Characterization and identification of calmodulin and calmodulin binding proteins in hemocyte of the black tiger shrimp (Penaeus monodon)

Developmental and Comparative Immunology 50 (2015) 87–97

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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i

Characterization and identification of calmodulin and calmodulin binding proteins in hemocyte of the black tiger shrimp (Penaeus monodon) Panjana Sengprasert a, Piti Amparyup b,c, Anchalee Tassanakajorn b, Ratree Wongpanya a,* a

Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Pahonyothin, Bangkok 10900, Thailand Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand c National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand b

A R T I C L E

I N F O

Article history: Received 29 October 2014 Revised 4 February 2015 Accepted 5 February 2015 Available online 10 February 2015 Keywords: Calmodulin Penaeus monodon Shrimp Immune response Signaling protein

A B S T R A C T

Calmodulin (CaM), a ubiquitous intracellular calcium (Ca2+) sensor in all eukaryotic cells, is one of the well-known signaling proteins. Previously, CaM gene has shown a high transcriptional level in hemocyte of the pathogen infected shrimp, suggesting that shrimp CaM does not only regulate Ca2+ metabolism, but is also involved in immune response cascade. In the present study, the CaM gene of shrimp Penaeus monodon was identified and the recombinant P. monodon CaM (rPmCaM) was produced and biochemically characterized. The identification of CaM-binding proteins was also performed. The PmCaM cDNA consisted of an open reading frame of 447 bp encoding for 149 amino acid residues with a calculated mass of 16,810 Da and an isoelectric point of 4.09. Tissue distribution showed that the PmCaM transcript was expressed in all examined tissues. The results of gel mobility shift assay, circular dichroism spectroscopy and fluorescence spectroscopy all confirmed that the conformational changes of the rPmCaM were observed after the calcium binding. According to the gene silencing of PmCaM transcript levels, the shrimp’s susceptibility to pathogenic Vibrio harveyi infection increased in comparison with that of the control groups. Protein pull-down assay and LC–MS/MS analysis were performed to identify rPmCaMbinding proteins involved in shrimp immune responses and transglutaminase, elongation factor 1-alpha, elongation factor 2 and actin were found. However, by computational analysis, only the first three proteins contained CaM-binding domain. These findings suggested that PmCaM may play an important role in regulation of shrimp immune system. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The black tiger shrimp, Penaeus monodon, is an important marine species that is widely cultivated especially in tropical countries. The shrimp productions increase every year in Asia and Latin America (Bachère, 2000). However, shrimp aquaculture has faced significant problems with disease outbreaks resulting in the production collapses and economic losses (Flegel, 2006). Like other invertebrates, shrimp defense mechanism is relied mainly on the innate immunity which consists of physical barrier, humoral and particularly cellular responses. In the humoral barrier, many antimicrobial peptides, pathogen recognition receptors (PRRs), prophenoloxidase

* Corresponding author. Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Pahonyothin, Bangkok 10900, Thailand. Tel.: +66 2 562 5555 ext 2012; fax: +66 2 561 4627. E-mail address: [email protected] (R. Wongpanya). http://dx.doi.org/10.1016/j.dci.2015.02.003 0145-305X/© 2015 Elsevier Ltd. All rights reserved.

(proPO) and clotting proteins are produced to eradicate pathogens (Aguirre-Guzman et al., 2009; Zhao et al., 2009). For the cellular response, hemocyte plays a crucial role in phagocytosis, encapsulation and nodule formation (Aguirre-Guzman et al., 2009; Li and Xiang, 2012). However, these immune systems function in a concerted fashion for an effective response. Although the shrimp immune response has been extensively studied, co-operative working of each cascade via signaling pathway is still unclear. Therefore, the elucidation of proteins involved in the immune signaling system is required. Calcium (Ca2+) ions play crucial roles in cell signaling, helping the activation of many proteins for their various functions such as enzymes, transcription factors and transducers in cytosol. The signal transduction is induced by altering the intracellular concentration of Ca2+ ions which depends on various stresses such as external bioenergy, changes in membrane polarization, drug uptake and pathogen infection (Ji et al., 2011; Kiang et al., 2002; Pyrko et al., 2007; Vetter and Leclerc, 2003). Ca2+ ions can regulate proteins in

