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Molecular Immunology 57 (2014) 292– 301

Contents lists available at ScienceDirect

Molecular Immunology

jo u r n al homep age: www.elsev ier .com/ locate /mol imm

mmunological role of C4 CC chemokine-1 from snakehead murrelhanna striatus

rasanth Bhatta, Venkatesh Kumaresana, Rajesh Palanisamya, Mukesh Kumar Chaurasiaa

Annie J. Gnanamb, Mukesh Pasupuleti c, Jesu Arockiaraja,∗

Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University,attankulathur, Chennai 603 203, Tamil Nadu, IndiaInstitute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A4800, Austin, TX 78712, USALab PCN 206, Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031,ttar Pradesh, India

r t i c l e i n f o

rticle history:eceived 28 August 2013eceived in revised form 9 October 2013ccepted 15 October 2013vailable online 12 November 2013

eywords:hemokineurrel

pizootic ulcerative syndrome (EUS)hemotaxisell proliferation

a b s t r a c t

In this study, we have reported a cDNA sequence of C4 CC chemokine identified from snakehead murrel(also known as striped murrel) Channa striatus (named as CsCC-Chem-1) normalized cDNA library con-structed by Genome Sequencing FLXTM Technology (GS-FLXTM). CsCC-Chem-1 is 641 base pairs (bp) longthat contain 438 bp open reading frame (ORF). The ORF encodes a polypeptide of 146 amino acids with amolecular mass of 15 kDa. The polypeptide contains a small cytokine domain at 30–88. The domain car-ries the CC motif at Cys33-Cys34. In addition, CsCC-Chem-1 consists of another two cysteine residues at C59

and C73, which, together with C33 and C34, make CsCC-Chem-1 as a C4-CC chemokine. CsCC-Chem-1 alsocontains a ‘TCCT’ motif at 32–35 as CC signature motif; this new motif may represent new characteristicfeatures, which may lead to some unknown function that needs to be further focused on. Phylogenitically,CsCC-Chem-1 clustered together with CC-Chem-1 from rock bream Oplegnathus fasciatus and Europeansea bass Dicentrarchus labrax. Significantly (P < 0.05) highest gene expression was noticed in spleen andis up-regulated upon fungus (Aphanomyces invadans), bacteria (Aeromonas hydrophila) and virus (polyI:C) infection at various time points. The gene expression results indicate the influence of CsCC-Chem-1in the immune system of murrel. Overall, the gene expression study showed that the CsCC-Chem-1 is a

capable gene to increase the cellular response against various microbial infections. Further, we clonedthe coding sequence of CsCC-Chem-1 in pMAL vector and purified the recombinant protein to studythe functional properties. The cell proliferation activity of recombinant CsCC-Chem-1 protein showeda significant metabolic activity in a concentration dependant manner. Moreover, the chemotaxis assayshowed the capability of recombinant CsCC-Chem-1 protein which can induce the migration of spleen

owe

leukocytes in C. striatus. H

. Introduction

Innate immune system is the first line defense of the host’sght against the foreign particle until the adaptive immuneystem recognizes the pathogen and destroys it (Sinyakov et al.,002). In fish, the innate immune system is a major and necessaryomponent for killing the pathogen, because the adaptive immuneystem has its own limitations (Magnadottir, 2006). Chemokines8–15 kDa), a pro-inflammatory cytokine (otherwise known as

hemotactic cytokine), is an important component of the innatemmune system (Whyte, 2007). Schall (1991) reported that thehemokines are produced in response to variety of pathogens such

∗ Corresponding author. Tel.: +91 44 27452270; fax: +91 44 27453903.E-mail address: jesuaraj@hotmail.com (J. Arockiaraj).

161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.molimm.2013.10.012

ver, this remains to be verified further at molecular and proteomic level.© 2013 Elsevier Ltd. All rights reserved.

as bacteria, virus, parasites and mycobacteria. It acts as a key reg-ulator in various biological processes including immune response,bridging innate and adaptive immunity, promoting leukocytemigration, differentiation of recruited cells (Esche et al., 2005),neurological development and function (Gordon et al., 2009),organogenesis and germ cell migration (Devries et al., 2006). Inaddition, other researchers (Oppenheim et al., 1991; Schall andBacon, 1994; Baggiolini and Dahinden, 1994; Bacon and Schall,1996) demonstrated that the chemokines are also involved inmany physiological and biochemical functions including integrinactivation, chemotaxis, lipid mediator biosynthesis, superoxideradical production and granule enzyme release.

The super family of chemokines is grouped into four divisionsnamely, CXC (�), CC (�), C (�) and CX3C (�), based on the positionof the first two cysteine residues. CXC and CC chemokines are thetwo major divisions; the former is characterized by the presence

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P. Bhatt et al. / Molecular I

f one amino acid separating the two cysteines, whereas theater is characterized by the presence of two cysteines residuesontinuously (Baggiolini et al., 1994). Laing and Secombes (2004a)tated that CC chemokines are considered to act on mononuclearells rather than on neutrophils unlike CXC chemokines. Further,epending on the structure and function, Laing and Secombes2004a) categorized CC chemokines into various subcategoriesamely allergenic, pro-inflammatory, haemofiltrate CC chemokineHCC), developmental and homeostatic chemokines. Among these,he CC chemokines from allergenic, pro-inflammatory and HCCubcategory members are demonstrated as inducible chemokines,hereas, the CC chemokines from developmental and homeostatic

ubcategory members are constitutively expressed (Laing andecombes, 2004b).

