An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity

Accepted Manuscript An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity Zhihao Jia, Tao Zhang, Shuai Jiang, Mengqiang Wang, Qi Cheng, Mingzhe Sun, Lingling Wang, Linsheng Song PII:

S0145-305X(15)30019-7

DOI:

10.1016/j.dci.2015.07.014

Reference:

DCI 2436

To appear in:

Developmental and Comparative Immunology

Received Date: 15 June 2015 Revised Date:

16 July 2015

Accepted Date: 16 July 2015

Please cite this article as: Jia, Z., Zhang, T., Jiang, S., Wang, M., Cheng, Q., Sun, M., Wang, L., Song, L., An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity, Developmental and Comparative Immunology (2015), doi: 10.1016/ j.dci.2015.07.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity

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Qi Chengc,

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Tao Zhanga,d,

Mingzhe Suna,d,

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Lingling Wanga,

Linsheng Songb,*

Key laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese

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Academy of Sciences, Qingdao 266071, China

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Key Laboratory of Mariculture & Stock enhancement in North China’s Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, China

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Abstract

Dalian Polytechnic University, Dalian, 116034, China

University of Chinese Academy of Sciences, Beijing 100049, China

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Mengqiang Wanga,

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Shuai Jianga,

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Zhihao Jiaa,d,

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Integrins are a family of cell adhesion molecules which play important roles in the

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regulation of cell adhesion, migration, proliferation, apoptosis and phagocytosis. In

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the present study, the immune function of an integrin from the oyster Crassostrea

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gigas (designated CgIntegrin) was characterized to understand the regulatory

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mechanism of hemocyte phagocytosis toward different microbes. The full-length

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cDNA of CgIntegrin was 2571 bp with an open reading frame (ORF) of 2397 bp,

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encoding a polypeptide of 799 amino acids. The mRNA transcripts of CgIntegrin

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were predominantly detected in hemocytes, gonad and adductor muscle, while lowly in

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hepatopancreas, mantle and gill. The mRNA expression level was up-regulated at 6 h

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ACCEPTED MANUSCRIPT post lipopolysaccharide (LPS) stimulation (p<0.01), while no significant change was

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observed after peptidoglycan (PGN) stimulation. The oyster hemocytes with relative

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high CgIntegrin expression level exhibited different phagocytic abilities towards

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different microorganism and particles, such as Gram-positive bacteria Vibrio

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splendidus, Gram-negative bacteria Staphylococcus aureus and latex beads.

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Moreover, the phagocytic rate towards V. splendidus was significantly decreased

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after the blockade of CgIntegrin using the polyclonal antibody. The recombinant

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CgIntegrin (rCgIntegrin) displayed agglutinating activity towards V. splendidus but

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not S. aureus and Y. lipolytica. It also exhibited a higher binding affinity towards

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LPS (compared to rTrx group) in a dose-dependent manner with the apparent

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dissociation constant (Kd) of 5.53 × 10-6 M. The results indicated that CgIntegrin

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served as a pattern recognition receptor with LPS binding activity, which could

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directly bind to V. splendidus and enhance the phagocytosis of oyster hemocytes.

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Keywords:

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Crassostrea gigas; Innate immunity; Integrin; Phagocytosis; Liposaccharides; Vibrio splendidus

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1. Introduction

Multiple adhesion molecules on cell membrane participate in the cell-to-cell

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communication, and they are also involved in innate cellular immune responses such

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as phagocytosis, nodulation and encapsulation (Kerr, 1999; Zhuang, 2008). There are

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several gene families encoding cell adhesion molecules, such as integrins,

ACCEPTED MANUSCRIPT immunoglobulin, cadherins and selectins (Deborah Cooper, 1994). Among these

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molecules, integrins are a kind of pluripotent cell adhesion molecules which are

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heterodimeric transmembrane while restricted to metazoan especially in vertebrates

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and invertebrates.

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The structures and biological functions of integrins have been well-studied in

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vertebrates (Hynes, 2002). Based on extensive searches of the human and mouse

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genomic sequences, 18 non-covalently attached α and 8 β subunits were found to

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assemble into at least 24 distinct types of integrins (Hynes, 1992). In invertebrates,

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different types of integrin homologues have been found ranging from Caenorhabditis

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elegans to Drosophila melanogaster, and most of them were reported to be involved

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in early embryonic development and cell-mediated immune responses (Gettner, 1995;

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Hu et al., 2010; Levin et al., 2005; Marsden, 1997; Zhuang et al., 2008). For instance,

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β integrin domains from tobacco hornworm moth Manduca sexta and soybean looper

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moth Pseudoplusia includens could participate in pathogen adhesion, encapsulation,

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and phagocytosis (Hynes, 1992; Levin et al., 2005). In crustaceans, an integrin β

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subunit from freshwater crayfish Pacifastacus leniusculus was demonstrated to be

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involved in immune response, and an integrin from Chinese mitten crab Eriocheir

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sinensis was investigated to function as an cellular receptor mediator in the response

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against bacterial infection (Holmblad et al., 1997; Huang et al., 2015). So far, at least

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26 α subunits and 17 β subunits of integrin have been identified from invertebrate,

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which played important roles in regulating cell adhesion, migration, proliferation and

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apoptosis (Marsden and Burke, 1997; O'Reilly et al., 2008; Yee, 1993).

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ACCEPTED MANUSCRIPT Phagocytosis is a fundamental process of innate immune response, playing key

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roles in recognition, ingestion and elimination of invaders. The occurrence of

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phagocytosis needs the presence of multiple cell adhesion molecules and some other

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receptors such as integrins (Canesi et al., 2002; Song et al., 2010). Recently, some

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integrins have been proved to be important participators of phagocytosis as well as the

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innate immune responses in invertebrates. For instance, the β integrin domains from

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Ostrinia furnacalis, Pseudoplusia includens and Manduca sexta participated in cell

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adhesion, encapsulation and phagocytosis (Lavine and Strand, 2003; Moita et al.,

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2006; Soderhall, 1998). An integrin from shrimp Litopenaeus vannamei was found to

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mediate microbial agglutination (Zhang et al., 2012). In addition, an integrin from

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Chinese mitten crab Eriocheir sinensis was detected to have LPS binding activity

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(Huang et al., 2015). But the roles of integrins in phagocytosis of invertebrates are

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still far from well understood .

