Molecular characterization of a pattern recognition protein LGBP highly expressed in the early stages of mud crab Scylla paramamosain

Accepted Manuscript Molecular characterization of a pattern recognition protein LGBP highly expressed in the early stages of mud crab Scylla paramamosain

Xiaowan Ma, Weibin Zhang, Fangyi Chen, Kejian Wang, Hui Peng PII: DOI: Reference:

S1095-6433(18)30154-5 doi:10.1016/j.cbpa.2018.08.017 CBA 10373

To appear in:

Comparative Biochemistry and Physiology, Part A

Received date: Revised date: Accepted date:

26 April 2018 31 August 2018 31 August 2018

Please cite this article as: Xiaowan Ma, Weibin Zhang, Fangyi Chen, Kejian Wang, Hui Peng , Molecular characterization of a pattern recognition protein LGBP highly expressed in the early stages of mud crab Scylla paramamosain. Cba (2018), doi:10.1016/ j.cbpa.2018.08.017

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ACCEPTED MANUSCRIPT Molecular characterization of a pattern recognition protein LGBP highly expressed in the early stages of mud crab Scylla paramamosain Xiaowan Maa,1 , Weibin Zhang a,1 , Fangyi Chen b,c,*, Kejian Wang a,b,c, Hui Peng b,c a

State Key Laboratory of Marine Environmental Science, College of Ocean&Earth Science s, Xiamen

University, Xiamen, Fujian 361102, PR China Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological

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b

Resources, Xiamen University, Xiamen, Fujian 361102, PR China

State-Province Joint Engineering Laboratory of Marine Bioproducts and Technology, Xiamen

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c

University, Xiamen, Fujian 361102, PR China These authors made equal contributions.

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1

* Corresponding author:

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Fangyi Chen

[email protected] (F.-Y. Chen)

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Abstract

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College of Ocean and Earth Sciences, Xiamen University, Xiamen, Fujian 361102, PR China.

The early developmental stages of the mud crab Scylla paramamosain suffer from h igh mo rtality by

pathogen

infections;

however,

few

immune

associated

factors

are

known.

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caused

Lipopolysaccharide and β-1,3-g lucan-binding protein (LGBP) functions as a typical pathogen recognition receptor and plays an important role in the innate immune system of invertebrates. In this

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study we characterized a LGBP gene (Sp LGBP) wh ich was highly expressed in the late embryonic, zoea I larval stage and hepatopancreas of S. paramamosain.. It encodes 364 amino acids, composed of several conserved domains like the bacterial g lucanase motif. The reco mb inant SpLGBP protein (rSp LGBP) was obtained through the E.coli expression system, in wh ich two 6◊His-tags were added to both C and N terminals during vector construction for the improvement of purificat ion efficie ncy. In vivo the study showed that the Sp LGBP mRNA was significantly up-regulated under Vibrio

parahaemolyticus and a lipopolysaccharide (LPS) challenge in the hemocytes and hepatopancreas. The ELISA b inding assay in vitro indicated that the rSpLGBP was capable of binding to LPSs and peptidoglycan (PGN). The rSpLGBP could agglutinate both G+ and G- bacteria in the presence of Ca 2+. 1

ACCEPTED MANUSCRIPT Our results suggest that SpLGBP may play an immunological role against pathogenic infection in the early developmental stages of S. paramamosain. Key words: Ca2+ dependent, lipopolysaccharide and β-1,3-g lucan-binding protein (LGBP), LPS, recognition, Scylla paramamosain, Vibro parahaemolyticus

Introduction

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1.