So far, many CC chemokine sequences from rainbow troutDixon et al., 1998; Liu et al., 2002; Laing and Secombes, 2004b),ommon carp (Fujiki et al., 1999), Japanese flounder (Kono et al.,003; Khattiya et al., 2003, 2004, 2007), cichlid fish (Kuroda et al.,003), channel catfish and blue catfish (He et al., 2004; Peatmant al., 2005), zebrafish (Peatman and Liu, 2006), salmon (Peatmannd Liu, 2007), large yellow croaker (Zhang and Chen, 2008), miiuyroaker (Xu et al., 2011), cobia (Su et al., 2012) and rock bream (Kimt al., 2013) have been reported. However, these CC chemokinesre varied. Moreover, knowledge on the structural and functionalole of fish CC chemokines in the defense process is limited. It isnteresting to note that the information on CC chemokine-1 fromnakehead fish (or striped murrel) Channa striatus is nil. Hence, it ismperative to provide sound information on CC chemokine-1 fromtriped murrels.

Snakehead murrel C. striatus, a freshwater air breathing fishs highly regarded for its food value in South and Southeastsian countries (Arockiaraj et al., 1999; Marimuthu et al., 2001a).rockiaraj et al. (2003) and Marimuthu et al. (2001b, 2009) indi-ated many instances of C. striatus being traditionally used foredicinal purposes. However, murrel culture industry is encoun-

ering a risk of infectious disease like epizootic ulcerative syndromeEUS), a fungus (Aphanomyces invadans) causing disease. Hence its important to study immune aspect of C. striatus. Cheng et al.2011a) stated that a significant progress toward the interpretationf defense process is molecular identification, characterization andts expression that are managed in response to pathogenic infec-ion. Hence, to gain insight into the characterization of C. striatus CChemokine-1 (named as CsCC-Chem-1) and its role in C. striatus, weeport the bioinformatics analysis and tissue specific mRNA expres-ion of CsCC-Chem-1 upon fungal, bacterial and viral infection.urthermore, the coding sequence of CsCC-Chem-1 was expressedn an Escherichia coli expression vector system and its recombinantrotein were purified. The purified recombinant protein was exam-

ned by different biological activities including cell proliferative andhemotactic assays. This finding would supply basic informationn this potentially capable molecule and lead possible ways forurther focus at molecular and proteomic level in murrel defenseystem.

. Materials and methods

.1. Establishment of C. striatus cDNA library and CsCC-Chem-1dentification

A normalized C. striatus cDNA library was constructed using totalNA isolated from spleen, liver, kidney, muscle and gills of murrel.

he detailed procedure on C. striatus cDNA library establishmentas explained in our earlier studies (Arockiaraj et al., 2013; Abirami

t al., 2013). A cDNA encoding CsCC-Chem-1 was identified from C.triatus cDNA library during screening. The obtained CsCC-Chem-1

ology 57 (2014) 292– 301 293

cDNA sequence and its coding and non-coding region and aminoacid sequences were analyzed on DNAssist (ver. 2.2) as suggestedby Patterton and Graves (2000).

2.2. In silico analysis of CsCC-Chem-1

Sequence similarities were searched on BLAST program (http://blast.ncbi.nlm.nih.gov/Blast) at NCBI Database. Domains andmotifs were analyzed on PROSITE Database (http://prosite.expasy.org/scanprosite/). The N-terminal transmembrane sequencewas determined by DAS Transmembrane Prediction Program(http://www.sbc.su.se/∼miklos/DAS). Signal peptide analysiswas conducted using the SignalP (http://www.cbs.dtu.dk). Mul-tiple sequence alignment was carried out on ClustalW (ver. 2)(http://www.ebi.ac.uk/Tools/msa/clustalw2/) and the alignmentwas edited on Bioedit (ver. 7.1.3.0). The phylogentic tree wasconstructed using Neighbor-Joining Method at MEGA 5.05. Furtherthe genetic distances were calculated using the Poisson CorrectionMethod (Uinuk-Ool et al., 2003).

2.3. Experimental fish

Healthy C. striatus (average body weight of 50 g) were obtainedfrom Surya Agro Farms, Erode, Tamil Nadu, India. The fisheswere transported to Division of Fisheries Biotechnology & Molecu-lar Biology, SRM University in oxygenated polythene bags. Theywere maintained in 15 flat-bottomed plastic containers (150 L)with aerated and filtered de-chlorinated freshwater (water qual-ity: dissolved oxygen, 5.8 ± 0.2 mg/L; water temperature, 28 ± 1 ◦Cand pH, 7.2 ± 0.2) in the laboratory. All fishes were acclimatizedfor 1 week before being challenged to various immune stimu-lants. A maximum of 15 fish tank−1 were maintained during theexperiment. During acclimatization period, the fishes were fed adlibitum two times daily at 0900 and 1600 hours with a commer-cially available fish feed (Cargill Animal Nutrition, Andhra Pradesh,India).

2.4. Immune challenge

To study the relative gene expression of CsCC-Chem-1 uponinfection, C. striatus were injected with fungus, bacteria and virusintraperitoneally. PBS (1×) were prepared and served as control(100 �l/fish).

For fungus infection, 50 g fish were injected with 100 �l of A.invadans (a primary causative agent of EUS) at a concentration of102 spores. We have clearly explained in our earlier findings on A.invadans isolation from the infected C. striatus muscle, culture inthe laboratory, identification and injection to the fishes (Arockiarajet al., 2013; Abirami et al., 2013).

For bacterial challenge, the fish was injected with Aeromonashydrophila (a secondary causative agent of EUS) (5 × 106 CFU/ml)suspended in 1× phosphate buffer saline (100 �l/fish). A. hydrophilawas also isolated and identified from the muscle sample of EUSinfected C. striatus as described by Dhanaraj et al. (2008).

For virus challenge, poly I:C [polyinosinic-polycytidylic acidsodium salt], a synthetic analog of double-stranded RNA (dsRNA),a molecular pattern associated with viral infection [poly I:C iscomposed of a strand of poly(I) annealed to a strand of poly(C)]were injected to fishes (dosage: 150 �g/50 g fish, 100 �l/animal,�-irradiated, Sigma–Aldrich, USA).

