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0h

Molecular Immunology 56 (2013) 497– 506

Contents lists available at SciVerse ScienceDirect

Molecular Immunology

jo ur nal home p age: www.elsev ier .com/ locate /mol imm

ish lily type lectin-1 contains �-prism architecture: Immunologicalharacterization

birami Arasua,b, Venkatesh Kumaresana, Akila Sathyamoorthia,c,ajesh Palanisamya, Nagaram Prabhaa,c, Prasanth Bhatta, Arpita Royd,uthukumaresan Kuppusamy Thirumalaid, Annie J. Gnaname,ukesh Pasupuletid, Kasi Marimuthuf, Jesu Arockiaraja,∗

Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University,attankulathur 603 203, Chennai, Tamil Nadu, IndiaDepartment of Microbiology, SRM Arts & Science College, Kattankulathur 603 203, Chennai, IndiaDepartment of Biotechnology, SRM Arts & Science College, Kattankulathur 603 203, Chennai, IndiaSRM Research Institute, SRM University, Kattankulathur 603 203, Chennai, IndiaInstitute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A4800, Austin, TX 78712, USADepartment of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling Bedong, 08100 Bedong, Kedah, Malaysia

r t i c l e i n f o

rticle history:eceived 10 May 2013eceived in revised form 17 June 2013ccepted 27 June 2013

eywords:hanna striatusily type lectinene expression

a b s t r a c t

In this study we report a full-length lily type lectin-1 (CsLTL-1) identified from striped murrel, Channastriatus. CsLTL-1 was identified from the established C. striatus cDNA library using GS-FLXTM genomesequencing technology and was found to contain 354 nucleotide base pairs and its open reading frame(ORF) encodes a 118 amino acid residue. CsLTL-1 mRNA is predominately expressed in the gills and isup-regulated upon infection with fungus (Aphanomyces invadans) and bacteria (Aeromonas hydrophila).Hemagglutination studies with recombinant CsLTL-1 show that, at 4 �g/ml agglutinates occurs in acalcium independent manner and is inhibited in the presence of d-mannose (50 mM) and d-glucose(100 mM). The CsLTL-1 sequence was completely characterized using various bioinformatics tools. CsLTL-

emagglutination assayugar binding specificity

1 peptide contains a mannose binding site at 30–99 along with its specific motif of �-prism architecture.The phylogenetic analysis showed that CsLTL-1 clustered together with LTL-1 from Oplegnathus fascia-tus. CsLTL-1 protein 3D structure was predicted by I-Tasser program and the model was evaluated usingRamachanran plot analysis. The secondary structure analysis of CsLTL-1 reveals that the protein con-tains 23% �-sheets and 77% coils. The overall results showed that CsLTL-1 is an important immune geneinvolved in the recognition and elimination of pathogens in murrels.

© 2013 Elsevier Ltd. All rights reserved.

. Introduction

Immune system is classified into two main types i.e., innatemmunity and adaptive immunity. The innate immune system isresent in both invertebrates and vertebrates, while the adap-ive immunity responses are found in only in higher vertebratesTsutsui et al., 2006a). Among the both, the innate immunity is a

ore generalized and robust response whereas the other is a highlypecific response to infectious pathogens. Fishes lack specialized

ymphatic organs, and their adaptive immune system is also notully developed or effective at low environmental temperature (Blynd Clem, 1992). Therefore, fish species largely depend on their

∗ 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.2013.06.020

innate immunity rather than adaptive immunity for protectionsagainst pathogens. Moreover, instead of depending up on antibod-ies for pathogen recognition, innate immunity depends largely onthe pattern-based recognition, often through the arrangement ofsugars on their surfaces. The innate immunity components whichidentify these sugars are called as lectins. Extracellular and solublelectins can recognize specific carbohydrate on the pathogen sur-faces and bind to them, followed by phagocytosis by macrophagesand complement-mediated cell lysis. Lectins have been shown toplay an important role in the innate immune system of fish, espe-cially in the absence of acquired or antibody-mediated immunitysystem (Ewart et al., 2001; Fujita, 2002; Jack and Turner, 2003;

Turner, 2003; Gadjeva et al., 2004).

Previously, lectins were classified according to the carbohydratespecificity to which they bind (e.g., �-galactoside-binding lectins).With the advent of molecular tools a more consistent classification

4 mmun

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98 A. Arasu et al. / Molecular I

merged, which is based upon the amino acid sequence homologynd evolutionary relatedness of the lectins. All lectins possess ateast one non-catalytic domain, which binds reversibly to a specificarbohydrate which is widely distributed in bacteria, fungi, plants,nvertebrates and vertebrates, either in a soluble or membrane-ound form (Peumans and Van Damme, 1995; Sharon and Lis,972; Barondes, 1988). Lectins such as F-type lectins, intelectins,annose-binding protein (MBP) are known to play an important

ole in innate immunity and disease resistance (Holmskov et al.,994; Malhotra and Sim, 1995; Ni and Tizzard, 1996; Turner, 1996;u, 1997). Mannose binding lectin (MBL) is an acute-phase pro-ein produced by liver hepatocytes that increases in response ton infection or inflammation conditions. MBL acts as an opsoninor phagocytosis by macrophages (Hoffmann et al., 1999; Suckalet al., 2005; Kindt et al., 2007). Numerous researchers have beeneported MBL type lectins in salmon, rainbow trout, carp, rohu,hannel catfish and blue catfish (Jensen et al., 1997a; Ewart et al.,999; Vitved et al., 2000; Mitra and Das, 2002; Ourth et al.,007).

In higher vertebrates lectins is found in serum, plasma, sur-ace mucus, egg surfaces and components (Jensen et al., 1997a,b;ttinger et al., 1999), although the skin mucus of several animal

pecies, including fish is also assumed to be a rich source of novelnd new unreported lectins (Jensen et al., 1997b). Suzuki et al.2003) have identified a novel and new type of lily type lectin fromuffer fish and named it as pufflectin-s. Lily type lectin (LTL) hashree d-mannose binding sites and it has high similarity with other

annose binding lectin, skin mucus lectin and intestine mucusectin (Kamiya and Shimizu, 1980; Oda et al., 1984; Al-Hassant al., 1986; Shiomi et al., 1987, 1989, 1990; Kamiya et al., 1988;oto-Nance et al., 1995; Toda et al., 1996; Tasumi et al., 2002,004). Interestingly, Chandra et al. (1999) and Tsutsui et al. (2003)eported a lectin from plants, snowdrop and garlic which consistf three mannose binding sites similar to that of lily type lectin.owever, it is uncertain whether all skin mucus lectins belong to

he lily type lectin or some other lectins. In aspect the available lit-rature on molecular as well as biochemical details of LTL is verycanty.

The striped murrel (otherwise called snakehead) Channa stria-us is an economically important freshwater finfish. Worldwidenland fish culture industry is suffering from massive economicosses due to epizootic ulcerative syndrome (EUS) and fish basedathogens. The available literature indicate that infection fromsh pathogens like bacteria (Aeromonas hydrophila and Aeromonasobria), fungus (Aphanomyces invadans) and viruses can causetunted growth and severe mortalities in the C. striatus (Chinabutt al., 1995; Catap and Munday, 1998; Blazer et al., 1999). Weelieve that information related innate immune genes character-

zation is necessary to control the diseases at the molecular level.o far, many immune-related genes or proteins and their func-ions have been identified in different finfishes, but the detailsn striped murrel are still poorly understood. Due to its signif-cant commercial importance, studies related to the alterationsn the gene expression especially innate immunity genes underhe infection conditions are urgently needed. According to ournowledge, the corresponding work on gene characterization oftriped murrel lily type lectin-1 has not been reported to date.n this study, a striped murrel (C. striatus) lily type lectin-1ene (designated as CsLTL-1) was identified from the constructedDNA library of C. striatus using Genome Sequence FLXTM (GSLXTM) Technology. Quantitative real-time PCR (qRT-PCR) wasarried out to evaluate tissue distribution and the response to

mmune stimulants such as bacteria (A. hydrophila) and fun-us (A. invandans). The protein was over expressed, purified andhe purified protein was then investigated in biological activi-ies.

ology 56 (2013) 497– 506

2. Materials and methods

2.1. Fish

Healthy C. striatus (average body weight of 40 g) were obtainedfrom the Surya Agro Farms, Erode, Tamil Nadu, India. Fishes weremaintained in flat-bottomed plastic tanks (150 l) with aerated andfiltered freshwater at 29 ± 2 ◦C in the laboratory. All fishes wereacclimatized for 1 week before being challenged to A. invadans andA. hydrophila. A maximum of 15 fishes per tank were maintainedduring the experiment.