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The Pacific oyster Crassostrea gigas is one of dominant aquaculture bivalves

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worldwide. The recently released whole genome sequence of oyster provided

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important information for better understanding of its immune system, and useful clues

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to mine the multiple cell adhesion molecules involved in cell-mediated innate immune

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response. An integrin homologue (designated CgIntegrin) was previously identified

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from C. gigas (Terahara et al., 2005), and it was observed to be up-regulated upon

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secondary challenge with Vibrio splendidus (Zhang et al., 2014). The aim of our pre

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sent study was to (1) investigate the expression level of CgIntegrin mRNA in

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hemocytes post different PAMPs stimulation, (2) clone the coding sequence of

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ACCEPTED MANUSCRIPT extracellular domain of CgIntegrin and obtain the recombinant proteins, (3)

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characterize its immunological function in innate immunity, hopefully make

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contributions to the further understanding of the innate immune system of C. gigas.

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2. Methods and Materials

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2.1 Ethics statement

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The oysters used in the present study were marine cultured animals with average

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length of 10-15 cm and weight of 150-200 g, they were acclimated in aerated

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seawater at 18 °C for two weeks prior to use. All the experiments were conducted

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according to the regulations of local and central government. The animal experiments

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were approved by the local animal care and use committee.

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2.2 The stimulations with PGN and LPS

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After cultured in filtered aerated seawater at 18 °C for a week, three hundred and

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sixty oysters were randomly divided into three stimulation groups as well as one

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untreated group. Oysters received an injection of 100 µL of lipopolysaccharide (LPS

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in sterile sea water, 100 mg mL-1) from Escherichia coli 0111:B4 (Sigma-Aldrich),

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100 µL of peptidoglycan (PGN in sterile sea water, 100 mg mL-1) from

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Staphylococcus aureus (Sigma-Aldrich) were employed as treatment groups,

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respectively. Oysters received the injection of same volume of sterile sea water were

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employed as control group. The oysters were continued to be cultured in water tanks

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after treatment. Eight individuals from each group were randomly collected at 3, 6, 9,

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12, 24 and 48 h after injection, respectively. The hemolymph from these oysters were

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collected and centrifuged at 800 g, 4 °C for 10 min to harvest the hemocytes. The

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hemocytes were stored in Trizol reagent (Invitrogen) at -80 °C for RNA extraction.

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2.3 RNA isolation and cDNA synthesis Gonad, adductor muscle, mantle, gill, hemocytes and hepatopancreas were obtained

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from six healthy adult oysters. Trizol reagent was used to extract the total RNA from

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tissue samples according to the manufacturer's protocol. The RNase-free DNase I

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(Promega) was used to digest the genomic DNA from the total RNA, and first-strand

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cDNA synthesis was carried out based on Promega M-MLV reverse transcriptase using

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oligo (dT)-adaptor as primer (Table 1). The reverse transcription reaction was

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performed at 42 °C for 1 h, and terminated by heating at 95 °C for 5 min. The cDNA

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was diluted to 1:40 and stored at -80 °C for the following gene cloning and SYBR

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Green fluorescent quantitative real-time RT-PCR.

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2.4 Cloning and sequence analysis of cDNA

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Sequence information of CgIntegrin (AB066348.1) was retrieved from the National

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Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). A pair of

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sequence specific primers P1 and P2 (Table 1) were designed to clone part of the

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extracellular domain of CgIntegrin. After gel-purification, the PCR products were

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cloned into pMD 18-T vector (TaKaRa) and sequenced by sequencing primers (Table

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1).

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2.5 Real-time PCR analysis of CgIntegrin expression

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The mRNA expression of CgIntegrin was detected by SYBR Green fluorescent

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quantitative real-time PCR (qRT-PCR). A pair of specific primers P3 and P4 (Table 1)

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for CgIntegrin were used to amplify a fragment of 188 bp. The oyster Elongation Factor

ACCEPTED MANUSCRIPT (EF) fragment, amplified by primers P5 and P6 (Table 1), was set as the internal

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reference. The SYBR Green real-time PCR assay was carried out on an ABI PRISM

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7500 Sequence Detection System (Applied Biosystems). Dissociation curve analysis of

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amplification products was performed at the end of each PCR to confirm that only one

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PCR product was amplified and detected. After the PCR program, data were analyzed

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by the SDS 2.0 software (Applied Biosystems). The relative expression of CgIntegrin

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was analyzed by the 2-∆∆CT method (Schmittgen and Livak, 2008). All the data were

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given in terms of relative mRNA expression as mean ± SD (N = 4).

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2.6 Prokaryotic expression and purification of recombinant CgIntegrin

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The cDNA fragment encoding extracellular fragment of integrin β domain was

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amplified with the primers P7 and P8 (Table 1). Nco I and Xhol I site sequences were

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added to the 5＇end of primer P7 and P8, respectively. The PCR fragment was digested

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by restriction endonucleases Nco I and Xhol I (NEB), and then ligated into expression

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vector pET-32a+ (Novagen) which was digested by the same restriction endonucleases.

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The recombinant plasmid (pET-32a-CgIntegrin) was transformed into E. coli BL21

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(DE3) pLysS (Novagen). Positive transformants were incubated in LB medium

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containing 50 µg mL-1 ampicillin at 37 °C with shaking at 220 rpm for 4 h. When the

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culture mediums reached OD600 of 0.3-0.6, IPTG was added to the LB medium at a final

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concentration of 1 mM and incubated at 16 °C with shaking at 180 rpm for 20 h. After

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induction, the bacteria was harvested by centrifugation at 6000 g for 15 min, and

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resuspended in PBS, sonicated to lyse the bacteria, and centrifuged to get the

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supernatant.

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ACCEPTED MANUSCRIPT The recombinant protein of CgIntegrin (rCgIntegrin) was purified by Ni-NTA

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Sepharose column (Roche), and the purified protein was extensively dialyzed to

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eliminate imidazole at 4°C for three times. The protein was separated by reducing 12%

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SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized with Coomassie

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Bright Blue R250. The concentration of purified soluble protein was quantified by

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bicinchoninic acid (BCA) method.

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2.7 Preparation of polyclonal antibody, Elisa and Western blotting analysis

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For the antibody generation assay, six weeks old rats were immunized with

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recombinant CgIntegrin to acquire polyclonal antibody as previously description

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(Cheng et al., 2006). Briefly, 100 µL rCgIntegrin (1 mg mL-1) was mixed with freund’s

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adjuvant to immunize each female rat with a weight of approximate 120 g for four

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times. The blood was taken out from the heart of the rats. And after tipped at 4 °C for

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4 h, the blood was centrifuged at 5000 rpm/min for 30 min to harvest the serum.