The mud crab (Scylla paramamosain) is an important aquaculture species with a high price and

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market demand in South China. Over the last decade, the mud crab industry has developed rapidly. In 2016, the mud crab g lobal production is 134,783 tons, which includes 45,396 tons of capture

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production and 89,390 tons of aquaculture production. The aquaculture value is $8909 per ton (http://www.fao.org/fishery/topic/16140/en). However, crab aquaculture has been perplexed by some

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intricate p roblems, such as the disease epidemics caused by high densities in ponds culture and high mortality caused by disease during development (Hamasaki et al., 2011). So a comprehensive

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understanding of its immune system is needed to help solve these problems. The mud crab, like other invertebrates, relies only on an innate immune system to defend against

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pathogenic microorganisms and lacks an adaptive immune system. Th rough pathogen recognition receptors (PRRs), organisms first recognize invading microorganis ms by the pathogen-associated

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mo lecular patterns (PAMPs) on their surface, and subsequently trigger downstream cellu lar or humoral immune react ions. Proteins functioning as PRRs in invertebrates include the β -glucan recognition protein (β GRP), peptidoglycan binding protein (P GBP), LPSs and g lucan binding protein (LGBP),

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Gram-negative bacteria binding protein (GNBP), thioester-containing protein, C-type lectin, galactoside-binding lectin (galectin ), scavenger receptor and fib rinogen -like do main immunolectin (Wang and Wang, 2013).

LGBP, also known as GNBP, was first purified fro m Bo mbyx mori (Lee et al., 1996) and functioned as a biosensor for LPSs fro m Gram-negative bacteria and β-1,3-g lucan (β G) fro m fungi. Later, the same function was affirmed in Drosophila melanogaster (Kim et al., 2000). Normally, LGBPs share several common mot ifs for carbohydrates, such as two potential polysaccharide recognition motifs, a polysaccharide binding motif (PsBM), a β -glucan recognition motif (β GRM ) and a glucanase motif (GM). In crustaceans, several LGBPs have been cloned and their function 2

ACCEPTED MANUSCRIPT characterized in innate immune recognition (Cheng et al., 2005; Du et al., 2007; Lin et al., 2008). However, little is known about S. paramamosain specifically. In this study, a LGBP gene of S. paramamosain was cloned and characterized. The expression profile o f this gene in the hemocytes and hepatopancreas upon the LPS and Vibrio challenge was determined by using relative quantitative real-time PCR (qPCR). In addition, Sp LGBP mRNA

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transcripts in different developmental stages were investigated. The recombinant SpLGBP p rotein was generated by prokaryotic expression and affinity chromatography purification. We analyzed the ability

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of rSpLGBP to b ind to LPSs and PGN. An agglutination assay was carried out to reveal whether or not

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it could agglutinate some bacteria.

Material and Methods

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2.1 Animal collection, bacterial challenge and tissue preparation

Healthy mud crabs (300 ± 30 g each) were purchased from a co mmercial market in Xiamen,

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Fujian province, China. The crabs were acclimated in seawater (salin ity 30‰, temperature 25-26°C) for a week before challenging and sampling. To investigate the SpLGBP mRNA expression level at

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different develop mental stages, the emb ryonic stages from egg lay ing until hatching out as zoea I larva were sampled daily (n = 3), quickly frozen in liquid nitrogen and stored at -80℃ for later use.

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After using sterile syringes (5 mL) to ext ract the hemo ly mph fro m the arthrodial membrane

between the dactyl and propodus of the third pereopod, the hemoly mph was immed iately mixed with an anticoagulation agent then centrifuged to harvest the hemocytes . Thereafter, various tissues of mud

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crabs (n = 3) including hepatopancreas, gill, mid-gut, muscle, heart, stomach and thorax ganglion were dissected, flash frozen by liquid nitrogen, and stored at -80℃ for RNA extraction. For the bacterial and LPS challenge experiment, 150 crabs were randomly separated into 3 groups. Two experimental groups were infected. One was injected with 100 μ L LPS (2.5 mg / mL) fro m Escherichia coli (L2880, Sig ma) and another with 100 μ L V. parahaemolyticus live bacteria (approximately 2.5 × 107 cfu/mL) dissolved in saline. The third group was used as a control and received an in jection of an equal volu me of saline solution. Tissue (n= 3) were samp led at 0 h (fro m the untreated crab) and at 3, 6, 12, 24 and 48 h post-injection and frozen at -80°C for later RNA extraction.