2.2. Construction of C. striatus cDNA library and identification ofCsLTL-1

A full-length cDNA of CsLTL-1 was identified from the con-structed C. striatus cDNA library by the GS FLXTM technology.Briefly, total RNA was isolated using Tri ReagentTM (Life Tech-nologies) from the tissue pool including spleen, liver, kidney,muscle and gills of healthy C. striatus. The mRNA was thenpurified using a mRNA isolation kit (Miltenyi Biotech). Thefirst strand cDNA synthesis and normalization were carriedout with CloneMinerTM cDNA library construction kit (Invitro-gen) and Trimmer Direct Kit: cDNA Normalization Kit (BioCatGmbH). Thereafter, the GS-FLXTM sequencing of C. striatuscDNA was performed according to the manufacturer’s pro-tocol (Roche). The raw data was processed with the Rochequality control pipeline using the default settings. Seqclean(http://compbio.dfci.harvard.edu/tgi/software/) software was usedto screen for and remove normalization adaptor sequences,homopolymers and reads shorter than 40 bp prior to assembly. Fur-ther, the sequences were subjected to assemble by MIRA program(version 3.2.0) (Chevreux et al., 2004) into full-length cDNAs. Fromthe established cDNA library of C. striatus sequence database, a LTL-1 gene was identified through BLAST annotation program on NCBI(http://www.blast2go.com/b2ghome).

2.3. Bioinformatics analysis of CsLTL-1

The full-length CsLTL-1 sequence was compared with othersequences available in NCBI database (http://blast.ncbi.nlm.nih.gov/Blast) and the similarities were analyzed. The open readingframe (ORF) and amino acid sequence of CsLTL-1 was obtainedby using DNAssist (version 2.2.). Characteristic domains or motifswere identified using the PROSITE profile database (http://prosite.expasy.org/scanprosite/). Identity, similarity and gap percent-ages were calculated using FASTA program (http://fasta.bioch.virginia.edu/fasta www2/fasta www.cgi). The N-terminal trans-membrane sequence was determined by DAS transmembraneprediction program (http://www.sbc.su.se/∼miklos/DAS). Signalpeptide analysis was done using the SignalP (http://www.cbs.dtu.dk). The domain and motif were analyzed using the PROSITEprogram (http://kr.expasy.org/prosite). Multiple sequence align-ment was carried out on ClustalW (version 2) (http://www.ebi.ac.uk/Tools/msa/clustalw2/) program to find out the evolutionarilyconserved residues among the different organisms. The sequenceswere aligned using BLOSUM50 method with a gap extension value0.5 and gap open value and gap distance value of 5. The alignedsequences were analyzed on Bioedit (version 7.1.3.0). In graphicview, the threshold limit was set to 100% to obtain the exactmatches in the aligned sequences (Hall, 1999). The evolutionaryhistory of CsLTL-1 was inferred using the Neighbor-Joining method.

The evolutionary distances were computed using the Poisson cor-rection method (Uinuk-Ool et al., 2003). All positions containinggaps and missing data were eliminated. The phylogentic tree wasconducted in MEGA 5.

A. Arasu et al. / Molecular Immunology 56 (2013) 497– 506 499

Table 1Details of primers used in this study.

Name Target Sequence (5′–3′ direction)

CsLTL-1 (F1) q RT-PCR amplification CCAAACACGGTGAAGCAATATGCsLTL-1 (R2) q RT-PCR amplification GAATAATGACGGTCAGCTGGT�-actin (F3) q RT-PCR internal control TCTTCCAGCCTTCCTTCCTTGGTA

lm1(afrepdwtoh2hfdadc1miaTdRtcgs

2

ilmppb(w(Dipwiw4ssc

�-actin (R4) q RT-PCR internal control

CsLTL-1 (F5) ORF amplification

CsLTL-1 (R6) ORF amplification

The secondary structure of the CsLTL-1 protein was ana-yzed using SOPMA program and is constructed on Polyview

ethod (http://polyview.cchmc.org). The 3D structure of the CsLTL- deduced amino acid sequence was predicted by I-Tasser programhttp://zhanglab.ccmb.med.umich.edu/I-TASSER). I-Tasser gener-tes full-length model of proteins by excising continuous fragmentsrom threading alignments and then reassembling them usingeplica-exchanged Monte Carlo simulations (Zhang, 2008; Ambrisht al., 2010). The quality of the five 3D structures of CsLTL-1redicted by the I-Tasser program was assessed using Ramachan-ran plot analysis to obtain the best structure. The analysisas carried out to find the amino acid residues dispersion in

he favored, allowed and disallowed region. The phi psi plotf the predicted 3D structure of CsLTL-1 was constructed usingttp://mordred.bioc.cam.ac.uk/∼rapper/rampage.php (Lovell et al.,002). Further, all the five models were evaluated usingttp://modbase.compbio.ucsf.edu/modeval/ (Shen and Sali, 2006)

or discrete optimized protein energy (DOPE) and root mean squareistance (RMSD) score to obtain the best structure of CsLTL-1mong the five predicted 3D models of I-Tasser program. The pre-icted 3D structure of CsLTL-1 was further analyzed for structuralharacterization using PyMol Molecular Graphics System (version.5). The validated model of CsLTL-1 was used for the analysis. Theodel was viewed in Mesh view and surface view. Domain regions

n the sequence were identified by Pfam and SMART databases,long with motif prediction by PRINTS and motif search databases.he PDB structure obtained from I-Tasser program was used to pre-ict the position of the sugar binding sites. Moreover, to predict theNA structure of CsLTL-1, the cDNA of CsLTL-1 was converted tohe corresponding RNA sequence using http://dnatorna.com/. Theonverted RNA sequence was submitted in RNA fold server pro-ram (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) to predict thetructure of CsLTL-1 RNA with minimum free energy (MFE).

.4. Fungus and bacterial infection

In order to analyze the mRNA expression level, the fish werenjected with fungus A. invadans (102 spores). A. invandans were iso-ated from the EUS infected C. striatus muscle sample. The infected

uscle sample was taken from the EUS infected C. striatus and waslaced in a Petri dish of algal boost GP medium with 100 units ml−1

enicillin G and 100 �g/ml streptomycin. The GP medium was incu-ated at 25 ◦C for 12 h and examined under a binocular microscopeNikon-Eclipse E400 microscope, Germany). The fungal speciesere identified according to the description of Caster and Cole

Caster and Cole, 1990) using potato dextrose agar and Czapekox agar (Himedia, Mumbai). For bacterial challenge, the fish were

njected intraperitonealy with A. hydrophila (5 × 106 CFU/ml) sus-ended in 1× phosphate buffer saline (100 �l/fish). A. hydrophilaere also isolated and identified from the muscle sample of EUS

nfected C. striatus as described by Dhanaraj et al. (2008). Samplesere collected before (0 h), and after injection (3, 6, 12, 24 and

8 h) and were immediately snap-frozen in liquid nitrogen andtored at −80 ◦C until total RNA was isolated. Using a sterilizedyringe, the blood (0.5–1.0 ml per fish) was collected from the fishaudal fin and immediately centrifuged at 4000 × g for 10 min at

GACGTCGCACTTCATGATGCTGTT(GA)3GAATTCATGAGTAAGAACTACTTGTCCAGAEcoRI(GA)3CTGCAGCCAAAGGTTCAAAGAAATGAHindIII

4 ◦C to allow blood cell collection for total RNA extraction. PBS (1×)were prepared and served as control (100 �l/fish). All samples wereanalyzed in three duplications and the best representative data isexpressed here. The methodology followed for this, is describedelsewhere (Livak and Schmittgenm, 2001).

2.5. Total RNA isolation and first strand cDNA conversion

Total RNA from the control and infected fish were isolated usingTri ReagentTM (Life Technologies), according to the manufacture’sprotocol with slight modifications (Arockiaraj et al., 2012a,b). Using2.5 �g of RNA, first strand cDNA synthesis was carried out using aSuperScript® VILOTM cDNA Synthesis Kit (Life technologies) withslight modifications (Arockiaraj et al., 2012c,d). The isolated cDNAwas stored at −20 ◦C for further analysis.

