An opioid growth factor receptor (OGFR) for [Met5]-enkephalin in Chlamys farreri

Fish & Shellfish Immunology 34 (2013) 1228e1235

Contents lists available at SciVerse ScienceDirect

Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

An opioid growth factor receptor (OGFR) for [Met5]-enkephalin in Chlamys farreri Ying Guo a, b, Lingling Wang a, Zhi Zhou a, Mengqiang Wang a, Rui Liu a, Leilei Wang a, b, Qiufen Jiang a, b, Linsheng Song a, * a b

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Rd., Qingdao 266071, China University of Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 October 2012 Received in revised form 4 February 2013 Accepted 4 February 2013 Available online 24 February 2013

Opioid growth factor receptor (OGFR) is a receptor for [Met5]-enkephalin and plays important roles in the regulation of cell growth and embryonic development. In the present study, a cDNA of 2381 bp for the scallop Chlamys farreri OGFR (designated as CfOGFR) was identified by rapid amplification of cDNA ends (RACE) approach and expression sequence tag (EST) analysis. The complete cDNA sequence of CfOGFR contained an open reading frame (ORF) of 1200 bp, which encoded a protein of 399 amino acids. The amino acid sequence of CfOGFR shared 33e64% similarity with other OGFRs. There was a low complexity domain and a conserved OGFR_N domain at the N-terminal of CfOGFR. The mRNA transcripts of CfOGFR were constitutively expressed in the tested tissues with the highest expression level in hepatopancreas. During the early embryonic development, the mRNA transcripts of CfOGFR could be detected in different development stages, where the expression level presented a downward trend as a whole. The stimulations of LPS, Glu and poly (I:C) significantly induced the expression of CfOGFR mRNA in hemocytes (P < 0.05), while PGN stimulation exerted no influence. Co-IP and western blot results revealed that the CfOGFR in hemocytes displayed high affinity and specificity to [Met5]-enkephalin. Exogenous [Met5]-enkephalin was observed to inhibit the proliferation of HEK293T cells transfected with pcDNA3.1(þ)-CfOGFR in a time and dosage dependent manner. These results collectively indicated that CfOGFR, as a homolog of OGFRs in C. farreri, played an important role in cells proliferation, and might be involved in the immune response of scallops. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Chlamys farreri [Met5]-enkephalin Opioid growth factor receptor Cell proliferation Immune response

1. Introduction [Met5]-enkephalin is a native opioid peptide, and it plays an important role in cell proliferation and tissue organization during development other than serving as a neuromodulator in the nervous system [1e3]. To distinguish its role as an inhibitory growth factor, [Met5]-enkephalin is also termed as opioid growth factor (OGF) [4]. OGF exhibits a broad spectrum of biological activities mediated by opioid growth factor receptor (OGFR), which is initially expressed on the periphery of outer nuclear envelope and detaches from it after binding to OGF [5,6]. The complex OGF-OGFR is then rapidly shift to the para-nuclear cytoplasm and enters the nucleus to exert functions [7]. The natural activity of OGFR is to regulate cell growth [8e11], and it is also involved in embryonic development [12], cellular renewal and wound repair [13].

* Corresponding author. Tel.: þ86 532 82898552; fax: þ86 532 82880645. E-mail address: [email protected] (L. Song). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.02.002

Both OGF and OGFR are present in an extensive range of the phylum Chordata, including mammalian, aves, reptilia, amphibia and osteichthyes [14], and the multifunction of OGF-OGFR system has been widely reported in rat, mouse, and human cells and tissues [15e17]. The up-regulation of OGFR could stimulate the interfacing of OGF with OGFR, which in turn enhanced the suppression of cell proliferation of various cancer cells [18]. OGFR was involved in the injury repair of the corneal epithelium in the rabbit [19] and the renewal of epithelium in murine tongue [20]. It was also identified from embryonic derivatives including ectoderm, mesoderm, and endoderm of the rat on gestation day 20 and certified to restrain DNA synthesis in a wide variety of organ systems during prenatal life [12]. Imiquimod, an immune response modifier with potent antiviral and anti-tumor properties, might not only suppress tumor growth but also increase tumor immunogenicity by up-regulating the OGFR expression [21]. Though there are fewer reports about OGFRs in invertebrates, it is fascinating to find that OGF could specifically bind to the bacterial strain Staphylococcus aureus [22]. It is speculated that OGF and OGFR might have appeared as early as 2 billion years ago, the time corresponding to the evolution of