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For ELISA, 100 µL of rCgIntegrin (0.2 µg µL-1, diluted in 50 mmol

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L-1carbonate-bicarbonate buffer, pH 9.6) was used to coat the 96-well microtiter plate

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(Costar) and incubated at 4 °C overnight. The plate was washed three times with

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PBST (135 mmol L-1 NaCl, 4.7 mmol L-1 KCl, 10 mmol L-1 Na2HPO4, 2 mmol L-1

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NaH2PO4, 0.1% Tween-20, pH=7.4) and followed by blocking with 200 µL of 5%

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BSA in PBST at 37°C for 1 h. Immunized rat serum was then added into each well with

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the presence of 3% BSA. After 1 h of incubation at 37°C, the plate was washed by 200

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µL of PBST for three times, and 100 µL of Hrp-labeled goat-anti-rat antibody (Abcam,

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diluted 1:3000) was added for incubating at 37°C for 1 h. The plate was then washed

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ACCEPTED MANUSCRIPT for four times with 200 µL of PBST, and 100 µL of 0.1% (w/v) p-nitrophenyl

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phosphate (pNNP, Sigma) in 50 mmol-1 carbonate buffer (PH=9.8) containing 0.5 mmol

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L-1 MgCl2 was added to each well and incubated at room temperature in dark for 15 min.

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The reaction was stopped by adding 50 µL H2SO4 (2 mol L-1) per well and the

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absorbance was measured with Microplate spectrometer at 450 nm (Molecular

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Devices).

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For the western blotting assay, rCgIntegrin was separated by SDS-PAGE, and

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electrophoretically transferred onto nitrocellulose membrane. The membrane was then

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blocked with 5% milk (dissolved in PBST) at room temperature for 1 h. After washed

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with PBST for three times, the membrane was incubated with polyclonal antibody

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(diluted at 1:1,000) at 4 °C overnight. The membrane was then washed three times with

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PBST and incubated with goat-anti-rat IgG (Beyotime; 1:10,000) for 1 h at room

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temperature. After final three times of washing in PBST, the membrane was incubated

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in Western lighting ECL substrate system (Thermo Scientific, USA) before exposure

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to KODAK X-OMAT AR X-ray film (Eastman Kodak, USA).

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2.8 Immunohistochemistry analysis of CgIntegrin

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Immunohistochemistry of tissues and hemocytes was performed according to the

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previous description (Jemaa et al., 2014; Jiang et al., 2013) with some modification.

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Tissues, including hepatopancreas, gill, heart, mantle and gonad, were fixed using

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Bouin’s fixative (Saturated picric acid solution: formaldehyde: glacial acetic

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acid=15:5:1) at room temperature overnight before faded in 70% ethanol for 2 h for

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2-4 times, and then dehydrated in 80%, 95% and 100% successive ethanol baths.

ACCEPTED MANUSCRIPT After dehydrated with Xylene/ethanol (1:1), Xylene, respectively, all the tissue

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samples were embedded in paraffin, and the cross-sections were prepared by

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RM-2016 microtome (LEIKA, Germany). Paraffin was eliminated in Xylene bath and

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the sections were rehydrated in successive 95 to 30% ethanol baths and finally in

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distilled water. Antigens were refolded in sodium citrate-hydrochloric acid buffer

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before the sections were incubated with primary antibody diluted with 3% BSA in

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PBS at 37°C for 1 h, and then incubated with Alexa Fluor 488-labeled goat-anti-rat

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antibody (diluted 1:500 in 3% BSA in PBS with Evans blue dye) as the second antibody

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at 37°C for 50 min. After three times of washing in PBST, sections were covered by

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cover slide and observed under fluorescence microscopy (Olympus).

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For Immunohistochemistry in hemocytes, hemolymph were collected from six

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healthy oysters and immediately centrifuged at 800 × g, 4 °C for 10 min to harvest the

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hemocytes. Modified Leibovitz’s L-15 mediums (Gibco, M-L15 for short) (Cao et al.,

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2003) were used to suspend the hemocytes, and the suspension was added into cell

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culture dishes. After incubated at room temperature for 3 h, the supernatant was

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discarded and 4% PFA (Paraformaldehyde diluted in TBS) was added to fix the

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hemocytes for 15 min. After three times of washing in TBST (20 mmol L-1 Tris-HCl,

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150 mmol L-1 NaCl, 0.1% Tween 20, pH 7.5), the dishes were blocked with 3% BSA in

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PBS at room temperature for 30 min. Then the supernatant was removed and the dishes

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were incubated with 500 µL antibody of rCgIntegrin (diluted 1:500 in blocking buffer)

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as the primary antibody at room temperature for 1 h. After that, the dishes were then

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incubated with Alexa Fluor 488-labeled goat-anti-rat antibody (diluted 1:1000 in

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ACCEPTED MANUSCRIPT locking buffer) as the second antibody at 37 °C for 1 h. DAPI (diluted 1:10,000 in PBS)

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was added into the dishes to stain the nucleus while DIL (diluted 1:10,000 in PBS) was

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used to stain the membrane. After the last three times of wash, 1 mL of PBS was added

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into each dish before observation under Laser Scan Confocal Microscope (ZEISS).

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2.9 Phagocytosis assay

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Phagocytosis assay was performed according to previous description with some

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modification. Briefly, hemolymph was collected from oysters and mixed

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immediately with equal volume of pre-chilled sodium citrate anticoagulant buffer,

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then the mixture was centrifuged at 800 g for 10 min to harvest hemocytes.

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Hemocytes were resuspended with M-L15 medium and incubated with 20 µL of

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FITC-labeled V. splendidus, S. aureus and Y. lipolytica or Latex beads (2 µm,

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Sigma) at room temperature for 1 h with rotation, respectively. To detect the

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distribution of CgIntegrin, antibody against rCgIntegrin was added into the

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suspension as the primary antibody and Alexa Fluor 594-labeled goat-anti-rat IgG

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(Life Technologies) (diluted 1:1000 in M-L15 medium) was employed as the

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secondary antibody. The phagocytic rates of hemocytes and expression level of

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CgIntegrin were analyzed on an FACS Arial II flow cytometer (Becton, Dickinson

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and Company).

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2.10 Antibody blocking assay of CgIntegrin

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Hemocytes were obtained as described above and incubated with polyclonal

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antibody of rCgIntegrin (diluted 1:500) for an hour at room temperature with rotation.

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After three times of washing with M-L15 medium, 20 µL FITC-labeled V.

ACCEPTED MANUSCRIPT splendidus, S. aureus, Y. lipolytica and Latex beads (2 µm, Sigma) were added

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into the suspension. The phagocytosis rate was evaluated by FACS Arial II flow

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cytometer (Becton, Dickinson and Company). Rat IgG was used as negative

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control and hemocytes without antibody were set as blank control. There were 3

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repeats in every group.

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2.11 Bacterial agglutinating assay

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Twenty five microliters of rCgIntegrin (1 mg mL-1) was incubated with 20 µL

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FITC-labeled V. splendidus, S. aureus and Y. lipolytica (1× 106 CFU ) at room

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temperature in dark with rotation for 1 h, and the samples were observed under

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fluorescence microscopy (Olympus). rTrx was used as negative control while TBS was

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set as blank control.