2.5. Sample collection

Tissue samples (blood, liver, spleen, heart, intestine, kidney,head kidney, muscle, skin, gill and brain) were collected before(0 h), and after injection (3, 6, 12, 24, 48 and 72 h) and were

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94 P. Bhatt et al. / Molecular I

mmediately snap-frozen in liquid nitrogen and stored at −80 ◦Cntil the total RNA was isolated. Using a sterilized syringe, blood0.5–1.0 mL fish−1) was collected from the fish caudal fin and imme-iately centrifuged at 4000 × g for 10 min at 4 ◦C to allow blood cellollection for total RNA extraction.

.6. Total RNA isolation and cDNA conversion

Total RNA from the control and infected fish were isolated usingri ReagentTM (Life Technologies), according to the manufacturer’srotocol with slight modifications (Arockiaraj et al., 2011a,b). Using.5 �g of RNA, first strand cDNA synthesis was carried out using auperScript® VILOTM cDNA Synthesis Kit (Life Technologies) withlight modifications (Arockiaraj et al., 2011a, 2012a). The isolatedDNA was stored at −20 ◦C for further analysis.

.7. Tissue specific CsCC-Chem-1 gene expression by qRT-PCR

The gene expression of CsCC-Chem-1 in various tissues wasetermined by quantitative real time polymerase chain reactionqRT-PCR). qRT-PCR was carried out using a ABI 7500 Real-timeetection System (Applied Biosystems) in 20 �l reaction volumeontaining 4 �l of cDNA from each tissue, 10 �l of Fast SYBR® Greenaster Mix, 0.5 �l of each primer (20 pmol/�l) (CsCC-Chem-1 F1,

CA GAC AGA CAA GGG TCT ATT C and CsCC-Chem-1 R1, GGA GACGG AGA CAA AGT AGT G) and 5 �l dH2O. The qRT-PCR cycle pro-le was 1 cycle of 95 ◦C for 10 s, followed by 35 cycles of 95 ◦C for

s, 58 ◦C for 10 s and 72 ◦C for 20 s and finally 1 cycle of 95 ◦C for5 s, 60 ◦C for 30 s and 95 ◦C for 15 s. The same qRT-PCR cycle profileas used for the internal control gene, �-actin. �-actin of C. striatusrimers (�-actin F2, TCT TCC AGC CTT CCT TCC TTG GTA and �-actin4, GAC GTC GCA CTT CAT GAT GCT GTT) were designed from theequence of GenBank Accession No. EU570219. After the PCR pro-ram, data were analyzed with ABI 7500 SDS software (Appliediosystems). To maintain the consistency, the baseline was setutomatically by the software. The comparative CT method (2−��CT

ethod) was used to analyze the expression level of CsCC-Chem-1Livak and Schmittgenm, 2001). All samples were analyzed in threeuplications and the results are expressed as relative fold of oneample as mean ± standard deviation.

.8. Cloning of CsCC-Chem-1 into vector

All of the cloning experiments were carried out according toambrook et al. (1989) with slight modifications (Arockiaraj et al.,012b). The primer set of CsCC-Chem-1 F5: (GA)3GAATTC ATGTC AGC TGC AGA AGC CTC TTC EcoRI and CsCC-Chem-1 R6:

GA)3CTGCAG CCG TCT CAG AAG ATA AGG AGT AGA CCG GC PstIas designed with the corresponding restriction enzyme sites at

he N- and C-termini for CsCC-Chem-1, in order to clone the cod-ng sequence into the expression vector, pMAL-c2X (BioLabs Inc.,).sing plasmid DNA of CsCC-Chem-1 as a template and Taq DNAolymerase (Qiagen), PCR was carried out to amplify the codingequence. The PCR product was purified using the GeneJETTM Gelxtraction Kit (Thermo Scientific). After PCR, the amplified prod-ct was purified by using GeneJETTM (Thermo Scientific). Further,he cloned products were sequenced using the ABI Prism-Bigdyeerminator Cycle Sequencing Ready Reaction kit by using the samerimers used for the amplification and analyzed using an ABI 3730equencer. Then, both insert and vector were digested with theespective restriction enzymes. The ligated product was trans-

ormed into XL1 blue cells and the correct recombinant product (asonfirmed by restriction enzyme digestion and sequencing) wasransformed into competent E. coli BL21 (DE3) cells for proteinxpression.

ology 57 (2014) 292– 301

2.9. CsCC-Chem-1 protein over expression and purification

Transformed E. coli BL21 (DE3) cells were incubated in ampi-cillin (100 �g/mL) Luria broth (LB) overnight. This culture was thenused to inoculate 100 mL of LB broth in 0.2% glucose-rich mediumwith ampicillin at 37 ◦C until cell density reached 0.7 at OD600.E. coli BL21 (DE3) harboring pMAL-c2x-CsCC-Chem-1 was inducedfor over expression with 1 mM isopropyl-�-thiogalactopyranoside(IPTG) and incubated at 15 ◦C for 5 h. Cells were harvested bycentrifugation (4000 × g for 20 min at 4 ◦C). E. coli BL21 (DE3) unin-duced culture was used as a negative control. Then the cells werere-suspended in column buffer (Tris–HCl, pH 7.4, 200 mM NaCl)and frozen at −20 ◦C overnight. After thawing on ice, cells were dis-rupted by sonication. The crude CsCC-Chem-1 fusion protein fusedwith maltose binding protein (MBP) was purified using pMALTM

protein fusion and purification system protocol (BioLabs). Furtherthe fusion protein was digested and purified as described in ourearlier report (Arockiaraj et al., 2012c). The purified protein wassubjected to MS/MS analysis to determine the quality of recombi-nant CsCC-Chem-1 protein following the methodology of Mann andAebersold (2003). Then the protein was verified by 12% SDS-PAGEand the molecular weight of target protein was evaluated usingprotein molecular weight standards. Proteins were visualized bystaining with 0.05% Coomassie blue R-250. The concentration ofthe purified protein was determined via the Bradford method, usingbovine serum albumin (BSA) as the standard (Bradford, 1976). Thepurified protein was kept at −80 ◦C until determination of biologicalactivity.

