Fish & Shellfish Immunology 43 (2015) 200e208

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Characterization of a gC1qR from the giant freshwater prawn, Macrobrachium rosenbergii Ting Ye a, 1, Xin Huang b, 1, Xian-Wei Wang c, 1, Yan-Ru Shi b, Kai-Min Hui b, Qian Ren b, * a

College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China c MOE Key Laboratory of Plant Cell Engineering and Germplasm Innovation/Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Sciences, Shandong University, Jinan, Shandong 250100, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 August 2014 Received in revised form 22 December 2014 Accepted 23 December 2014 Available online 30 December 2014

gC1qR, as a multicompartmental and a multifunctional protein, plays an important role in innate immunity. In this study, a gC1qR homolog (MrgC1qR) in the giant freshwater prawn, Macrobrachium rosenbergii was identified. MrgC1qR, a 258-amino-acid polypeptide, shares high identities with gC1qR from other species. MrgC1qR gene was expressed in different tissues and was highest expressed in the hepatopancreas. In addition, the MrgC1qR transcript was significantly enhanced after 6 h of white spot syndrome virus (WSSV) infection or post 2 h, 24 h of Vibrio anguillarum challenge compared to appropriate controls. Moreover, recombinant MrgC1qR (rMrgC1qR) had bacterial binding activity, the result also revealed that rMrgC1qR could bind pathogen-associated molecular patterns (PAMPs) such as LPS or PGN, suggesting that MrgC1qRmight function as a pathogen-recognition receptor (PRR). Furthermore, glutathione S-transferase (GST) pull-down assays showed that rMrgC1qR with GST-tag could bind to rMrFicolin1 or rMrFicolin2 with His-tag. Altogether, these results may demonstrate a role for MrgC1qR in innate immunity in the giant freshwater prawns. © 2014 Elsevier Ltd. All rights reserved.

Keywords: MrgC1qR Innate immunity Ficolins Macrobrachium rosenbergii

1. Introduction C1q is part of the C1 complex, which plays a crucial role in the induction of primary immune response. C1q is composed of two major structural and functional domains: 6 globular “heads” (gC1q) and a collagen-like “stalk” (cC1q). gC1qR, a heterodimer glycoprotein, binds to gC1q [1e3]. gC1qR is a single-chain, multiligand binding protein which migrates with an apparent molecular mass of 33 kDa by SDS-PAGE, but as a multimer of 97.2 kDa by gel filtration under nondissociating conditions. gC1qR is a ubiquitously expressed, biological important, multifunctional and multicompartmental protein. It was found that gC1qR could be identified in various cellular compartments, including nucleus, cytoplasm, mitochondria and plasma membrane [4e6]. Although gC1qR has been localized predominantly to mitochondria, variably low cell surface expression has been observed also. gC1qR, which is

* Corresponding author. Tel.: þ86 25 85891955; fax: þ86 25 85891526. E-mail address: [email protected] (Q. Ren). 1 Ting Ye, Xin-Huang and Xian-Wei Wang are equally contributed to this paper. 1050-4648/© 2014 Elsevier Ltd. All rights reserved.

expressed on cell surfaces, can function as docking receptors for a number of structurally or functionally molecules [7,8]. However, as only a few invertebrate gC1qR have yet been identified, the physiological functions and their underlying molecular mechanisms remain to be elucidated. gC1qR has been shown to bind to a broad range of molecular patterns, including antibodyeantigen complexes, bacteria, and viruses. Up to date, gC1qR has been indicated to bind a number of pathogen proteins, including HIV1 Rev [9], adenovirus core protein V [10], herpes simplex virus open reading frame protein P [11], EBNA-1 of EpsteineBarr virus [12], protein A of Staphylococcus aureus [13,14], Listeria monocytogenes internalin B [15], and protein P22 of Hepatitis B virus [16,17]. The data suggests a gC1qR trigger strategy to the host immune response. Numerous reports have demonstrated that gC1qR may play an important role in innate immunity, however, physiological roles of these proteins in invertebrates, especially in crustaceans, remain to be elucidated. In the previous research, FcgC1qR from Fenneropenaeus chinensis might be involved in anti-bacterial infection [18]. PmC1qBP, also a gC1qR, had cross-binding activity to the mouse C1q and it was upregulated by Vibrio vulnificus challenge [19]. PlgC1qR from