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2.12 PAMPs binding assay

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For the PAMPs binding assay, the 96-well microliter plates (Costar) were coated

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with 20 µg of LPS, PGN, Mannan (Sigma-Aldrich) in 100 µL of carbonate-bicarbonate

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buffer (50 mmol L-1, pH 9.6), respectively, and incubated at 4°C overnight. After three

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times of washing with PBST, the wells were blocked with 200 µL of 5% BSA in PBS at

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37°C for 1 h, and then incubated with 100 µL of rCgIntegrin at different concentration

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with the presence of 0.1 mg mL-1 BSA. After incubated at 37°C for 1 h, the plate was

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washed three times with PBST, and 100 µL of mouse anti-his tag monoclonal antibody

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(1:1000 dilution) was added. After another incubation at 37°C for 1 h, the wells were

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incubated with 100 µL of Hrp-labeled goat-anti-rat IgG (Abcam, 1:3000 dilution) as

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the second antibody for 1 h. The wells were washed four times with TBST for 5 min,

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ACCEPTED MANUSCRIPT and 100 µL of 0.1% (w/v) pNNP (Sigma) in 50 mmoL-1 carbonate buffer (PH=9.8)

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containing 0.5 mmol L-1 MgCl2 was added to each well, and then incubated at room

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temperature in dark for 15 min. The reaction was stopped by adding 50 µL of 2 mol L-1

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H2SO4 per well and the absorbance was measured with an ELISA reader at 450 nm

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(Molecular Devices). The wells filled with 100 µL of TBS were used as negative

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control (blank). The apparent dissociation constant (KD) values were calculated using

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Prism 5.00 software (GraphPad software) with a one-site binding model and nonlinear

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regression analysis where ∆A450 is the absorbance at 450 nm.

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2.13 Statistical analysis

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All the data were expressed as mean ± standard deviation (N = 3 or 4), and analyzed

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by Statistical Package for Social Sciences (SPSS) 16.0. The significant differences

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among groups were tested by one-way analysis of variance (ANOVA) and multiple

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comparisons. Differences were considered significant at p<0.05 and extremely

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significant at p<0.01.

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3. Results

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3.1 The sequence characteristics and phylogeny of CgIntegrin

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The coding sequence of CgIntegrin was retrieved from NCBI (GenBank accession

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NO. AB066348). There is an open reading frame (ORF) encoding a polypeptide of 799

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amino acids with a predicted weight of 88.84 kDa which contains an Integrin β domain

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(amino acids 48 to 468), an EGF domain (amino acids 610 to 640), a transmembrane

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domain (amino acids 731 to 753) and Integrin β cytoplasm domain (amino acids 754 to

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799) in CgIntegrin. (Fig. 1). BLAST analysis revealed significant sequence similarity between CgIntegirn and

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β-Integrins from other species. High conserved features of D165D167K168 and special

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determining loop (SDL (K198-G223)) (Arnaout et al., 2005) were identified in

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CgIntegrin by Multiple alignment (Fig. 2). A phylogenetic tree of 32 β-Integrins was

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constructed by the neighbor-joining method while all the members were distinctly

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separated into two distinct groups of vertebrate and invertebrate (Fig. 3). CgIntegrin

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was clustered into the invertebrates group and formed an independent branch with

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β-Integrin from Biomphalaria glabrata.

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3.2 Predicted tertiary structure of CgIntegrin

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The tertiary structure of CgIntegrin (N-terminal portion of extracellular region)

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was predicted using I-TASSER methods (Roy et al., 2010) based on the template of

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β-Integrin from Homo sapiens (PDB: 3fcsb) (Xiong et al., 2009). The predicted

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spatial structure of CgIntegrin contained four main parts, a βA domain, a Hybrid

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domain, a plexin-semaphorin-integrin (PSI) domain, and an integrin IE (EGF-like)

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domain (Fig .4). The βA domain was consisted of six β sheets which were surrounded

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by eight α helices while the Hybrid domain was similar to the I-set of Ig domain.

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3.3 Tissue distribution of CgIntegrin mRNA

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The expression level of CgIntegrin mRNA in the different tissues were examined

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by SYBR Green Real-time PCR analysis (Fig. 5). CgIntegrin transcripts were

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detected in all the tested tissues including hemocytes, gonad, adductor muscle, mantle,

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gill, and hepatopancreas The highest expression level was detected in gonad which

ACCEPTED MANUSCRIPT was 5.22-fold of that in hepatopancreas. The expression level of CgIntegrin mRNA

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was relatively higher in adductor muscle and hemocytes, while lower in mantle and

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gill.

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3.4 The mRNA expression pattern of CgIntegrin in hemocytes after LPS and PGN

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stimulation

The expression level of CgIntegrin mRNA in hemocytes from oysters was detected

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at 0, 3, 6, 9, 12, 24 and 48 h post LPS or PGN stimulation. The mRNA expression

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level of CgIntegrin was significantly up-regulated (p<0.01) at 6 h after LPS

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stimulation (Fig. 6A) compared with that in control group, while no significant

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change was detected in PGN group (Fig. 6B), indicating that CgIntegrin was probably

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involved in the immune response against Gram-negative bacteria but not

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Gram-positive bacteria.

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3.5 The recombinant protein of CgIntegrin and polyclonal antibodies

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The 639 bp cDNA fragment encoding the amino acids sequences from A55 to E266

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from the extracellular part of CgIntegrin was cloned using primer P7 and P8 (Table 1).

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The recombinant plasmid (pET-32a-CgIntegrin) was transformed into E. coli BL21

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(DE3) pLysS (Novagen). The supernatant of the whole cell lysate was collected after

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the positive transformants were cultured and inducted with IPTG (final concentration

327

of 1 mmol L-1). rCgIntegrin was purified by Ni-NTA affinity chromatography, and

328

analyzed by SDS-PAGE. A distinct band was observed with a molecular weight of 43.8

329

kDa (Fig. 7).

330

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The purified rCgIntegrin was employed to prepare polyclonal antibody and the titer

ACCEPTED MANUSCRIPT of the polyclonal antibody was determined by ELISA (1:1,000). A clear band was

332

revealed by western blotting assay, indicating the high recognition specificity of the

333

polyclonal antibody against CgIntegrin (Fig 7). Pre-immune serum was used as

334

negative control and no bands were detected. The specificity of the polyclonal antibody

335

was detected and there was only one band in hemocytes (data not shown).