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the signal transduction by binding directly to the proteins or binding to a Ca2+ binding protein that subsequently binds to a target protein (Aslam et al., 2012; Clough et al., 2002; Xu et al., 2012). Calmodulin (CaM) is one of the several proteins reported as a calcium binding protein. CaM, a ubiquitous intracellular calcium (Ca2+) sensor in all eukaryotic cells, is one of the well-known signaling proteins. The amino acid sequence of CaM is highly conserved through the evolution. This protein can bind and regulate proteins in various cascades. Structural analysis of CaM from various organisms has been characterized (Chattopadhyaya et al., 1992; Symersky et al., 2003). It was found that the CaM contains N- and C-terminal lobes linking with a flexible central linker. Each lobe is composed of two EF hand motifs with two Ca2+ binding sites (Babu et al., 1988; Ban et al., 1994; Chou et al., 2001). The binding of Ca2+ ions causes the conformational change of CaM by bringing two helices of each lobe to more perpendicular conformation and exposing hydrophobic residues on their surface. Therefore, CaM is able to interact with specific target proteins and regulate their functions (Meador et al., 1992; Wu et al., 2012). In marine invertebrate, not only CaM does regulate Ca2+ metabolism of intracellular processes, but also plays a crucial role in extracellular processes such as biomineralization in mussel leading to shell formation (Zeng et al., 2012) and the regulation of molting in crayfish, Procambarus clarkii (Gao et al., 2009). In shrimp, CaM was firstly isolated from Crangon crangon and its biological and physiological properties were similar to other invertebrate CaMs (Michael et al., 1992). Recently, it was revealed that CaM gene was highly expressed in Vibrio harveyi infected shrimp hemocyte (Wongpanya et al., 2007). Additionally, the expression levels of CaM in shrimp hemocytes of Litopenaeus vannamei and P. monodon were altered after the pathogen infection (Ji et al., 2011; Somboonwiwat et al., 2010). This indicated that CaM might relate in shrimp pathogen defense mechanism. Although, CaM-mediated processes have been extensively studied, the information of CaM involved in shrimp pathogen defense mechanism is still limited. In this present study, P. monodon CaM gene was identified and the effect of CaM gene knockdown was elucidated. Moreover, a recombinant P. monodon CaM (rPmCaM) was produced and biochemically characterized and CaM-binding proteins were also identified. 2. Material and methods 2.1. Sample preparation Healthy shrimps (P. monodon) were obtained from a commercial farm in Chantaburi, Thailand. The shrimps (20 g) were reared in a water system with a salinity of 20 ppt at 25 °C for 3 days before experiments. Hemolymph was first withdrawn from the ventral sinus at the first abdominal segment using 10% (w/v) sodium citrate as an anticoagulant. The sample was then centrifuged for 20 min at 10,000 rpm, 4 °C. Hemocyte pellet was collected for RNA extraction. Subsequently, various tissues including gills, hepatopancreas, hearts, lymphoid organs and intestine were dissected and their RNA extractions were carried out. 2.2. Total RNA extraction and sequence analysis Total RNA was extracted from hemocyte using RNeasy mini Kit (Qiagen). The total RNA was then treated with RNase-free-DNase I (Promega) to eliminate contaminant DNA. First-strand cDNAs were synthesized using oligo (dT) primers and the ImProm-IITM Reverse Transcriptase System kit (Promega), according to the manufacturer’s instruction. The cDNA was subsequently used as a template for open reading frame (ORF) amplification. Specific primers were designed based on EST database from Chulalongkorn University. The PCR product was determined by agarose gel electrophoresis and

purified by HiYield Gel/PCR DNA Fragment Extraction Kit (RBC Bioscience). The purified PCR product was cloned into pGEM®-T Easy vector (Promega), generating pGEM-PmCaM. The sequence of CaM was confirmed using T7 and SP6 primers (Macrogen). The PmCaM gene was analyzed using BLASTX program with GenBank database. Multiple sequence alignments of various organism CaMs were performed using the ClastalW2 analysis program (http://www.ebi.ac.uk/Tools/clustalw2). Domain architecture was predicted by PROSITE Scan (https://www.ebi.ac.uk/Tools/ pfa/ps_scan/). 2.3. Tissue-specific expression The mRNA expression level of PmCaM in various tissues of healthy shrimp was determined using RT-PCR. Total RNA was extracted from hemocyte, gills, hepatopancreas, hearts, lymphoid organs and intestines as described previously. The specific primers, PmCaM-F and PmCaM-R, were used to amplify PmCaM. An elongation factor 1-α (EF1α) amplified by EF1α-F and EF1α-R primers (Table 1) was used as an internal control. PCR thermocycling conditions were as follows 95 °C for 5 min; 30 cycles of 95 °C for 1 min, 55 °C for 30 s and 72 °C for 1 min, and a final heating at 72 °C for 10 min. The RT-PCR product was analyzed by 1.2% (w/v) agarose gel electrophoresis. 2.4. Synthesis of double-stranded RNA Double stranded RNA (dsRNA) corresponding to the PmCaM sequence was generated by in vitro transcription as described by Charoensapsri et al. (2009). Briefly, DNA template for PmCaM dsRNA preparation was amplified by PCR using gene-specific primers, PmCaM-F and PmCaM-R (Table 1). The PCR product was then used as a template for the synthesis of dsRNA using primers consisting of the same primer sequences flanked by T7 promoter sequence at 5′ end. Sense and anti-sense DNA templates were generated separately using two pairs of primer, PmCaMT7-F and PmCaM-R and PmCaM-F and PmCaMT7-R, respectively (Table 1). For an exogenous gene, green fluorescent protein (GFP) gene amplification was carried out using pEGFP-1 vector as a template with specific primers, GFPT7-F and GFP-R (Table 1) for the sense strand template and GFP-F and GFPT7-R for the anti-sense strand template (Table 1). A proper amount (0.5 μg) of each template was used in an in vitro transcription using the T7 RiboMAXTM Express Large Scale RNA Production Systems (Promega), according to the manufacturer’s instruction. Equal amounts of sense and anti-sense single stranded RNA were annealed to produce dsRNA. To get rid of the remaining DNA template in the solution, the mixture was treated with RNase-free DNase I. The quality and amount of dsRNAs were determined by 1.2% (w/

Table 1 Primer sequences used for RT-PCR analysis. Primer

Sequence (5′–3′)

PmCaMexp-F

5′-CATGCCATGGGCCATCATCATCATCATCATATGGCGGATCAGC TGACCGAAG-3′ 5′-ATAAGAATGCGGCCGCTCACTTCGAGGTCATCATC-3′ 5′-ACAGTCATGAGGTCCTTGGG-3′ 5′-TCTCTCCGAGGTTGGTCATC-3′ 5′-GGATCCTAATACGACTCACTATAGGGACAGTCATGAGGTCCTT GGG-3′ 5′-GGATCCTAATACGACTCACTATAGGGTCTCTCCGAGGTTGGTC ATC-3′ 5′- ATGGTGAGCAAGGGCGAGGA-3′ 5′- TTACTTGTACAGCTCGTCCA-3′ 5′-TAATACGACTCACTATAGGGATGGTGAGCAAGGGCGAGGA-3′ 5′-TAATACGACTCACTATAGGGTTACTTGTACAGCTCGTCCA-3′ 5′-GGTGCTGGACAAGCTGAAGGC-3′ 5′-CGTTCCGGT GATCATGTTCTTGAT-3′