Y. Guo et al. / Fish & Shellfish Immunology 34 (2013) 1228e1235

bacteria [9]. It seems interesting to find out if OGFRs in invertebrates exert similar functions to vertebrate type counterparts. Mollusk is one of the most spectacular animal phyla with great taxonomic diversity and morphological disparity [23], and many opioid peptides including OGF and opioid peptide precursors have been identified from mollusk [24e26]. Zhikong scallop, Chlamys farreri, is an important fishery and aquaculture mollusk species as well as an ideal model for studying neurobiology. Characterization of the OGFR in scallops would provide a better understanding of the origin and evolutionary process of OGFRs, and the knowledge on its potential immune functions might contribute to the health management and disease control in scallop aquaculture. The purposes of the present study were (1) to identify and characterize OGFR from scallop C. farreri (designated as CfOGFR), (2) to detect its mRNA expression in different stages of ontogenesis and different tissues of adult scallops, (3) to monitor its temporal expression in hemocytes of scallops stimulated by pathogen associated molecular patterns (PAMPs), (4) to investigate the potential functions of CfOGFR in regulating cell proliferation and immune response. 2. Materials and methods 2.1. Immune stimulation of scallops and tissue collection Healthy scallops with an average shell length of 55 mm were collected from a local farm in Qingdao, China. They were acclimated in the aerated seawater for a week before processing. Two hundred and forty scallops employed for the PAMPs stimulation experiment were randomly divided into 6 equivalent groups. Five groups of them received an injection of 50 ml phosphate buffered saline (PBS; 0.14 M sodium chloride, 3 mM potassium chloride, 8 mM disodium hydrogenphosphate dodecahydrate, 1.5 mM potassium phosphate monobasic, pH 7.4), lippolysaccharides (LPS) from Escherichia coli 0111:B4 (SigmaeAldrich, USA; 0.5 mg ml1 in PBS), peptidoglycan (PGN) from S. aureus (SigmaeAldrich, USA; 0.8 mg ml1 in PBS), bglucan (Glu) from Saccharomyces cerevisiae (SigmaeAldrich, USA; 1.0 mg ml1 in PBS), and polyinosinic-polycytidylic acid (Poly (I:C)) (SigmaeAldrich, USA; 1.0 mg ml1 in PBS), respectively. The untreated scallops were employed as blank. After injection, scallops were returned to water tanks and five individuals of each group were randomly sampled at 3, 6, 12, 24 and 48 h post injection. The hemolymphs were collected and centrifuged at 800 g, 4  C for 10 min to harvest the hemocytes for RNA preparation. Six kinds of tissues including kidney, gonad, mantle, gill, adductor muscle and hepatopancreas from six healthy adult scallops were collected as parallel samples to determine the distribution of CfOGFR mRNA. Haemolymphs from the corresponding scallops were also gathered from the adductor muscle and immediately centrifuged at 800 g, 4  C for 10 min to harvest the hemocytes. For the temporal expression profile of CfOGFR mRNA during the ontogenesis of scallops, oocytes, embryos and larvae were sampled according to the previous report [27]. The samples were collected from fifteen development stages, including oocytes, fertilized eggs, 2-cell embryos, 4-cell embryos, 8-cell embryos, 16-cell embryos, 32-cell embryos, morula, blastula, gastrula, trochophore, D-hinge larvae, early veliger larvae, late veliger larvae and eyespots period. All the sampled tissues were stored at 80  C after addition of 1 ml Trizol reagent (Invitrogen, USA) for subsequent RNA preparation. 2.2. RNA extraction and cDNA synthesis Total RNA was extracted from the tissues of scallops using Trizol reagent (Invitrogen, USA). The synthesis of the first-strand cDNA was carried out according to the method described by Wang et al.

1229

[28]. The cDNA mixture was diluted to 1:100 and stored at 80  C for subsequent SYBR Green fluorescent quantitative real-time PCR (qRT-PCR) assay. 2.3. EST analysis and cloning of the full-length CfOGFR cDNA BLAST analysis of the EST sequences from scallop C. farreri [29] revealed that one EST of 264 bp (rscag0_002606) was homologous to the OGFRs identified previously. Eight specific primers (Table S1) were designed based on the EST sequence to clone the full-length cDNA of CfOGFR by rapid amplification of cDNA ends (RACE) approach. PCR amplification to obtain the 30 end of CfOGFR was carried out using sense primer P1, P2, P3, P4 and antisense primer Oligo (dT)-adaptor P9, while sense primer Oligo (dG)adaptor P10 and antisense primer P5, P6, P7 and P8 were used to get the 50 end of CfOGFR. All the PCR amplifications were performed in a PCR Thermal Cycle (Takara, Japan), and the amplified specific PCR products were gel-purified and cloned into pMD18-T simple vector (Takara, Japan). After transformed into the competent cells of E. coli Top10, the positive recombinants were identified through anti-ampicillin selection and PCR screening with primers RV-M and M13-47 (Table S1). The positive clones were sequenced on an ABI 3730 XL Automated Sequencer (Applied Biosystems, USA). To verify the sequence of CfOGFR, a reciprocal amplification was carried out using primers P11 and P12. The sequencing results were verified and subject to cluster analysis. 2.4. Sequence analysis The homology search of the cDNA and protein sequences of CfOGFR was conducted with BLAST algorithm (http://blast.ncbi. nlm.nih.gov/Blast.cgi). The Expert Protein Analysis System (http:// www.expasy.org) was employed to analyze the deduced amino acid sequence. SignalP 3.0 program was used to predict the presence and location of signal peptides, and the cleavage sites in amino acid sequence (http://www.cbs.dtu.dk/services/SignalP). The protein domains were predicted using the simple modular architecture research tool (SMART) version 5.1 (http://smart.embl-heidelberg. de/). Multiple sequences alignment of the CfOGFR with other OGFRs was created by the ClustalW multiple alignment program (http://www.ebi.ac.uk/Tools/clustalw2/) and multiple sequences alignment show program (http://www.biosoft.net/sms/index. html). An unrooted phylogenic tree was constructed based on the deduced amino acid sequence of CfOGFR and other OGFRs by the neighbor-joining (NJ) algorithm using the MEGA 5 software. To derive the confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. 2.5. qRT-PCR analysis of CfOGFR mRNA expression The qRT-PCR analysis was conducted to investigate the expression of CfOGFR during the ontogenesis, the distribution of its mRNA transcripts in different tissues of adult scallops, and its temporal expression profile in the hemocytes of scallops post PAMPs stimulation. A PCR product of 116 bp was amplified with the primers P15 and P16 (Table S1) from cDNA template and sequenced to verify the PCR specificity. Two primers for CfEF1a (elongation factor 1 alpha from scallop C. farreri), P13 and P14 (Table S1), were used to amplify an 86 bp fragment, which acted as an internal control to verify the successful reverse transcription and to calibrate the cDNA template for corresponding scallop samples. The qRT-PCR assay was carried out in an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, USA) as described by Zhang et al. [30]. Dissociation curve analysis of amplification products was performed to confirm that only one PCR product was amplified and detected. The 2DDCt