336

3.6 Immunohistochemistry localization of CgIntegrin

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Immunohistochemistry assay was performed to detect the localization of

338

CgIntegrin in hemocytes and tissues. The positive signal was green, which was

339

dominantly located at the edge of heart and mantle, while relative low in gonad. No

340

positive signal was detected in hepatopancreas and gill (Fig. 8A). In hemocytes,

341

CgIntegrin was located on the cell membrane as well as cytoplasma and represented

342

an aggregate distribution (Fig. 8B).

343

3.7 Effect of CgIntegrin on phagocytic activity of oyster hemocytes

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Flow cytometry analysis revealed that CgIntegrin was expressed on the

345

membrane of different hemocytes. The hemocytes with a relative high CgIntegrin

346

expression level were designated as Integrinhi hemocytes (Fig. 9A) compared

347

with the control group (Fig. 9B). The percentage of Integrinhi hemocytes was

348

about 32.6%. Different microrganisms and particle were used to detect the

349

phagocytic ability of Integrinhi hemocytes. The phagocytes were separated by

350

latex beads, V. splendidus and S. aureus, respectively. In corresponding kinds of

351

phagocytes, the percentage of Integrinhi hemocytes with latex beads, V.

352

splendidus and S. aureus were 56.6%, 17.8%, 10.5%, respectively (Fig. 9C).

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ACCEPTED MANUSCRIPT In another antibody blockade assay, the phagocytic rate of hemocytes towards

354

V. splendidus in the blockade, IgG and blank group were 46.1%, 42.4% and

355

33.6%, respectively. The result suggested that the blockade of CgIntegrin

356

significantly decreased the phagocytic rate towards V. splendidus (p < 0.05). No

357

significant changes were detected in S. aureus, Y. lipolytica and latex beads groups

358

(Fig. 10).

359

3.8 V. splendidus agglutination and LPS recognition activity of CgIntegrin

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After incubation with rCgIntegrin at room temperature for 1 h, agglutination of V.

361

splendidus was observed, while no agglutination was observed for Y. lipolytica

362

and S. aureus (Fig. 11). In the PAMPs binding assay, CgIntegrin displayed a higher

363

binding affinity towards LPS (compared to rTrx group) in a dose-dependent manner

364

(Fig. 12A). The apparent dissociation constant (Kd) of CgIntegrin to LPS was 5.53 ×

365

10-6 M calculated from the saturation curve fitting according to the one-site specific

366

binding model. CgIntegrin showed no binding activity to PGN and Mannan (Fig.

367

12B).

368

4. Discussion

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Integrins are a family of molecules that play crucial roles in cell-cell and

370

cell-matrix adhesion, integrins are also of fundamental importance in cell migration,

371

proliferation and apoptosis (Burke, 1999; Canesi et al., 2002; Yee, 1993). In the

372

present study, an integrin (CgIntegrin) from the Pacific oyster C. gigas (Terahara et

373

al., 2005) was selected to further investigate its role in the innate immune response.

374

CgIntegrin shared high similarities with other β-Integrins from arthropods and

ACCEPTED MANUSCRIPT mammalian, and the conserved special determining loop (SDL (K198-G223)) was also

376

found in CgIntegrin. The predicted spatial structure of CgIntegrin contained four main

377

parts, a βA domain, a Hybrid domain, a plexin-semaphorin-integrin (PSI) domian, and

378

an integrin IE (EGF-like) domain, which shared high similarity with other known

379

β-Integrins. The βA domain was consisted of six β sheets which were surrounded by

380

eight helices. The integrin performs tertiary and quaternary structural rearrangements

381

when it is interacted with ligands essential for cell signaling (Xiong et al., 2002). The

382

βA domain is a major ligand-binding site mediating the protein-protein interaction.

383

The binding of βA domain needs divalent cations which contact the ligand as well as

384

stabilize the binding surface. The hybrid domain is similar to the I-set of Ig domain

385

while it is responsible for the extensive contact with βA domain to affect its

386

hydrophilic and hydrophobic nature (Xiong et al., 2001). The predicted structure of

387

CgIntegrin indicated that it could have functions in the binding of extracellular

388

molecules and triggering the intracellular signaling pathways to regulate the immune

389

response.

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Integrins have been found to be involved in many immunological processes such

391

as early embryogenesis, complement receptor-dependent phagocytosis and regulation

392

of cell proliferation in invertebrates (Miyazawa and Nonaka, 2004). In the present

393

study, the mRNA transcripts of CgIntegrin was detected to be highly expressed in

394

hemocytes, gonad and adductor muscle. After the stimulation of LPS, the expression

395

level of CgIntegrin in hemocytes was significantly up-regulated at 6 h while no

396

significant change was observed after PGN stimulation. In addition, CgIntegrin was

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ACCEPTED MANUSCRIPT 397

reported to be up-regulated upon secondary challenge with V. splendidus (Zhang et

398

al., 2014). It suggested that CgIntegrin could act as an acute-phase protein in response

399

to stimulation and was involved in pathogen resistance to Gram-negative bacteria. Phagocytosis of hemocytes plays a crucial role in the host defense of bivalves

401

(Wootton et al., 2003), in which the presence of cell-adhesion molecules as well as

402

certain cell membrane receptors is essential for the recognition of pathogens and

403

migration of phagocytes. Integrins, a kind of pluripotent cell adhesion molecules,

404

serve as very important receptors in the ligand-binding phagocytosis to activate the

405

down-stream signaling which are involved in the elimination of pathogens (De

406

Arcangelis and Georges-Labouesse, 2000). In the present study, CgIntegrin was

407

mainly expressed on the membrane of oyster hemocytes and 32.6% hemocytes were

408

detected with relative high CgIntegrin expression level. Moreover, the oyster

409

hemocytes with relative high CgIntegrin expression level exhibited different

410

phagocytic abilities towards different microorganism and particles, such as

411

Gram-positive bacteria V. splendidus, Gram-negative bacteria S. aureus and latex

412

beads. In order to further investigate the potential function of CgIntegrin in

413

phagocytosis, the blockade of CgIntegrin was performed reference to the previous

414

report, in which the blockade of β-Integrin could inhibit the invasion of V. splendidus

415

in C. gigas (Duperthuy et al., 2011). In the present study, when CgIntegins on the

416

surface of hemocytes were blocked by polyclonal antibody against CgIntegrin, the

417

phagocytic rate of the whole hemocytes towards V. splendidus was significantly

418

down-regulated from 46.1% to 33.6%. It was inferred that CgIntegrin might be

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ACCEPTED MANUSCRIPT involved in both the invasion of V. splendidus and the phagocytosis towards V.

420

splendidus in oyster. Interestingly, no significant changes of phagocytic rate were

421

detected for S. aureus, Y. lipolytica and latex beads after the blockade of

422

CgIntegrins. These results suggested that CgIntegrin might be important participators

423

of phagocytosis in oyster and it could probably mediate the specific phagocytosis of

424

hemocytes towards V. splendidus.