PmCaMexp-R PmCaM-F PmCaM-R PmCaMT7-F PmCaMT7-R GFP-F GFP-R GFPT7-F GFPT7-R EF1α-F EF1α-R

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v) agarose gel electrophoresis and UV spectrophotometry, respectively. 2.5. In vivo PmCaM gene silencing P. monodon (2 g, fresh weight) was intramuscularly injected with 10 μg of either PmCaM or GFP dsRNA (5 μg dsRNA per 1 g shrimp) dissolved in 30 μl saline solution (150 mM NaCl), using a 1-ml insulin syringe with a 27-gauge needle. Control shrimp was injected with 30 μl saline solution. The injection of dsRNA or saline solution was repeated at 24 h after the first dsRNA injection. Total RNA of three sample groups was collected and extracted from the hemocyte of shrimp after 48 h second post-infection of the dsRNA. First-strand cDNA was synthesized using the ImProm-IITM Reverse Transcriptase (Promega) according to the manufacturer’s instruction. The PmCaM transcription level was determined by RT-PCR using gene-specific primers, PmCaM-F and PmCaM-R (Table 1). EF1α gene was amplified and used as an internal control. PCR conditions were as follows 95 °C for 5 min; 25 cycles of 95 °C for 1 min, 55 °C for 30 s and 72 °C for 1 min, and a final heating at 72 °C for 10 min. The RT-PCR product was analyzed by 1.2% (w/v) agarose gel electrophoresis and visualized under UV transilluminescence. 2.6. Cumulative mortality assay Shrimp pathogenic bacterium, Vibrio harveyi 639, was prepared as described by Charoensapsri et al. (2009) and used for cumulative mortality assay. Black tiger shrimp (2 g in size) was intramuscularly injected with 10 μg of PmCaM dsRNA or 30 μl 150 mM NaCl as a control. Injection of GFP dsRNA with the same amount was included as a sequence-independent dsRNA control. After 24 hours of the first injection, 10 μg of dsRNAs or 30 μl 150 mM NaCl was repeatedly injected along with V. harveyi (2 × 105 CFUs). Cumulative mortality rate was recorded twice a day for 7 days after bacterial challenge. This experiment was performed in triplicate with 10 healthy shrimps in each group. Statistical analysis was performed using one-way ANOVA test.

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6000 rpm for 30 min, 4 °C. The supernatant was then applied to 1 ml HiTrap Phenyl FF connected to FPLC which was previously equilibrated with 50 mM Tris–HCl pH 8.0 containing 2.3 M (NH4)2SO4. The protein was eluted with a linear gradient of 50 mM Tris–HCl pH 8.0. The purified protein was dialyzed against dialysis buffer (50 mM Tris– HCl pH 8.0 and 0.5 M NaCl) containing 10 mM EGTA. Protein concentration was determined by Bradford assay (Bradford, 1976).

2.8. Western blot analysis Purified protein was separated on 12% SDS–PAGE and electroblotted onto a PVDF membrane (Amersham Biosciences) with a constant current of 180 mA for 1.15 h (ATTA system). The membrane was blocked with 3% (w/v) BSA in TBS-T buffer (50 mM Tris– HCl pH 7.4, 150 mM NaCl and 0.2% (v/v) Tween20) for 1 h at room temperature. The membrane was then washed twice with TBS-T buffer. After that the membrane was probed with 1:5000 PentaHisTM HRP antibody (Qiagen) in the TBS-T buffer for 1 h at room temperature and washed twice with the same buffer. The immunodetection pattern was examined by chemiluminescence using ECL Western blotting reagent (GE Healthcare) and developed on autoradiography film according to the manufacturer’s instruction.

2.9. Ca2+-dependent mobility shift assay Ca2+-dependent mobility shift assay was performed according to the method of Ren et al. (2013) with some modifications. The purified rPmCaM (30 μg) was incubated in 20 mM Tris–HCl pH 8.0 containing either 2 mM CaCl2 or 2 mM EGTA with shaking for 1 h at room temperature. The sample was then subjected to 15% SDS– PAGE, which was run with a constant current of 30 mA in Tris– glycine buffer (12 mM Tris, 95 mM glycine and 0.1% (w/v) SDS). Gel mobility shift of rPmCaM was visualized by Coomassie blue R-250 staining.

2.7. Recombinant protein expression and purification

2.10. Fluorescence binding study

The full length CaM gene was sub-cloned from the plasmid pGEM-PmCaM into pET28b expression vector via the NcoI/NotI restriction sites. The recombinant plasmid, pET28b-PmCaM, encoded for a fusion protein containing a N-terminal His6-tag, was confirmed by sequencing. After transformation, E. coli BL21 (DE3) star codon harboring pET28b-PmCaM was selected for rPmCaM expression. Cells were grown in Luria–Bertani (LB) medium supplemented with 50 μg/ml kanamycin and 34 μg/ml chloramphenicol at 37 °C until an O.D.600 reached 0.6. Protein expression was induced by supplementation with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37 °C for 4 h. Cells were harvested by centrifugation at 6000 rpm for 30 min at 4 °C. The cells were resuspended in lysis buffer (1 M Tris–HCl pH 8.0, 0.5 M NaCl, 5 mM imidazole, 1 mM PMSF, 2 mM β-mercaptoethanol and 1% Triton X-100) and disrupted by sonication (Sonics Vibracell VCX750). Cell lysate was centrifuged at 10,000 rpm for 30 min at 4 °C. The supernatant was applied onto a 1 ml HiTrap Chelating HP column connected to FPLC (ÄKTA, GE Healthcare) which was previously equilibrated with five column volumes of buffer A (50 mM Tris–HCl pH 8.0, 0.5 M NaCl and 30 mM imidazole). Unbound proteins were washed out using the same buffer. Bound proteins were then eluted with linear gradient of buffer B (50 mM Tris–HCl pH 8.0, 0.5 M NaCl and 0.5 M imidazole). The eluted fractions containing rPmCaM were subjected to ammonium sulfate precipitation and further purification on the hydrophobic column chromatography. Protein precipitation using 50% (NH4)2SO4 (or 2.3 M) was firstly carried out followed by centrifugation at

