highest expression level in hemocytes,

32

especially in granulocytes. The mRNA expression level of CgAP-1 gene in hemocytes was

33

significantly up-regulated (8.53-fold of that in PBS group, p < 0.01) at 6 h after LPS

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stimulation. CgAP-1 protein could be translocated into the nucleus of oyster hemocytes after

35

LPS stimulation. The mRNA transcripts of interleukin17s (CgIL17-4 and CgIL17-5) in the

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hemocytes of CgAP-1-RNAi oysters decreased significantly at 24 h after LPS stimulation,

37

which were 0.37-fold (p < 0.05) and 0.17-fold (p < 0.01) compared with that in EGFP-RNAi

38

oysters, respectively. The results suggested that CgAP-1 played an important role in the

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immune response of oyster by regulating the expression of CgIL17s.

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Key words: Activator protein-1; Interleukin 17s; Crassostrea gigas; Lipopolysaccharide

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stimulation; Immune responses

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43

Introduction

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The activator protein-1 (AP-1) belongs to the class of basic region leucine zipper (bZIP)

45

transcription factors and plays crucial roles in multiple immune responses [1]. AP-1 can be

46

phosphorylated by mitogen-activated protein kinase (MAPK) pathway after receiving the

47

stimulus signal [2], and the activated AP-1 forms homologous dimer or heterodimer, which is

48

then transferred from the cell cytoplasm into nucleus. AP-1 in nucleus binds the DNA

49

sequence motif and then regulates the transcription of downstream genes [3].

50

An increasing number of AP-1 family members have been identified in both vertebrates and

51

invertebrates. Mammal AP-1 family contains Jun and Fos proteins. Jun proteins are mainly

52

composed of c-Jun, Jun B and Jun D encoding by c-Jun, Jun B, and Jun D genes, respectively

53

[4, 5], while Fos proteins are consisted of c-Fos, Fra-1, Fra-2 and Fos-B [5, 6]. Jun proteins

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commonly contain a DNA binding domain (bZIP) and a transcriptional activation domain

55

(Jun) [7]. The bZIP forms homo- and/or heterodimers through their leucine zipper motif and

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then binds to the target genes to regulate gene transcription [8], and the Jun domain can be

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phosphorylated and activated by Jun N-terminal kinase (JNK) [9]. The phosphorylation of

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Fos domain is mediated by extracellular signal-regulated kinase 5 (ERK5) [10]. The AP-1

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homologs identified in invertebrates share the similar structural characteristics with AP-1

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family members in vertebrates. In Drosophila, two AP-1 homologs (designed as dFRA and

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dJRA) were identified to have the structural properties in common with mammalian Fos and

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Jun, respectively [11, 12]. c-Fos and c-Jun homologs were also identified in shrimp

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Litopenaeus vannamei [13]. In mollusks, AP-1 homologs were found in disk abalone Haliotis

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discus discus (AbJun) [14] and Pinctada fucata (PfAP-1) [15]. Two AP-1 homologs (ChJun

65

and ChFos) were also identified in C. hongkongensis [16]. The Jun and bZIP domains in

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many invertebrate AP-1s are relatively conserved in comparison with those in mammalian

67

ones, indicating the functional conservation of AP-1 in vertebrates and invertebrates.

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AP-1 can bind the DNA sequence motif to regulate the expression of downstream genes, such

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as inducing the production of various immune effectors. In vertebrates, AP-1 family members

70

were reported to mediate the release of different cytokines and antimicrobial peptides (AMPs).

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For instance, AP-1 could induce the expression of beta-defensin-2 (hBD-2) gene in human

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intestinal epithelial cells [17]. In mononuclear cells and smooth muscle cells, AP-1 could

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regulate the production of IL-6, IL-8 [18] and IL-1β [19, 20], respectively. JunB was reported

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to play a crucial role in the development of T cells by facilitating IL-2 signaling [21]. Jun D

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could control IL-1β levels rather than IL-6 and TNF-α (tumor necrosis factor-α) levels in the

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brain tissue of mice [22]. In invertebrates, AP-1 family members are involved in inducing the

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expression of multiple AMPs [23]. In Drosophila, Jun and Fos (defined as D-Jun and D-Fos)

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functioned as transcription factors to regulate the expressions of AMP genes, including

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Attacin A, Cecropin A, Drosomycin, Defensin, and Metchnikowin [13, 24]. Lvc-Fos and

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Lvc-Jun in Pacific white shrimp Litopenaeus vannamei could induce the expression of

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penaeidins [13, 23]. In Mercenaria mercenaria, the activation of AP-1 led to the production of

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lysozyme and big defensin [25]. However, the detailed mechanism of AP-1 activation and its

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function in regulating cytokine production are still not very clear in invertebrates.