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Integrins participate in the interaction of the cells and extracellular matrix.

426

They also act as an important mediator in the innate immune response, such as

427

phagocytosis encapsulation and nodulation (Zhuang et al., 2008). It was reported

428

that β-Integrin from C. gigas could mediate the invasion of V. splendidus in an

429

RGD-dependent manner with the presence of CgEcSOD (Duperthuy et al., 2011).

430

Similarly, in another invertebrate, Pacifastacus leniusculus, an extracellular SOD

431

could associate with the cell surface, bind an adhesive/opsonic protein and may be

432

involved in phagocytosis (Johansson et al., 1999). In the present study, the bacterial

433

agglutination assay and PAMP binding assay were performed to further investigate

434

the interaction between integrin and microrganisms. CgIntegrin could agglutinate V.

435

splendidus rather than S. aureus and Y. lipolytica. It was also reported that an

436

β-Integrin from shrimp Litopenaeus vannamei could mediate microbial agglutination

437

(Zhang et al., 2012). The agglutination activity towards V. splendidus indicated that

438

CgIntegrin might be involved in the specific immune response against V.

439

splendidus. Gram-negative bacteria V. splendidus was identified from Yesso

440

scallop (Patinopecten yessoensis) with LPS as the main PAMP on its surface (Liu

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ACCEPTED MANUSCRIPT et al., 2013). Interestingly, CgIntegrin exhibited LPS binding activity, while no

442

binding activities were detected for PGN and Mannan, which was consistent with

443

the specific agglutination activity of CgIntegrin towards V. splendidus. Together

444

with the observation in the blockade assay, it was suspected that the phagocytosis

445

of hemocytes towards V. splendidus might be mediated by the binding of

446

CgIntegrin towards bacterial LPS. Further investigation in the relationship

447

between LPS-integrin binding and the phagocytosis is needed to verify this

448

observation. The results provided evidence for the distinct interaction between

449

integrin and microorganisms via a PRR-like manner in invertebrates. Collectively,

450

considering the participation of CgIntegrin in the specific recognition,

451

phagocytosis and agglutination towards V. splendidus, we are encouraged to

452

suggest that CgIntegrin plays critical roles in pathogen recognition, elimination and

453

cell-cell interactions during immune response.

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In conclusion, an integrin was identified from Pacific oyster C. gigas with

455

conserved features of βA domain, the Hybrid domain, a plexin-semaphorin-integrin

456

(PSI) domian, and integrin IE (EGF-like) domain. The expression of CgIntegrin was

457

up-regulated post LPS stimulation while no significant change was detected in PGN

458

group. After the blockade of CgIntegrin, the phagocytic rate of hemocytes towards V.

459

splendidus was down-regulated. In addition, rCgIntegrin could agglutinate V.

460

splendidus and exhibited direct binding activity to LPS. Collectively, these

461

findings indicated that CgIntegrin could mediate the phagocytosis of V.

462

splendidus possibly by directly binding to LPS, which provide novel insight into

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ACCEPTED MANUSCRIPT 463

the underlying mechanism of the innate immune response of oysters.

464 465

Acknowledgement The authors are grateful to all the laboratory members for continuous technical

467

advice and helpful discussions. This research was supported by the High Technology

468

Project (863 Project, No. 2014AA093501) from the Chinese Ministry of Science and

469

Technology, earmarked fund (CARS-48) for Modern Agro-industry Technology

470

Research

471

National & Local Joint Engineering Laboratory of Ecological Mariculture

472

Taishan Scholar Program of Shandong, China.

fund

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473

from and

the

Reference

475

Arnaout, M., Mahalingam, B., Xiong, J.P., 2005. Integrin structure, allostery, and

478 479 480

Burke, R.D., 1999. Invertebrate Integrins: Structure, Function, and Evolution. Int.

EP

477

bidirectional signaling. Annu. Rev. Cell Dev. Biol. 21, 381-410.

Rev. Cytol. 191, 257-284.

AC C

476

TE D

474

Canesi, L., Gallo, G., Gavioli, M., Pruzzo, C., 2002. Bacteria-hemocyte interactions and phagocytosis in marine bivalves. Microsc. Res. Tech. 57, 469-476.

481

Cao, A., Mercado, L., Ramos-Martinez, J.I., Barcia, R., 2003. Primary cultures of

482

hemocytes from Mytilus galloprovincialis Lmk.: expression of IL-2Rα subunit.

483

Aquaculture 216, 1-8.

484

Cheng, S., Zhan, W., Xing, J., Sheng, X., 2006. Development and characterization of

ACCEPTED MANUSCRIPT 485

monoclonal antibody to the lymphocystis disease virus of Japanese flounder

486

Paralichthys olivaceus isolated from China. J. Virol. Methods 135, 173-180.

487

De Arcangelis, A., Georges L, E., 2000. Integrin and ECM functions: roles in

489 490

vertebrate development. Trends Genet. 16, 389-395.

RI PT

488

Deborah Cooper, C.M.B., Michael C. Berndt, Mathew A. Vadas, 1994. P-selectin interacts with a beta 2-integrin to enhance phagocytosis. J. Immunol. 153, 3199.

Duperthuy, M., Schmitt, P., Garzon, E., Caro, A., Rosa, R.D., Le Roux., et al., 2011.

492

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune

493

cells by the oyster pathogen Vibrio splendidus. Proc. Natl. Acad. Sci. U. S. A.

494

108, 2993-2998.

M AN U

SC

491

Gettner, S.N.K., Kenyon C., Reichardt, Louis F, 1995. Characterization of betapat-3

496

heterodimers, a family of essential integrin receptors in C. elegans. J. Cell Biol.

497

129, 1127.

TE D

495

Holmblad, T., Thörnqvist, P.O., Söderhäll, K., Johansson, M.W., 1997. Identification

499

and cloning of an integrin β subunit from hemocytes of the freshwater crayfish

500

Pacifastacus leniusculus. J. Exp. Zool. 277, 255-261.

AC C

EP

498

501

Hu, J., Zhao, H., Yu, X., Liu, J., Wang, P., Chen, J., et al., 2010. Integrin beta1

502

subunit from Ostrinia furnacalis hemocytes: molecular characterization,

503 504

expression, and effects on the spreading of plasmatocytes. J. Insect Physiol. 56, 1846-1856.

505

Huang, Y., Zhao, L., Feng, J., Zhu, H., Huang, X., Ren, Q., et al., 2015. A novel

506

integrin function in innate immunity from chinese mitten crab (eriocheir

ACCEPTED MANUSCRIPT

508 509 510 511

sinensis). Dev. Comp. Immunol. 52, 155-165 Hynes, 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25. Hynes, R.O., 2002. Integrins: Bidirectional,Allosteric Signaling Machines. Cell 110,

RI PT

507

673–687.