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ACCEPTED MANUSCRIPT 2.2 Total RNA Extraction and cDNA Synthesis Total RNA was isolated from different tissues including hemocytes using TRIzol reagen t (Invitrogen, USA). Then 1 μg total RNA was further treated with 1 μL RNase-free DNaseI (Pro mega, USA) to eliminate the contamination o f DNA and later RNA was quantified using NanoDrop Spectrophotometer (Thermo, USA) at A260/ 280 with ratio between 1.8-2.0. 1 μg total RNA was reverse transcribed into first-strand cDNA using SuperScript™ III reverse transcriptase (Invitrogen,

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USA) in a final volu me of 20 μL with 4 μL 5 × Primes scrip Buffer, 1 μL Prime script RT Enzy me Mix

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I, 1 μL Oligo dT prime (50 μM), 1μL Radom 6 mers (100 μM) and treated under 37°C for 15 min.

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2.3 Isolation and Cloning of SpLGBP Gene

A transcriptome library of early develop mental stages of the mud crab was constructed previously

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in our lab (unpublished). A 1354 bp sequence in the database showing high similarity to the Gen Bank database for Portunus tritubercμlatus LGBP was named after SpLGBP. A couple of specific primers

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(Sp LGBP-F/ R) (Supplementary Table 1) were designed to amp lify the coding reg ion of Sp LGBP using

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the cDNA of hemocytes.

2.4 Sequence Analysis

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The Sp LGBP gene sequence was compared with LGBPs fro m other organis ms. The ho mologue search for the sequences was in the GenBan k database using the BLAST algorith m at NCBI (http://www.ncbi.nlm.nih.gov/).

We

used

the

SignalP

4.0

program

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(http://www.cbs.dtu.dk/services/SignalP/) to predict the signal peptide region, and the Expert Protein Analysis System to predict the mot ifs (https://prosite.expasy.org/prosite.html), molecu lar weight and calculation theory (http://web.expasy.org/protparam/) of the deduced amino acid sequence. The mu ltip le

align ment

was

(http://www.ebi.ac.u k/clustalw/).

analyzed

by

ClustalW

The

secondary

structure

Multiple of

Alignment

SpLGBP

was

program detected

(https://www.predictprotein.org/home). A neighbor-joining (NJ) phylogenetic tree was constructed using MEGA6 (Tamu ra et al., 2013). The reliability of the branching was tested using bootstrap resampling (1000 pseudo replicates).

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ACCEPTED MANUSCRIPT 2.5 qPCR analysis of SpLGBP mRNA expression To analyze the relative expression level of Sp LGBP in d ifferent develop mental stages and tissues, or under the challenge of V. parahaemolyticus or LPS, q PCR was monitored in a 7500 Real-Time PCR System (Applied Biosystems) with a reaction volume o f 20 μL containing 10 μL Power SYBR Green PCR Master Mix (Roche), 10 μM primers, and 10 ng cDNA. The standard cycling procedure was as follows: 50℃ for 2 min, 95℃ for 10 min, fo llo wed by 40 temperature cycles (95℃ for 15 s, 60℃ for 1

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min). β-actin was used as an internal control to determine the relative exp ression levels of SpLGBP

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mRNA. The primers specific for Sp LGBP amp lifying 170 bp product and endogenous gene-β-actin are designed and listed in Suplementary Table 1. The specificity of the primers had been confirmed using

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melting curve analysis and the amplification efficiency was calculated within 95 -105%. The 2−ΔΔCt method was used to calculate the qPCR data (Livak and Sch mittgen, 2001). At least three replications

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were conducted in all assays. SpLGBP transcripts levels in other tissues were normalized to that in heart, while in different developmental stages were normalized to that in the 7th day after laying

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considered to be significant with P < 0.05.