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The recently released genome sequence of the Pacific oyster C. gigas provides convenience

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for the study of molluscan innate immunity [26]. Cytokines play critical roles in the innate

86

immune system, and the proinflammatory cytokines such as IL-1, IL-6 and IL17 can mediate

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the proliferation and differentiation of multiple immune cells, as well as various immune

88

responses [27-29]. Recently, six IL17s (designated as CgIL17-1 to CgIL17-6) had been

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characterized from oyster C. gigas [30, 31]. CgIL17-4 and CgIL17-5 of them were found to

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play crucial roles in the immune responses [32]. In the present study, an AP-1 was identified

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from C. gigas (designated as CgAP-1) and its temporal alteration of mRNA expression,

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subcellular localization of CgAP-1 proteins in oyster hemocytes as well as its function in

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mediating the production of CgIL17-4 and CgIL17-5 were investigated after LPS stimulation.

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The results would be helpful for understanding the activation mechanism of AP-1 and its

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function in regulating IL17 production in invertebrates.

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2. Material and methods

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2.1 Animals

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The oysters C. gigas (about 13.0 cm in shell length) were collected from a farm in Dalian,

100

Liaoning Province, China, and raised in seawater at 15-18 ℃ for two weeks. The seawater was

101

exchanged to fresh seawater daily.

102 103

2.2 Immune challenge and tissue collection

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Different tissues (gonad, adductor muscle, mantle, gills, hemocytes and hepatopancreas) were

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collected from nine untreated oysters to examine the mRNA distribution of CgAP-1 gene. A

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total of 100 oysters were equally separated into two groups for immune treatment. The oysters

107

in the two groups received individual intramuscular injections with 100 µL 0.01 M

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phosphate-buffered saline PBS (0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM

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KH2PO4) and 100 µL of LPS from Escherichia coli (O222:B44, Sigma Aldrich, USA)

110

dissolved in PBS, respectively. Nine oysters were randomly sampled from each group at 0, 6,

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12, 24 and 48 h after PBS and LPS stimulations (PBS was used as control). The hemolymphs

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from three oysters were pooled together as one sample, and there were three samples for each

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time point. The hemocytes were harvested by centrifugation at 1500 rpm, 4 ℃ for 8 min. All

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the samples were stored at −80 °C for subsequent RNA extraction by using Trizol reagent

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(Thermo Fisher Scientific, USA) [33]. The full open reading frame (ORF) of CgAP-1 was

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cloned with the primers of CgAP-1-F and CgAP-1-R (Table 1) in a PCR Thermal Cycle

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(TaKaRa).

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2.3 cDNA synthesis

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Total RNA was isolated from hemocytes using Trizol reagent (Thermo Fisher Scientific, USA)

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according to its protocol. The extracted RNA was quantified by Nanodrop 2000 (Thermo

122

Fisher, USA). The cDNA synthesis was conducted with the total RNA as template according

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to the direction of manufacturer (Takara, China). The reaction mixtures were incubated at

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42 ℃ for 1 h and then terminated by heating at 95 ℃ for 5 min, which was then stored at

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-80 ℃.

126 127

2.4 qRT-PCR analysis

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Quantitative real-time PCR (qRT-PCR) was performed to detect the tissue distribution of

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CgAP-1 mRNA by the specific primers CgAP-1-RT-F1 and CgAP-1-RT-R1 (Table 1). CgEF

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(Accession No. NP_001292242.2) amplified with the primers of CgEF-RT-F and CgEF-RT-R

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(Table 1) was used as control. The temporal mRNA expression profiles of CgAP-1 were also

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detected in the hemocytes by qRT-PCR after LPS stimulation, and the PBS group was set as

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the control. The mRNA transcripts of CgIL17-4 (GenBank accession KJ531895) and

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CgIL17-5 (KJ531896) were detected in hemocytes via qRT-PCR by specific primers

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(CgIL17-4-RT-F and CgIL17-4-RT-R, CgIL17-5-RT-F and CgIL17-5-RT-R, respectively).

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The thermal profile for qRT-PCR program was 95 ℃ for 5 min, followed by 40 cycles of 95 ℃

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for 5 s, 60 ℃ for 31 s. The relative mRNA expression levels of CgAP-1, CgIL17-4 and

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CgIL17-5 were analyzed by comparative Ct method (2-∆∆Ct method) [34, 35]. Vertical bars

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represent the mean ± S.D. (N = 3).

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2.5 Sequence and phylogenetic analysis of CgAP-1

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The protein sequences of CgAP-1 and AP-1s from different species acquired from the

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National

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http://www.ncbi.nlm.nih.gov/ebinet.htm) databases were aligned by Clustal X v2.0 program

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and GeneDoc software (http://www.nrbsc.org/gfx/genedoc/ebinet.htm). The domains of

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CgAP-1

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(http://smart.embl-heidelberg.de/). The phylogenetic tree was constructed based on the AP-1

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protein sequences by employing MEGA 7.0 software with the neighbor-joining (NJ) method

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[36].