2.6. CsLTL-1 gene expression studies

The relative expression of CsLTL-1 in blood, gills, liver, heart,spleen, intestine, head kidney, kidney, skin, muscle and brain weremeasured by quantitative real-time polymerase chain reaction(qRT-PCR). qRT-PCR was carried out using a ABI 7500 Real-timeDetection System (Applied Biosystems) in 20 �l reaction volumecontaining 4 �l of cDNA from each tissue, 10 �l of Fast SYBR® GreenMaster Mix, 0.5 �l of each primer (20 pmol/�l) and 5 �l dH2O. TheqRT-PCR cycle profile was 1 cycle of 95 ◦C for 10 s, followed by 35cycles of 95 ◦C for 5 s, 58 ◦C for 10 s and 72 ◦C for 20 s and finally 1cycle of 95 ◦C for 15 s, 60 ◦C for 30 s and 95 ◦C for 15 s. The same qRT-PCR cycle profile was used for the internal control gene, �-actin.�-actin of C. striatus primers were designed from the sequence ofGenBank Accession No. EU570219. The primer details are given inTable 1. After the PCR program, data were analyzed with ABI 7500SDS software (Applied Biosystems). To maintain the consistency,the baseline was set automatically by the software. The compara-tive CT method (2−��CT method) was used to analyze the expressionlevel of CsLTL-1 (Livak and Schmittgenm, 2001). All samples wereanalyzed in three duplications and the best representative data isexpressed here. The methodology followed for this, is describedelsewhere (Livak and Schmittgenm, 2001).

2.7. Cloning of CsLTL-1 into pMAL-c2X vector

All the cloning experiments were carried out according toSambrook et al. (1989) with slight modifications (Arockiaraj et al.,2012d,e). In order to clone the CsLTL-1 gene in pMAL-c2X (BioLabsInc.), the forward and reverse primer were designed with corre-sponding restriction sites (Table 1). The isolated cDNA was usedas template to amplify the CsLTL-1 gene in PCR. After PCR, theamplified product was purified by using the GeneJETTM (ThermoScientific). Further the cloned products were sequenced using theABI Prism-Bigdye Terminator Cycle Sequencing Ready Reaction kitand analyzed using an ABI 3730 sequencer. The eluted product was

then digested with respective restriction enzymes, which is presentin forward and reverse primers. To achieve the protein expres-sion level, the ligated product was transformed into Escherichia coliBL21.

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00 A. Arasu et al. / Molecular I

.8. Over expression and purification of recombinant CsLTL-1rotein

Transformed E. coli BL21 (DE3) cells were incubated in ampi-illin (100 �g/ml) Luria broth (LB) overnight. At 0.7 OD the geneas overexpressed with the final concentration of 1 mM IPTG and

ncubated at 12 ◦C for 6 h. Cells were harvested by centrifuga-ion (4000 × g for 20 min at 4 ◦C). E. coli BL21 (DE3) uninducedulture was used as a negative control. The cell pellet was thene-suspended in column equilibration. Cells were sonicated andentrifuged at 8000 × g for 30 min at 4 ◦C. The crude CsLTL-1 fusionrotein fused with maltose binding protein (MBP) was purifiedsing pMALTM protein fusion and purification system protocol (Bio-abs). Further, the fusion protein was digested and purified asescribed in our earlier report (Arockiaraj et al., 2013). The purifiedrotein was analyzed by 12% SDS-PAGE and the molecular weightas determined using protein standard marker. The purified pro-

ein was visualized by staining with 0.05% Coomassie blue R-250.he purified fractions were pooled together and the protein con-entration was determined using the Bradford method (Bradford,976). The purified protein was kept at −80 ◦C until determinationf biological activity.

.9. Hemagglutination assay

The hemagglutinating activity (HA) of CsLTL-1 was testedsing 2% (v/v) erythrocytes from mouse according to the methodescribed by Wei et al. (2010) and Luo et al. (2003). Erythrocytesere washed three times with TBS-Ca buffer (50 mM Tris–HCl,H 7.5, 150 mM NaCl, 10 mM CaCl2) and then suspended at 2%v/v) in TBS-Ca buffer. Two-fold serial dilutions (25 �l) of CsLTL-

in TBS-Ca buffer were mixed with 25 �l of the erythrocytes in6-well microtiter plate. The lowest dilution that caused distinctemagglutination was determined after 1 h at room temperature.rythrocytes mixed with serial dilutions of bovine serum albuminBSA, Sigma) and in TBS-Ca buffer were processed in parallel withhe above-described erythrocytes to serve as controls.

To test whether calcium is required for the hemagglutinationctivity of CsLTL-1, 12.5 �l of serial dilutions of EDTA in TBS-Cauffer was mixed with 12.5 �l of CsLTL-1, 25 �l 2% trypsin-treatedouse erythrocytes were added, and the mixture was incubated

or 1 h at room temperature. Hemagglutination of erythrocytes wasbserved. Assays were performed in triplicate.

.10. Sugar binding specificity of CsLTL-1

The sugar binding specificity of CsLTL-1 was determined by annhibitory agglutination assay (indirect binding assay) (Wei et al.,010). Serial dilutions (12.5 �l) of various carbohydrates (Sigma) inBS-Ca buffer, including d-glucose, lactose, sucrose, d-galactose, d-annose, maltose, d-fructose and xylose were mixed with 12.5 �l

f CsLTL-1 and incubated for 30 min at 4 ◦C. Then 2% trypsin-treatedouse erythrocytes were added, and the mixture was incubated for

h at room temperature. An inhibitory effect was expressed as theinimum concentration of a carbohydrate required for complete

nhibition of the hemagglutinating activity of CsLTL-1. This assayas performed in triplicate.

.11. Bacterial agglutination assay

The bacterial recognition of CsLTL-1 was assessed by a bacte-ial agglutination test following the methodology of Tunkijjanukij

nd Olafsen (1998) and Yang et al. (2007). The Gram negativeacteria E. coli, A. hydrophila, Vibrio vulnificus and Vibrio alginolyti-us and Gram positive bacteria Bacillus mycoides, Streptococcusyogenes and Staphylococcus aureus were suspended in a Tris

ology 56 (2013) 497– 506

buffered saline (TBS) (50 mM Tris–HCl, 100 mM NaCl, pH 7.5) at5.0 × 109 cells ml−1. A 10 �l of bacteria suspension was added to40 �l of recombinant CsLTL-1 in serial dilution (5, 25, 50, and100 �g/ml) by TBS containing 10 mM CaCl2, or only TBS cal-cium chloride solution as a control. The mixtures were incubatedovernight at 4 ◦C. Cells were observed in light microscopy.

2.12. Statistical analysis

For comparison of relative CsLTL-1 mRNA expression, statisticalanalysis was performed using one-way ANOVA and mean compar-isons were performed by Tukey’s Multiple Range Test using SPSS11.5 at the 5% significance level.

3. Results

3.1. CsLTL-1 cDNA analysis

The nucleotide and deduced amino acid sequences of theCsLTL-1 from C. striatus are given in supplementary material. TheCsLTL-1 nucleotide sequence has been deposited in EMBL GenBankdatabase under accession number HF571337. The full-length CsLTL-1 cDNA is 357 bp with an open reading frame (ORF) of 354 bp that istranslated into a putative peptide of 118 amino acid (aa) residues.The CsLTL-1 peptide has a theoretical molecular mass of 13 kDawith 9.1 as isoelectric point (pI).

3.2. Domain and motif analysis of CsLTL-1

CsLTL-1 aa sequence do not have a signal peptide nor a trans-membrane region, but contains Bulp-type mannose binding lectin(B-lectin) domain between 3 and 113 aa (total of 111 aa sequence).A long dimerization interface site is also available within the B-lectin domain profile between aa 4 and 112 (total of 109 aa).Further within the B-lectin, a mannose binding site is availablebetween aa 30 and 99 with its specific motif of QxDxNxVxY inthree-fold internal repeat (�-prism architecture). The repeat 1 at:Gln30-Asp32-Asn34-Val36-Tyr38, repeat 2 at: Gln59-Asp61-Asn63-Val65-Tyr67and repeat 3 with slight changes (TxNxDxQxV) in theaa positions at Thr91-Asp93-Lys95-Val97-Tyr99 is available respec-tively. This �-prism architecture motif is involved in �-D mannoserecognition. Other than these, there are another 5 high probabil-ity occurrence motifs present in the aa sequence of CsLTL-1 (datashown in supplementary material).

3.3. Homologous analysis and multiple sequence alignment

The CsLTL-1 amino acid identity and similarity percentageswere calculated using FASTA program. We analyzed the CsLTL-1sequence identity with other lectin superfamily members includinglily type lectin, skin mucus lectin and mannose binding lectin fromdifferent fishes (data shown in supplementary material). Resultsshow that the CsLTL-1 has higher identity (56%) and similarity(73%) to the lily type lectin-1 from rock bream Oplegnathus fas-ciatus compared to the other lectin super family members selectedin this study. In addition, CsLTL-1 was aligned with other homol-ogous lectin members from O. fasciatus, orange-spotted grouperEpinephelus coioides, Spotnape ponyfish Leiognathus nuchalis, bar-tail flathead Platycephalus indicus and puffer fish Takifugu rubripes inClustalW multiple analysis (Fig. 1). The results revealed that CsLTL-

1 is bit longer in length (118 aa) than other species, even though thelength of the amino acids varied from species to species, conservedmotifs were observed among the sequences taken for the analysis,thus confirming the identity of the gene as LTL.