1230

Y. Guo et al. / Fish & Shellfish Immunology 34 (2013) 1228e1235

method was used to analyze the expression level of CfOGFR mRNA [31]. All data were given in terms of the relative mRNA expression. 2.6. Recombinant and protein purification of CfOGFR The cDNA fragment encoding the mature CfOGFR was recombined and expressed as described by Zhou et al. [32] with primers P11, P12, T7 and T7ter (Table S1). The recombinant protein CfOGFR (designated as rCfOGFR) was purified by Ni2þ chelating sepharose column [33], separated electrophoretically on reducing 12% SDSPAGE, and visualized with coomassie bright blue R-250. The rCfOGFR was stored at 80  C for subsequent experiments. 2.7. Preparation of antiserum to rCfOGFR and western blot assay For antiserum preparation, the renatured rCfOGFR protein was dialyzed continually against ddH2O before it was freeze concentrated. The rCfOGFR was immuned to a 6-week old SPF KM female rat (Institute for Drug Control in Qingdao, China) to prepare polyclonal antibodies as described by Cheng [34]. The blood of the immuned rat was collected and allowed to clot at 4  C overnight and then centrifuged at 3000 g, 4  C for 20 min. The antiserum in the supernatant was divided into small aliquots and stored at 80  C for subsequent experiments after being tested via western blot according to the previous report [35]. 2.8. OGF-CfOGFR binding assay Co-immunoprecipitation (Co-IP) was conducted to evaluate the binding ability of CfOGFR to OGF according to the previous report [36]. The haemolymphs of five scallops (about 0.5 ml per individual) were collected using a syringe, which contained the same volume of pre-cooled (4  C) modified Alsever solution (0.12 M glucose, 0.03 M sodium citrate, 9 mM EDTA and 0.38 M sodium chloride, pH 7.2) as an anticoagulant. The haemolymphs were pooled together and mixed homogeneously, and then immediately divided into three equivalent groups, named as the experimental group, control group A and control group B, respectively. Firstly, OGF was added to the experimental group and control group B (0.5 mg ml1), respectively, and then the haemolymphs of the three groups were centrifuged at 800 g, 4  C for 10 min to harvest the hemocytes after being incubated at room temperature with shaking at 100 rpm for 2 h. Secondly, the hemocytes were washed with sterile PBS three times, and then dissociated by Cell Lysis Buffer for Western and IP analysis (Beyotime, China). The lysate was centrifuged at 12,000 g, 4  C for 5 min to collect the supernatant. Thirdly, 5 ml of normal rabbit serum and 20 ml protein G sepharose were mixed with 1 ml of the collected supernant and incubated at 4  C for 2 h. Then the mixture was centrifuged at 1200 g, 4  C for 1 min to collect the supernatant. Fourthly, 5 ml of anti-Met-Enkephalin antibody (Abcam, USA) together with 40 ml of protein G sepharose was added to the supernatant of the experimental group and control group A, whereas 5 ml of normal rabbit serum and 40 ml of protein G sepharose were added to control group B. Finally, after being set at 4  C overnight, the incubated mixture was centrifuged at 1200 g, 4  C for 1 min and the supernatant was discarded. The protein G sepharose was washed with sterile PBS for five times and resuspended in 100 ml of loading buffer for SDS-PAGE. The resuspended matter was boiled for 5 min and the supernatant was employed for western blot with the antiserum to rCfOGFR as the test antibody. 2.9. Cell proliferation assay The cell proliferation assay was performed to verify the property of CfOGFR as a viable receptor for OGF. The recombinant