Center

protein

were

for

predicted

Biotechnology

by

the

Information

website

of

SMART

(NCBI,

sites

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2.6 Plasmid constructions

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Primers CgAP-1-F and CgAP-1-R (Table 1) were designed in accordance with the sequence

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information of CgAP-1 (XP_019928182.1) acquired from the NCBI database and used to

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clone the open reading frame (ORF) of CgAP-1 from C. gigas. After gel-purification with

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MiniBest Agarose Gel DNA Extraction Kit Ver.4.0 (Takara, Japan), the products were inserted

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into pMD19-T vector (Transgen Biotech, China) and sequenced in both directions with

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M13-47 and M13-RV (Table 1). The recombinant plasmid (pMD19-T-CgAP-1) were purified

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and confirmed through sequencing after transformed into competent cells of Escherichia coli

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Trans5α (TransGen Biotech, China).

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2.7 Recombinant expression and purification of CgAP-1

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PCR was performed using primers CgAP-1-ExF and CgAP-1-ExR with BamH ℃ and Hind ℃

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sites (Table 1), which were designed according to the ORF sequence of CgAP-1. The PCR

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procedure was conducted as follows: one cycle at 95 ℃ for 3 min, 35 cycles at 94 ℃ for 30 s,

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55 ℃ for 30 s, 72 ℃ for 1 min (35 cycles), and 72 ℃ for 10 min. The PCR fragments were

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digested by restriction enzymes and inserted into expression vector pET28a (Novagen,

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Germany) using T4 DNA ligase. The recombinant plasmid (pET-28a-CgAP-1) was

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transformed into E. coli Transetta (DE3) (TransGen Biotech, China). The positive

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transformant of E. coli Transetta (DE3) with pET-28a-CgAP-1 was incubated in LB medium

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(containing 50 mg/mL kanamycin) at 37 ℃ with shaking at 180 rpm for about 4 h. When the

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culture medium reached OD600 of 0.4-0.6, the cells were incubated for four additional hours

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with the induction of Isopropyl β-D-Thio-galactoside (IPTG). The recombinant CgAP-1

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protein (rCgAP-1) was purified by His-tag purification resin (Sangon Biotech, China), and

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pooled by elution with 400 mmol/L imidazole under denatured condition (8 mol/L urea).

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rCgAP-1 was refolded against gradient urea-TBS glycerol buffer (50 mmol/L Tris-HCl, 50

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mmol/L NaCl, 15% glycerol, 2 mmol/L reduced glutathione, 0.2 mmol/L oxide glutathione, a

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gradient urea concentration of 6, 5, 4, 3, 2, 1, and 0 M, pH 8.0, each gradient at 4 ℃ for 12 h).

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The purified protein was separated by 12% SDS-poly-acrylamide gel electrophoresis

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(SDS-PAGE), and visualized with Coomassie bright blue R250 [37]. The purified rCgAP-1

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was quantified with bicinchoninic acid (BCA) method [38], and stored at -80 ℃ for

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subsequent experiments.

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2.6 Western blot

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Six-week old mice were immunized by using the purified rCgAP-1 to acquire the polyclonal

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antibody according to the previous description [37]. The specificity of antibody was identified

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by western blot. The protein samples extracted from oyster hemocytes were separated by 12%

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SDS-PAGE and then diverted onto nitrocellulose membranes [39]. The membranes were

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blocked with 5% non-fat milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 1% Tween-20, pH

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8.0) for 1 h, and then incubated with 1/100 diluted antiserum against CgAP-1 with 5% non-fat

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milk for 4 h. Afterward, the membranes were incubated with alkaline phosphatase-conjugated

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AP-labeled Goat anti-mouse IgG (Beyotime, China, 1:1000 diluted in TBS) for 3 h after three

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times of washing to remove the free non-specifically binding antiserum. After washed three

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times, the membranes were steeped in the reaction system (10 mL of TBS with 45 and 35 µL

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of NBT and BCIP, respectively, Sangon Biotech, China) in the dark for 5 min and then

194

stopped by washing with distilled water.

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2.7 Immunocytochemical assay and the flow cytometry (FCM) analysis

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Immunocytochemical assay was performed to detect the translocation of CgAP-1 in the

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hemocytes at 2 h after LPS stimulation with PBS as control. The hemolymphs from the

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oysters were fixed with 1 mL of a mixture containing an anticoagulant (510 mM NaCl, 100

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mM glucose, 200 mM citric acid, 30 mM sodium citrate, 10 mM EDTA·2Na, pH 7.4) and 4%

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paraformaldehyde (1:1 in volume) for 15 min and centrifuged at 600 g at 4 ℃ for 4 min to

202

collect the hemocytes. The collected hemocytes were washed three times with PBS and then

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deposited onto glass slides. The hemocytes on the glass slides were washed six times with