A. Arasu et al. / Molecular Immunology 56 (2013) 497– 506 501

10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|

c.striatus MSKNYLSRNDELRKGD YLM SENGN YKAVFQ GDGNFV VYAWSP IWATNTHGKNPYKI LLQQDGNLVMYPKH 70 o.fasciatus MSKNYLSRNDELRKGD YLI SNNGQ WKAIFQ DDANFV IYGWKP VWASDTWGSDAVRL CMQTDSNLVMYNQC 70 E.coioides MSRNFLSRNDELRRGD YLI SNNGE YKAVFQ EDGNFV IYGWKS VWESETKGTDAQRL CMQADCNLVMYNKC 70 L.nuchalis MSKNYLSKNDELRKGD FLI SNNRQ FKAVFQ EDGNFV VYGWKP MWFSGTNGADVERL CMQADCNLVMYNAC 70 P.indicus MSKNFLSKNDELRRGD YLL SNNGQ WKAIFQ DDGNFV IYGWSP VWDSKTHGSDVFRL CMQADCNLVMYNKT 70 T.rubripes MSVTVLENGSELKRGDSVLSKNSQWIALFQHDGNFVVYRTEPVWASDTSGMDPTRLCMQEDCNLVMYNDE 70

D-Mannose Binding Site-1 D-Mannose Binding Site-2

80 90 100 110 ...|....|....|....|....|....|....|....|....|...

c.striatus GEAIWSTGTYSNQSCCRMRLTLNNDGQLVLERDGKTIWNTADSKGSKK 118 o.fasciatus DTPRWATDS-NRSECNMCRLQLTDDGKLV VYRKSEE LWSSADSKGSKK 117 E.coioides DEPRWHTNS-AKSDCNMCRLQLTDDGKLVLSRECDEIWSSAKSKGMK- 116 L.nuchalis DEPKWHSNS-SKPKPNMCRLQLTDTGDLV VYRECEE IWRSTNDK---- 113 P.indicus GSPKWHTNS-SKGDCNMCRLELTNDGKLV LYKESDQ IWSSDDNNGMK - 116 T.rubripes DKPRWHTNT-SKGGRNTCVLSLTDEGKLV LKKDCQQ LWNSDRDHGMK - 116

D-Mannose Binding Site-3

QXDXNXVXY QXDXNXVXY

TXNXDXQXV

Fig. 1. Multiple sequence alignment of CsLTL-1 with other homologous genes lily type lectin-1 from O. fasciatus, lily type lectin from Epinephelus coioides, L. nuchalis and P.i alonr shes.

m

3

1fsflOot

3

Ttc(dagrmatdmmmscFif

ndicus and skin mucus lectin from Takifugu rubripes. The amino acids are numberedesidues are shaded in black and grey respectively. Deletions are indicated by daaterial. The three d-mannose binding sites are highlighted.

.4. Phylogenetic tree

In order to elucidate the evolutionary relations between CsLTL- and other species, phylogenetic analysis was performed usingull-length amino acid sequence of CsLTL-1 along with 12 repre-entatives from LTL and 1 from mannose binding lectin. The treeormed three distinct clades, i.e., fish LTLs, plant mannose bindingectin and bacterial mannose binding lectin. CsLTL-1 and LTL-1 from. fasciatus were clustered together and formed a sister group withther fish LTL. Overall the phylogenetic tree is in agreement withhe traditional taxonomy (Fig. 2).

.5. Structural analysis of CsLTL-1

CsLTL-1 protein 3D structure was predicted by I-Tasser program.he secondary structure analysis of CsLTL-1 reveals that the pro-ein contains 23% (27 residues) �-sheets and 77% (89 residues)oils. No �-helix region is presented in the secondary structuredata shown in supplementary material). I-Tasser program pre-icted five different 3D models of CsLTL-1 protein. The quality ofll the five 3D models of CsLTL-1 predicted by the I-Tasser pro-ram was assessed using Ramachandran plot analysis and the bestepresentation of psi phi graph analysis is given in supplementaryaterial. The analysis clearly shows the proline and glycine favored

nd allowed regions in psi phi graph. Moreover, the analysis showshe amino acid residues dispersion in the favored, allowed andisallowed region of all the five models and presented in supple-entary material. Further the zDOPE and RMSD value of the fiveodels were calculated to obtain the best score (supplementaryaterial). Based on the score, best model was selected and pre-

ented in supplementary material. The overall structure of CsLTL-1

onsisted of 12 �-sheets indicated by patches, turns and loops.urther structural analysis indicated that the overall structures composed of three sub-domains connected by pseudo-three-old symmetry. And each sub-domain is composed of 4-stranded

g the right margin as well the top of the sequences. Conserved and semi-conservedThe GenBank accession numbers of the homologous are given in supplementary

anti-parallel �-sheet. The mannose-binding sites were located inthe clefts formed by the three bundles of �-sheets. The sugar bind-ing site possessed the motif ‘QxDxNxVxY’ of two repeats and thethird repeats with slight modification ‘TxNxDxQxV’. The selectedbest 3D structure of CsLTL-1 was further analyzed for structuralcharacterization. The surface view and mesh view analysis of CsLTL-1 shows that the three mannose binding sites are arranged in a�-prism-like shape. The molecular surface CsLTL-1 looks highlyintact (data shown in supplementary material). The predicted RNAfold structure of CsLTL-1 with minimum free energy (MFE) is givenin supplementary material. The MFE of the predicted RNA struc-ture of CsLTL-1 is −98.95 kcal/mol. The predicted mRNA structureshows that the RNA is mostly paired and very few nucleotides areleft unpaired.

3.6. Tissue distribution of CsLTL-1

To determine the tissue-specific CsLTL-1 mRNA expression pro-file, quantitative real-time PCR was carried out using gene-specificprimers designed from the CsLTL-1 coding sequence. The relativemRNA expression of each tissue was calculated using C. striatus�-actin as a reference gene and the result was further comparedwith brain expression level to determine the relative tissue-specificexpression profile (Fig. 3A). CsLTL-1 mRNA was constitutivelyexpressed in all eleven selected tissues including blood, gills, liver,heart, spleen, intestine, head kidney, kidney, skin, muscle and brain.Further analysis showed that CsLTL-1 mRNA expression was sig-nificantly higher (P < 0.05) in the gills, liver, intestine and skin;moderate in kidney, muscle and spleen; and poorly expressed in

blood, head kidney, heart and brain. Furthermore, in all tissues, theexpression level was significantly (P < 0.05) varied in the brain. Thisresult indicates that CsLTL-1 showed tissue specific variation in C.striatus tissues.

502 A. Arasu et al. / Molecular Immunology 56 (2013) 497– 506

Epinephelus coioides LTL

Larimichthys crocea LTL

Leiognathus nuchalis LTL

Platycephalus indicus LTL

Oplegnathus fasciatus LTL -1

Channa striatus LTL-1

Allium ampeloprasum MBL

Annona squamosa MBL

Allium sativum MBL

Diplachne fusca L

Mycobacterium sp. MBL

Brevebacillus laterosporus D-MBL

Arthrobacter aurescens D-MBL

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Fig. 2. Phylogentic analysis of CsLTL-1 with other species was reconstructed by the Neighbor-Joining Method. The tree is based on an alignment corresponding to full-lengthamino acid sequences using ClustralW and MEGA (5.05). The numbers at the branches denote bootsrap majority concensus values on 1000 replicates. The GenBank accessionnumbers are given in supplementary material. The scale bar represents a genetic distance 0.1 as the frequency of substitutions in pair wise comparison of two sequences.LTL, lily type lectin; LTL-1, lily type lectin-1; MBL, mannose binding lectin; L, lectin and d-MBL, d-mannose binding lectin.

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Fig. 3. Relative quantification of CsLTL-1 gene expression by real-time PCR. (A) Results of tissue distribution analysis of CsLTL-1 from various organs of C. striatus. Data aregiven as a ratio to CsLTL-1 mRNA expression in brain. (B and C) The time course of CsLTL-1 mRNA expression in gills at 0, 3, 6, 12, 24, and 48 h post-injection with A. invadansand A. hydrophila respectively.