2.10. Proliferation assay

The proliferation assay was performed as described by Kimet al. (2013) with slight modifications. The assay was performedusing spleen leucocytes which were prepared as described byGraham and Secombes (1988) and Montero et al. (2011). C. striatusspleen leucocytes (1 × 107 cells/well) were prepared by centrifuga-tion process (500 × g for 30 min at 4 ◦C). Further, the proliferationactivity was conducted using WST-1 cell proliferation assay kit(Clontech) as suggested by the manufacturer. The cells were furtheradjusted to 2 × 104 cells/well and re-suspended in Roswell ParkMemorial Institute (RPMI) 1640 medium (Life Technologies) alongwith recombinant CsCC-Chem-1 protein at various concentrations(1, 5, 10, 25, 50 and 100 �g/�L). Followed by the proliferation activ-ity, 10 �L of the WST-1 reagent (Clontech) was added into each welland incubated at 25 ◦C for 12 h. After incubation, the optical densitywas measured in a microplate reader (Promega) at 450 nm. Blankwith RPMI 1640 medium was treated as control. The assay wasperformed in three duplications.

2.11. Chemotaxis assay

This assay was conducted based on the methodology of Zhangand Chen (2008). The assay was performed in a neuro probe 48-well micro chemotaxis chamber (Neuro Probe). Briefly, 1 mL ofRPMI 1640 medium along with 5% fetal bovine serum (FBS) (LifeTechnologies) and 100 U penicillin–streptomycin (Life Technolo-gies) were incubated for 90 min in 5% CO2 at 25 ◦C with recombinantCsCC-Chem-1 protein at various concentrations (1, 5, 10, 25, 50 and100 �g/�L). A polycarbonate filter (3 mm diameter) was placed inthe lower wells of the chemotaxis chamber. After incubation, thetotal number of cells that migrated into the lower chamber wascounted using a binocular microscope (Nikon). Blank with RPMI1640 medium was treated as control. The chemotaxis index was

calculated by comparison between number of cells that migratedby recombinant CsCC-Chem-1 protein induction and number ofcells that migrated by RPMI 1640 medium induction. The assay wasperformed in three replications.

P. Bhatt et al. / Molecular Immunology 57 (2014) 292– 301 295

5’ctctctggagaagaagggacaagaagaaccagcagagattatttactatttatttgttca 60 atagaatatttatcct tcct tgca aaaatggtcagctgc agaa gcctcttcaaaagt gtg 120

M V S C R S L F K S V 11

gtggttgccatcgtgc tggt ggtc ctga ccgagtccgga tcag cagc tgaaaaactggca 180

V V A I V L V V L T E S G S A A E K L A 31

acctgctgtaccaaagtcaccaaagaggagctaacagatccaatctcaggatacttggtc 240

T C C T K V T K E E L T D P I S G Y L V 51

cagaaaaatgccgtag ctcc gtgt gtta gagctgtcatc ttcc agacagacaagggtcta 300

Q K N A V A P C V R A V I F Q T D K G L 71

ttctgcagctacgtaa gagc tccc tggg tcgcccgcaag atta gggctttcgagaaagaa 360

F C S Y V R A P W V A R K I R A F E K E 91

aaaagcaagagcacta cttt gtct ccag tctccccctct ggag tgtcgctcctctccata 420

K S K S T T L S P V S P S G V S L L S I 111

atcacgtcgactgctt cccc gtcg tcat ctttcacgtca tctc gtcccgttttctcctcg 480

I T S T A S P S S S F T S S R P V F S S 131

acatccaaggctgctg ttgg tgaa accg tctcagaagat aagg agtagaccggcatcatg 540

T S K A A V G E T V S E D K E * 146

tctgcctccagc tgaa acaa agaa gaccaaaatctaagt aaac actgatggtctcaa aac 600 tgagaccatgtaaatg caag atag agca gttgaaaaact ta3’ 641

Fig. 1. Nucleotide and deduced amino acid sequences of CsCC-Chem-1. The nucleotide and amino acids are numbered along the right margin. The coding region is highlightedin gray shade. The signal sequence is given in box. Small cytokine domain (total of 59 aa sequence) along with its cysteine motifs at Cys33, Cys34, Cys59 and Cys73 (highlightedin circle) is underlined. Microsatellite sequences (553GAA555, 561GAA563 and 632GAA634) were highlighted in yellow color. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

CsCC-Che m-1 MVSCRSL FKSVVVAIVL VVLTESG-SAAEKLA TCCTKVTKEE LTDPISG-YL VQKNAVAP 5 8Chem-1 O.f asciatu s MVNCGSL LKSALVILAL VAVVQPG-SAPEKLA SCCKTVSNQK ITEPILG-YL VQ-TATPS 5 7Chem-1 D.l abrax MANCGTL MKSALVAIVL MAVIHSG-SADWKLA SCCKKVNKLE ITEPITG-YM IQ-KPNRE 5 7Chem-1 G. morhua MTTCGTL TKSMLLLAVV VALTGQGFAADEKFY KCCTTVSREE ITETIVD-YM VQ-LRKKN 5 8Chem-2 O. mykiss ------- ---------- ----------AEKLV SCCKTVSRTE VNDPITG-YW IQ-NYNAP 3 1Chem-1 H.s apiens MQIITTA LVCLLLAGMW PEDVDSK-SMQVPFS RCCFSFAEQE I--PLRA-IL CYRNTSSI 5 6