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Pacifastacus leniusculus could inhibit WSSV replication in vitro and in vivo [20]. However, WSSV-induced formation of a calreticulin/ gC1qR complex could inhibit apoptosis of hematopoietic tissue cell culture (Hpt) and help WSSV replication [21]. Although there are some research focusing on function of gC1qR in crustaceans, -no information of prawns gC1qR could be obtained up to date. The giant freshwater prawns, a commercially important species in aquaculture, has been greatly challenged by diseases [22]. The outbreak and spread of pathogen have resulted in the high mortality of farmed prawns and huge economic losses [23,24]. Due to the lack of a true adaptive immune response system, prawns completely depend on the innate immune system against pathogen invasion [23e25]. So it is imperative to further understand the innate immunity of prawns and explore new approaches against the diseases. In this study, we have focused on the cDNA cloning, characterization, and the response to microbial infection of gC1qR in Macrobrachium rosenbergii in order to get further insight of prawns immune defense mechanism. The expression patterns following pathogen challenge, microorganisms binding assay, and protein interaction in vitro were also analyzed in order to understand their functions in host defense against pathogens. This study demonstrates the roles of gC1qR in innate immunity of the giant freshwater prawns. 2. Materials and methods 2.1. Prawns, immune challenge, samples collection The giant prawns, M. rosenbergii (about 15 g each) were purchased from an aquaculture market in Hangzhou, Zhejiang Province, China. The prawns were cultured at room temperature (25  C) with circulating water. The prawns were then divided into2 groups: WSSV-challenged group and Vibrio anguillarum-challenged group. The WSSV inoculum was prepared following the previous paper [26]. About 100 mL of filtrate (105 WSSV copies/mL) was injected into the lateral area of the fourth abdominal segment of the prawns using a syringe with 29-gauge needle. At 0, 2, 6, 12 and 24 h post WSSV or Vibrio challenge, the hepatopancreas was isolated from the prawns for RNA extraction. Other samples of hemocytes, gills, stomach and intestine from unchallenged prawns were also collected for RNA isolation. For hemocytes isolation, hemolymph was first collected from 3 prawns (untreated) by mixing with 1/10 volume anticoagulant buffer (0.14 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, and 10 mM EDTA, pH 4.6). Then, the hemolymph was centrifuged at 800 g to isolate the hemocytes. 2.2. RNA isolation, cDNA synthesis and gene cloning of MrgC1qR The total RNA was extracted from the above-mentioned samples using EasyPure RNA Kit (TransGen, Beijing, China) following the manufacturer's protocols. The concentration of RNA was measured using Nanodrop (Thermo). The first-strand cDNA was produced for the qRT-PCR analysis using a PrimeScript™ RT Reagent Kit (Perfect Real Time) (Takara, Dalian, China) with an oligo dT primer. For gene cloning of the 3'end of MrgC1qR, the first strand cDNA was synthesized using 30 -Full RACE Core Set Ver. 2.0 (Takara, Dalian, China) with 30 RACE Adaptor primer. A gene specific forward primer (MrgC1qR-F: 50 -ATGAGTCTGTTTAGTCGGG CC-30 ) was designed based on the EST sequence obtained from the hepatopancreas transcriptome data.30 fragment was amplified from the hepatopancreas using MrgC1qR-F and 30 RACE Outer Primer (50 -TACCGTCGTTCCACTAGTGATTT-30 ). The full