A. Arasu et al. / Molecular Immun

Fig. 4. SDS-PAGE of over expressed recombinant CsLTL-1 in E. coli BL21 (DE3) cellsawp

3b

awinBctt

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EHIcopfps

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oiwdbi

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nd purified recombinant fusion protein. M, protein marker; In, before inductionith IPTG; Un, after IPTG induction at 10 ◦C for 18 h; FP, CsLTL-1 fusion protein; P,urified recombinant CsLTL-1 protein.

.7. Transcriptional responses of CsfLTL-1 after fungal andacterial infection

Quantitative real time-PCR was performed to examine the rel-tive expression of the CsLTL-1 in C. striatus gill tissue challengedith fungus (A. invandans) and bacteria (A. hydrophila). In fungus

nfected gill tissue, the relative expression level of CsLTL-1 was sig-ificantly (P < 0.05) up-regulated at 24 h (Fig. 3B) post-injection.acteria infected C. striatus CsLTL-1 mRNA expression was signifi-antly increased (7 fold; P < 0.05) at 12 h post-injection comparedo the PBS injected control (Fig. 3C). The expression almost reachedo basal level at 48 h post-injection of bacteria.

.8. Recombinant CsLTL-1 protein expression and purification

The putative mature CsLTL-1 molecule cDNA was expressed in. coli BL 21 (DE3) cells after cloning the cDNA into the EcoRI andindIII restriction sites of pMAL-c2x-CsLTL-1 expression vector,

PTG driven expression of CsLTL-1 was done in E. coli BL 21 (DE3)ells. The recombinant CsLTL-1 was purified from the supernatantf induced cells. The molecular mass of the CsLTL-1 with fusionrotein was found to be 55.5 kDa as shown by SDS-PAGE (42.5 kDaor maltose binding protein and 13 kDa for CsLTL-1). In order tourify the CsLTL-1 from the maltose binding fusion protein, DEAEepharose anion exchange chromatography was performed (Fig. 4).

.9. Biological property of recombinant CsLTL-1 protein

.9.1. Hemagglutination assayThe hemagglutination activity was tested with CsLTL-1 protein

n mouse erythrocytes. CsLTL-1 at 4 �g/ml with 10 mM CaCl2 couldnduce the hemagglutination of the erythrocytes. No agglutination

as observed in the BSA control experiments under the same con-itions. The hemagglutination activity of CsLTL-1was not inhibitedy the addition of 12.5 �l of 10 mM EDTA, indicating that CsLTL-1

nduced agglutination does not require calcium.

.9.2. Sugar binding specificity of CsLTL-1The sugar binding specificity of CsLTL-1 was examined by

nhibitory hemagglutination assay (indirect binding assay). Eightugars were taken for the inhibitory agglutination studies with

ouse erythrocytes. Carbohydrates were pre-incubated with

sLTL-1, and the mixtures were then added to mouse erythrocytes.oreover, CsLTL-1 protein had its own sugar specificity. Among the

arbohydrates tested, d-mannose (50 mM) and d-glucose (100 mM)

ology 56 (2013) 497– 506 503

effectively inhibited the hemagglutination activity of CsLTL-1. Theother tested carbohydrates did not show any inhibition until theconcentration of the sugars reached 200 mM.

3.9.3. Bacterial agglutination assayThe Gram negative bacteria E. coli, A. hydrophila, V. vulnificus and

V. alginolyticus and Gram positive bacteria B. mycoides, S. pyogenesand S. aureus were selected to test CsLTL-1 bacterial agglutinationactivity. The microbes were incubated with the CsLTL-1 protein.CsLTL-1 showed no agglutination activity against any of the sevenbacteria tested.

4. Discussion

In the present study, we have characterized lily type lectin-1, a novel lectin from the striped murrel, C. striatus. The CsLTL-1amino acid sequence showed homology to other lily type lectinfrom O. fasciatus, Larimichthy scroace, E. coioides, P. indicus and L.nuchalis. This CsLTL-1 interacts specifically with d-mannose, likepufflectin (Tsutsui et al., 2003). Furthermore, this LTL also showsmore than 50% similarity to the mannose binding lectin of mono-cotyledonous plants including Allium ampeloprasum, A. sativum andAnnona squamosa as reported by Tsutsui et al. (2003). Moreover,CsLTL-1 possesses three mannose-binding motifs (QxDxNxVxY),which are conserved among mannose-specific lectins of lily typelectin (Tsutsui et al., 2003), skin mucus lectin (Suzuki et al.,2003; Tsutsui et al., 2006b) intestine mucus lectin (Tsutsui et al.,2005, 2006a) and monocots (Smeets et al., 1997). These sitesare present at Gln30-Asp32-Asn34-Val36-Tyr38, Gln59-Asp61-Asn63-Val65-Tyr67and site 3 with slight changes (TxNxDxQxV) in the aapositions as Thr91-Asp93-Lys95-Val97-Tyr99 in CsLTL-1 and they arelikely to be involved in the specific binding of d-mannose sugar.

In several mammalian and bird species, mannose-bindinglectins (MBL) in the serum are known to activate the lectin path-way of the complement system (Turner, 1996; Leursen et al., 1998).Because cDNAs encoding MBL homologues were detected in thefamily Cyprinidae (Vitved et al., 2000) bony fishes are also assumedto possess MBL and the lectin pathway (Nakao and Yano, 1998).CsLTL-1 mannose binding site amino acids have 63% similarity tothe MBL from Salmo salar. A phylogenetic tree was constructedbased on the amino acid sequence of CsLTL-1 and other knownlectin sequences from bacteria, plants and fishes. Phylogenetic treeanalysis revealed that CsLTL-1 had high similarity with lily typelectin-1 from O. fasciatus together with the structural features, indi-cating that CsLTL-1 might be a lily type lectin.

The secondary structure analysis showed that the CsLLT-1 con-tains more coils due to the presence of high amount of glycine,which interfere the �-helix formation. Ramachandran plot analy-sis showed that the 3D model of CsLLT-1 protein has more of itsresidues in the favored region (88.80%) and very limited residuesin the outlier or disallowed region (4.30%). According to Horst et al.(2012) and David et al. (2008) the reliable protein model shouldhave a zDOPE value < −0.5 and the least RMSD score. Similarlyour CsLLT-1 protein model has zDOPE value of −0.708 and leastRMSD value of 4.018. The predicted 3D structure of CsLTL-1 showsthat �-sheets, connected with turns and coils, occurred predom-inantly in the structure of CsLTL-1. The structural organization of�-sheets is found to be similar with Scilla campanulata (Wrigh et al.,1999) and mannose binding tuber lectin from Typhonium divari-catum (Luo et al., 2007). The ExPASy analysis indicated that theoverall structure is composed of three carbohydrate recognition

domain (CRD) connected by turns and loops. Each CRD is composedof four �-sheets. The mannose-binding sites were located in theclefts formed by the three bundles of �-sheets. Interestingly, ourCsLTL-1 is very similar to the 3D structure of many other mannose

5 mmun

bCXrts

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Cps(tornfvCln

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04 A. Arasu et al. / Molecular I

inding lectins (Barre et al., 1996). The predicted RNA structure ofsLTL-1 scored a minimum free energy value of −98.95 kcal/mol.iong and Waterman (1997) reported that the paired base pairseceive negative MFE value and unpaired base pairs receive posi-ive MFE value. Hence, it is possible to suggest that CsLTL-1 RNA istable as reported by Xiong and Waterman (1997).

In healthy C. striatus, CsLTL-1 mRNA transcript is distributedainly in the gill followed by skin, liver, intestine, kidney, muscle,

pleen, blood, head kidney, heart and brain. The gills are involvedn a continuous water exchange from outside environment in fishnd are more susceptible to pathogen infection (Arockiaraj et al.,013). The tissue distribution of CsLTL-1 suggests that it may offerhe initial response to an invasion of marine pathogeneic microor-anisms. Similarly, pufflectin mRNA was also widely expressed inill followed by oral cavity wall, esophagous and skin (Tsutsui et al.,003). The mucosal lectins (designated as congerins), a mannoseinding lectin from conger eel is expressed in skin, gill and upperlimentary canal (Nakamura et al., 2001). The direct injection of. invandans and A. hydrophila significantly increased the CsLTL-1RNA expression and attained the maximum transcript level in

he gills of C. striatus at 24 h (23 fold) and 12 h (7 fold) respectively.herefore, CsLTL-1 is inducible and sensitive to fungal and bacterialnfection.