CsCC-Chem-1 CVR-AVIFQTDKG-LFCSYVRAPWVARKIRAFEKEKSKSTTLSPVSPSGVSLLSIITSTA 116Chem-1 O.f asciatu s CVR-AVI FQTEAG-L FCSQVTAPWVRRKIVAF ERAKALAIPS SVVPSSTVSL LSIITSTA 1 15Chem-1 D.l abrax CVP-AII LQTEKG-L FCSPVRAEWVRRKVVAF EKAKAQATTS SVVPSSTVSL LSIITSTA 1 15Chem-1 G. morhua CVN-AIIFQTESGNLYCSKHDEPWVMTKVRQ---PR------------------------ 90Chem-2 O. mykiss CVR-AVI FETKKG-L FCSYHKQPWVRRKIHQF EMARLSSTFL SLSIPNSLTS TSTPTTTS 8 9Chem-1 H.s apiens CSNEGLI FKLKRGKE ACALDTVGWVQRHRKML RHCPSKRK-- ---------- -------- 9 6

CsCC-Che m-1 S-PSSSF TSSRPVFSST SKAAVGETVSEDKE- 146Chem-1 O.f asciatu s S-PSS-- SSI------- --------------- 122Chem-1 D.l abrax SSPSS-- SSLPSSLSTS EMP-ADETFSETDDE 144Chem-1 G. morhua ------- ---------- --------------- 90Chem-2 O. mykiss L-PSSPPSSLFP-LSSSS-----PSVPS---- 110Chem-1 H.s apiens ------- ---------- --------------- 96

Fig. 2. Multiple sequence alignment of CsCC-Chem-1 with other CC chemokines from rock bream Oplegnathus fasciatus (BAM34025), European sea bass Dicentrarchus labrax(CAM32188), Atlantic cod Gadus morhua (AAU81657), rainbow trout Oncorhynchus mykiss (AAR19791) and human Homo sapiens (P22362). The amino acids are numberedalong the right margin. The signal peptide region is highlighted in gray shade. The small cytokine domain caring CC motif is underlined. Deletions are indicated by dashes.

296 P. Bhatt et al. / Molecular Immunology 57 (2014) 292– 301

Mus musculus CC-Che m-6 (P277 84)

Mus musculus CC-Chem-9 (P51670)

Homo sapi ens CC-Chem-23 (P5 577 3)

Homo sapiens CC-Chem-14 (Q16627)

Homo sapiens CC-Chem-18 (P55774)

Gall us gal lus CC-Chem-4 (Q9082 6)

Homo sapi ens CC-Chem-5 (P13501)

Homo sap ien s CC-Chem-3 (P10147 )

Homo sapi ens CC-Chem-4 (P13236)

Channa stria tus CC-Chem-14 (JX46 9849 )

Homo sapiens CC-Chem-1 (P2 2362)

Homo sapiens CC-Chem-11 (P51671)

Homo sapi ens CC-Chem-8 (P80 075)

Homo sap iens CC-Chem-17 (Q925 83)

Channa st riatus CC-Chem-19 (JX469 851)

Homo sapiens CC-Chem-19 (Q9 9731)

Homo sapien s CC-Che m-20 (P7 8556)

Mus musculus CC-Chem-20 (NP 058 656)

Channa str iatu s CC-Che m-20 (JX4 69848)

Gadu s morhu a CC-Che m-1 (A AU816 57)

Sparus aura ta CC-Chem-3 (ADE58986 )

Oncorhynchus myk iss CC-Che m-2 (A AR19791)

CsCC-Chem-1

Oplegn athu s fasciatus CC-Che m-1 (B AM34025 )

Dicen tra rchus Labra x CC-Chem-1 (CA M32188 )3970

72

46

98

99

99

99

54

36

27

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44

40

30

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Fig. 3. Phylogentic tree of CsCC-Chem-1 with other species was reconstructed by the Neighbor-Joining Method. The tree is based on an alignment corresponding to fulll e brana distan

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ength amino acid sequences using ClustralW and MEGA (5.05). The numbers at thccession numbers are given in the parentheses. The scale bar represents a genetic

.12. Statistics

For comparison of CsCC-Chem-1 gene expression, cell prolif-ration activity and chemotaxis activity, statistical analysis waserformed using one-way ANOVA and mean comparisons wereerformed by Tukey’s Multiple Range Test using SPSS 11.5 at the% significance level.

. Results

.1. CsCC-Chem-1 sequence analysis

A cDNA encoding CsCC-Chem-1 was identified from C.triatus normalized cDNA library constructed using Genomeequence FLXTM Technology (GS-FLXTM). The obtained CsCC-Chem-

sequence was submitted to NCBI GenBank Database under theene accession number JX469847. The physico-chemical propertyf CsCC-Chem-1 sequence was analyzed using DNAssist Program.

he results revealed that CsCC-Chem-1 was 641 bp in length.he sequence contains 87 bp 5′ untranslated region (UTR), 438 bppen reading frame (ORF) and 116 bp 3′ UTR. The ORF encoded

polypeptide of 146 amino acids. Further analysis showed that

ches denote bootsrap majority consensus values on 1000 replicates. The GenBankce 0.1 as the frequency of substitutions in pair wise comparison of two sequences.

the CsCC-Chem-1 polypeptide has an isoelectric point of 9.3 and atheoretical molecular weight of 15 kDa. The signal prediction pro-gram exhibited CsCC-Chem-1 to possess a signal peptide regionbetween 1 and 26, indicating that CsCC-Chem-1 may be an extracel-lular protein. Moreover, the domain search program showed a smallcytokine domain at 30–88 of CsCC-Chem-1 polypeptide (Fig. 1). Thisdomain carries the CC motif of CsCC-Chem-1 at Cys33-Cys34. In addi-tion to these, CsCC-Chem-1 protein also contains 9 high probabilitymotifs including 1 serine rich region (93–133), 4 protein kinase Cphosphorylation sites (3–5, 67–69,124–126 and 132–134), 3 caseinkinase II phosphorylation sites (25–28, 38–41 and 140–143) and 1N-myristoylation site (70–75).