Fluorescence measurements were performed on a luminescence spectrometer LS508 (Perkin-Elmer). The purified rPmCaM (20 μM) in 20 mM Tris–HCl pH 8.0 was incubated with 100 μM 8-anilinonapthalene-1-sulfonic acid (ANS) (Sigma Aldrich) either in the presence of 2 mM CaCl2 or absence of calcium ions (incubating with 2 mM EGTA for 30 min). The emission spectra from 400 to 650 nm with an excitation at 370 nm for ANS were recorded. Data were plotted as fluorescence intensity versus emission wavelengths.

2.11. Far-UV circular dichroism spectroscopy Secondary structure of the protein in various conditions was elucidated using circular dichroism (CD) spectrophotometer (Jasco J-815). The CD spectra were measured at a protein concentration of 0.3 mg/ml in 20 mM Tris–HCl pH 8.0 in the presence and absence of 2 mM CaCl2. The quartz cuvette with a path length of 0.1 cm was used for far-UV CD measurement. Spectra were recorded from 190 to 250 nm at rate of 20 nm/min, a step resolution of 0.5 and a bandwidth of 1.0 nm. The baseline was corrected by subtracting the spectrum of a buffer obtained under the identical condition. The results were converted to per-residue molar absorption units, [θ] (deg cm2 mol−1) and the secondary structure content was analyzed with the K2D2 program. To evaluate the protein stability, the CD spectra of 0.3 mg/ml rPmCaM in 20 mM Tris–HCl pH 8.0 were recorded at various temperatures.

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2.12. Pull down assay of CaM binding proteins

3.2. Tissue specific expression of PmCaM

Hemolymph was withdrawn from the ventral sinus with an anticoagulant solution (10% (w/v) sodium citrate) and centrifuged at 10,000 rpm, for 20 min at 4 °C. The hemocyte pellet was homogenized in phosphate buffer saline (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, and 18 mM KH2PO4). Hemocyte lysate supernatant (HLS) was obtained by centrifugation of the homogenate at 10,000 rpm for 20 min at 4 °C. Total protein in the HLS was firstly incubated with rPmCaM (100 μg) for 1 h. Ni-NTA resin was then added and the mixture was incubated for 2 h at 4 °C. The resin–rPmCaM–HLS complex was recovered by centrifugation at 13,000 rpm for 30 min at 4 °C. The pellet was washed twice with washing buffer (50 mM Tris–HCl pH 8, 0.5 M NaCl and 5 mM imidazole). Thereafter, bound proteins were eluted from the resin using elution buffer (50 mM Tris– HCl pH 8, 0.5 M NaCl and 0.5 M imidazole) and analyzed by 15% SDS– PAGE. HLS incubated with Ni-NTA resin and without rPmCaM was used as a negative control. The gel was stained with silver staining solution. After visualization, expected bands for rPmCaMbinding protein were excised and identified by LC/MS/MS (Proteomic Service Center, Mahidol University).

Expression of PmCaM transcript in various tissues was determined by semi-quantitative RT-PCR. EF1α was used as an internal control for normalizing of the RNA sample. PmCaM were found to be expressed in all examined tissues including gills, hemocyte, lymphoid, intestine, heart and hepatopancreas (Fig. 2). This finding implied that PmCaM might be involved in many different biological processes of shrimp.

2.13. Computational analysis Putative CaM binding proteins obtained from LC/MS/MS analysis were subjected for computational analysis. A potential CaMbinding site was predicted from their amino acid sequences using Calmodulin Target Database. Additionally, CaM-binding motif of the putative CaM binding proteins was elucidated using Pepwheel program. Homology modeling of PmCaM was also constructed by Swiss-Model (Guex et al., 2009) using the structure of fruit fly CaM– CaM kinase I complex (PDB code 1MXE) as a template. Protein model qualities were subsequently evaluated by Procheck. In addition, in silico protein–protein docking was performed to predict PmCaM– protein targets interaction using ZDOCK server (version 3.0.2) (Pierce et al., 2011, 2014). Structural model and interaction data were verified using Discovery Studio 4.0 visualizer software (Accelrys Inc., CA, USA).

3. Results 3.1. PmCaM sequence analysis DNA sequence of PmCaM contained an ORF of 447 bp encoding for 149 amino acid residues with a predicted molecular mass of 16.8 kDa and a calculated isoelectric point of 4.09 (Fig. 1A). The sequence analysis with BLAST algorithm revealed that the deduced amino acid sequence of PmCaM showed high identities to CaMs of pearl oyster, crayfish and fruit fly (99%–100%) and very similar to human, rabbit and mouse CaM (97%–98%). PmCaM consisted of four EF-hand (helix–loop–helix) domains with four putative Ca2+-binding sites. Those domains, except the third one, showed complete conservative sequences among others, Drosophila melanogaster (AFH08031.1), Caenorhabditis elegans (CAA10601.1), Oryctolagus cuniculus (NP_001182569.1), Homo sapiens (AAD45181.1), Mus musculus (AAA66181.1), Procambarus clarkii (ACI15835.1) and Pinctada fucata (AAQ20043.1). Amino acid alignment showed that only three amino acid residues (F100, T144 and S148) of the PmCaM are different from vertebrate CaMs. The F100 and S148 residues could be found in other invertebrates while Y100 and A148 have found in vertebrate CaMs (Fig. 1B). However, PmCaM shared more than 97% amino acid sequence identity with the other CaMs, suggesting that the PmCaM might play key functions similar to the other CaMs.