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PBS. Then, the samples on the glass slides were blocked with 3% bovine serum albumi (BSA)

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at 37 ℃ for 30 min and incubated with anti-CgAP-1 antibody (1:300 in 3% BSA) at 4 ℃

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overnight. After washed for three times with PBS, the samples were incubated with Alexa

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Fluor 488-conjugated second antibody (Solarbio life sciences, China, diluted 1:1000 (v/v) in

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3% BSA) at 37 ℃ in dark for 1h. Then the slides with hemocytes were filled with

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4ʹ-6-diamidino-2-phenylindole dihydrochloride (DAPI, Beyotime China, 1 µg/mL in PBS) for

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10 min and washed by PBS for six times in the dark. The treated slides were stored at

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glycerinum and observed under inverted fluorescence microscope (Axio Imager A2, ZEISS).

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The flow cytometric morphology analysis of different cells was conducted according to their

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relative size (forward scatter, FSC) and complexity (side scatter, SSC) using a FACS Arial II

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flow cytometry (Becton Dickinson Biosciences). The collected untreated oyster hemocytes

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were fixed in 4% paraformaldehyde (PFA) for 10 min to keep intact cell morphology. The

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hemocytes incubated with anti-CgAP-1 polyclonal antibody were analyzed and sorted by flow

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cytometry [40].

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2.8 RNA interference

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CgAP-1 RNA interference fragment was amplified by PCR to composite cDNA of CgAP-1

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and EGFP with the primers (CgAP-1-Fi, CgAP-1-Ri, EGFP-Fi and EGFP-Ri, Table1). The

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dsRNAs were synthesized by using T7 polymerase according to the instruction of

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manufacture (Takara, China). Twelve oysters were separated into two groups, and the oysters

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in experiment group (AP-1-RNAi) received injection of CgAP-1 dsRNA, while those in

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control group (EGEF group) received injection of EGFP dsRNA. The dsRNAs of CgAP-1

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and EGFP were injected into the adductor muscle of each oyster and the second injection was

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carried out at 12 h after the first injection. The hemocytes were collected from the treated

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oysters at 24 h after the second injection. qRT-PCR was used to evaluate the efficiency of

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RNAi with the primers CgAP-1-RT-F2 and CgAP-1-RT-R2 (Table 1).

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Other eighteen oysters were used to detect the expressions of CgIL17s in CgAP-1-RNAi

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oysters after LPS stimulation. The oysters were separated into three groups averagely,

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including one experiment group, one control group (EGFP group), and one blank group. At

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24 h after twice injection of dsRNAs, respectively, LPS was injected into CgAP-1-RNAi and

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EGFP-RNAi oysters. The total RNA from hemocytes was collected to detect expression

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levels of CgIL17-4 and CgIL17-5 mRNA via qRT-PCR by specific primers (Table 1) at 24 h

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after LPS injection. Differences in the unpaired sample t-test were considered significant at p

237

< 0.05 and extremely significant at p < 0.01. Vertical bars represent the mean ± S.D. (N = 3).

238 239

3. Results

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3.1 Molecular characteristic, phylogenic relationship, and multiple sequence alignment of

241

CgAP-1

242

The full-length cDNA sequence of CgAP-1 was of 1492 bp with an open reading frame (ORF)

243

of 873 bp, encoding a polypeptide of 290 amino acids with an isoelectric point of 8.82. The

244

sequence was deposited in GenBank under accession number CGI_10006579. CgAP-1

245

contained a conserved Jun protein kinase catalytic domain and a basic region leucin zipper

246

(bZIP) domain (Fig. 1). CgAP-1 shared similarities ranging from 22.7% to 98.6% with

247

previously identified AP-1 from other species, such as 22.7% similarity with that from

248

Branchiostoma belcheri (AAL02138.1), 98.6% similarity with that from C. hongkongensis

249

(AHF51977.1). Seventeen AP-1s from various species in vertebrates and invertebrates were

250

selected for phylogenetic analysis, which were separated clearly into vertebrate and

251

invertebrate branches. CgAP-1 was clustered with ChAP-1 from C. hongkongensis, and then

252

assigned into the invertebrate branch of the phylogenetic tree (Fig. 2).

253 254

3.2 Expression pattern of CgAP-1 mRNA

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The mRNA transcripts of CgAP-1 gene were detected in all the tested tissues, including

256

hemocytes, hepatopancreas, adductor muscle, gonad, gills and mantle with relatively higher

257

expression levels in hemocytes, mantle and gills (8.25-fold, 5.87 and 4.72-fold of that in

258

gonad, p < 0.05, respectively). The mRNA expressions of CgAP-1 gene in adductor muscle

259

and hepatopancreas were 3.80-fold (p < 0.05) and 3.10-fold (p < 0.05) of that in gonad,

260

respectively (Fig. 4A).