length of the MrgC1qR cDNA was obtained by overlapping the 30 fragment and EST sequence. 2.3. Sequence analysis and construction of the phylogenetic tree Multiple alignments of MrgC1qR were performed with BLASTX ( cDNA translation and deduced protein prediction were obtained using ExPASy ( Domain composition was predicted by SMART ( A phylogenetic tree of MrgC1qR was constructed using MEGA 5.05 [27]. 2.4. Tissue distribution and expression pattern of MrgC1qR Real-time PCR was performed to detect the tissue distribution of MrgC1qR in hemocytes, hepatopancreas, gills, stomach, and intestine. The expression pattern of MrgC1qR in hepatopancreas post-pathogen challenge was also studied using real-time PCR. The primers used in the qRT-PCR analysis are as follows: MrgC1qRRT-F: 5'-AAGTCTATGTCTTCTGGCTTGCC-30 , MrgC1qR -RT-R:50 eTCTTCTGCTAAAGCTTCCCCTGG-3'. MrGAPDH gene was also amplified as a reference gene and the primers used were: MrGAPDH-RT-F: 5’eACCACCAACTCAATCACCATGTC-30 , MrGAPDH-RT-R: 5’eAGAGCA CCGTCCTCAGCCTT-3’. PCR conditions were as follows: 94  C for 2 min, followed by 40 cycles of 94  C for 20 s, 60  C for 1 min. A 2  Premix Ex Taq™ (ROX plus) (Takara, Dalian, China) was used in the qRT-PCR experiment according to the manufacturer's instructions. The qRT-PCR experiment was conducted on the Stepone Real-Time PCR System (ABI, American). All samples were repeated in triplicates in the qRT-PCR analysis. The data was calculated using 2DDCt methods [28] and then subjected to statistical analysis. An unpaired sample t-test was conducted, and the difference was considered significant if P < 0.05. 2.5. Recombinant expression of MrgC1qR-GST or MrgC1qR-His For recombinant expression in Escherichia coli, the MrgC1qR gene was amplified and cloned into the pGEX-4T-1 or pET30avector. The primers used for the recombinant expression of MrgC1qR were as follows: Ex-MrgC1qR-F: 50 -TACTCAGAATTCATGAGTCTGTTTAGTCGGGCC-30 , with an EcoR I site (italic), Ex-MrgC1qR-R: 50 -TACTCACTCGAGTTATTCTCTCTCTACAAAATCCTG30 , with an Xho I site (italic). The amplified fragments were cloned intopET30a or pGEX-4T-1 vector and then were sequenced to ensure the correct open reading frame (ORF). Afterward, the recombinant plasmids pET30a-MrgC1qR or pGEX-4T-1-MrgC1qR were transformed to Transetta (DE3) cells (Transgen, Beijing) for IPTG-induced recombinant expression. The recombinant MrgC1qRGST or MrgC1qR-His proteins were then purified using Glutathione Sepharose 4 Fast Flow (Amersham Biosciences, USA) or His Mag Sepharose™ Ni (GE, USA) respectively. The methods were followed according to the manufacturer's instruction. The purified MrgC1qRHis proteins were used as antigens to rabbit to prepare antibodies. The titres of antisera were approximately 1:5000 as determined by enzyme-linked immunosorbent assay (ELISA). The immunoglobulin (IgG) fraction was purified by proteinA-Sepharose (Bio-Rad, USA) and stored at 70  C. 2.6. Binding assay for recombinant MrgC1qR-His proteins to microorganisms Gram-positive bacteria (Bacillus thuringiensis, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, S. aureus, and Micrococcus luteus) and Gram-negative bacteria (E. coli, V. anguillarum, V. harveyi, A. hydrophila) were used to detect binding activity of the


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Fig. 1. The full length cDNAs and deduced amino acid sequences of MrgC1qR from M. rosenbergii. The signal peptides and MAM33 domain of MrgC1qRis shown with gray. The Polyadenylation signal (AATAAA) was aslo marked with gray.

recombinant MrgC1qR-His proteins. After overnight culturing, the bacteria were spun down (4000 g) at 4  C, then washed and resuspended in TBS to OD600 of 1.0. The purified recombinant MrgC1qR (1 mg/mL; 100 mL) in binding buffer (50 mmol/L TriseHCl(pH 8.0)) was incubated with 100 mL of bacteria (2  107 cells/ml) for 1 h at 37  C. The bacteria were pelleted, washed four times with TBS, and subjected to elution with 7% SDS for 1 min [28]. Eluted proteins were collected and analyzed by 12.5% SDS-PAGE under reducing conditions. Recombinant

MrgC1qR-His was detected by MrgC1qR antibody using Western blot. 2.7. LPS and PGN binding assay Lipopolysaccharide (LPS) and peptidoglycan (PGN) binding activity of recombinantMrgC1qR-His was determined by ELISA as previously described [29]. In brief, sugar was dissolved in water (80 mg/ml) and 50 ml of sugar solution was added into each well of a

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T. Ye et al. / Fish & Shellfish Immunology 43 (2015) 200e208