3.3. PmCaM gene silencing and cumulative mortality assay To elucidate the potential role of PmCaM in the shrimp immune system, gene silencing mediated by RNAi was carried out. After 48 h of the second post-injection, a specific reduction in the transcription level of PmCaM transcript was found by the PmCaM dsRNA (Fig. 3A). For the control groups, the injection of the GFP dsRNA or NaCl did not affect the mRNA levels of PmCaM and the expression of the control gene, EF1α, was stable in all experiments, indicating that the gene silencing is sequence-specific. In addition to monitoring the transcriptional level of PmCaM transcript, the effect of PmCaM silencing on cumulative mortality assay was determined. PmCaM silenced shrimps were then challenged with virulent pathogenic bacteria, V. harveyi 639 (2 × 105 CFUs). The PmCaM silenced shrimp shows a cumulative mortality of 80% within 7 days of determination, as compared to 20% and 18% cumulative mortality in the control GFP dsRNA and NaCl shrimp (Fig. 3B), respectively. The significant increase in mortality rate examined with PmCaM silenced shrimp was supported by statistical values using one-way ANOVA (p < 0.05) compared to those of the control groups. 3.4. Protein purification and characterization rPmCaM was successfully expressed as a soluble form in bacterial system. Protein purification was also accomplished using an affinity and hydrophobic means. A major band was observed on 15% SDS–PAGE with the molecular mass of ~17 kDa. Western blot analysis also confirmed that the ~17 kDa was rPmCaM (data not shown). For Ca2+-dependent mobility shift assay, the result showed that the presence of Ca2+ ions affected the mobility of the rPmCaM on gel (Fig. 4A). Without calcium ion, the mobility of rPmCaM was slower than that of Ca2+ binding condition. This implied that Ca2+ ions induced conformational changes of the rPmCaM, generated a more compact form of the protein, so Ca2+–rPmCaM complex can move faster than that of apoprotein. It is important to note that before dialysis against EGTA, the mobility of rPmCaM was similar to that of Ca2+–rPmCaM. This indicated that the purified rPmCaM from bacterial system was produced as a Ca2+ bound rPmCaM. Conformational changes of rPmCaM induced by Ca2+ ion-binding was further investigated by fluorescence spectroscopy using hydrophobic fluorophore, ANS. The fluorescence emission of ANS is high in the hydrophobic environment but quenched in the hydrophilic environment. Therefore, ANS can be a useful fluorophore for probing the conformational change in proteins. The result showed that in the absence of Ca2+ ions the ANS emission intensity in rPmCaM was less than that of Ca2+ bound rPmCaM and the maximum emission intensity was found at 480 nm in a presence of CaCl2 (Fig. 4B). However, the fluorescence intensity of ANS in the purified rPmCaM, before dialysis against EGTA, was detected, suggesting that there are some Ca2+ ions in the purified rPmCaM and ANS can therefore bind to the protein. This result was in good agreement with the gel mobility shift assay. Secondary structure of rPmCaM predicted using PSIPRED program contained mostly α-helices with four Ca2+-binding sites forming a random coil (Fig. 5A). To confirm this result, the secondary structure integrity of rPmCaM was also investigated using CD

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Fig. 1. PmCaM sequence analysis. (A) ORF and deduced amino acid sequence of PmCaM. Four calcium binding domains are highlighted in gray. The stop codon is indicated with an asterisk. (B) Multiple sequence alignment of PmCaM with other CaMs. Asterisks (*) represent identical amino acid residues, and colons (:) represent non identical amino acid residues. Lines enclose the EF-hand motifs and the calcium binding domains are highlighted in gray. The dark gray shows non-conserved residues.

spectroscopy. The CD data analyzed by K2D2 program are shown in Table 2. After dialysis against EGTA, apo-rPmCaM showed positive peak at 190 nm, and two negative peaks at 208 and 222 nm, indicating high content of α-helix structure (84.73%) (Fig. 5B). However, when CaCl2 was added, the α-helical content of rPmCaM was increased to 87.59%. Again, the purified rPmCaM without the dialysis against EGTA showed the similar content of α-helix structure (87.95%) to that of Ca2+ bound form. This result therefore confirmed that the purified protein was in Ca2+ bound form and Ca2+

Fig. 2. Tissue-specific expression of PmCaM transcript by RT-PCR analysis in various tissues of shrimp. G, gills; Hep, hepatopancreas; Hem, hemocytes; H, heart; I, intestine; and Lym, lymphoid. EF1α was used as an internal control.