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embryo. Anova comparison test was used to analyze by SPSS software (version 16.0). Values were

2.6 Expression and Purification of Recombinant SpLGBP (rSpLGBP) Proteins

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A pair o f primers (Supplementary Table 1) was designed to amplify the gene SpLGBP. To enhance the combination efficiency of the His -tag to the Ni-colu mn by adding t wo His -tags to both the C and N teminals. The gene was cloned into Pet-32a (+) using the restriction enzy mes Ba mHI and XhoI, then

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transformed into E. coli BL21 (DE3) plysS (Novagen, USA). The reco mbinant strain was cultured in LB b roth at 37℃ until OD600 reached 0.4-0.6. Then IPTG (0.05mM) was added and the cells were cultured for 17 h at 18°C. Cells were harvested by centrifugation, resuspended in a 20 mM phosphate buffer (pH=7.4), sonicated and the soluble fraction was collected in the supernatant by centrifugation. The supernatant was applied to a Ni-Sepharose column (GE healthcare, USA), pre -equilibrated with buffer I (20 mM PB, 500 mMNaCl, pH=7.4), follo wed by buffer I with 25 mM imidazo le. Then the protein was eluted with buffer I with 50 mM , 100 mM, 250 mM and 500 mM imidazole. The purity and mo lecular weight of the rSp LGBP p rotein was evaluated through SDS-PA GE on a 15% (w/v) resolving gel. The concentration of the rSpLGBP protein was quantified by the Bradford assay 5

ACCEPTED MANUSCRIPT according to manufacturer’s instructions.

2.7 Binding Assay of rSpLGBP ELISA was used to analyze the binding ability of rSp LGBP to carbohydrates (Chen et al., 2016). Briefly, each type of carbohydrate LPS (E. coli 055:B5) and PGN (69554) was first dissolved in MilliQ water at a concentration of 1 mg/ mL, then diluted with coating buffer to 80 μg/mL. 50 μL was used to

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coat each well of a 96-microwell plate and dried at 60°C for 3 h. 200 μL bovine seru m albu min BSA

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(10 mg/ mL) in PBST (pH = 7.5) was used to block the reaction at 37°C fo r 2 h. After washing 3 t imes with 200 μL PBST, 50 μL rSpLGBP (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110 μg/ mL in PBST

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and 0.1 mg/ mL BSA) was added into the well and incubated at 28°C fo r 2 h, then washed with PBST three times. 100 μL anti-His-tag mouse antibody (diluted 5000-fold in PBST) was added and incubated

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at 37°C fo r 1 h, then washed. 100 μL Horseradish Pero xidase HRP -conjugated goat anti-mouse IgG (diluted 10000-fold in PBST) was added and incubated at 37°C for 1 h. After washing, 100μL

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3,3’,5,5’-tetramethylbenzidine (So larbio) liquid was added and incubated at room temperature for 20 min. 100 μL 2M H2 SO4 was used to stop the reaction. Absorbance at 450 n m was measured with an

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Infinite M200 Pro.

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2.8 Agglutination Assay of rSpLGBP

The agglutination ability of rSp LGBP was examined using Gram-positive (Staphylococcus aureus, Bacillus cereus and Micrococcus lysodeikticus) and Gram-negative bacteria (Pseudomonas aeruginosa,

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Aeromonas hydrophila and Shigella flexneri). The

microorganisms were collected in the

mid-logarith mic phase by centrifugation at 6000r/ min for 5 min. After washing twice with TBS (10 mM Tris–HCl, 150 mM NaCl, pH=7.5), the microorganisms were resuspended in TBS at 1 OD. In the presence or absence of 10 mM CaCl2 , 25 μL microorganisms and 25 μL rSp LGBP (300 μg/ mL) were incubated together at room temperature for 1 h, then observed under a microscope. BSA (300 μg/mL) was used as a negative control.

3.

Results

3.1 Sequence Characterization and phylogenetic analysis of SpLGBP 6

ACCEPTED MANUSCRIPT The open reading frame (ORF) of Sp LGBP comp rised 1092 nucleotide sequences coding a mature protein of 364 amino acid residues (Fig 1 A). It predicted that a signal peptide sequence existed in the first 15 amino acids of the N-terminal region. The mature peptide had a calculated molecular mass of 41.41 kDA and an estimated isoelectric point (pI) of 4.5. The sequence of Sp LGBP was submitted to NCBI GenBank under the accession number MH036753.