Fig. 3. Phylogenetic analysis of M. rosenbergii gC1qRgC1qR with other known gC1qR of sequences from GenBank. NJ tree was produced with MEGA 4. Litopenaeus vannamei gC1qR (GenBank accession no. AGO21477), Fenneropenaeus chinensis gC1qR (GenBank accession no. AFJ59951), Penaeus monodon gC1qR (GenBank accession no. ADV18978), Pacifastacus leniusculus gC1qR (GenBank accession no. AEC50078), B. mori gC1qR (GenBank accession no. ABD36320), Zootermopsis nevadensis gC1qR (GenBank accession no. KDR12558), A. aegypti gC1qR (GenBank accession no. XP_001661410), M. domestica gC1qR (GenBank accession no. XP_005186565), C. formosanus gC1qR (GenBank accession no. AGM32084), P. xuthus gC1qR (GenBank accession no. BAM18194), C. quinquefasciatus gC1qR (GenBank accession no. XP_001850674), Anopheles gambiae PEST gC1qR (GenBank accession no. XP_318437), A. darlingi gC1qR (GenBank accession no. ETN58839), Daphnia pulex gC1qR (GenBank accession no. EFX69077), Ceratitis capitata gC1qR (GenBank accession no. XP_004520309), Nasonia vitripennis gC1qR (GenBank accession no. XP_008206466), Harpegnathos saltator gC1qR (GenBank accession no. EFN75748), Riptortus pedestris gC1qR (GenBank accession no. BAN21443), Lepeophtheirus salmonis gC1qR (GenBank accession no. ACO12436), Pediculus humanus corporis gC1qR (GenBank accession no. XP_002423727), Cerapachys biroi gC1qR (GenBank accession no. EZA55849), Acromyrmex echinatior gC1qR (GenBank accession no. EGI59094), Ixodes scapularis gC1qR (GenBank accession no. XP_002400595), Tribolium castaneum gC1qR (GenBank accession no. XP_967206), Camponotus floridanus gC1qR (GenBank accession no. EFN62645), Bombus impatiens gC1qR (GenBank accession no. XP_003489258), Apis dorsata gC1qR (GenBank accession no. XP_006621860), Apis mellifera gC1qR (GenBank accession no. XP_397201.2), Apis florea gC1qR (GenBank accession no. XP_003696723).

microtitre plate. Afterward, the microtitre plate was incubated at 37  C overnight and heated at 60  C for 30 min. Then, it was blocked with 50 ml of bovine serum albumin (BSA; 1 mg/ml) at 37  C for 2 h and then washed with TBS for 4 times. The purified proteins with gradient dilution, 0e30 mg/ml in TBS containing 0.1 mg/ml BSA, were added to the wells and incubated at 28  C for 3 h. The wells were washed 4 times; 100 ml of antiserum against rMrgC1qR (1/300 diluted in 0.1 mg/ml BSA) was then added and incubated at 37  C for 1 h. Each well was washed 4 times and then incubated with 100 ml of peroxidase-conjugated goat anti-rabbit IgG (1/3000

diluted) for 1 h at 37  C. The plate was washed 4 times with TBS and developed with 0.01% 3,30 ,5,50 -tetramethylbenzidine (Sigma). Approximately 2 M H2SO4 was used to stop the reaction. The absorbance was read and noted at 450 nm. The assays were performed in triplicate. 2.8. Western-blot After being separated in a 12% SDS-PAGE gel, proteins were transferred onto a nitrocellulose membrane (Bio-Rad, America).

Fig. 2. Multiple alignments of M. rosenbergii gC1qR with gC1qR of other species. gC1qR from Litopenaeus vannamei (GenBank accession no. AGO21477), Fenneropenaeus chinensis (GenBank accession no. AFJ59951), Penaeus monodon (GenBank accession no. ADV18978), Pacifastacus leniusculus (GenBank accession no. AEC50078), M. domestica (GenBank accession no. XP_005186565), A. aegypti (GenBank accession no. XP_001661410), A. darlingi (GenBank accession no. ETN58839), B. mori (GenBank accession no. ABD36320), C. formosanus (GenBank accession no. AGM32084), C. quinquefasciatus (GenBank accession no. XP_001850674), P. xuthus (GenBank accession no. BAM18194) Identities were shaded darker.

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Fig. 4. Relative expression of MrgC1qR in different tissues of F. chinensis. The data determined by quantitative Real-time RT-PCR. Total RNA was extracted from hepatopancreas, hemocyte, intestine, stomach and gill of healthy shrimp respectively. The experiment is representative of three independent experiments. Each column represented the mean of triplicate assays within ±1% standard deviation. The statistically differences between interested treatments were represented with star (*P < 0.05, **P < 0.01).