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Fig. 3. In vivo gene silencing of PmCaM. (A) Specific-gene silencing of PmCaM transcripts in shrimp hemocyte. (B) Cumulative mortality of PmCaM silenced shrimp challenged with V. harveyi. Shrimp were injected with V. harveyi 639 (2 × 105 CFU) following the dsRNA injection. Control groups were shrimps injected with GFP dsRNA and saline buffer (150 mMNaCl). Shrimp mortality was recorded twice each day for 7 days. Percent mortality in each experimental group (10 shrimps/group) is presented as the means of triplicate independent experiments. Bars indicate mean of standard deviation.

ions could induce some secondary structural changes of rPmCaM. In addition, the protein stability at various temperatures was also investigated. The result showed that, at 80 °C the α-helical contents of the protein in the presence of Ca2+ ions and EGTA were 62.60% and 10.45%, respectively. Therefore, Ca2+ ions could stabilize the helical content by 52%. 3.5. Identification of CaM binding proteins CaM-binding proteins in shrimp hemocyte were examined by protein–protein pull-down assay (Fig. 6A). The eluted fraction (lane 8) revealed three bands of CaM-binding protein as indicated by arrows. To identify the protein targets, these three bands were

subsequently sequenced by LC/MS/MS. From the MS/MS analysis, there were 4 matched sequences with MASCOT database, including transglutaminase (TG) and elongation factor 2 (EF2) from P. monodon, elongation factor 1-α (EF1α) from Marsupenaeus japonicus and actin protein from Litopenaeus vannamei. A possible CaM-binding site of TG, EF1α, EF2 and actin was next analyzed using an algorithm available in the Calmodulin Target Database. The result showed that only TG, EF1α and EF2, contained CaM-binding site. The predicted CaM-binding site could be found between amino acid residues 514 and 530 for TG, amino acid residues 165 and 182 for EF1α and amino acid residues 316–330 for EF2. These three putative CaM-binding sites were then subjected to Pepwheel program to predict a helical organization. The putative CaM-binding site of TG, EF1α and EF2 formed an amphipathic α-helix with hydrophobic and hydrophilic residues grouped on the opposite faces (Fig. 6B). This suggested that rPmCaM might interact with TG, EF1α and EF2 via the putative CaM-binding site. To understand the possible interactions between rPmCaM and the putative CaM-binding proteins, homology modeling and molecular docking were employed. The three dimensional structures of rPmCaM and three putative CaM-binding peptides were first modeled. The rPmCaM structure was created using the crystal structure of the fruit fly CaM–CaM kinase I complex (PDB code 1MXE) as a template. The 3D structure of TG was constructed using crystal structure of human coagulation factor XIII (PDB code 1f13A) as a template. Homology modeling of EF1α structure was performed using yeast elongation factor complex EF1A:EF1BA (PDB code 1f60A) as a template and the crystal structure of fruit fly EF2 (PDB code 3j38Z) was used as a template for EF2 modeling. Next, to examine potentially informative interactions between the rPmCaM and the putative CaM-binding peptides of TG, EF1α and EF2, the modeled structures of these peptides were docked into the rPmCaM structure using ZDOCK server. All putative CaM-binding peptides were buried in hydrophobic pocket between N-lobe (F13, A16, L19, F20, V36, M37, L40, M52, A56 and M73) and C-lobe (I101, M110, M125, I126, A129, V137, F142, M145 and M146) of the rPmCaM. For the CaM-binding motif of TG, side chains of G514, R516, S521, N524 and T529 could form H-bond with D51, Q42, E85 and M125 of rPmCaM (Fig. 7A). In addition to H-bonding, hydrophobic interactions were also found. For the EF1α, H-bonding was formed as shown in Fig. 7B. Nevertheless, some amino acid residues (F167, I170, V174 A176, Y177, V178, V181 and Y183) of the EF1α could interact with a

Fig. 4. Effect of Ca2+ binding. (A) Electrophoretic mobility shift assay. Lane M: standard protein marker; lane 1: purified rPmCaM; lane 2: rPmCaM dialyzed against EGTA (called rPmCaMd); lane 3: rPmCaMd with 2 mM CaCl2; lane 4: rPmCaMd with 2 mM EGTA. (B) Emission fluorescence spectra of ANS in various conditions at 25 °C. The X-axis represents fluorescence intensity while the Y-axis represents emission wavelength.

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Fig. 5. rPmCaM secondary structure determination. (A) Secondary structure of rPmCaM predicted by PSIPRED program. Pink barrels represent α-helices, black lines indicate random coils. (B) Far-UV CD spectra of rPmCaM in the presence of CaCl2 or EGTA at 25 °C. (C) Far-UV CD spectra of rPmCaM with CaCl2 at various temperatures. (D) Far-UV CD spectra of rPmCaM with EGTA at various temperatures. X- and Y-axes represent molar ellipticity [θ]n and wavelength, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

hydrophobic surface of rPmCaM that might stabilize the protein interaction. For rPmCaM–EF2 complex, L305, I307, M326, W329 and L330 of EF2 could form hydrophobic interaction with M73, F93, V109 and M110 of rPmCaM. Also, Q306, K308, Q310, R314, K323 and R327 of the putative CaM-binding motif of EF2 formed H-bonding with S82, E85, E12, M145, E8 and R327 of rPmCaM, respectively (Fig. 7C). According to these data, both H-bonds and hydrophobic interactions were involved in the protein–protein binding, stabilizing rPmCaM–peptide complexes.

4. Discussion CaM is a Ca2+-binding protein that plays an essential function in signaling pathways of eukaryotic cells. In invertebrates, CaM does not only play function in signal transduction but also could be responsible for pathogen infections (Li et al., 2014; Wongpanya et al., 2007). However, the function of CaM in immune responses through Ca2+-dependent signaling pathway in invertebrates is still unclear. In the present study, CaM from P. monodon was identified and

Table 2 Secondary structure of rPmCaM in various conditions determined by CD spectroscopy. Secondary structure

α-Helix β-Sheet Random-coil

% secondary structure rPmCaM

rPmCaMd

rPmCaMd–CaCl2

rPmCaMd–EGTA

87.59 0.48 11.93

84.73 0.57 14.7

87.59 0.48 11.93

84.73 0.57 14.7

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Fig. 6. Identification of rPmCaM-binding protein. (A) Silver staining of protein pull-down assay. Lane M, standard protein marker; lane 1, purified rPmCaM; lane 2, total protein in HLS; lanes 3–5, flow-through fraction, wash fraction and eluted fraction of Ni-NTA-HLS, respectively; lanes 6–8, flow-through fraction, wash fraction and eluted fraction of Ni-NTA-rPmCaM-HLS, respectively. Arrows indicates the putative targets. (B) Helical wheel diagrams of putative CaM-binding site of TG, EF1α and EF2. Hydrophobic residues are marked by a circle, whereas charged residues are labeled accordingly. The peptide sequences are indicated on top.