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A conserved domain classified as glucanase hydrolase family 16 exists at amino acids between positions 79–262. Sp LGBP co mprises two integrin binding motifs of RGD (Arg-Gly-Asp) at positions

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104 and 155, a protein kinase C phosphorylation site (SAR) at positions 131 -133, a cleavage of β-1,3-or β-1,4-g lucosidic lin kages of bacterial g lucanase (GM) with four amino acid residues (Trp 175,

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Glu 180, Ile 181, Asp 182), a β-glucan recognition motif (β GRM) and a polysaccharide recognition motif for the polysaccharide binding motif (PsBM ) (Fig 1A). In addition, two putative N-g lycosylation

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residues (NRS; NDS) at positions 64 and 316 were also obs erved in the sequences of SpLGBP (Fig 1A).

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Using the LGBP sequences of different organis ms, a phylogenetic tree was reconstructed by the NJ method. Phylogenetic tree analysis showed two distinct nodes. Mollusk LGBPs were grouped into

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one cluster, while crustacean LGBPs were grouped into the other. It is likely that SpLGBP belongs to

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the family of crustacean LGBPs (Fig 1B).

3.2 The high expression level of SpLGBP mRNA in late embryonic stages, zoea I larval stage and hepatopancreas and the induction under V. parahaemolyticus or LPS challenge

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qPCR was emp loyed to show the distribution of the Sp LGBP transcript in the different t issues we examined and the time course exp ression level of Sp LGBP mRNA fro m the day o f egg laying to hatching. The SpLGBP mRNA was first detected 8 days after laying and up-regulated significantly after 9 and 10 days (Fig. 2A). A mong the different tissues we examined, SpLGBP mRNA showed the highest expression level in the hepatopancreas in healthy adult crabs (Fig. 2B). The expression level of Sp LGBP mRNA in the hemocytes and hepatopancreas after the LPS (Fig. 3A and C) or V. parahaemolyticus (Fig. 3B and D) challenge were determined using qPCR. After injecting with LPS, Sp LGBP mRNA increased significantly after 12 h then declined in the hemocytes. A significant up-regulation occurred at 3 h post-challenge followed by a trend of down-regulat ion after 7

ACCEPTED MANUSCRIPT challenging with V. parahaemolyticus. After the V. parahaemolyticus challenge, a sharp increase was observed in the hepatopancreas at 3 h post-challenge, then declined and a second up-regulation was observed at 24 and 48 h post-challenge (Fig. 3D).

3.3 The Recombinant expression and purification of rSpLGBP

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The rSp LGBP was expressed in E. coli BL21 (DE3). After IPTG induction for 17 h, whole cell lysate was analyzed by SDS-PA GE. We found that the rSp LGBP mostly expressed in the form of

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inclusion bodies with an extra His -tag at the N-terminal (17.7 kDa). After purify ing the insoluble fraction using a Ni-NTA colu mn at the optimal concentration of imidazo le (250 mM), a single band

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with a molecular mass of ~60 kDa was achieved in SDS-PAGE (Fig 4A and B).

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3.4 The binding property of rSpLGBP to LPS and Peptidoglycan

To examine the b inding activity of rSp LGBP with PAMPs, we investigated two polysaccharides

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using ELISA. The results showed that rSpLGBP binds to LPSs and PGN (Fig 5).

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3.5 The agglutinating ability of rSpLGBP to both Gram positive and negative bacteria in the absence of Ca 2+

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In order to determine the agglutinating activity of purified rSp LGBP, we observed six microorganis ms (three Gram- bacteria and three Gram+ bacteria) incubated with rSpLGBP in the presence or absence of Ca 2+ (Fig 6). In the presence of Ca2+, purified rSp LGBP agglutinated all tested

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microorganisms, while in the absence of Ca 2+ no agglutination was observed.