plasmid pcDNA3.1(þ)-CfOGFR was constructed according to Zhou [32], with the primers P17, P18, M13F, M13R, BGH rev, T7 and T7ter (Table S1). HEK293T, human embryonic kidney cell (ATCC NO. CRL1573), was purchased from the cell bank of CAS in Shanghai, China, and cultured in DMEM medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA), 100 IU ml1 penicillin (Invitrogen, USA), 100 IU ml1 streptomycin (Invitrogen, USA) at 37  C in a humidified incubator with 5% CO2. The cells in the logarithmic phase were transiently transfected with the pcDNA3.1(þ)-CfOGFR plasmid or the blank expression vector pcDNA3.1 using Lipofectamine LTXÔ Reagent (Invitrogen, USA), respectively. Twenty four hours later, the transfected cells were collected and seeded at equivalent amounts (104 per well) into two 24-well plates (Costar, USA) for two sets of individual experiments. In experiment I, 20 ml per well of [Met5]-enkephalin (Sigmae Aldrich, USA; prepared in sterile PBS, 102 M) or sterile PBS was added to the 24-well plate at 6 h, 18 h and 30 h after seeding, respectively. The processed cells in the plate were observed under a microscope (Carl Zeiss, USA) at 42 h after seeding and resupplied with fresh culture medium containing 10% (v/v) WST-1 solution (Beyotime, China). The plate was incubated for an additional 4 h and then the OD450 and OD690 were measured. Each treatment was performed three times for statistical analysis. In experiment II, 5, 10, or 20 ml per well of [Met5]-enkephalin (102 M) was added to the transfected cells at 6 h after seeding, respectively. The plate was processed as the former one after 24 h incubation with [Met5]enkephalin. 2.10. Statistical analysis All data were given as means  S.D. and subjected to one-way analysis of variance (one-way ANOVA) followed by a multiple comparison (S-N-K). Differences were considered significant at P <0.05. 3. Result 3.1. The molecular features of CfOGFR A fragment of 2361 bp representing the complete cDNA of CfOGFR was obtained by overlapping EST rscag0_002606 with the amplified fragments. The sequence was deposited in GenBank under accession No. JX000476. The complete cDNA sequence of CfOGFR contained an open reading frame (ORF) of 1200 bp that encoded a protein of 399 amino acids (Fig. S1). The 50 untranslated region (UTR) was of 81 bp and the 30 UTR was of 1080 bp with a poly (A) tail. The predicted molecular weight of the deduced amino acid of CfOGFR was 45.98 kDa and its theoretical isoelectric point was 4.98. An OGFR_N domain was identified in the N-terminal of CfOGFR protein sequence, with a low complexity domain observed upstream it. 3.2. The homology of CfOGFR with other OGFRs BLAST analysis revealed that the deduced amino acid sequence of CfOGFR shared 33e64% similarity with other OGFRs and the higher similarity was observed in the N-terminals by the alignment of CfOGFR with other OGFRs (Fig. S2). Based on the amino acid sequences of OGFRs, a phylogenic tree was constructed by the Mega 5 program using neighbor-joining (NJ) method with 1000 bootstrap test (Fig. 1). CfOGFR was closest to OGFR from Crassostrea gigas, and they two were first clustered with OGFRs from Xenopus tropicalis and vertebrates, then gathered with OGFR from Tetrahymena thermophila, and finally clustered with OGFRs from bacteria.

Y. Guo et al. / Fish & Shellfish Immunology 34 (2013) 1228e1235

1231

3.4. The temporal expression profile of CfOGFR during the ontogenesis of scallops The mRNA transcripts of CfOGFR in the early development stages were detected by qRT-PCR and its expression was detectable in all the tested development stages from oocytes to eyespots period. The relative mRNA expression level of CfOGFR exhibited a downward trend during the whole ontogenesis whereas there was a little regression in the late veliger larvae. The first sharp drop appeared in the fertilized eggs (0.64-fold to the level of oocytes) and the second appeared in the 16-cell embryos (0.05-fold to the level of oocytes). The lowest expression level of CfOGFR mRNA was observed in the blastula (0.016-fold to the level of oocytes) (Fig. 3). 3.5. The response of CfOGFR mRNA in hemocytes against PAMPs stimulation Fig. 1. Neighbor-joining tree based on the amino acid sequences of different OGFRs. The numbers at the forks indicate the bootstrap. The species of OGFRs used for phylogenetic analysis include: Homo sapiens (NP_031372), Rattus norvegicus (NP_445792), Mus musculus (NP_113550), Crassostrea gigas (EKC24098), Bos taurus (NP_001070487), Gallus gallus (XP_425708), Cricetulus griseus (EGW11059), Danio rerio (XP_003201204), Salmo salar (CAJ90908), Taeniopygia guttata (XP_002194613), Vibrio parahaemolyticus (ZP_05121006), Oscillatoria sp. (ZP_07110015), Pan troglodytes (XP_001146234), Pseudomonas syringae pv. (EGH82438), Macaca mulatta (XP_001087181), Tetrahymena thermophila (XP_001027192), Xenopus (Silurana) tropicalis (NP_001090756), Anolis carolinensis (XP_003220721).

3.3. The tissue distribution of CfOGFR mRNA The qRT-PCR was employed to investigate the tissue distribution of CfOGFR mRNA with CfEF1a as the internal control. There was only one peak at the corresponding melting temperature of CfOGFR and CfEF1a genes in the dissociation curve analysis, indicating that the PCR was specifically amplified (data not shown). The mRNA transcripts of CfOGFR were detected in all the tested tissues including kidney, gonad, hemocytes, mantle, gill, adductor muscle and hepatopancreas. The highest expression level was observed in hepatopancreas, which was about 146-fold of that in kidney (P < 0.05). It was higher expressed in adductor muscle and gill (P < 0.05), which were about 17.5-fold and 11.5-fold of that in kidney, respectively. The expression levels were comparatively low in mantle, hemocytes, gonad and kidney (Fig. 2).