The membrane was blocked in PBS containing 5% non-fat dried milk for 1 h, and incubated with the primary antibody at 4  C overnight. Subsequently, the membrane was washed in TBS containing 0.05% Tween and incubated with HRP-conjugated secondary antibody. Two hours later, proteins in the membrane were detected with the reaction mixture (1 mL of 4-chloro-1-naphthol in methanol (6 mg/mL), 9 mL of TBS, and 6 mL of H2O2) in the dark for 10 min. 2.9. GST-pull down In order to indicate the interaction between MrgC1qR and MrFico1 or MrFico2, GST-pull down assay was performed as follows: GST or MrgC1qR-GST was incubated with Glutathione Sepharose 4 Fast Flow, followed by washing with10 ml PBS buffer and then MrFico1 or MrFico2 with His-tag were added and incubated at 4  C for 2 h. After washing thoroughly with 10 ml PBS buffer, the proteins were eluted by adding PBS containing 10 mM reduced glutathione and then analyzed on 12.5% SDSePAGE.

Fig. 5. Quantitative real-time PCR analysis of MrgC1q R expression in hepatopancreas (Hep) in response to WSSV or V. anguillarum. (A) Quantitative real-time PCR analysis of MrgC1qRinhepatopancreas of shrimp at 2, 6, 12 and 24 h post WSSV infection. (B), Quantitative real-time PCR analysis of MrgC1qRin hepatopancreas of shrimp challenged with V. anguillarum at 2, 6, 12 and 24 h. Each column indicated the mean of triplicate assays. The statistically differences between interested treatments were represented with star (*P < 0.05, **P < 0.01).

3. Results 3.1. Sequence analysis of MrgC1qR gene from the freshwater prawns and phylogenetic tree The full length of MrgC1qR (KP259729) gene was 1153 bp in length, including a 99 bp 50 untranslated region (UTR), a 774 bp ORF encoding a 258 amino acids protein, and a 280 bp 30 UTR with the polyadenylation signal (AATAAA), 11 bp upstream of polyA tail. SMART analysis indicated that MrgC1qR protein had a signal peptide, a MAM33 domain with 182 amino acids (Fig. 1). The molecular weight and pI of MAM33 of MrgC1qR was 20.54 kDa and 4.11, respectively. To elucidate the conserved of MrgC1qR, multiple alignments of amino acid sequences were conducted with other species. The analysis revealed that MrgC1qR had high similarity with gC1qR from other species (Fig. 2), suggesting that the function of MrgC1qR might be preserved during animal evolution. The

amino acid sequence of MrgC1qR shared 72.24%, 72%, 71.10%, 69.23%, 45.15%, 42.18%, 42.16%, 44%, 41.7%, 41.09%, 40.94% identities with those of Litopenaeus vannamei, F. chinensis, Penaeus monodon, P. leniusculus, Musca domestica, Aedes aegypti, Anopheles darlingi, Bombyx mori, Coptotermes formosanus, Culex quinquefasciatus, Papilio xuthus, respectively. MrgC1qR and other gC1qR from different species was used for phylogenetic analysis (Fig. 3). The phylogenetic tree showed that MrgC1qR, together with gC1qR proteins from P. leniusculus, L. vannamei, F. chinensis,P. monodon, Daphnia pulex, Lepeophtheirus salmonis, belong to crustacean group. 3.2. Tissue distribution and qRT-PCR analysis of MrgC1qR expression RT-PCR was used to detect MrgC1qR expression indifferent shrimp tissue. From the results, MrgC1qR was a widely distributed


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Fig. 6. Binding activity of MrgC1qR. (A) Direct binding of recombinant MrgC1qR to bacterial. Purified recombinant MrgC1qR in buffer A (50 mM TriseHCl, pH 8.0) was incubated with bacterial at37  C for 1 h. Bacteria were pelleted, washed four times with TBS and then subjected to elution with 7% SDS for 1 min. Recombinant MrgC1qR was detected by MrgC1qR antibody. Lane headings represented the different bacterium. (B) LPS and PGN binding assay. LPSor PGN was coated at 5 mg/ml in bicarbonate buffer (0.1 M, pH 9.6) on 96well flat-bottom microtiter plate (at 4  C overnight. After washing and blocking with 1% BSA in PBS, purified recombinant MrgC1qR proteins (10 mg/ml, 20 mg/ml, 30 mg/ml) were incubated for 2 h at 37  C, respectively. Bound proteins were detected by MrgC1qR antibody, followed measured at 405 nm by ELISA microplate reader. All the assays were repeated for three times.