biochemically characterized. The PmCaM cDNA encoded for 149 amino acids with a very high identity to other CaMs including D. melanogaster, P. clarkii, H. sapiens, M. musculus and O. cuniculus. In comparison with other CaMs, only two amino acid residues of PmCaM (F100 and S148) differed from vertebrate CaMs (Y100 and A148). The highly conserved sequence indicated that all residues of CaM are indispensable. However, in vertebrate CaMs the residue Y100 is phosphorylated, leading to the increase in binding affinity for Ca2+ ions and target proteins (Corti et al., 1999). For PmCaM, this position was replaced by phenylalanine (F100), which may cause some alterations of Ca2+ binding affinity. This may affect to the binding of target proteins and functions of PmCaM in shrimp. For tissue specific expression, CaM expressed ubiquitously in various tissues similar to other aquatic animals (Ji et al., 2011; Li et al., 2014). However, in freshwater crayfish, CaM mRNA is abundant in excitable tissues (such as muscle and nerve) than in any epithelia (gill, antennal gland and digestive) suggesting that CaM plays a crucial role in signaling cascade (Gao et al., 2009). Moreover, in Chinese mitten crab, CaM gene expression was significantly up-regulated in hemocytes, gill, hepatopancreas, intestine and muscle in response to bacterial challenges. It is suggested that the CaM is an important stress and immune response gene (Li et al., 2014). In crustaceans, hemocyte is essential in immunity that contains several immune proteins such as prophenoloxidase (proPO), antimicrobial peptide (AMP), crustins and lipopolysaccharide and β- 1,3glucan-binding protein (LGBP). These molecules were expressed in response to pathogen infections (Amparyup et al., 2009, 2012; Donpudsa et al., 2014). However, the intracellular signal transduction of these molecules for pathogen response is still limited. The

expression of CaM in hemocyte might be involved in important regulations and signal transductions. The significantly increased mortality in PmCaM-silenced shrimp after V. harveyi challenge implied that the PmCaM might play an important role in pathogen defense system. Our result corresponds to Maningas et al. (2008) in that the silencing of transglutaminase gene, one of enzyme in immune cascade, could increase mortality rate of WSSV and Vibrio penaecida challenged shrimp. According to our study, the increase in PmCaM-silenced shrimp mortality rate after Vibrio infection implied that PmCaM may be involved in shrimp immune response. Although, CaM is an essential signaling protein that plays several functions in organisms, the gene knockdown does not affect their viability. Our silencing PmCaM without the pathogen infection showed no effect on shrimp mortality in comparison with the control shrimps. Similarly, in sea lamprey larvae, CaM gene silencing did not effect on the animal mortality (Heath et al., 2014). For other signaling proteins, silencing of Rab7 gene in P. monodon caused less than 5% shrimp mortality in comparison with GFP dsRNA and NaCl injection groups (Ongvarrasopone et al., 2008). Even though PmCaM is not directly involved in shrimp immunity, it is possibly a part of some shrimp immune response cascades. For the characterization of the rPmCaM, the effect of Ca2+binding on rPmCaM conformational change and CaM-binding proteins were examined. Since CaM has a high affinity to Ca2+ ion with the dissociation constant of 10-−6 M, rPmCaM, which was produced in Ca2+-bound form, was therefore dialyzed against high concentration of EGTA before the experiments to determine its native conformation (Stigler and Rief, 2012). In electrophoretic mobility shift assay, Ca2+ bound rPmCaM moved faster than the apo-protein.

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Fig. 7. H-bonding between PmCaM and putative CaM-binding peptides, TG (A), EF1α (B) and EF2 (C). These structures were visualized by Discovery Studio 4.0. Amino acids of rPmCaM and putative CaM-binding peptides are labeled in blue and red, respectively. H-bonds are represented by green dash lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

This observation was similar to that of pearl oyster and freshwater mussel CaMs (Li et al., 2005; Ren et al., 2013). The binding of Ca2+ ions could induce a conformational change of CaM to be compact, while apo-CaM may be in a less compact form, resulting in the different electrophoretic mobility (Zhang and Lou, 2012). This result was also supported by CD experiment, in that Ca2+ ions increased α-helical content of rPmCaM when compared to that of the apoprotein. The stability study suggested that the Ca2+ ions probably stabilized salt bridges between positively and negatively chargeamino acids, inducing a thermostability of rPmCaM secondary structure. This result was in close agreement with the experiment of Brzeska et al. in 1983 which found that, in the presence of Ca2+, the conformation of calmodulin was changed after temperature reached above 90 °C, while Ca2+-free form was more susceptible to thermal unfolding (Brzeska et al., 1983). Additionally, the conformation of Ca2+-CaM could still regain its native conformation after reducing the temperature but the conformation of Ca2+-free CaM was irreversible at such a high temperature (Guerini and Krebs, 1983). The conformational change after Ca2+-binding was also investigated by fluorescence spectroscopy using extrinsic fluorescent