Fig. 2. Tissue distribution of the CfOGFR mRNA. The expression levels of gonad, hemocytes, mantle, gill, adductor muscle and hepatopancreas are normalized to that of kidney. Each group values are shown as mean  S.D. (N ¼ 6), and bars with different letters are significantly different (P < 0.05).

The temporal expression of CfOGFR mRNA in hemocytes of scallops after PAMPs simulation was monitored by qRT-PCR (Fig. 4). In the LPS stimulation group, the mRNA expression of CfOGFR was significantly up-regulated from 24 h post stimulation, and reached the peak value (6.4-fold compared to blank group) at 48 h post stimulation (P < 0.05). The same temporal expression profile of CfOGFR mRNA was observed in the poly (I:C) stimulation group, whose peak value (3.5-fold compared to blank group) also appeared at 48 h post stimulation (P < 0.05). The injection of Glu significantly induced the expression of CfOGFR mRNA from 3 h post stimulation, and the peak value (8.9-fold compared to blank group) was observed at 24 h post stimulation (P < 0.05). No obvious change of CfOGFR mRNA expression was observed in the PGN stimulation group (P > 0.05). In the control group, there was no significant change (P > 0.05) of CfOGFR mRNA expression during the whole experiment period after PBS stimulation. 3.6. The rCfOGFR and its antiserum After IPTG induction, the whole cell lysate of E. coli BL21 (DE3) with pEASY-E1-CfOGFR was analyzed by SDS-PAGE, and a distinct band with a molecular weight of w52 kDa was revealed (Fig. 5). A clear reaction band of rCfOGFR was revealed by western blot,

Fig. 3. Temporal expression profile of the CfOGFR mRNA in the ontogenesis of scallop C. farreri. The fifteen detected stages are: oocytes (O), fertilized eggs (F), 2-cell embryos (E2), 4-cell embryos (E4), 8-cell embryos (E8), 16-cell embryos (E16), 32-cell embryos (E32), morula (M), blastula (B), gastrula (G), trochophore (T), D-hinge larvae (D), early veliger larvae (Evl), late veliger larvae (Lvl) and eyepots period (Ep). Each group values are shown as mean  SD (N ¼ 6), and bars with different letters are significantly different (P < 0.05).

1232

Y. Guo et al. / Fish & Shellfish Immunology 34 (2013) 1228e1235

Fig. 4. The response of CfOGFR mRNA in hemocytes against PAMPs stimulation. Each group values are shown as mean  S.D. (N ¼ 5), and bars with different letters are significantly different (P < 0.05).

validating the specificity of autonomous antiserum to rCfOGFR (Fig. 6). 3.7. The binding ability of CfOGFR to OGF Co-IP assay was employed to verify the binding ability of CfOGFR to OGF. A clear reaction band of CfOGFR was revealed in the experimental group whereas no visible reaction band was detected in control groups (Fig. 7), indicating that CfOGFR bond to OGF with high affinity and specificity. 3.8. The inhibitory effect of CfOGFR on cell proliferation Transfection and WST-1 assay were employed to mirror the effect of OGF and CfOGFR on cell proliferation. The morphological structure of transfected HEK293T cells incubated with either [Met5]-enkephalin or sterile PBS closely resembled that of the normal ones under a microscope (Carl Zeiss, USA). Exogenous [Met5]-enkephalin induced a significant inhibition on the proliferation of transfected cells in a time and dosage dependent manner. The cell proliferation level began to decline at 12 h after [Met5]enkephalin treatment and the descending trend lasted to the end of the experiment (Fig. 8A). The lowest level of cell proliferation was observed at 36 h after [Met5]-enkephalin treatment, which was 0.37-fold to that of the group without [Met5]-enkephalin treatment. The proliferation level of the transfected cells incubated with 0.25, 0.5, 1 mM [Met5]-enkephalin was 0.90-fold, 0.32-fold and 0.24fold to that of the group without [Met5]-enkephalin at 24 h after [Met5]-enkephalin treatment, respectively (Fig. 8B). 4. Discussion OGFR gene has been identified in mammals such as human, rat and mouse, and its molecular structure has no resemblance to that of classical opioid receptors or significant homologies to known domains or functional motifs, except for a bipartite nuclear localization signal (NLS) [9]. The greatest identity and similarity for the amino acid sequences of OGFRs in mammals occurred at their Nterminals, while the variation of repeated sequences in the C-terminals made various OGFRs distinguished [37]. In the present study, an OGFR gene was identified from scallop C. farreri with an ORF of 1200 bp, a 50 UTR of 81 bp, a 30 UTR of 1080 bp. BALST

Fig. 5. SDS-PAGE assay of rCfOGFR. Lane A: bacteria lysate without IPTG induction. Lane B: bacteria lysate with IPTG induction. Lane C: purified rCfOGFR.

analysis revealed that the deduced amino acid sequence of CfOGFR shared 33e64% similarity with that of other OGFRs. A low complexity domain and a conserved OGFR_N domain were identified in the deduced amino acid sequence of CfOGFR, whereas the NLS and the imperfect repeats conserved in mammalian OGFRs were not observed [9,14]. Similarly, the NLS and imperfect repeats were not found in the amino acid sequences of OGFRs from C. gigas and T. thermophila. In fish Atlantic salmon, the imperfect repeats were also absent from the C-terminal of OGFR even it shared 62% similarity with its human isoform [38]. It was speculated that the NLS and the imperfect repeats possibly appeared gradually in the process of evolution. In the phylogenic tree, CfOGFR together with its closest homolog, OGFR from C. gigas, were firstly clustered with OGFRs from X. tropicalis and vertebrates, then T. thermophila, and finally clustered with OGFRs from bacteria. These results collectively indicated that CfOGFR was a homolog of OGFR in C. farreri. As a receptor for OGF, OGFR from mammals such as mouse, rat and human displayed specific and saturable binding to OGF [9]. In