To understand the specific biological activity of CsLTL-1, theecombinant CsLTL-1 was expressed in E. coli and the HA assay wasested. The results show that, in the presence of 10 mM CaCl2, CsLTL-

protein at 4 �g/ml can induce the agglutination of the mouserythrocytes. As expected in the presence of EDTA, we did notbserve any agglutination, indicating that CsLTL-1 activity is cal-ium independent. Similar results were reported by Tsutsui et al.2003) for pufflectin also. Wei et al. (2010) reported that someectins do not require calcium to function. For instance, lectinurified from the eggs of shishamo smelt, Osmerus (Spirinchus)

anceolatus (Hosono et al., 2005), tobacco hornworm, Manduca sextaYu et al., 2006) and webworm moth, Hyphantria cunea (Shin et al.,000) do not require Ca2+ for agglutinating activity. On the otherand, lectin from Amphioxus, require calcium for the hemagglu-ination as well as microbial aggregation activity, but not for the

icrobial binding and/or growth suppression activities (Yu et al.,007). Strangely, Congerin from conger eel, can bind to sugar in

Ca2+ dependent manner, however, it showed Ca2+ independentctivity in its yeast-binding (Tsutsui et al., 2007). In this study, weound that CsLTL-1 do not require calcium for agglutinating activity.

The sugar binding specificity of CsLTL-1 was determined by theirRD structure. Based on the structural features, CsLTL-1 shouldossess the mannose-binding activity which was verified by theugar-inhibition experiments. d-mannose (50 mM) and d-glucose100 mM) could inhibit the CsLTL-1 mediated agglutination, whilehe other six sugars including lactose, sucrose, d-galactose, malt-se, d-fructose and xylose failed to do so. Never the less ouresults are in correlation to the result obtained with the recombi-ant protein pufflectin from pufferfish (Tsutsui et al., 2003) lectin

rom Fenneropenaeus chinensis (Sun et al., 2008) and Litopenaeusanameii (Zhang et al., 2009). Mannose and glucose could inhibit thesLTL-1 mediated agglutination, while the other sugars, including

actose, sucrose, d-galactose, maltose, d-fructose and xylose couldot inhibit the agglutination activities (Sun et al., 2008).

CsLTL-1 did not induce agglutination in any of the seven bacte-ial species investigated in this study. Similar results obtained fromufflectin (Tsutsui et al., 2003), showed no agglutination activityith the Gram negative bacteria (E. coli, A. hydrophila, V. vulnificus

nd V. alginolyticus) and Gram positive bacteria (B. mycoides, S. pyo-

enes and S. aureus). Tsutsui et al. (2006a) demonstrated that thegglutination activity of pufflectin against a total of 120 bacterialsolates from fugu fish skin and rearing water. Among the 120 bac-erial isolates, they found only a few bacteria have the agglutination

ology 56 (2013) 497– 506

activity, mainly from a few species of genus Vibrio, suggesting thatpufflectin contributes to a self-defense mechanism on the skin sur-face of pufferfish. Moreover, they (Tsutsui et al., 2006a) reportedthat the agglutination activity was significantly higher in bacterialisolates derived from rearing water than those from pufferfish skin.It indicates that pufflectin is effective in excluding environmentalbacteria from the skin surface rather than that are present in thewater.

In this study, CsLTL-1 was not able to agglutinate seven testedbacteria. As suggested by Tsutsui et al. (2003) this indicates thatvirulent bacteria can attach to the gill surface, colonize, and inducedisease since they are not agglutinated by the lily type lectin. Todate, there is no report of immunological function in LTL exceptpufflectin. Therefore, here we are discussing about a few skin mucuslectin which is around 63–73% similarity to the CsLTL-1. Kamiya andShimizu (1980) first described a fish skin mucus lectin as a defensefactor, observing that windowpane flounder lectin agglutinatesa marine yeast Metschnikowia reukaufii and a marine bacteriumMicrocyclus marina. Congerin from conger eel was shown to agglu-tinate, but not inhibit, the growth of V. anguillarum (Kamiya et al.,1988). In Japanese eel, AJL-2 agglutinates E. coli and suppresses itsgrowth (Tasumi et al., 2002), and AJL-1 is the first lectin demon-strated to agglutinate a Gram-positive bacterium, S. difficile (Tasumiet al., 2004). However, it is notable that skin mucus lectins in fish,including lily type lectin, cannot agglutinate many bacteria, butexhibited specific activity towards bacterial species (Tsutsui et al.,2003). In other words, most bacteria might have evading mecha-nisms from lectin-associated self-defence system of fish.

Interestingly, Tsutsui et al. (2003) demonstrated that pufflectincan bind to metazoan parasite Heterobothrium okamotoi, suggestingthat LTL may play a defense role against parasites. This recogni-tion between the parasite and host may be involved by the bindingbetween H. okamotoi carbohydrate and pufflectin as discussed byBuchmann (2001) and Jorndrup and Buchmann (2005). Given thesimilarities in the activities between pufflectin and CsLTL-1, it istempting to speculate that CsLTL-1 might have antiparasitic activ-ity.

In conclusion, CsLTL-1 was identified and characterized fromC. striatus, using GS-FLXTM technology. CsLTL-1 mRNA is predom-inantly expressed in gill, skin, liver and intestine. Its expressionis highly regulated by fungal and bacterial challenge. In vitrostudies with recombinant CsLTL-1 show calcium is not requiredfor its biological activity. The CsLTL-1 hemagglutination activitywas inhibited in the presence very high concentration (50 and100 mM−1) of d-mannose and d-glucose respectively. Given thisconvincing data we speculate that that the new member CsLTL-1 from the lectin super family might be an important moleculeinvolved in pattern recognition and pathogen elimination in theinnate immunity of C. striatus.

Acknowledgements

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

Appendix A. Supplementary data

Supplementary material related to this article can be found,in the online version, at http://dx.doi.org/10.1016/j.molimm.2013.06.020.

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eferences

l-Hassan, J.M., Thomson, M., Summers, B., Criddle, R.S., 1986. Purification and prop-erties of a hemagglutination factor from Arabian Gulf catfish (Arius thalassinus)epidermal secretion. Comparative Biochemistry and Physiology Part B 85, 31–39.

mbrish, R., Alper, K., Yang, Z., 2010. I-TASSER: a unified platform for automatedprotein structure and function prediction. Nature Protocols 5, 725–738.

rockiaraj, J., Avin, F.A., Vanaraja, P., Easwvaran, S., Singh, A., Othman, R.Y., Bhassu,S., 2012a. Immune role of MrNFkBI-a, an IkB family member characterized inprawn M. rosenbergii. Fish and Shellfish Immunology 33, 619–625.

rockiaraj, J., Vanaraja, P., Sarasvathi, E., Arun, S., Othman, R.Y., Bhassu, S., 2012b.Molecular functions of chaperonin gene, containing tailless complex polypep-tide 1 from Macrobrachium rosenbergii. Gene 508, 241–249.

rockiaraj, J., Easwvaran, S., Vanaraja, P., Singh, A., Othman, R.Y., Bhassu, S., 2012c.Effect of infectious hypodermal and haematopoietic necrosis virus (IHHNV)infection on caspase 3c expression and activity in freshwater prawn Macro-brachium rosenbergii. Fish and Shellfish Immunology 32, 161–169.

rockiaraj, J., Easwvaran, S., Vanaraja, P., Singh, A., Othman, R.Y., Bhassu, S., 2012d.Immunological role of thiol-dependent peroxiredoxin gene in Macrobrachiumrosenbergii. Fish and Shellfish Immunology 33, 121–129.

rockiaraj, J., Vanaraja, P., Sarasvathi, E., Arun, S., Othman, R.Y., Bhassu, S.,2012e. Gene expression and functional studies of small heat shock protein 37(MrHSP37) from Macrobrachium rosenbergii challenged with infectious hypo-dermal and hematopoietic necrosis virus (IHHNV). Molecular Biology Reports39, 6671–6682.

rockiaraj, J., Annie, J.G., Dhanaraj, M., Mukesh, P., James, M., Arun, S., 2013. Anupstream initiator caspase 10 of snakehead murrel Channa striatus, containingDED, p20 and p10 subunits: molecular cloning, gene expression and proteolyticactivity. Fish and Shellfish Immunology 34, 505–513.

arondes, S.H., 1988. Bifunctional properties of lectins: lectins redefined. Trends inBiochemical Sciences 13, 480–482.

arre, A., Van Damme, E.J.M., Peumans, W.J., Rouge, P., 1996. Structure–functionrelationship of monocot mannose binding lectins. Plant Physiology 112,1531–1540.