3.2. Homologous analysis

Sequence similarity analysis showed that CsCC-Chem-1 isclosely related to the CC-Chem-1 from European sea bass Dicen-trarchus labrax (74%) followed by rock bream Oplegnathus fasciatus

(71%) (data not shown). Multiple sequence alignment of CsCC-Chem-1 with other known CC chemokines is presented in Fig. 2.The length of the amino acid sequences taken for multiple sequencealignment ranged between 90 and 144 excluding CsCC-Chem-1

P. Bhatt et al. / Molecular Immunology 57 (2014) 292– 301 297

Fig. 4. Relative expression of CsCC-Chem-1 gene quantified by real time PCR. (A) Results of tissue specific mRNA expression of CsCC-Chem-1 from various tissues of C. striatus.D of CsCa P < 0.0i

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ata are given as a ratio to CsCC-Chem-1 expression in brain. (B–D) The time courset 0, 3, 6, 12, 24, 48 and 72 h post-injection respectively. The significant difference (ndicated with asterisks.

146 amino acids). Further, the results revealed that the four cys-eine residues at C33, C34, C59 and C73 are conserved among theequences taken for analysis. The conserved cysteine residues at33 and C34 make the CC motif. Moreover, the other two cysteineesidues at C59 and C73 together with C33 and C34, make CsCC-hem-1 as a C4-CC chemokine.

.3. Phylogenetic tree

Phylogenetic tree of CsCC-Chem-1 was constructed along withther known CC chemokines sequences from mammals, avian andeleosts. CsCC-Chem-1 clustered together with CC chemokine from. fasciatus and D. labrax, which was closest to the CC chemokinesroup 1 (Fig. 3). The expression pattern (inducible/constitutive) ofC chemokines from human Homo sapiens has been extensivelytudied. But this detail is still missing in teleost fishes. Hence, weere interested to study the expression pattern of CsCC-Chem-

by comparing this sequence with other earlier reported CChemokines from H. sapiens. The results show that the CsCC-Chem-

fall within the clade of constitutive chemokines.

.4. Tissue specific mRNA expression

To study the tissue specific mRNA expression of CsCC-Chem-1,otal RNA isolation followed by first strand cDNA were synthesized.he synthesized cDNA were used as a template for quantificationf gene expression and subjected to qRT-PCR analysis. The resultf tissue specific mRNA expression is given in Fig. 4A. The anal-

sis exhibit that the CsCC-Chem-1 transcript is expressed in allhe tissues taken for examination. Significantly (P < 0.05) highestxpression was noticed in spleen followed by head kidney, kidney,eart, blood, gill, liver, skin, intestine, muscle and brain.

C-Chem-1 mRNA expression upon fugal, bacterial and poly I:C challenge in spleen5) of CsCC-Chem-1 expression between the challenged and the control group were

3.5. Tissue specific mRNA expression upon pathogenic infection

Based on the results of tissue specific mRNA expression,CsCC-Chem-1 mRNA expression in C. striatus was inducted inspleen following infection with fungus (A. invadans), bacteria (A.hydrophila) and virus (poly I:C). In fungus injected C. striatus, nosignificant expression was observed until 24 h post-injection (p.i.),but a significant (P < 0.05) up-regulation of CsCC-Chem-1 mRNAexpression was noticed (12-fold) at 48 h p.i., when compared tothe control group (Fig. 4B). In bacteria injected C. striatus, theexpression increased from 3 h p.i. and reached the highest (P < 0.05)expression at 24 h p.i. (10-fold increment compared to the con-trol) (Fig. 4C). The relative expression pattern of CsCC-Chem-1 inthe spleen after poly I:C injection is presented in Fig. 4D. Similar tobacterial expression pattern, the virus induced CsCC-Chem-1 mRNAexpression also increased from 3 h p.i. and reached the peak at 24 hp.i. (P < 0.05) (9-fold increment compared to the control). Overall,the results indicate the influence of CsCC-Chem-1 in the immunesystem of murrel.

3.6. Expression and purification of recombinant CsCC-Chem-1protein

The coding sequence of CsCC-Chem-1 was cloned into an E.coli vector. The cloned product was expressed and its recombinantprotein was purified. IPTG driven expression of CsCC-Chem-1 wasconducted in E. coli BL 21 (DE3) cells. The recombinant CsCC-Chem-1 protein was purified from the supernatant of induced cells. Theresults of SDS-PAGE showed a single band with molecular weight

around 57.5 kDa (42.5 kDa for MBP and 15 kDa for recombinantCsCC-Chem-1 protein). Further, the protein has been purified fromthe MBP fusion protein using DEAE-Sepharose ion exchange chro-matography method, and finally the recombinant CsCC-Chem-1

298 P. Bhatt et al. / Molecular Immunology 57 (2014) 292– 301

Fig. 5. SDS-PAGE of over expressed recombinant CsCC-Chem-1 in E. coli BL21 (DE3)cells and purified recombinant fusion protein. M: protein marker; Un: before induc-tion with IPTG; In: after IPTG induction at 15 ◦C for 5 h; FP: fusion protein; P: purifiedrecombinant CsCC-Chem-1 protein.

Fig. 6. Cell proliferation activity of recombinant CsCC-Chem-1 protein. C. striatusspleen leucocytes were incubated at 25 ◦C for 12 h with recombinant CsCC-Chem-1protein at various concentrations (1, 5, 10, 25, 50 and 100 �g/�L) using WST-1 cellproliferation assay kit. Data were given here as an average representation of threeraC

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Fig. 7. Chemotaxis activity of recombinant CsCC-Chem-1 protein. The chemotaxisindex was calculated in a neuro probe 48-well micro chemotaxis chamber bycomparison between number of cells that migrated by recombinant CsCC-Chem-1 protein induction at various concentrations (1, 5, 10, 25, 50 and 100 �g/�L) andnumber of cells that migrated by RPMI 1640 medium induction. Data were givenhere as an average representation of three replicates ± standard deviation. The sig-

eplicates ± standard deviation. The significant difference (P < 0.05) of cell prolifer-tion activity between the control and the various concentration of recombinantsCC-Chem-1 protein were indicated with asterisks.

rotein gave a major single band with molecular mass of 15 kDaFig. 5).