Discussion

LGBP is a typical pattern recognition receptor protein, with high ho mology with Gram-negative bacteria binding protein (GNBP) and β-1,3-glucan binding proteins (βGBPs). Studies have shown that this protein play an important role in non-self recognition and induction of antimicrobial peptides regulation pathway (Kim et al., 2000). Characterization of LGBP in S. paramamosain will provide us valuable information in the immune system of the mud crab. Like other crustaceans, SpLGBP contains several conserved domains, including two N-lin ked 8

ACCEPTED MANUSCRIPT glycosylation sites, two integrin-binding motifs, a kinase C-phosphorylation site, a bacterial glucanase motif and a β-1,3-linkage of a polysaccharides recognition motif. N-linked glycosylation sites were observed in several shrimp LGBPs (Chaosomboon et al., 2017; Du et al., 2007). The bacterial glucanase motif is considered to be essential in bacteria g lucanase catalytic mechanis ms (Cheng et al., 2005; Du et al., 2007; Jiang et al., 2004). Although the glucanase activity has not yet been found, it is

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hypothesized that LGBP lost its glucanase activity during evolution fro m the β -1,3-glucanase but maintains the glucan binding property and plays a key role in immune processes (Lee et al., 2000). In

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addition, the four amino acid residues of the active sites of the β -1,3-lin kage to the polysaccharides recognition motif is conserved in different organisms (Cheng et al., 2005; Rou x et al., 2002;

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Sritunyalucksana et al., 2002). Integrin-binding mot ifs (RGD) are the co mmon cell adhesion and mo lecular recognition sites shared by the extracellular matrix and a variety of adhesion protein

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mo lecules (Johansson et al., 1999). LGBP is considered to be a receptor p rotein in the p lasma, b in ding with the cell memb rane through RGD motifs, inducing a series of immune reactions. Meanwhile, RGD

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also exists in pero xinectin with a protein adhesion function to regulate the cell adhesion protein binding sites (Ruoslahti, 1996). Many integrins could identify and combine with RGD. RGD mot ifs were also

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observed in other invertebrates such as in L. vannamei (Vaseeharan, 2012), while in M. japonicus the integrin-binding motif is LGD (Lin et al., 2008). Since conserved domains exist in the sequence of S.

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paramamosain, the SpLGBP might recognize the invasion pathogens as a pattern recognition receptor, like other crustacean LGBPs.

The specific tissue distributions of LGBPs mRNA have been demonstrated in several other

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organisms, where they are predo minantly detected in the hepatopancreas (Chaosomboon et al., 2017; Hou et al., 2015) and hemocytes (Sivakamavalli et al., 2015). In crustaceans, the hepatopancreas and hemocytes are considered to be the key organs for producing the immune associated factors (Gross et al., 2001; Rou x et al., 2002). Although plenty of wo rk has been done in the adult invertebrates, little is known about LGBPs in early developmental stages. In our in vivo experiment, the Sp LGBP mRNA was only detected in the late emb ryonic stages and showed an increasing trend until hatching out as zoea I larva. It is likely that the innate immune system is gradually established during this period, as the transcription of immune associated factors begins. In addition, without the physical protection of the egg envelope, the zoea I larva would be surrounded by a large amount of microorganisms after 9

ACCEPTED MANUSCRIPT hatching out. As a result, the high expression level of the SpLGBP transcript before and during the zoea I stage might increase the ability of mud crabs to recognize the pathogens and better protect themselves. The expression pattern of Sp LGBP was observed when challenged with immunostimu lants such as the important marine pathogen V. parahaemolyticus and the Gram-negative bacteria cell wall

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component LPS. In general, noticeable t ime -course changes of Sp LGBP transcripts in the hemocytes and hepatopancreas were similar under the stimulat ion of LPS or V. parahaemolyticus. The Sp LGBP

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mRNA was induced under both challenges . However, our experiment showed that the expression level of Sp LGBP mRNA declined sharply in the first few hours after the LPS challenge. This could be

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explained by a significant decrease in the total hemocyte counts in crustaceans caused by LPS inject ion (Ji et al., 2009). The hemocytes might be d ispatched to the wound to defend against the bacteria. Later,

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as more hemocytes were released fro m hemocytopoietic tissue or other sites (Sritunyalucksana and Söderhäll, 2000), the SpLGBP mRNA expression level was up-regulated. Therefore, we speculate that

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SpLGBP was induced in response to bacterial challenge to combat the invading pathogens. PRRs are considered to have an ability to recognize invading microorganisms and the PA MPs on