261

The mRNA transcripts of CgAP-1 gene in the hemocytes of oysters were further detected

262

after LPS stimulation. The expression levels of CgAP-1 mRNA increased significantly

263

(8.53-fold of that in PBS group, p < 0.01) at 6 h, returned to the ordinary level at 12, 24 and

264

48 h after LPS stimulation, which was 1.12-fold, 1.05-fold and 1.21-fold of that in PBS group

265

(p > 0.05), respectively (Fig. 4B).

266 267

3.3 Recombinant expression and purification of CgAP-1 protein

268

The recombinant CgAP-1 protein (rCgAP-1) was purified by using the Ni-NATA affinity

269

chromatography and examined by 15% SDS-PAGE. An evident band with a molecular weight

270

about 35 kDa was observed (Lane 3 in Fig. 5A). The specificity of polyclonal antibody

271

against CgAP-1 was examined with the hemocyte protein from oysters by Western blot. A

272

single band about 35 kDa with the high specificity was revealed (Fig. 5B), which was

273

identical to the prediction of molecular mass of CgAP-1.

274 275

3.4 The expression of CgAP-1 protein in hemocytes

276

By using the flow cytometry assay, CgAP-1 was mainly detected in granulocytes (G). The

277

fluorescence intensity for granulocytes, agranulocytes (A) and semi-granulocytes (SG) were

278

114053.90, 9576.51 and 46714.05, respectively (Fig. 5C-D). The immunocytochemical assay

279

was conducted by fluorescence microscope to detect the subcellular localization of CgAP-1

280

protein in hemocytes at 1 h after LPS stimulation with PBS as control. The positive green

281

fluorescence signals of CgAP-1 were mainly located in cytoplasm of hemocytes in the PBS

282

control group, while they were mainly distributed in the hemocyte nucleus at 2 h after LPS

283

stimulation (Fig. 6).

284 285

3.5 The mRNA transcripts of CgIL-17 in CgAP-1-RNAi oysters after LPS stimulation.

286

The mRNA transcripts of CgIL-17s in hemocytes were detected after CgAP-1 was knocked

287

down by RNAi. The expression level of CgAP-1 mRNA in the hemocytes decreased

288

significantly (0.41-fold of that in EGFP-RNAi oysters, p < 0.05) at 24 h after the injection of

289

CgAP-1 dsRNA (Fig. 7A). In CgAP-1-RNAi oysters, the transcript levels of CgIL17-4 and

290

CgIL17-5 in hemocytes decreased significantly at 24 h after LPS stimulation, which were

291

0.37-fold (p < 0.05) and 0.17-fold (p < 0.01), compared with that in EGFP-RNAi oysters,

292

respectively (Fig. 7B-C).

293 294

4. Discussion

295

AP-1 is a transcription factor usually consisted of the Jun and Fos subfamilies and plays

296

crucial roles in multiple immune responses in vertebrates and invertebrates [5]. AP-1 in

297

mammals could induce cytokine expression to participate in the immune response [41].

298

However, the involvement of invertebrate AP-1 in the regulation the of cytokine expressions

299

is still not well understood. Recently, several IL17s were identified from oyster [41], and

300

IL17-4 and IL17-5 of them were found to play crucial roles in the antibacterial immunity [32].

301

In the present study, CgAP-1 was identified from C. gigas with the objective to and its

302

activation and immune function in mediating the expression of CgIL17-4 and CgIL17-5.

303

AP-1 is a family of transcription factor proteins belonging to a class of basic leucine zipper

304

transcription factor. c-Jun and Fos are the major AP-1 family members in mammals. c-Jun

305

contains a Jun domain and a bZIP domain [7], and Fos contains a Fos basic domain and a

306

bZIP domain [42]. Jun and Fos proteins exist a random-coil structures in the absence of DNA

307

and form α-helical structures in the presence of DNA [43]. They exhibit different DNA

308

binding activities depending on the bZIP domain, and they could form homodimer or

309

heterodimer mediated by the leucine-zipper [44]. There are also some AP-1 homologs

310

identified in invertebrates. For example, AP-1 members (dFRA and dJRA) identified from

311

Drosophila were in common with mammalian Fos and Jun, respectively [11, 12]. ChFos from

312

oyster C. hongkongensis contained a leucine-zipper region and a Fos basic domain, which

313

displayed the typical structural characteristics of Fos family proteins [16]. Oyster ChAP-1

314

was composed of 290 amino acid residues with a Jun and bZIP domain, which was similar to

315

that of known AP-1 proteins [45]. In the present study, CgAP-1 contained a Jun domain and a

316

bZIP domain, indicating that it shared relatively conservative domain architecture with the

317

c-Jun proteins from other species. In the phylogenetic tree, all the selected AP-1s were

318

divided into vertebrate and invertebrate branches, and CgAP-1 was firstly clustered with

319

ChAP-1 and dropped into the invertebrate branch. The results suggested that CgAP-1

320

belonged to AP-1 family in molluscs and might share similar functions with c-Jun proteins

321

from other species.