Y. Guo et al. / Fish & Shellfish Immunology 34 (2013) 1228e1235

1233

Fig. 6. Western blot assay of rCfOGFR. The left lane: protein molecular standard (kDa). The right lane: rCfOGFR reacting with the antiserum.

the present study, Co-IP and western blot assays were conducted to verify the binding ability of CfOGFR to OGF. A clear reaction band with high specificity was revealed, and its manifested molecular weight was consistent with that of rCfOGFR, indicating that the protein bond to OGF was actually CfOGFR. The result suggested that

Fig. 7. Western blot assay for the binding ability of CfOGFR to OGF. Lane marker: protein molecular standard (kDa). Lane A: Control group A. Lane B: Control group B. Lane C: Experimental group.

Fig. 8. A Cell proliferation assay with different [Met5]-enkephalin incubation time. Each group values are shown as mean  S.D. (N ¼ 3), and bars with different letters are significantly different (P < 0.05). B Cell proliferation assay with different [Met5]enkephalin concentrations. Each group values are shown as mean  S.D. (N ¼ 3), and bars with different letters are significantly different (P < 0.05).

CfOGFR protein was constitutively expressed in hemocytes and it should share similar domain with mammalian OGFRs to bind OGF. It has been reported that the regulation of human cell proliferation by OGFR is dependent on nucleocytoplasmic translocation, and this process requires the integrity of two NLSs in OGFR to interact with transport receptors [7]. As there was no classical NLS in the deduced amino acid sequence of CfOGFR, it needs to be ascertained whether CfOGFR could inhibit the cell proliferation as well. In the present study, the valid recombinant plasmid, pcDNA3.1(þ)-CfOGFR, was constructed and transfected into HEK293T cells. The exogenous OGF was found to inhibit the proliferation of the transfected cells in a time and dosage dependent manner. It was speculated that the OGF-OGFR system in vertebrates was able to inhibit DNA synthesis, directly toward the G1eS interface of the cell, and enhance the cyclin-dependent kinase inhibitory pathways [11,39,40]. Though the present study could not provide the specific mechanism about the interaction of OGF with CfOGFR, it exhibited that CfOGFR inhibited the transfected cells proliferation through communicating with other human cell elements synergistically, indicating the conservatism in the functional domains of OGFRs. The absence of NLS and imperfect repeats in the CfOGFR protein sequence somewhat reduced its structure specificity, meanwhile, perhaps facilitated the multiplicity of its functions.

1234

Y. Guo et al. / Fish & Shellfish Immunology 34 (2013) 1228e1235

Moreover, during the early embryonic development, the mRNA transcripts of CfOGFR were detectable in different development stages and its level presented a downward trend as a whole. Given its ability to inhibit cell proliferation, the down-regulation of CfOGFR during the development stages was probably the result of scallop’s self-regulation, which in turn promoted the tissue development of scallop larvae. OGF and OGFR were reported to be present in normal human ovarian surface epithelial (HOSE) cells, benign ovarian cysts, and ovarian cancers. However, the OGF and OGFR protein levels were significantly reduced 29% and 34%, respectively, in ovarian cysts, and significantly decreased 58% and 48%, respectively, in ovarian cancers, suggesting that OGF and OGFR might be associated with ovarian cancer carcinogenesis [41]. These phenomena tempted us to suspect that the cell proliferation might be out of control without the normal regulation of OGF and OGFR. The presence of CfOGFR during the development stages prevented the abnormal proliferation, while its down-regulation facilitated the tissue development. The mRNA transcripts of CfOGFR were constitutively expressed in the tested tissues of adult scallop, and the highest expression level was observed in hepatopancreas. As hepatopancreas was considered as one of the main immune tissues of scallops [42,43], the higher mRNA expression of CfOGFR in hepatopancreas suggested that CfOGFR might be associated with the immune regulation. Circulating hemocytes in scallops are main immunocytes responsible for the immune defense, and they recognize and eliminate pathogens mainly by phagocytosis, encapsulation, nodulation and oxidative killing [44e46]. To gain the preliminary insight of its effects in the immune response of scallops, the temporal expression profile of CfOGFR mRNA in hemocytes was monitored after four typical PAMPs stimulations. In comparison with hepatopancreas, even the expression level of CfOGFR mRNA was relatively low in hemocytes, it was markedly enhanced by LPS, Glu and poly (I:C) stimulations (P < 0.05), suggesting that CfOGFR could be efficiently induced by some specific pathogen stimulations and might be involved in the immune response of scallops. It has been proved that the recognition of specific pathogens by hemocytes would induce the production of cytokines such as INF-a and TNF-a, and result in an enhanced immune response. In addition, IFN-a was reported to up-regulate the expression of OGFR mRNA in two basal cell carcinoma cell lines and keratinocytes to inhibit cell proliferation [21]. In the present study, LPS, Glu and poly (I:C) were suspected to be recognized by hemocytes and then induce the production of IFN-a like molecules to up-regulate the expression of OGFR, which in turn might depress the immune response of scallops to prevent its overactivation. However, further investigation is still needed to better understand the functions of OGFR, especially its involvement in the ontogenesis and immune response of invertebrates. Acknowledgments The authors would thank all of the colleagues in our lab for helpful discussion and technical advice. This research was supported by National Basic Research Program of China (973 Program, No. 2010CB126404) from the Chinese Ministry of Science and Technology, and grants (No. 41276169 to L.W. and No. 30925028 to L.S.) from National Science Foundation of China, and Shandong Provincial Natural Science Foundation (No. JQ201110 to L.W.). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2013.02.002.