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their cell walls, and init iate the downstream immune system. The ELISA assay showed that the rSp LGBP exh ibited a binding potential to both LPSs and PGN, wh ich ind icates that rSpLGBP could

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respond to the main membrane components of both Gram negative and Gram positive bacteria. Additionally, the rLGBPs of several other invertebrates also bind to β -1,3-glucan (Yang et al., 2010; Zhang et al., 2016), which confirms that LGBP as a PRR could respond to the membrane structure of

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fungi. Interestingly, the binding potency of rLGBPs might have different preferences in d ifferent invertebrates. For examp le, the LGBP of E. sinensi could comb ine with LPSs and β-1, 3-g lucan but not to PGN (Zhang et al., 2016), while the rLGBP of F. merguiensis has a higher binding ability to β-1,3-g lucan than LPSs and lipoteichoic acid (LTA) (Chaosomboon et al., 2017). Considering the observed induction under the LPS challenge, it seems likely that the binding to LPSs increased the transcription of SpLGBP mRNA and possibly triggered the downstream immune pathways to better combat the foreign invaders.

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In the bacteria agglutination assay, rSpLGBP induced agglutinate activity of Gram-positive and negative bacteria only under

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calcium-dependent conditions, which suggests that the presence of

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Ca2+ might be essential for the agglutination ability of rSpLGBP.

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Even though the calcium-binding site has not been identified,

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agglutination ability enhanced by the presence of Ca 2+ has been

the presence of Ca2+ also pl ays a key role in the process of

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al., 2018). In other cases,

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reported in several LGBPs (Chaosomboon et al., 2017; Phupet et

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early recog nition of pathogen challenge, and induces the downstream innate immune

reactions. For example, the phenol oxi das e acti vi ty was induced in the presence of b-1,3-glucan

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5.

pl us Ca2+ and in the freshwater crayfish, hemolymph clotting is also depended on the presence

of Ca2+ (Vazquez et al., 2009). However, the exact bi nding site of LGBP to the

Ca2+ is not

clear yet. It is acknowledged that Ca2+ is necessary in the

bindi ng

process of C-type lectin wi th carbohydrates where the conserved site on the CRD structure is

considered to be responsi ble for Ca2+ bindi ng and sugar coordinated bindi ng (Zelensky and 11

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Gready, 2005). In addi tion, the presence of Ca

2+

induced the structural change

of protein (Zelensky and Gready, 2005). Whether or not it was the same case in S pLGBP

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needs further work to prove. In the case of mud crab, when the li ve bacteria invaded the

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organism, several i mmune actions might work together to defend the pathogen infecti ons. It

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might invol ve the reactions of S pLGBP, such as binding with the ligands on the bacteria,

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inducing aggluti nation of bacteria, acti vati ng the downstream innate immune pathways and

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triggering immune related components to protect the crab from infections. Conclusion

To our knowledge, this is the first analysis of the LGBP gene in S. paramamosain. The Sp LGBP

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was induced under the stimulation of LPSs o r V. parahaemolyticus, indicating that it might be involved in the immune response towards G+/ G- bacteria via binding and agglutination with

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polysaccharides. In conclusion, SpLGBP appears to play a key role as a PRR in pathogenic infections, and this study provides a basis for further experimental investigation of PRRs in the immune response

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against infections.

Acknowledgements

This work was supported by a Fundamental Research Funds for the Central Un iversities [20720180100], Grants [U1205123, 41676158] fro m the Nat ional Natural Science Foundation of China (NSFC), a grant [2017NZ0004/2017NZ0004-1] from the Fujian Science and Technology Department.

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1 A. cDNA sequence and i ts deduced amino aci d sequence of S pLGBP. The stop codon is marked by the asterisk; the signal peptide is underlined; putative N-glycosylation sites are gray shaded; the glycosyl hydrolase domain (RGD) is marked with a b lack bo x; SA R is underlined with dots; GM is marked with red box. B. Phylogenetic analysis of LGBPs from S.paramamosain and other

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organisms. The tree was generated by the neighbor-join ing method produced by MEGA 6. A mino acid sequences of LGBPs fro m shrimp, crabs and scallops were selected according to the alignment.