.7. Cell proliferation and chemotaxis activities

The proliferation and chemotaxis assays were conducted totudy the effect of recombinant CsCC-Chem-1 protein in C. striatuspleen leukocyte activity. Hence C. striatus spleen leukocytes werereated with recombinant CsCC-Chem-1 protein at various concen-rations and its cells proliferation capacity and chemotaxis activityas tested. The results of both the proliferation and chemotaxis

ctivities showed that the spleen leukocytes treated with recom-inant CsCC-Chem-1 protein at various concentrations exhibited

greater cell proliferation activity (Fig. 6) and chemotaxis activ-ty (Fig. 7) at various concentrations. Further, we observed thatn both cases the recombinant CsCC-Chem-1 protein functionedn a concentration dependant manner (P < 0.05) i.e., the activityncreases according to the concentration of the recombinant prod-ct increases.

. Discussion

In this study, we presented a detailed description on bioinfor-atics characterization and gene expression of CsCC-Chem-1 at

ormal as well as diseased state. Most of the earlier reported (Zhang

nificant difference (P < 0.05) of chemotaxis activity between the control and thevarious concentration of recombinant CsCC-Chem-1 protein were indicated withasterisks.

and Chen, 2008; Cheng et al., 2011a; Xu et al., 2011; Su et al., 2012;Kim et al., 2013) CC chemokines are proinflammatory chemokines.But, our CsCC-Chem-1 was not a proinflammatory chemokine dueto the absence of mRNA instability motif at 3′ UTR. Moreover, in thephylogenetic tree also, CsCC-Chem-1 fell within the clade of consti-tutive chemokines. Hence, based on the bioinformatics analysis, it ispredicted that CsCC-Chem-1 was a developmental and homeostaticchemokine as suggested by Laing and Secombes (2004b). More-over, CsCC-Chem-1 at 3′ UTR contained 3 microsatellite sequences(553GAA555, 561GAA563 and 632GAA634), which are necessary forgenome mapping due to their characteristic features of polymor-phism (Serapion et al., 2004). Similar findings were reported fromMiichthys miiuy CC chemokine (Cheng et al., 2011a).

CsCC-Chem-1 amino acid had a high isoelectric point (9.3) sim-ilar to other CC chemokine from Japanese flounder Paralichthysolivaceus (Khattiya et al., 2007), miiuy croaker M. miiuy (Xu et al.,2011) and O. fasciatus (Kim et al., 2013). Kim et al. (2013) stated thatthis high pI value is due to the presence of many lysine residues andarginine residues of the CC chemokine. In this study, we observed12 (8%) lysine residues and 6 arginine residues (4%) from CsCC-Chem-1 amino acid. Moreover, we observed that the CsCC-Chem-1is a secreted molecule due to the presence of signal sequence at theN-terminal region.

Li et al. (2011) reported that many CC chemokines contain anN-terminal ‘DCCL’ motif, and further suggested that this motif isthe signature motif (CC signature motif) of the CC chemokinesand this motif express the characteristic features of the chemokinesequence. Interestingly, in our findings we observed ‘TCCT’ motif at32–35 as CC signature motif at the N-terminal region of the CsCC-Chem-1 sequence. As Li et al. (2011) suggested, this variation inthe signature motif may represent new characteristic features ofthe CsCC-Chem-1 sequence. Hence it is possible to suggest that thenew features of CsCC-Chem-1 may lead to some unknown function,which needs to be focused further.

The multiple sequence alignment of CsCC-Chem-1 and humanshowed that most of the conserved amino acid residues are presentin the mature peptide sequence. Xu et al. (2011) stated that theseconserved sequences help to make the secondary structure ofthe molecule. The homologous analysis showed that the CsCC-

Chem-1 shared maximum similarity with fish CC chemokines andminimum with mammalian CC chemokines. Huising et al. (2003)and Bao et al. (2006) suggested that the low sequence similarityamong chemokines from various species resulted in conservative

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ubstitutions of amino acids to suitable function-specific diversifi-ation in the early evolutionary stages and extensive fish-specificene duplication of chemokine genes. For example, Zhang et al.2008) observed a ‘RGD’ motif in CC chemokine from large yellowroaker, but the similar motif from our sequence is substituted as1RAV63. It is interesting to note that the second residue from bothRGD’ and ‘RAV’ motif, G (Gly) and A (Ala) are non-polar aminocid (hydrophobic), so it is a perfect substitution. But the thirdesidue from these motifs D (Asp) and V (Val) have some sim-larities and dissimilarities. The dissimilarities of these residuesre Asp is a non-essential and negatively charged amino acid,hereas Val is an essential and non-polar amino acid. The simi-

arities among these residues, being that both are �-amino acidnd proteinogenic amino acid. So, based on these similarities, its possible to suggest that the third residue from both RGD andAV motifs are also a perfect substitution. Hence, in accordanceith the earlier suggestion of Huising et al. (2003) and Bao et al.

2006) RGD motif is substituted by RAV in CsCC-Chem-1 and pro-eeding the function of ‘RGD’. According to Barnes et al. (1983)his motif frequently interacts with integrin cell adhesion receptorsnd these interactions are considered to be involved in cell signal-ng, homeostatic regulation, phagocytosis, cell migration, cellularrafficking and lymphocyte recognition as reported by David et al.1990).