322

AP-1 is a ubiquitous protein distributing in different tissues of vertebrates and invertebrates.

323

Mammal AP-1s were found to be mainly expressed in lymphoid tissues including thymus,

324

lymph nodes, and tonsils [46]. In invertebrates, most of AP-1s were relatively higher

325

expressed in immune tissues, including gill, intestine and hemocytes, and mantle. For

326

example, Lvc-Jun in shrimp L. vannamei was mainly expressed in gill and intestine [13]. In

327

the present study, CgAP-1 could be detected in all the tested tissues, including hemocytes,

328

hepatopancreas, adductor muscle, gonad, gill and mantle with relatively higher expression

329

levels in hemocytes, mantle, and gills. Similarly, the expression level of ChAP-1 in C.

330

hongkongensis was relatively higher in gill, hemocytes, and mantle [16]. The hemocytes in

331

aquatic invertebrates are considered as one of the main immune components and play crucial

332

roles in mediating host cellular and humoral immunity [41]. In oyster C. gigas, granulocytes,

333

semi-granulocytes and agranulocytes were characterized as three major types of hemocytes

334

[47], and granulocytes were found to be the main immunocompetent hemocytes of oysters

335

with relatively higher level of the phagocytic capacity and production of immune effectors. In

336

the present study, CgAP-1 protein was found to be higher expressed in the granulocytes,

337

which was 2.44-fold (p < 0.01) and 11.90-fold (p < 0.01) higher than that in

338

semi-granulocytes and agranulocytes, respectively. These results collectively suggested that

339

CgAP-1 might play vital roles in the innate immune response of oysters mediated by the

340

granulocytes.

341

AP-1 can be activated in response to cytokines, growth factors and stress factors during cell

342

differentiation, tumor formation, or mitogenic response in vertebrates [15]. The expressions

343

of c-Jun, c-Fos and JunB in human lung A549 cells could be induced by LPS stimulation [48].

344

In gastric epithelial cells, the mRNA transcripts of AP-1 gene increased after Helicobacter

345

pylori infection [49]. It has been reported that bacterial infection can also induce AP-1

346

production in invertebrates. In shrimp Penaeus monodon, both infections with Vibrio harveyi

347

and Streptococcus agalactiae can trigger an outstanding up-regulation of Pmc-Jun transcripts

348

[50]. Lvc-Jun in L. vannamei exhibits obvious up-regulation after white spot syndrome virus

349

and V. parahaemolyticus infection [13]. In the present study, the mRNA transcripts of CgAP-1

350

gene in hemocytes were significantly up-regulated after LPS stimulation. Similarly, VpAP-1

351

in clam Venerupis philippinarum could be activated significantly after V. anguillarum

352

stimulation [51]. These results suggested that CgAP-1 might be involved in the antibacterial

353

immune responses of oysters. In mammals, AP-1 could be translocated into the nucleus in

354

monocytes [47], platelets [52] and macrophages [53, 54] after immune stimulation, and even

355

co-translocated with NF-κB into the nucleus under neuromedin B stimulation [55]. In the

356

present study, CgAP-1 protein was found to be translocated into nucleus of oyster hemocyte

357

after LPS stimulation. MAPKs including ERK1/2, ERK5, JNK and p38 play an essential role

358

in transducing extracellular signals to cytoplasmic and nuclear effectors [10]. MAPK cascades

359

are responsible to regulate both the expression and post-translational modifications of AP-1

360

proteins in mammals [56]. It was demonstrated that c-Fos could be activated via the ERK

361

pathway in NP cells [57, 58]. Jun could be translocated into the nucleus when it was activated

362

by JNK [59] and the receptor activator of nuclear factor kappa-B ligand (RANKL) [60].

363

Mammalian AP-1 can be phosphorylated by MAPK pathway, and the activated AP-1 forms

364

homodimer or heterodimer to be transferred into nucleus [2]. Although the components of

365

MAPK pathway have been reported in oysters [61], the activation mechanism of AP-1 in

366

molluscs is still not well understood. The results indicated that the activated CgAP-1 could be

367

transferred into nucleus of oyster hemocytes to be involved in the immune responses against

368

pathogen infection.