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GenBank accession numbers of each protein are shown. The SpLGBP is indicated by a diamond. The

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bar (0.05) represents the genetic distance.

Fig. 2 Expression profile of S pLGBP in different tissues (A). HP: hepatopancreas; HC: hemocytes;

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GI: gill; M G: mid-gut; MU: muscle; HT: heart; ST: stomach; TG: thorax ganglion. Expression profile of S pLGB P in earl y developmental stages (B). 1-11: each day of emb ryo post laying; 12: zoea I

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Fig. 3 Time course mRNA expression of S pLGBP in hemocytes ( A, B) and hepatopancreas (C, D) after LPS (A, C) or V. parahaemolyticus (B, D) challenge. β-actin was used as a house-keeping gene.

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Fig. 4 SDS-PAGE anal ysis of the purified recombi nant S pLGB P (rSpLGBP). A. M: molecu lar weight markers; Lane 1 : crude sample; Lane 2: eluant, Lane 3-7: b uffer I with 25, 50, 100, 250 and

500 mM imidazole. B. M: mo lecular weight markers; Lane 1: purified reco mbinant LGBP fro m SpLGBP (rSpLGBP).

Fig. 5 Bindi ng of rS pLGBP to LPS (A) and PGN (B ) determined by ELISA. Each point represents the mean of three individual measurements ± SEM.

Fig. 6 B acterial and fungal agglutinati on induced by rS pLGBP. Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus and Micrococcus lysodeikticus) and Gram-negative bacteria 15

ACCEPTED MANUSCRIPT (Pseudomonas aeruginosa, Aeromonas hydrophila and Shigella flexneri) agglutination induced by purified rSp LGBP (300 μg/ mL) in the presence or absence of 10 mM CaCl 2 . BSA was used instead of

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1 A. cDNA sequence and i ts deduced amino aci d sequence of S pLGBP. The stop codon is marked by the asterisk; the signal peptide is underlined; putative N-glycosylation sites are gray shaded; the glycosyl hydrolase domain (RGD) is marked with a b lack bo x; SA R is underlined with dots; GM is marked with red box. B. Phylogenetic analysis of LGBPs from S.paramamosain and other

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organisms. The tree was generated by the neighbor-join ing method produced by MEGA 6. A mino acid sequences of LGBPs fro m shrimp, crabs and scallops were selected according to the alignment.

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GenBank accession numbers of each protein are shown. The SpLGBP is indicated by a diamond. The

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Fig. 2 Expression profile of S pLGBP in different tissues (A). HP: hepatopancreas; HC: hemocytes;

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GI: gill; M G: mid-gut; MU: muscle; HT: heart; ST: stomach; TG: thorax ganglion. Expression profile of S pLGB P in earl y developmental stages (B). 1-11: each day of emb ryo post laying; 12: zoea I

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Fig. 3 Time course mRNA expression of S pLGBP in hemocytes (A, B) and hepatopancreas (C, D) after LPS (A,C) or V. parahaemolyticus (B , D) challenge. β-actin was used as a house-keeping gene.

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The bars show standard errors of mean values. Asterisks represent significant differences (*p < 0.05,

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Fig. 4 SDS-PAGE anal ysis of the purified recombi nant S pLGB P (rSpLGBP). A. M: molecu lar weight markers; Lane 1 : crude sample; Lane 2: eluant, Lane 3-7: b uffer I with 25, 50, 100, 250 and

500 mM imidazole. B. M: mo lecular weight markers; Lane 1: purified reco mbinant LGBP fro m SpLGBP (rSpLGBP).

Fig. 5 Bindi ng of rS pLGBP to LPS (A) and PGN (B ) determined by ELISA. Each point represents the mean of three individual measurements ± SEM.

Fig. 6 B acterial and fungal agglutinati on induced by rS pLGBP. Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus and Micrococcus lysodeikticus) and Gram-negative bacteria (Pseudomonas aeruginosa, Aeromonas hydrophila and Shigella flexneri) agglutination induced by 24

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