In the phylogentic tree, the inducible and constitutive CChemokines formed two separate clades. It is interesting to notehat CC-Chem-1 of C. striatus fell in the clade of constitutivehemokines. In contrast, CC-Chem-1 of H. sapiens fell in the clade ofnducible chemokines. This is in accordance with report that Mount2004) stated that these kinds of sequences assembled into theame function, but via different evolutionary paths, hence theseequences fell in different clades. Moreover, the phylogenetic anal-sis showed that some fish CC chemokines do not include otherpecies such as mammals and avian CC chemokines. It indicateshat most of the fish CC chemokines have been formed after theivergence of other mammalian and avian CC chemokines (Zhangt al., 2008).

Tissue specific mRNA expression analysis showed that CsCC-hem-1 was expressed in all the tissues tested and significantlyighest expression was observed in spleen. It is interesting to notehat the CC chemokine expression was not similar in all species.or example, the expression was noticed in the following tissues:lood (Zhang et al., 2008), kidney (Zhang and Chen, 2008), spleenTian et al., 2010; Li et al., 2011), fin (Xu et al., 2011), muscle (Chengt al., 2011b), gill (Su et al., 2012) and intestine (Cheng et al., 2011a;im et al., 2013). All these researchers suggested that the possibleeason for this variation in the expression is chemokines have multioles in homeostasis immune surveillance. The earlier studies sug-ested that the CC chemokines exhibit various tissue distributionatterns and dissimilar response to immune stimulants, thus show-

ng their functional variations in immune responses. In our study,e observed the highest CsCC-Chem-1 gene expression in spleen,hich is due to spleen being the major site of innate and adaptive

mmunity in fishes (Chaves-Pozo et al., 2005; Trede et al., 2004). Thepleen contains a huge number of immune cells including lympho-ytes and macrophages, which could be a reason why CsCC-Chem-1as highly expressed in it. Moreover, in C. striatus infected with fun-

us, bacteria and virus, the expression was up-regulated in spleent various time points. It indicates that the gene expression wasifferentially modulated by microbial pathogens. Zhang and Chen2008) stated that the variations in the gene expression at variousime points reflect the functional differences of the gene’s immune

esponse. This result suggests that CsCC-Chem-1 is involved in themmune responses against fungus, bacteria and virus.

In this study, we confirmed the CsCC-Chem-1 sequence usingMAL-c2x vector system. Further, the sequence was expressed in

ology 57 (2014) 292– 301 299

E. coli and obtained as a fusion protein. Finally, the CsCC-Chem-1 was separated from its MBP fusion protein and obtained as apurified protein. The molecular weight of the recombinant CsCC-Chem-1 protein was approximately 15 kDa on 12% SDS-PAGE gel,in line with earlier findings (Zhang and Chen, 2008; Zhang et al.,2008; Montero et al., 2011; Li et al., 2011; Kim et al., 2013). Sofar, many chemokines have been reported from fishes, but only afew studies have shown its functional activities and especially frommurrel CC chemikine-1 is nil. Hence, we characterized the puri-fied recombinant CsCC-Chem-1 protein by in vitro assays includingproliferation activity and chemotaxis activity. The result of theproliferation assay not only showed the functional importance ofCsCC-Chem-1, but also indicates its importance in modulating theimmune response of the targeted cells as demonstrated by Escheet al. (2005). In addition, our findings showed the possibility forthe activation of cells which resembles antigen-presenting cells asreported by Li et al. (2012). Moreover, we observed that the cell pro-liferation activity of recombinant CsCC-Chem-1 protein showed asignificant metabolic activity in a concentration dependant manneras found in CC chemokine from O. fasciatus (Kim et al., 2013).

Chemotactic assay showed the capability of recombinantCsCC-Chem-1 protein which can induce the migration of spleenleukocytes in C. striatus. Kono et al. (2003) reported that this assayis a good index to examine the capacity of macrophages to carryout non-specific defense function. In this study, we observed thatrecombinant CsCC-Chem-1 protein was functioning on concentra-tion dependant manner toward chemotaxis activity. Hence, it ispossible to suggest that recombinant CsCC-Chem-1 protein foldedproperly during chemotaxis. In contrast, Zhang et al. (2008) andKim et al. (2013) addressed improper folding of chemokines duringchemotaxis activation due to either high or low activity in responseto the recombinant concentration. Therefore, based on our chemo-taxis activity, it may be possible to report that CsCC-Chem-1 possessfunctions which are specific to chemotactic activity. However, thisdata is not enough to explain the complete molecular mechanism ofCsCC-Chem-1. Further research is necessary to clarify the recom-binant CsCC-Chem-1 protein mechanism connected to biologicalproperties of C. striatus leukocytes.

Conclusively, we have characterized a CsCC-Chem-1 cDNAsequence using various bioinformatics tools. Moreover, we stud-ied the tissue specific CsCC-Chem-1 mRNA expression pattern andexamined the expression profile in response to fungus, bacterialand viral pathogens. The expression profiles after disease chal-lenge showed that the CsCC-Chem-1 induced significant increasein spleen at various time points. The gene expression studiesshowed that the CsCC-Chem-1 is a capable gene to increase thecellular response against various microbial infections. We suc-cessfully expressed the CsCC-Chem-1 from C. striatus through anE. coli expression vector and acquired a highly purified functionalprotein. The purified recombinant CsCC-Chem-1 protein has beencharacterized by in vitro assays including proliferation activityand chemotaxis activity. The proliferation activity of recombinantCsCC-Chem-1 protein showed a significant metabolic activity ina concentration dependant manner. Moreover, the chemotacticactivity showed the capability of recombinant CsCC-Chem-1 pro-tein which can induce the migration of spleen leukocytes in C.striatus. However, this remains to be verified further at molecularand proteomic level.

Acknowledgement

This research is supported by DBT’s Prestigious Rama-lingaswami Re-entry Fellowship (D.O.NO.BT/HRD/35/02/2006),Department of Biotechnology, Ministry of Science and Technology,Government of India, New Delhi.

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