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probe, ANS, which can bind to hydrophobic surface of target protein, resulting in an increase in fluorescence intensity. The result showed that fluorescence intensity of ANS was increased in the presence of Ca2+ ions, but dramatically dropped in the presence of EGTA. This result indicated that Ca2+ ions could induce conformational change of the rPmCaM leading to the exposure of hydrophobic residues, particularly methionine, onto the protein surface (Gifford et al., 2011; Zhang and Vogel, 1994). This hydrophobic patch on rPmCaM might be involved in the interactions with its target proteins such as nitric oxide synthase and Rac1 GTPase, resulting in a regulation of protein functions in organisms (Wu et al., 2012; Xu et al., 2012). More than 300 CaM-binding proteins have been identified that could provide CaM roles in organisms. In 2011, Alvarado and Wasserman successfully isolated GASP180, α-tubulin and pyruvate phosphate dikinase from tropozoite stage of Giardia intestinalis that were calcium-dependent proteins. The first two proteins were classified in cytoskeleton proteins and the other was an essential enzyme in glycolysis pathway, suggesting the function of CaM in motility and energetic metabolism (Alvarado and Wasserman, 2012). However, the function of CaM, especially in signal transduction and immune system, is still unclear. In this report, we identified rPmCaMbinding proteins in shrimp hemocyte using pull-down assay and LC–MS/MS. Four proteins, which were actin, TG, EF1α and EF2, were found as candidate CaM-interacting proteins. Our computational analysis showed that there is no CaM binding motif in this shrimp actin; however, there was no report on interactions between actin and CaM before. We speculated that the dynamic of actin might be indirectly mediated by CaM-linker proteins such as Rho GTPase, Rac1 and myosin. This actin dynamic promoted membrane movement that involved in phagocytic uptake in innate immunity (Bahloul et al., 2004; Xu et al., 2012). TG is an essential enzyme in shrimp clotting cascade, plays crucial functions in shrimp immune system and is highly synthesized in a proliferative tissue of black tiger shrimp (Huang et al., 2004). In crustacean, clotting system is not a proteolytic cascade; however, the process is mediated by Ca2+ dependent TG activation (Kopacek et al., 1993). The enzyme is released from hemocyte and activates cross-linking between glutamine and lysine residues of clottable proteins, resulting in a polymerization reaction (Hall et al., 1999; Kopacek et al., 1993). The silencing of this gene could reduce transcription level of the other immune genes, including crustin, lysozyme, proPO, C-type lectin, penaeidin-3c precursor and clottable protein (Fagutao et al., 2012). TG silenced shrimp was found to be susceptible to pathogen infection (Maningas et al., 2008). Although, the activity of shrimp TG was reported to be Ca2+ dependence, but the enzyme lacks a specific Ca2+-binding motif (EFhand motif). TG could bind Ca2+ ion using a region rich in negatively charge amino acids (Huang et al., 2004; Liu et al., 2011). Due to the weak Ca2+-binding affinity, the enzyme is probably regulated by other Ca2+ sensor proteins. In human platelets and the chicken gizzard, CaM could activate TG by threefold increasing on protein polymerization in comparison with that of a control experiment (Puszkin and Raghuraman, 1985). This evidence supports our computational analysis result that TG contained the CaM binding motif and the function of TG may possibly be regulated by PmCaM. For EF1α, this elongation factor was reported as a Ca2+/CaM-binding protein and involved in regulation of Tetrahynemia cilia movement (Ueno et al., 2003). For EF2, it was involved in a production of tumor necrosis factor-α that mediated inflammatory process (Gonzalez-Teran et al., 2013). Also, the EF2 stimulated interferon-γ and interleukin12 production, promoting pathogen elimination (Kushawaha et al., 2011). Although there was no report on Ca2+ /CaM involved in EF2 function, this factor contained the CaM binding motif according to our computational prediction. Thus, the shrimp EF2 may interact and be regulated by CaM. The identification of CaM binding proteins could probably indicate the involvement of CaM in shrimp immune system.

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The computer analysis on the interactions of PmCaM and putative PmCaM-binding peptide of TG, EF1α and EF2 showed that the three putative target peptides were successfully fitted into hydrophobic pocket of PmCaM with a good energy and acceptable Z-score. Generally, hydrophobic pocket of CaM composes of important hydrophobic amino acid residues in N-lobe and C-lobe, especially methionine residues which are important for binding to target proteins (Liu et al., 2012). Therefore, the hydrophobic interaction was essential for CaM–protein complex. In this study, the hydrophobic interaction was found between the hydrophobic residues of PmCaM and the putative PmCaM-binding peptides of TG, EF1α and EF2. This result corresponded to human CaM that interacted with its target using M72 and M145 (Bhattacharyya and Banerjee, 2011; Liu et al., 2012). Moreover, M124 and E120 of bovine CaM, which were analogous to M125 and E121 of PmCaM, could interact with CaMdependent protein kinase (Stefan et al., 2012). Since the amino acid methionines were important residues for the protein binding, it could be therefore suggested that PmCaM could bind to TG, EF1α, EF2, and actin, and probably mediated their functions in shrimp. In conclusion, we identified a CaM, named PmCaM, from the black tiger shrimp, P. monodon. The result showed a very high conserved nucleotide sequence between PmCaM and other CaM organisms especially in the four EF-hand motifs. The PmCaM transcript was expressed in all examined tissues and the PmCaM silencing increased a cumulative mortality in V. harveyi injected shrimps. The expression and characterization showed that the rPmCaM was produced as a Ca2+-bound protein which can interact with TG, EF1α, EF2 and actin based on the pull down assay. Homology modeling and molecular docking analysis confirmed the possible binding between PmCaM and its targets. We suggested that PmCaM may have a functional role in regulating the shrimp immune response to shrimp pathogen. This finding about shrimp CaM function provides an insight into signaling cascade of shrimp immunity.

Acknowledgements This work was supported by Kasetsart University Research and Development Institute, Graduate School Kasetsart University (for Panjana Sengprasert), Faculty of Science, Kasetsart University, Thailand Research Fund (MRG5280048) and the King Prajadipok and Queen Rambhai Barni Memorial Foundation.

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