lazer, V.S., Vogelbein, W.K., Densmore, C.L., May, E.B., Lilley, J.H., Zwerner, D.E.,1999. Aphanomyces as a cause of ulcerative skin lesions of menhaden fromChesapeake Bay tributaries. Journal of Aquatic Animal Health 11, 340–349.

ly, J.E., Clem, L.W., 1992. Temperature and teleost immune mechanisms. Fish andShellfish Immunology 2, 159–171.

radford, M.M., 1976. A rapid and sensitive method for the quantification ofmicrogram quantities of protein utilizing the principle of protein–dye binding.Analytical Biochemistry 72, 248–254.

uchmann, K., 2001. Lectins in fish skin: do they play a role in host-monogeneaninteractions? Journal of Helminthology 75, 227–231.

aster, G.R., Cole, J.R., 1990. Diagnostic Procedures in Veterinary Bacteriology andMycology, 5th ed. Academic Press. Inc., CA.

atap, E.S., Munday, B.L., 1998. Effects of variations of water temperature and dietarylipids on the expression of experimental epizootic ulcerative syndrome (EUS) insand whiting Sillago ciliata. Fish Pathology 33, 327–335.

handra, N.R., Ramachandraiah, G., Bachhawat, K., Dam, T.K., Surolia, A., Vijayan,M., 1999. Crystal structure of a dimeric mannose-specific agglutinin from garlic:quaternal association and carbohydrate specificity. Journal of Molecular Biology285, 1157–1168.

hevreux, B., Pfisterer, T., Drescher, B., Driesel, A.J., Muller, W.E., et al., 2004. Usingthe miraEST assembler for reliable and automated mRNA transcript assemblyand SNP detection in sequenced ESTs. Genome Research 14, 1147–1159.

hinabut, S., Roberts, R.J., Willoughby, G.R., Pearson, M.D., 1995. Histopathology ofsnakehead, Channa striatus (Bloch), experimentally infected with the specificAphanomyces fungus associated with epizootic ulcerative syndrome (EUS) atdifferent temperatures. Journal of Fish Diseases 18, 41–47.

avid, E., Narayanan, E., Shen, M.Y., Andrej, S., 2008. How well can the accuracyof comparative protein structure models be predicted? Protein Science 17,1881–1893.

hanaraj, M., Haniffa, M.A., Ramakrishnan, C.M., Arunsingh, S.V., 2008. Microbialflora from the Epizootic Ulcerative Syndrome (EUS) infected murrel Channastriatus (Bloch, 1797) in Tirunelveli region. Turkish Journal of Veterinary andAnimal Sciences 32, 221–224.

wart, K.V., Johnson, S.C., Ross, N.W., 1999. Identification of pathogen-binding lectinin salmon serum. Comparative Biochemistry and Physiology Part C 123, 9–15.

wart, K.V., Johnson, S.C., Ross, N.W., 2001. Lectins of the innate immune system andtheir relevance to fish health. ICES Journal of Marine Sciences 58, 380–385.

ujita, T., 2002. Evolution of the lectin-complement pathway and its role in innateimmunity. Nature Reviews Immunology 2, 346–353.

adjeva, M., Takahashi, K., Thiel, S., 2004. Mannan-binding lectin-a soluble patternrecognition molecule. Molecular Immunology 41, 113–121.

oto-Nance, R., Watanabe, Y., Kamiya, H., Ida, H., 1995. Characterization of lectinsfrom the skin mucus of the loach Misugurunus anguillicaudatus. Fisheries Sci-ences 61, 137–140.

all, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor andanalysis program for Windows 95/98/NT. Nucleic Acid Symposium Series 41,95–98.

offmann, J.A., Kafatos, F.C., Janeway, C.A., Ezekowitz, R.A., 1999. Phylogenetic per-spectives in innate immunity. Science 284, 1313–1318.

olmskov, U., Malhotra, R., Sim, R.B., Jensenius, J.C., 1994. Collectins collagenousC-type lectins of the innate immune defense system. Immunology Today 15,67–74.

ology 56 (2013) 497– 506 505

Horst, J.A., Pieper, U., Sali, A., Zhan, L., Chopra, G., Samudrala, R., Featherstone, J.D.B.,2012. Strategic protein target analysis for developing drugs to stop dental caries.Advances in Dental Research 22, 86–93.

Hosono, M., Sugawara, S., Ogawa, Y., Kohno, T., Takayanagi, M., Nitta, K., 2005. Purifi-cation, characterization, cDNA cloning, and expression of asialofetuin-bindingC-type lectin from eggs of shishamo smelt (Osmerus [Spirinchus] lanceolatus).Biochimica et Biophysica Acta 1725, 160–173.

Jack, D.L., Turner, M.W., 2003. Anti-microbial activities of mannose-binding lectin.Biochemical Society 31, 753–757.

Jensen, L.E., Thiel, S., Petersen, T.E., Jensenius, J.C., 1997a. A rainbow trout lectinwith multimeric structure. Comparative Biochemistry and Physiology Part B116, 385–390.

Jensen, L.E., Hiney, M., Shields, D.C., Uhlar, C.M., Lindsay, A.J., Whitehead, A.S.,1997b. Acute phase protein in salmonids—evolutionary analyses and acutephase response. Journal of Immunology 158, 384–392.

Jorndrup, S., Buchmann, K., 2005. Carbohydrate localization on Gyrodactylus salarisand G. derjavini and corresponding carbohydrate binding capacity of their hostsSalmo salar and S. trutta. Journal of Helminthology 79, 41–46.

Kamiya, H., Shimizu, Y., 1980. Marine biopolymers with cell specificity II.Purification and characterization of agglutinins from mucous of window-pane flounder Lophopsetta maculata. Biochemica et Biophysica Acta 622,171–178.

Kamiya, H., Muramoto, K., Goto, R., 1988. Purification and properties of agglutininsfrom conger eel, Conger myriaster (Brevoort), skin mucus. Developmental andComparative Immunology 12, 309–318.

Kindt, T.J., Goldsby, R.A., Osborne, B.A., Kuby, J., 2007. Immunology, 6th ed. WHFreeman & Co., NY, Chapter 7.

Leursen, S.B., Dalgaard, T.S., Thiel, S., Lim, B.L., Jensen, T.V., Juul-Madsen,H.R., Takahashi, A., Hamana, T., Kawakami, M., Jensenius, J.C., 1998.Cloning and sequencing of a cDNA encoding chicken mannan-bindinglectin (MBL) and comparison with mammalian analogues. Immunology 93,421–430.

Livak, K.J., Schmittgenm, T.D., 2001. Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25,402–408.

Lovell, S.C., Davis, I.W., Arendall III, W.B., de Bakker, P.I.W., Word, J.M., Prisant, M.G.,Richardson, J.S., Richardson, D.C., 2002. Structure validation by Calpha geome-try: phi, psi and Cbeta deviation. Proteins: Structure, Function & Genetics 50,437–450.

Lu, J., 1997. Collectins: collectors of microorganisms for the innate immune system.Bioessays 19, 509–518.

Luo, T., Zhang, X., Shao, Z., Xu, X., 2003. PmAV, a novel gene involved in virus resis-tance of shrimp Penaeus monodon. FEBS Letters 551, 53–57.

Luo, Y., Xu, X., Liu, J., Li, J., Sun, Y., Liu, Z., Liu, J., Van Damme, E., Balzarini, J.,Bao, J., 2007. A novel mannose-binding tuber lectin from Typhonium divari-catum (L.) Decne (family Araceae) with antiviral activity against HSV-II andanti-proliferative effect on human cancer cell lines. Journal of Biochemistry andMolecular Biology 40, 358–367.

Malhotra, R., Sim, R.B., 1995. Collectins and viral infection. Trends in Microbiology3, 240–244.

Mitra, S., Das, H.R., 2002. A novel mannose-binding lectin from plasma of Labeorohita. Fish Physiology and Biochemistry 25, 121–129.

Nakamura, O., Watanabe, T., Kamiya, H., Muramoto, K., 2001. Galectin containingcells in the skin and mucosal tissues in Japanese conger eel, Conger myriaster:an immunohistochemical study. Developmental and Comparative Immunology25, 431–437.

Nakao, M., Yano, T., 1998. Structural and functional identification of complementcomponents of the bony fish, carp (Cyprinus carpio). Immunological Reviews166, 27–38.

Ni, Y., Tizzard, I., 1996. Lectin–carbohydrate interaction in the immune system.Veterinary Immunology and Immunopathology 44, 251–267.