References [1] Isayama T, McLaughlin PJ, Zagon IS. Endogenous opioids regulate cell-proliferation in the retina of developing rat. Brain Res Bull 1991;544: 79e85. [2] McLaughlin PJ. Regulation of DNA synthesis during prenatal heart development by the opioid growth factor, Met5-enkephalin. Circulation 1996;94:697. [3] Murgo AJ. Inhibition of B16-BL6 melanoma growth in mice by methionineenkephalin. J Natl Cancer Inst 1985;75:341e4. [4] Zagon IS, McLaughlin PJ. Identification of opioid peptides regulating proliferation of neurons and glia in the developing nervous system. Brain Res Bull 1991;542:318e23. [5] Leslie FM. Methods used for the study of opioid receptors. Pharmacol Rev 1987;39:197e249. [6] Zagon IS, Goodman SR, McLaughlin PJ. Characterization of zeta (z): a new opioid receptor involved in growth. Brain Res Bull 1989;482:297e305. [7] Cheng F, McLaughlin PJ, Verderame MF, Zagon IS. Dependence on nuclear localization signals of the opioid growth factor receptor in the regulation of cell proliferation. Exp Biol Med 2009;234:532e41. [8] Wu Y, McLaughlin PJ, Zagon IS. Ontogeny of the opioid growth factor, Met5enkephalin, preproenkephalin gene expression, and the zeta opioid receptor in the developing and adult aorta of rat. Dev Dyn 1998;211:327e37. [9] Zagon IS, Verderame MF, McLaughlin PJ. The biology of the opioid growth factor receptor (OGFr). Brain Res Bull 2002;38:351e76. [10] Malendowicz LK, Rebuffat P, Tortorella C, Nussdorfer GG, Ziolkowska A, Hochol A. Effects of Met5-enkephalin on cell proliferation in different models of adrenocortical cell growth. Int J Mol Med 2005;15:841e5. [11] Cheng F, McLaughlin PJ, Verderame MF, Zagon IS. The OGF-OGFr axis utilizes the p16(INK4a) and p21(WAF1/CIP1) pathways to restrict normal cell proliferation. Mol Biol Cell 2009;20:319e27. [12] Zagon IS, Wu Y, McLaughlin PJ. Opioid growth factor and organ development in rat and human embryos. Brain Res Bull 1999;839:313e22. [13] Sassani JW, Zagon IS, McLaughlin PJ. Opioid growth factor modulation of corneal epithelium: uppers and downers. Curr Eye Res 2003;26:249e62. [14] Zagon IS, Sassani JW, Allison G, McLaughlin PJ. Conserved expression of the opioid growth factor, Met5-enkephalin, and the zeta (z) opioid receptor in vertebrate cornea. Brain Res Bull 1995;671:105e11. [15] Zagon IS, Smith JP, McLaughlin PJ. Human pancreatic cancer cell proliferation in tissue culture is tonically inhibited by opioid growth factor. Int J Oncol 1999;14:577e84. [16] Zagon IS, Wu Y, McLaughlin PJ. The opioid growth factor, Met5-enkephalin, and the zeta (z) opioid receptor are present in human and mouse skin and tonically act to inhibit DNA synthesis in the epidermis. J Invest Dermatol 1996;106:490e7. [17] McLaughlin PJ, Levin RJ, Zagon IS. The opioid growth factor receptor in human head and neck squamous cell carcinoma. Int J Mol Med 2000;5:191e6. [18] Zagon IS, Donahue RN, Rogosnitzky M, McLaughlin PJ. Imiquimod upregulates the opioid growth factor receptor to inhibit cell proliferation independent of immune function. Exp Biol Med 2008;233:968e79. [19] Zagon IS, Sassani JW, McLaughlin PJ. Re-epithelialization of the rabbit cornea is regulated by opioid growth factor. Brain Res Bull 1998;803:61e8. [20] Zagon IS, Wu Y, McLaughlin PJ. Opioid growth factor inhibits DNA synthesis in mouse tongue epithelium in a circadian rhythm-dependent manner. Am J Physiol 1994;267:R645e52. [21] Urosevic M, Oberholzer PA, Maier T, Hafner J, Laine E, Slade H, et al. Imiquimod treatment induces expression of opioid growth factor receptor: a novel tumor antigen induced by interferon-alpha? Clin Cancer Res 2004;10: 4959e70. [22] Zagon IS, McLaughlin PJ. An opioid growth factor regulates the replication of microorganisms. Life Sci 1992;50:1179e87. [23] Glaubrecht M. On “Darwinian Mysteries” or molluscs as models in evolutionary biology: from local speciation to global radiation. Am Malacol Bull 2009;27:3e23. [24] Stefano GB, Leung M. Purification of opioid peptides from molluscan ganglia. Cell Mol Neurobiol 1982;2:347e52. [25] Leung M, Stefano GB. Isolation of molluscan opioid peptides. Life Sci 1983;33: 77e80. [26] Stefano GB, Martin R. Enkephalin-like immunoreactivity in the pedal ganglion of Mytilus edulis (bivalvia) and its proximity to dopamine-containing structures. Cell Tissue Res 1983;230:147e53. [27] Zhou Z, Wang L, Shi X, Yue F, Wang M, Zhang H, et al. The expression of dopa decarboxylase and dopamine beta hydroxylase and their responding to bacterial challenge during the ontogenesis of scallop Chlamys farreri. Fish Shellfish Immunol 2012;33:67e74. [28] Wang M, Yang J, Zhou Z, Qiu L, Wang L, Zhang H, et al. A primitive Toll-like receptor signaling pathway in mollusk Zhikong scallop Chlamys farreri. Dev Comp Immunol 2011;35:511e20. [29] Wang L, Song L, Zhao J, Qiu L, Zhang H, Xu W, et al. Expressed sequence tags from the Zhikong scallop (Chlamys farreri): discovery and annotation of hostdefense genes. Fish Shellfish Immunol 2009;26:744e50. [30] Zhang H, Song L, Li C, Zhao J, Wang H, Qiu U, et al. A novel C1q-domaincontaining protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity. Fish Shellfish Immunol 2008;25:281e9.