Jemaa, M., Morin, N., Cavelier, P., Cau, J., Strub, J.M., Delsert, C., 2014. Adult

513

somatic progenitor cells and hematopoiesis in oysters. J. Exp. Biol. 217,

514

3067-3077.

M AN U

SC

512

515

Jiang, Q., Zhou, Z., Wang, L., Wang, L., Yue, F., Wang, J., et al., 2013. A scallop

516

nitric oxide synthase (NOS) with structure similar to neuronal NOS and its

517

involvement in the immune defense. PloS one 8, e69158.

Johansson, M.W., Holmblad, T., Thornqvist, P.-O., Cammarata, M., Parrinello, N.,

519

Soderhall, K., 1999. A cell-surface superoxide dismutase is a binding protein for

520

peroxinectin, a cell-adhesive peroxidase in crayfish. J. Cell Sci. 112, 917-925.

521

Kerr, J.R., 1999. Cell adhesion molecules in the pathogenesis of and host defence

523 524

EP

against microbial infection. Mol. Pathol. 52, 220–230.

AC C

522

TE D

518

Lavine, M.D., Strand, M.R., 2003. Haemocytes from Pseudoplusia includens express multiple alpha and beta integrin subunits. Insect Mol. Biol. 12, 441-452.

525

Levin, D.M., Breuer, L.N., Zhuang, S., Anderson, S.A., Nardi, J.B., Kanost, M.R.,

526

2005. A hemocyte-specific integrin required for hemocytic encapsulation in the

527

tobacco hornworm, Manduca sexta. Insect Biochem. Mol. Biol. 35, 369-380.

528

Liu, R., Qiu, L., Yu, Z., Zi, J., Yue, F., Wang, L., et al., 2013. Identification and

ACCEPTED MANUSCRIPT 529

characterisation

530

(Patinopecten yessoensis) cultured in a low temperature environment. J.

531

Invertebr. Pathol. 114, 144-150.

534 535

Vibrio

splendidus

from

Yesso

scallop

Marsden, M., Burke, R., 1997. Cloning and characterization of novel β integrin subunits from a sea urchin. Dev. Biol. 181, 234-245.

RI PT

533

pathogenic

Miyazawa, S., Nonaka, M., 2004. Characterization of novel ascidian beta integrins as primitive complement receptor subunits. Immunogenetics 55, 836-844.

SC

532

of

Moita, L.F., Vriend, G., Mahairaki, V., Louis, C., Kafatos, F.C., 2006. Integrins of

537

Anopheles gambiae and a putative role of a new beta integrin, BINT2, in

538

phagocytosis of E. coli. Insect Biochem. Mol. Biol. 36, 282-290.

M AN U

536

O'Reilly, A.M., Lee, H.H., Simon, M.A., 2008. Integrins control the positioning and

540

proliferation of follicle stem cells in the Drosophila ovary. J. Cell Biol. 182,

541

801-815.

TE D

539

Roy, A., Kucukural, A., Zhang, Y., 2010. I-TASSER: a unified platform for

543

automated protein structure and function prediction. Nat. Protoc. 5, 725-738.

544

Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the

546 547

AC C

545

EP

542

comparative CT method. Nat. Protoc. 3, 1101-1108.

Soderhall, M.J.a.K., 1998. Isolation and purification of a cell adhesion factor from crayfish blood cells. J. Cell Biol. 16.

548

Song, L., Wang, L., Qiu, L., 2010. BIVALVE IMMUNITY. Invertebrate Immunity.

549

Terahara, K., Takahashi, K.G., Mori, K., 2005. Pacific oyster hemocytes undergo

550

apoptosis following cell-adhesion mediated

by integrin-like molecules.

ACCEPTED MANUSCRIPT 551

Comparative biochemistry and physiology. Comp. Biochem. Physiol. A. Mol.

552

Integr. Physiol. 141, 215-222. Wootton, E., Dyrynda, E., Ratcliffe, N., 2003. Bivalve immunity: comparisons

554

between the marine mussel Mytilus edulis, the edible cockle Cerastoderma edule

555

and the razor-shell Ensis siliqua. Fish Shellfish Immunol.15, 195-210.

RI PT

553

Xiong, J.-P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S.L., et al.,

557

2001. Crystal Structure of the Extracellular Segment of Integrin alphaVbeta3.

558

Science 294, 339.

M AN U

SC

556

559

Xiong, J.-P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S.L., et al.,

560

2002. Crystal structure of the extracellular segment of integrin αVβ3 in complex

561

with an Arg-Gly-Asp ligand. Science 296, 151-155.

Xiong, J.P., Mahalingham, B., Alonso, J.L., Borrelli, L.A., Rui, X., Anand, S., et al.,

563

2009. Crystal structure of the complete integrin alphaVbeta3 ectodomain plus an

564

alpha/beta transmembrane fragment. J. Cell Biol. 186, 589-600.

EP

566

Yee, G.H., RO, 1993. A novel, tissue-specific integrin subunit, beta nu, expressed in the midgut of Drosophila melanogaster. Developmental 118, 845-858.

AC C

565

TE D

562

567

Zhang, T., Qiu, L., Sun, Z., Wang, L., Zhou, Z., Liu, R., et al., 2014. The specifically

568

enhanced cellular immune responses in Pacific oyster (Crassostrea gigas) against

569

secondary challenge with Vibrio splendidus. Dev. Comp. Immunol. 45, 141-150.

570

Zhang, Y., Wang, L., Wang, L., Wu, N., Zhou, Z., Song, L., 2012. An Integrin from

571

Shrimp Litopenaeus vannamei Mediated Microbial Agglutination and Cell

572

Proliferation. PloS one 7, e40615.

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Zhuang, S., Kelo, L., Nardi, J.B., Kanost, M.R., 2008. Multiple alpha subunits of

574

integrin are involved in cell-mediated responses of the Manduca immune system.

575

Dev. Comp. Immunol. 32, 365-379.

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Figure Legends

581

Fig.1

582

The nucleotide and the deduced amino acid sequence of CgIntegrin. The putative

583

β-Integrin domain were in shade. The signal peptide was underlined. The EGF

584

domain was short dash-lined and the Integrin β cytoplasm domain was wavy line. The

585

asterisks indicated the stop codon.

586

Fig.2

587

Multiple sequence alignment by ClustalW of β-Integrin domain in CgIntegrin with

588

other identified integrins. Sequences filled in red showed the conserved amino acid

589

residues, and similar amino acids are in red. The species and the GenBank accession

590

numbers are as follow: C. gigas (BAB62173), L. vannamei (GU131148), Anopheles

591

gambiae

592

(XP_001662592),

593

(CAA67357), Homo sapiens (NP_002202) and Mus musculus (NP_034078). The

594

DDK amino acids in metal ion-dependent adhesion site (MIDAS) of CgIntegrin are

595

marked with ■, whereas SDL are bounded by black line.