gene and was detected in hepatopancreas, hemocytes, gills, stomach, and intestine (Fig. 4). The result also showed the MrgC1qR was highest expressed in hepatopancreas, while the expression in the other tissues (gill, hemolymph, stomach and intestine) were equal and relatively low. To further understand the time-course of MrgC1qR gene transcription upon stimulation with WSSV or V. anguillarum, the temporal expression of MrgC1qR in hepatopancreas was measured by using a real-time PCR method with b-actin as an internal control. The results revealed that the transcript of hepatopancreas MrgC1qR was upregulated after a 6 h WSSV challenge and then recovered to the basal level (Fig. 5A). qRT-PCR showed that hepatopancreasMrgC1qR was upregulated 2 h post V. anguillarum infection then declined. MrgC1qR was still higher after 24 h V. anguillarum challenge than in nonchallenged shrimp (Fig. 5B).

indicated that the binding activity was significantly increased with the increase of the MrgC1qR concentration at the range of 10e30 mg/ml (Fig. 6B). The results also displayed that MrgC1qR had stronger affinity for LPS than that of PGN. 3.4. The interaction between MrgC1qR and ficolins in the freshwater prawns In order to find out whetherMrgC1qR could interact with ficolins from the freshwater prawns, GST pull-down assay was performed. The results showed that the recombinant MrgC1qR-GST could bind to ficolins (MrFico1-His and MrFico2-His), while the control GST could not bound to ficolins, indicating the existence of the MrgC1qR e ficolin interaction in the freshwater prawns (Fig. 7). 4. Discussion

3.3. Binding activity of MrgC1qR To characterize the pattern recognition of MrgC1qR, the protein of MrgC1qR was expressed recombinantly and purified, followed by the detection of MrgC1qR binding activity. Direct binding assays showed that MrgC1qR could bind both the Gram-negative bacterium, E. coli, V. anguillarum, A. hydrophila and the Gram-positive bacterium B. megaterium, B. thuingiensis, B. subtilis, B. cereus, S. aureus, M. luteus (Fig. 6A). PGN forms the outer wall of Gram-positive bacteria, lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria. To further study the ability of MrgC1qR binding LPS and PGN, ELISA assay was performed. The results

Diseases are the greatest challenge to shrimp aquaculture. A better understanding of shrimp immune response will be helpful for disease control. Although gC1qRis a multi-compartmental and a multifunctional protein, knowledge of its roles in immune response still remains limited, particularly in invertebrates. In this study, it is revealed that the molecular characterization of gC1qR from the giant freshwater prawn, M. rosenbergii and its roles in immune response. The current study has revealed that the MrgC1qR shared high similarities with other species, suggesting the function of gC1qR is conservative. The MrgC1qR gene was a widely distributed in various examined tissues. Real-time PCR showed that the expression of

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Fig. 7. MrgC1qR interaction with ficolin. GST or MrgC1qR-GST was purified in glutathione agarose beads, followed incubated with MrFico1 or MrFico2 with His-tag respectively. After washing thoroughly PBS buffer, the proteins were eluted and analyzed on SDSePAGE.GST was used as control. Lane headings represented the different treatment.