369

It has been demonstrated that AP-1 can regulate the production of cytokines in vertebrates. In

370

LPS-induced mice THP-1 cells, AP-1 could translocate into nucleus to regulate the mRNA

371

and protein expressions of IL-1β and IL-6 [2, 31]. The activated AP-1 promoted the

372

expression of IL-8 in human head and neck squamous cell carcinomas (HNSCC) [62]. IL-17

373

mediated inflammatory reactions via p38/c-Fos and JNK/c-Jun activation in human nucleus

374

pulposus cells [63]. Recently, six IL17s, including CgIL17-1, CgIL17-2, CgIL17-3, CgIL17-4,

375

CgIL17-5 and CgIL17-6, have been identified from C. gigas. Among them, the mRNA

376

transcripts of CgIL17-4 and CgIL17-5 were found to be increased significantly after LPS

377

stimulation [30, 32]. In the present study, the mRNA transcripts of CgIL17-4 and CgIL17-5 in

378

EGFP-RNAi oysters were up-regulated significantly in hemocytes of oysters at 24 h after

379

LPS stimulation. Comparatively, the mRNA transcript levels of CgIL17-4 and CgIL17-5 in

380

CgAP-1-RNAi oysters decreased significantly after LPS stimulation, indicating the crucial

381

function of CgAP-1 in regulating the expression of CgIL17-4 and CgIL17-5. Several binding

382

sites for transcription factors, such as the AP-1, NF-kB and Oct-1, were found in the promoter

383

regions of CgIL17-5 [30], indicating that AP-1 could bind to the promoter of CgIL17-5 to

384

regulate its expression. Although no AP-1 transcription factor binding sites were identified in

385

the promoter regions of CgIL17-4, there were the binding sites for signal transducer and

386

transcription activator, such as NF-kB, GATA, and Oct-1. In mammals, the expression of

387

cytokines is regulated by the cooperation of various transcription factors. It was found that

388

AP-1 could interact with NF-kB transcription factor [55], and the cooperative and coordinate

389

involvements of NF-kB and AP-1 regulate the expression of IL-4 during T cell activation [64].

390

In the present study, CgIL17-4 and CgIL17-5 were found to be higher expressed in

391

granulocytes (unpublished data), which were considered as the main immunocompetent

392

hemocytes in C. gigas [40]. As an proinflammatory cytokine, CgIL17-5 was demonstrated to

393

mediate the clearance of extracellular bacteria in oysters [32]. All these results suggested that

394

CgAP-1 might be involved in regulating the expression of CgIL17-4 and CgIL17-5 in the

395

immune response of oysters.

396

In conclusion, CgAP-1 with a Jun domain and a bZIP domain was identified from oyster. Its

397

mRNA expression in hemocytes increased significantly after LPS stimulation. CgAP-1

398

protein was mainly located in the cytoplasm of hemocytes with highest level in granulocytes,

399

and it was translocated into nucleus of hemocytes after LPS stimulation. The mRNA

400

transcripts of CgIL17-4 and CgIL17-5 in CgAP-1-RNAi oysters decreased significantly after

401

LPS stimulation. All the results indicated that AP-1 plays critical role in inducing the

402

production of cytokines, which would provide insights for the further exploration about the

403

AP-1 activation and regulation mechanisms in invertebrates.

404 405

Acknowledgements

406

We are grateful to all the laboratory members for their technical advice and helpful

407

discussions. This research was supported by grants (No. U1706204, 31802336) from National

408

Science Foundation of China, National Key R&D Program (2018YFD0900504), Key R&D

409

Program of Liaoning Province (2017203004 to L.S.), earmarked fund (CARS-49) from

410

Modern Agro-industry Technology Research System, the Fund for Outstanding Talents and

411

Innovative Team of Agricultural Scientific Research, the Distinguished Professor of Liaoning

412

(to L. S.), AoShan Talents Cultivation Program Supported by Qingdao National Laboratory

413

for Marine Science and Technology (No. 2017ASTCP-OS13), Dalian High Level Talent

414

Innovation Support Program (2015R020), and the Research Foundation for Talented Scholars

415

in Dalian Ocean University (to L. W.).

416 417

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580 581 582

Figure legends

583

Fig. 1 Complete nucleotide and deduced structural domains of CgAP-1 protein. A. The Jun

584

domain and bZIP domain are in the frame. B. The predicted structural domains of Crassostrea

585

gigas are predicted by SMART (http://www.smart.embl-heidelberg.de/).

586 587

Fig. 2 Phylogenetic analysis of CgAP-1 with other AP-1 family members from different

588

animals using MEGA 7.0 software. CgAP-1 is marked with a circle.

589 590

Fig. 3 Multiple sequence alignment of CgAP-1 with AP-1s from other species by using

591

Clustal X and GeneDoc. Conserved amino acid residues in these sequences are shown in

592

black and relatively lower conservative amino acid residues are shown in gray. CgAP-1 is

593

marked with a circle. Proteins analyzed are listed below: Cg, Crassostrea gigas, Ch,

594

Crassostrea hongkongensis, Mu, Mus musculus, Bt, Bos taurus, Hs, Homo sapiens, Dr, Danio

595

rerio, Hdd, Haliotis discus discus, Rp, Ruditapes philippinarum, Pf, Pinctada fucata, Bg,

596

Biomphalaria glabrata, Ac, Aplysia californica, My, Mizuhopecten yessoensis, Mg, Mytilus

597

galloprovincialis, Bb, Branchiostoma belcheri, Lv, Litopenaeus vannamei, Ss, Salmo salar.