Y. Guo et al. / Fish & Shellfish Immunology 34 (2013) 1228e1235 [31] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2DDCT method. Methods 2001;25:402e8. [32] Zhou Z, Wang L, Wang M, Zhang H, Wu T, Qiu L, et al. Scallop phenylalanine hydroxylase implicates in immune response and can be induced by human TNF-a. Fish Shellfish Immunol 2011;31:856e63. [33] Leandro P, Rivera I, Lechner MC, de Almeida IT, Konecki D. The V388M mutation results in a kinetic variant form of phenylalanine hydroxylase. Mol Genet Metab 2000;69:204e12. [34] Cheng S, Zhan W, Xing J, Sheng X. Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China. J Virol Methods 2006;135:173e80. [35] Yang J, Wang L, Zhang H, Qiu L, Wang H, Song L. C-Type lectin in Chlamys farreri (CfLec-1) mediating immune recognition and opsonization. PLoS One 2011;6(2):e17089. [36] Klenova E, Chernukhin I, Inoue T, Shamsuddin S, Norton J. Immunoprecipitation techniques for the analysis of transcription factor complexes. Methods 2002;26:254e9. [37] Zagon IS, Verderame MF, Allen SS, McLaughlin PJ. Cloning, sequencing, chromosomal location, and function of cDNAs encoding an opioid growth factor receptor (OGFr) in humans. Brain Res Bull 2000;856:75e83. [38] Matejusova I, Felix B, Sorsa-Leslie T, Gilbey J, Noble LR, Jones CS, et al. Gene expression profiles of some immune relevant genes from skin of susceptible and responding Atlantic salmon (Salmo salar L.) infected with Gyrodactylus safaris (Monogenea) revealed by suppressive subtractive hybridisation. Int J Parasitol 2006;36:1175e83.

1235

[39] Cheng F, Zagon IS, Verderame MF, McLaughlin PJ. The opioid growth factor (OGF)-OGF receptor axis uses the p16 pathway to inhibit head and neck cancer. Cancer Res 2007;67:10511e8. [40] Zagon IS, Roesener CD, Verderame MF, Ohlsson-Wilhelm BM, Levin RJ, McLaughlin PJ. Opioid growth factor regulates the cell cycle of human neoplasias. Int J Oncol 2000;17:1053e61. [41] Fanning J, Hossler CA, Kesterson JP, Donahue RN, McLaughlin PJ, Zagon IS. Expression of the opioid growth factor-opioid growth factor receptor axis in human ovarian cancer. Gynecol Oncol 2012;124:319e24. [42] Wootton EC, Dyrynda EA, Ratcliffe NA. Bivalve immunity: comparisons between the marine mussel (Mytilus edulis), the edible cockle (Cerastoderma edule) and the razor shell (Ensis siliqua). Fish Shellfish Immunol 2003;15:195e210. [43] Zhao J, Song L, Li C, Zou H, Ni D, Wang W, et al. Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein. Mol Immunol 2007;44:1198e208. [44] Costa MM, Prado-Alvarez M, Gestal C, Li H, Roch P, Novoa B, et al. Functional and molecular immune response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes against pathogen-associated molecular patterns and bacteria. Fish Shellfish Immunol 2009;26:515e23. [45] Travers M-A, da Silva PM, Le Goic N, Marie D, Donval A, Huchette S, et al. Morphologic, cytometric and functional characterisation of abalone (Haliotis tuberculata) haemocytes. Fish Shellfish Immunol 2008;24:400e11. [46] Cao A, Ramos-Martinez J-I, Barcia R. In haemocytes from Mytilus galloprovincialis Lmk., treatment with corticotropin or growth factors conditions catecholamine release. Int Immunopharmacol 2007;7:1395e402.