MrgC1qR was highest in hepatopancreas. The hepatopancreas is a key organ involved in the immune response of shrimp and a primary site for the production of immune recognition molecules. The outer spaces of arterioles in hemal spaces of the hepatopancreas are distributed with the fixed phagocytes that are involved in eliminating the pathogens [30]. WSSV infection increased the expression level of MrgC1qR in hepatopancreas after 6 h of WSSV challenge, which may indicate an association with immune activities. The observation of MrgC1qR upregulation at 2 h post V. anguillarum infection also supports this supposition. Thus, a role in antipathogenic defense could be implicated by the upregulation of the MrgC1qR transcript in shrimp hepatopancreas following bacterial or virus challenge. FcgC1qR was also induced by bacteria and WSSV challenge [18]. PmC1qBP was upregulated by V. vulnificus challenge [19]. P. leniusculus PlgC1qR itself could inhibit WSSV replication and once formation of a calreticulin/gC1qR complex, it could inhibit apoptosis of hematopoietic tissue cell culture (Hpt) and help WSSV replication [20,21]. Taken together, it has shown that the expression level of MrgC1qR is the highest in hepatopancreas and upregulated after pathogen stimulation, suggesting thatMrgC1qR may play an important role in host defense. Innate immunity is classically considered to be the immediate response mounted through the recognition of common or conserved residues derived from pathogens. Such residues are referred to as pathogen-associated molecular patterns (PAMPs). Recognition of PAMPs is primarily via a restricted number of highly conserved, high-affinity receptors termed pattern recognition receptors (PRRs) [31,32]. In the present study, MrgC1qR could bind both the Gram-negative bacterium, E. coli, V. anguillarum, A. hydrophila and the Gram-positive bacterium B. thuingiensis, B. subtilis, B. cereus, S. aureus, B. megaterium, M. luteus. PGN forms the outer wall of Gram-positive bacterium, lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria [33,34], the results reveal that MrgC1qR bind PAMPs, LPS or PGN, suggesting MrgC1qR could take part in innate immunity. In this study, GST-pull down assay was showed rMrgC1qR-GST could bind MrFicolin1-His and MrFicolin2-His. In the previous research, it was found that both MrFicolin1 and MrFicolin2 could bind bacteria and also bind to LPS or PGN and also help clearance of bacteria [29]. DC-SIGN, a C-type lectin expressed on dendritic cell (DC), C1q, and gC1qR could form a trimolecular complex [35]. In

vertebrates, Ficolins share similar structure with complement C1q and ficolins have an N-terminal collagen-like domain and a C-terminal fibrinogen-like domain [36]. Both MrFicolin1 and MrFicolin2 have a coiled coil region at the N-terminal [29]. sghC1q, a novel C1q family member from half-smooth tongue sole (Cynoglossus semilaevis) also has a coiled coli region [37]. PmC1qBP, also a gC1qR, had cross-binding activity to the mouse C1q [19]. So, it could be speculated that MrgC1qR interact with MrFicolins through the same mechanism of gC1qR interaction with C1q. In all, MrgC1qR may participate in innate immunity through the interaction of MrFicolins. In conclusion, this study reports the first cloning of MrgC1qR from, the giant freshwater prawn, M. rosenbergii. Through expression pattern analysis upon pathogen challenge, it was revealed that MrgC1qR may play an important role in host defense. Recombinant MrgC1qR could bind bacteria, LPS, PGN and also could interact with MrFicolin1 and MrFicolin2. Altogether, it could be speculated that MrgC1qR might function as a PRR in the giant freshwater prawns. Acknowledgment The current study was supported by the National Natural Science Foundation of China (Grant Nos. 31302220, 31101926), Scientific Research Foundation of Zhejiang Sci-Tech University (13042164-Y), the Zhejiang Provincial Top Key Discipline of Biology (2012A11-C), The National Natural Science Foundation of Jiangsu Province (BK20131401), Natural Science Fund of Colleges and universities in Jiangsu Province (13KJB240002). References [1] Ghebrehiwet B, CebadaMora C, Tantral L, Jesty J, Peerschke EI. gC1qR/p33 serves as a molecular bridge between the complement and contact activation systems and is an important catalyst in inflammation. Current topics in complement. Springer; 2006. p. 95e105. [2] Kishore U, Reid K. C1q: structure, function, and receptors. Immunopharmacology 2000;49:159e70. [3] Kim K-B, Yi J-S, Nguyen N, Lee J-H, Kwon Y-C, Ahn B-Y, et al. Cell-surface receptor for complement component C1q (gC1qR) is a key regulator for lamellipodia formation and cancer metastasis. J Biol Chem 2011;286: 23093e101. [4] Fogal V, Zhang L, Krajewski S, Ruoslahti E. Mitochondrial/cell-surface protein p32/gC1qR as a molecular target in tumor cells and tumor stroma. Cancer Res 2008;68:7210e8.


T. Ye et al. / Fish & Shellfish Immunology 43 (2015) 200e208

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Characterization of a gC1qR from the giant freshwater prawn, Macrobrachium rosenbergii.

gC1qR, as a multicompartmental and a multifunctional protein, plays an important role in innate immunity. In this study, a gC1qR homolog (MrgC1qR) in ...
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