596

Fig.3

AC C

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580

(CAC00630), Bombyx

Eriocheir mori

sinensis

(AKJ26284),

(NP_001161754),

Aedes

Pacifastacus

aegypti

leniusculus

ACCEPTED MANUSCRIPT Consensus neighbor-joining tree was built based on the amino acid sequences of

598

CgIntegrin from different organisms. The numbers at the forks indicated the bootstrap

599

value. All the members were distinctly separated into two groups. In invertebrate

600

group, CgIntegrin was clustered with that from Biomphalaria glabrata and formed an

601

independent group.

602

Fig.4

603

The predicted three-dimensional structure of CgIntegrin contained two parts, βA

604

domain and hybrid domain. The βA domain with six β sheets which were surrounded

605

by eight helices, a MIDAS motif occupied a crevice at the top of central β strand and

606

hybrid domain similar to the I-set of Ig domain, as predicted by I-TASSER methods.

607

Fig.5

608

Real-time PCR analysis of CgIntegrin mRNA expression level in different tissues.

609

Comparison of the expression level of CgIntegrin mRNA (relative to CgEF) among

610

different tissues was normalized to hepatopancreas. Vertical bars represent the mean ±

611

S.D. (N = 6)

612

Fig.6

613

Temporal expression of the CgIntegrin transcripts in hemocytes after LPS or PGN

614

stimulation as measured by Real-time PCR. Comparison of the level of CgIntegrin

615

mRNA (relative to CgEF) was normalized to 0 h. Significant change (P < 0.01) was

616

detected at 6 h post LPS stimulation. Vertical bars represent the mean ± S.D. (N = 4).

617

**P < 0.01

618

Fig.7

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ACCEPTED MANUSCRIPT SDS-PAGE and Western-blot analysis of rCgIntegrin. Lane M: protein molecular

620

standard; lane 1: E. coli without IPTG induction; lane 2: Positive transformant E. coli

621

induced by IPTG; lane 3: Purified rCgIntegrin; lane 4: Western blot analysis of the

622

polyclonal antibody against rCgIntegrin.

623

Fig.8

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619

(A) Immunohistochemistry analysis of the distribution of CgIntegrin in different

625

tissues. The different tissues were fixed and the sections were blocked with 3% BSA

626

for 1 h, antibody against CgIntegrin was incubated, and the distribution of CgIntegrin

627

was visualized by Alexa Fluor 488-labeled goat-anti-rat antibody (upper panel), the rat

628

pre-immuned serum was used as control (lower panel), the tissues were stained with

629

Evans blue dye (red). Bar= 50 µm.

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(B) Localization of CgIntegrin in hemocytes. After the hemocytes were

631

incubated adhering to the cell culture dish, immunohistochemistry was performed to

632

detect the expression of CgIntergrin. 1. Nucleus was stained with DAPI (blue) 2.

633

CgIntegrin (green) 3. The cell membrane was stained with CM-DIL (red).

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Fig. 9

635

(A) Expression of CgIntegrin on the membrane of hemocytes was detected by flow

636

cytometry. Alexa Fluor 594-labeled goat-anti-rat antibody was used to mark

637

CgIntegrin. Two distinct groups of hemocytes were gated based on the expression

638

level of CgIntegrin, and the group with relative high CgIntegrin expression level was

639

named as Integrinhi hemocytes. (B) Rat IgG is set as control. (C) Phagocytic ability of

640

Integrinhi hemocytes towards latex beads, FITC labeled V. splendidus and S.

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634

ACCEPTED MANUSCRIPT 641

aureus were analyzed by flow cytometry. In the hemocytes performed

642

phagocytosis towards latex beads, V. splendidus and S. aureus, the percentage of

643

Integrinhi hemocytes were 56.6%, 17.8%, 10.5%, respectively.

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644

Fig.10 Phagocytic rates of hemocytes towards FITC labeled V. splendidus, S.

646

aureus and Y. lipolytica and latex beads after the blockade of CgIntegrin by

647

polyclonal antibodies were detected by flow cytometry. Rat IgG was set as negative

648

control and hemocytes without antibody was set as blank control. Vertical bars

649

represent the mean ± S.D. (N = 3). *P < 0.05

650

Fig.11 Agglutination of bacteria by rCgIntegrin. rCgIntegrin (line 1) could agglutinate

651

V. splendidus. No agglutination activity was detected towards S. aureus and Y.

652

lipolytica. rTrx (line 2) was set as negative and TBS (line 3) was set as blank

653

control.

654

Fig.12

655

(A) rCgIntegrin exhibited LPS binding activity in a dose-dependent manner.ELISA

656

assay was performed to determine the binding dissociated constant of rCgIntegrin

657

(0-12 µM) and LPS. Data are shown as the mean ± S.D. (N = 3). The data were

658

curve-fitted using a single-site binding model with R2=0.94 for LPS (Kd =5.53×10-6

659

M). (B) rCgIntegrin exhibited no PGN and Mannan binding activity (P/N<2.1).

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ACCEPTED MANUSCRIPT Table 1. Primers used in this paper. Primer

Sequence(5’—3’) GGCCACGCGTCGACTAGTACT17

Oligo(dT)-adaptor

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Clone primers P1(forward)

ATGTCTGATACTTTGACGCCTAC

P2(reverse)

ATTGACTTTGTCCCTGAAC

RT primers

CCTCGTAAAGAGCAGGGATG

P4

CCATTGAGTTTGAGAGGTCCAT

SC

P3

M AN U

EF primers P5 ( EF-RTF)

GAGCGTGAACGTGGTATCAC

P6 ( EF-RTR)

ACAGCACAGTCAGCCTGTGA

Recombination primers P7(forward)

CATGCCATGGATGTCTGATACTTTGACGCCTAC CCGCTCGAGATTGACTTTGTCCCTGAAC

Sequencing primers M13(forward)

AATTAACCCTCACTAAAGGG TGCGTCGGCTTTGCTCTG

AC C

EP

RV(reverse)

TE D

P8(reverse)

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights 1. CgIntegrin was a β-Integin form oyster Crassostrea gigas. 2. CgIntegrin was significantly up-regulated post Gram-negative bacteria challenge.

RI PT

3. CgIntegrin was expressed on the membrane of different hemocytes. 4. Integrinhi hemocytes displayed different phagocytic abilities towards diverse microorganisms.

AC C

EP

TE D

M AN U

SC

5. CgIntegrin had LPS binding activity and could directly bind to V. splendidus