598

The Jun domain is labeled with red frame, the bZIP domain is labeled with blue frame.

599 600

Fig. 4 The tissues distribution and temporal expression of the CgAP-1 detected by qRT-PCR.

601

A. The tissues distribution of CgAP-1 in the untreated oysters. EF was used as the internal

602

control. The transcript levels of CgAP-1 mRNA in mantle, hepatopancreas, gills, adductor

603

muscle and hemocytes were normalized to that of gonad. B. The temporal expression of the

604

CgAP-1. PBS group was used as control. The different letters showed that there existed

605

significant differences comparing with other groups (p < 0.05, Duncan). Asterisks indicated

606

significant differences (**: p < 0.01).

607 608

Fig. 5 The recombination protein of CgAP-1 and specificity detection for its polyclonal

609

antibodies. A. Lane M, standard protein molecular weight marker; Lane 1, negative control

610

(without induction); Lane 2, induced recombinant protein CgAP-1; Lane 3, purified

611

recombinant protein CgAP-1. B. Western blot with anti-CgAP-1-antibody in the hemocytes of

612

C. gigas. C-D. Three types of hemocytes separated by flow cytometry: agranulocytes (A),

613

semi-granulocytes (SG) and granulocytes (G). Anti-CgAP-1 conjugated to Alexa-fluor 488

614

was shown in green fluorescence signal. D. CgAP-1 protein in different type of hemocytes.

615

The different letters showed the significant differences comparing with other groups (p < 0.05,

616

Duncan).

617 618

Fig. 6 CgAP-1 protein translocated into hemocyte nucleus after LPS stimulation. The

619

subcellular localization of CgAP-1 protein in hemocytes was detected with CgAP-1 antibody

620

at 2 h after LPS stimulation and PBS was used as control. Green fluorescence signal was

621

corresponded to CgAP-1 and blue showed the nuclei of hemocytes stained with DAPI.

622 623

Fig. 7 The mRNA expressions of CgIL17-4 and CgIL17-5 in the hemocytes of CgAP-1-RNAi

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oysters after LPS stimulation. A. The efficiency of CgAP-1-RNAi in hemocytes was analyzed

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by qRT-PCR. EGFP-RNAi was used as control. B-C. The mRNA expression of CgIL17-4 and

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CgIL17-5 in CgAP-1-RNAi oysters after LPS stimulation by using qRT-PCR. EGFP-RNAi

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was used as the control. Asterisks indicated significant differences (*: p < 0.05, **: p < 0.01).

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Table 1. Sequences of the primers used in this study Primer

Sequence (5'-3')

RT-PCR primers CgAP-1-RT-F1

CTTCAGGTCCCCAGTCATTA

CgAP-1-RT-R1

GGGTAGGATTCCGTCAGTG

CgAP-1-RT-F2

TCACCACTACCCCGACACCAA

CgAP-1-RT-R2

GCCAATGCCTCCACGAACCC

CgEF-RT-F

AGTCACCAAGGCTGCACAGAAAG

CgEF-RT-R

TCCGACGTATTTCTTTGCGATGT

CgIL17-4-RT-F

ACTTGTCCCTGGGTTATGTGTAG

CgIL17-4-RT-R

TCCAAGAGGAACACGGAGAC

CgIL17-5-RT-F

TCTGGCTGACTCTCGTCCTTG

CgIL17-5-RT-R

GACCCTGTCGTTGTCCTCTACC

Clone primers CgAP-1-F

ATGGAAGCGATTGACCGCACGT

CgAP-1-R

TCATAGCTGCAAAGATGACGAAAT

Recombinant expression CgAP-1-ExF

CGCGGATCCGAAGCGATTGACCGCACGTT

CgAP-1-ExR

CCCAAGCTTTCATAGCTGCAAAGATGACGAAATC

M13-47

CGCCAGGGTTTTCCCAGTCACGAC

M13-RV

AGCGGATAACAATTTCACACAGGA

RNA interference CgAP-1-Fi CgAP-1-Ri

GATCACTAATACGACTCACTATAGGGGGATTTACTTGCTTCGCCCG GATCACTAATACGACTCACTATAGGGGGGACACTTTCTGGCAGCA AT

EGFP-Fi

GCGTAATACGACTCACTATAGGAGCACCCAGTCCGCCCTGAGC

EGFP-Ri

GCGTAATACGACTCACTATAGGCGTCGCCGTCCAGCTC

Highlights: 1. CgAP-1 identified in oyster was relatively higher expressed in hemocytes. 2. The mRNA expression of CgAP-1 in hemocytes was up-regulated after LPS stimulation. 3. CgAP-1 could translocate into hemocyte nucleus post LPS stimulation. 4. CgAP-1 could induce the expression of CgIL17-4 and CgIL17-5 after LPS stimulation.