Oda, Y., Ichida, S., Mimura, T., Maeda, K., Tsujikawa, K., Aonuma, S., 1984. Purifica-tion and characterization of a fish lectin from the external mucus of OphidiidaeGenypterus blacodes. Journal PharmacoBio-Dynamics 7, 614–623.

Ottinger, A.C., Johnson, S.C., Ewart, K.V., Brown, L.L., Ross, N.W., 1999. Enhance-ment of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar)macrophages by a mannose-binding lectin. Comparative Biochemistry andPhysiology Part C 123, 53–59.

Ourth, D.D., Narra, M.B., Simco, B.A., 2007. Comparative study of mannose-bindingC-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish(Ictalurus furcatus). Fish and ShellFish Immunology 23, 1152–1160.

Peumans, W.J., Van Damme, E.J., 1995. Lectins as plant defense proteins. Plant Phys-iology 109, 347–352.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Man-ual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,NY.

Sharon, N., Lis, H., 1972. Lectins: cell-agglutinating and sugar-specific proteins. Sci-ence 177, 949–959.

Shen, M.Y., Sali, A., 2006. Statistical potential for assessment and prediction of pro-tein structures. Protein Science 15, 2507–2524.

Shin, S.W., Park, D.S., Kim, S.C., Park, H.Y., 2000. Two carbohydrate recogni-

tion domains of Hyphantria cunea lectin bind to bacterial lipopolysaccharidesthrough O-specific chain. FEBS Letters 467, 70–74.

Shiomi, K., Takamiya, M., Yamanaka, H., Kikuchi, T., 1987. Purification of a lethalfactor in the skin secretion from the oriental catfish Pltosus lineatus. NipponSuisan Gakkaishi 53, 1275–1280.

5 mmun

S

S

S

S

S

S

T

T

T

T

T

T

T

T

Zhang, Y., 2008. I-TASSER server for protein 3D structure prediction. BMC Bioinfor-

06 A. Arasu et al. / Molecular I

hiomi, K., Uematsu, H., Yamanaka, H., Kikuchi, T., 1989. Purification and characteri-zation of a galactose-binding lectin from the skin mucus of the conger eel Congermyriaster. Comparative Biochemistry and Physiology Part B 92, 255–261.

hiomi, K., Uematsu, H., Ito, H., Yamanaka, H., Kikuchi, T., 1990. Purification andproperties of a lectin in the skin mucus of the dragonet Repomucenus richardsonii.Nippon Suisan Gakkaishi 56, 119–123.

meets, K., Damme, E.J.M.V., Verhaert, P., Barre, A., Rouge, P., Leuven, F.V., Peumans,W.J., 1997. Isolation, characterization and molecular cloning of the mannose-binding lectins from leaves and roots of garlic (Allium sativum L.). Plant MolecularBiology 33, 223–234.

uckale, J., Sim, R.B., Dodds, A.W., 2005. Evolution of innate immune systems. Bio-chemistry and Molecular Biology Education 33, 177–183.

un, Y.D., Fu, L.D., Jia, Y.P., Du, X.J., Wang, Q., Wang, Y.H., et al., 2008. Ahepatopancreas-specific C-type lectin from the Chinese shrimp Fenneropenaeuschinensis exhibits antimicrobial activity. Molecular Immunology 45, 348–361.

uzuki, Y., Tasumi, S., Tsutsui, S., Okamoto, M., Suetake, H., 2003. Molecular diversityof skin mucus lectins in fish. Comparative Biochemistry and Physiology Part B136, 723–730.

asumi, S., Ohira, T., Kawazoe, I., Suetake, H., Suzuki, Y., Aida, K., 2002. Primary struc-ture and characteristics of a lectin from skin mucus of the Japanese eel Anguillajaponica. Journal of Biological Chemistry 277, 27305–27311.

asumi, S., Yang, W.J., Usami, T., Tsutsui, S., Ohira, T., Kawazoe, I., Wilder, M.N., Aida,K., Suzuki, Y., 2004. Characteristics and primary structure of a galectin in the skinmucus of the Japanese eel Anguilla japonica. Developmental and ComparativeImmunology 28, 325–335.

oda, M., Goto-Nance, R., Muramoto, K., Kamiya, H., 1996. Characterization of thelectin from the skin mucus of the kingklip Genypterus capensis. Fisheries Science62, 138–141.

sutsui, S., Tasumi, S., Suetake, H., Suzuki, Y., 2003. Skin mucus lectin of puffer-fish (Fugu rubripes) homlogous to monocotyledonous plant lectin. Journal ofBiological Chemistry 278, 20882–20889.

sutsui, A., Takahashi, R., Sumida, T., 2005. Mannose binding lectin: genetics andautoimmune disease. Autoimmunity Reviews 4, 364–367.

sutsui, S., Okamoto, M., Tasumi, S., Suetake, H., Kikuchi, K., Suzuki, Y., 2006a. Novelmannose-specific lectins found in torafugu (Takifugu rubripes): a review. Com-parative Biochemistry and Physiology Part D 1, 122–127.

sutsui, S., Tasumi, S., Suetake, H., Kikuchi, K., Suzuki, Y., 2006b. Carbohydrate-

binding site of a novel mannose-specific lectin from fugu (Takifugu rubripes)skin mucus. Comparative Biochemistry and Physiology Part B 143, 514–519.

sutsui, S., Iwamoto, K., Nakamura, O., Watanabe, T., 2007. Yeast-binding C-typelectin with opsonic activity from conger eel (Conger myriaster) skin mucus.Molecular Immunology 44, 691–702.

ology 56 (2013) 497– 506

Tunkijjanukij, S., Olafsen, J.A., 1998. Sialic acid binding lectin with antibacterial activ-ity from the horse mussel: further characterization and immunolocalization.Developmental and Comparative Immunology 22, 139–150.

Turner, M.W., 1996. Mannose-binding lectin: the pluripotent molecule of the innateimmune system. Immunology Today 17, 532–540.

Turner, M.W., 2003. The role of mannose-binding lectin in health and disease. Molec-ular Immunology 40, 423–429.

Uinuk-Ool, T.S., Takezaki, N., Kuroda, N., Figueroa, F., Sato, A., Smote, I.E., Mayer, W.E.,Klein, J., 2003. Phylogeny of antigen-processing enzymes: catharsis of a cephalo-chordate, an Agatha and a bony fish. Scandinavian Journal of Immunology 58,436–448.

Vitved, L., Holmskov, U., Koch, C., Eisner, B., Hansen, S., Skolt, K., 2000. The homo-logue of mannose-binding lectin in the carp family cyprinid is expressed at highlevel in spleen, and the deduced primary structure predicts affinity for galactose.Immunogenetics 51, 955–964.

Wei, J., Xu, D., Zhou, J., Cui, H., Yan, Y., Ouyang, Z., Gong, J., Huang, Y., Huang, X.,Qin, Q., 2010. Molecular cloning, characterization and expression analysis of aC-type lectin (Ec-CTL) in orange-spotted grouper, Epinephelus coioides. Fish andShelfish Immunology 28, 178–186.

Wrigh, L.M., Reynolds, C.D., Rizkallah, P.J., Allen, A.K., Van Damme, E.J.M., Donovan,M.J., Peumans, W.J., 1999. Structural characterisation of the native fetuin-binding protein Scilla campanulata agglutinin: a novel two-domain lectin. FEBSLetters 468, 19–22.

Xiong, M., Waterman, M.S., 1997. A phase transition for the minimum free energyof secondary structures of a random RNA. Advances in Applied Mathematics 18,111–132.

Yang, H., Luo, T., Li, F., Li, S., Xu, X., 2007. Purification and characterization of acalcium-independent lectin (PjLec) from the haemolymph of the shrimp Penaeusjaponicus. Fish and Shellfish Immunology 22, 88–97.

Yu, X.Q., Ling, E., Tracy, M.E., Zhu, Y., 2006. Immulectin-4 from the tobacco horn-worm Manduca sexta binds to lipopolysaccharide and lipoteichoic acid. InsectMolecular Biology 15, 119–128.

Yu, Y.H., Yu, Y.C., Huang, H.Q., Feng, K.X., Pan, M.M., Yuan, S.C., et al., 2007. A short-form C-type lectin from Amphioxus acts as a direct microbial killing proteinvia interaction with peptidoglycan and glucan. Journal of Immunology 179,8425–8434.

matics 9, 40.Zhang, Y., Qiu, L., Song, L., Zhang, H., Zhao, J., Wang, L., Yu, Y., Li, C., Li, F., Xing, K.,

Huang, B., 2009. Cloning and characterization of a novel C-type lectin gene fromshrimp Litopenaeus vannamei. Fish and Shellfish Immunology 26, 183–192.