Developmental and Comparative Immunology 45 (2014) 269–277

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Developmental and Comparative Immunology

journal homepage: www.elsevier .com/locate /dci

Identification of 21 novel immune-type receptors in miiuy croakerand expression pattern of three typical inhibitory members

http://dx.doi.org/10.1016/j.dci.2014.03.0160145-305X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: tianjunxu@163.com (T. Xu).

Fanqiang Meng, Rixin Wang, Tianjun Xu ⇑Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan 316022, China

a r t i c l e i n f o

Article history:Received 21 November 2013Revised 21 March 2014Accepted 24 March 2014Available online 1 April 2014

Keywords:Miichthys miiuy (Miiuy Croaker)Novel immune-type receptor (NITR)Innate immunityFunctional domainsExpression pattern

a b s t r a c t

Novel immune-type receptor (NITR) genes belong to the immunoglobulin superfamily and are encodedby clusters of multigene families. NITRs encode type I transmembrane proteins and are only found in tel-eosts. In the current study, total 21 NITR genes are identified from miiuy croaker (Miichthys miiuy) andnamed as MmNITR1 to MmNITR21. Miiuy croaker NITR genes that encoded one or two extracellularimmunoglobulin (Ig) domains, a transmembrane (TM) region, an immunoreceptor tyrosine-based inhib-itor motif (ITIM) in the cytoplasmic (Cyt) region. The majority of MmNITRs possess cytoplasmic ITIM thatcan be classified as inhibitory receptors. However, a smaller number of NITRs (MmNITR8, MmNITR15 andMmNITR16) can be classified as activating receptors by the lack of cytoplasmic ITIMs and presence of apositively charged residue within their transmembrane domain. As typical inhibitory receptors,MmNITR1, MmNITR2 and MmNITR3 have different characteristics of the structure. In MmNITR1 gene,variable (V) and intermediate (I) domains are encoded by two separate exons. In contrast to MmNITR1,MmNITR3 gene encode V and I domains in a single exon. And MmNITR2 gene is characterized by the pres-ence of only one Ig-like (V-type) extracellular domain and lack of J or J-like motifs. Also MmNITR2 genedisplays an additional exon which is 48 bp long between the V domain and the TM region. Two and fourpotential N-link giycosylation sites (N-X-S/T) are present in the extracellular Ig domains. Real-time RT-PCR results showed that upon induction with Vibrio anguillarum, NITR gene expressions were inducedby bacteria in kidney, liver and spleen. Meanwhile, NITRs are also primarily detected in different tissues.Phylogenetic analyses of NITR V domains indicate that MmNITR1 and MmNITR2 are more similar thanMmNITR3.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Ever-increasing attention is being directed to the role of innateprocesses within the immune system, the diversity and variety ofimmune receptors which interact with other surface molecules ef-fect innate function, and the mechanisms whereby innate immunereceptors transduct of intracellular signals (Hoffmann et al., 1999).As an ancient form of host defense, the innate immune system re-lies upon a wide range of non-rearranging encoded receptorsincluding a large number of immunoglobulin superfamily (IgSF)receptors (Cannon et al., 2010; Janeway and Medzhitov, 2002). IgSFis an extensively diversified multigene family whose membersshare three conserved regions: an immunoglobulin (Ig) like do-main, a transmembrane (TM) region and a cytoplasmic tail (Cyt).Depending on the Cyt, they can be subdivided into activating and

inhibitory receptors. Generally speaking, short-tailed receptorsgenerate activating signals, whereas inhibitory receptors possesslong cytoplasmic tails which containing one or two immunorecep-tor tyrosine-based inhibitory motifs (ITIM). In the last decade, witha similar molecular structure to the killer Ig-like receptors (KIRs),two gene families that encoding non-rearranging receptors, havebeen discovered in teleost fish, the novel immunoglobulin-liketranscripts (NILTs) and the novel immune-type receptors (NITRs).As both KIRs and NITRs appear to stimulate the same signallingpathways, NITRs have been proposed to be functional homologsof mammalian KIRs (Yoder, 2009).

NITRs are a large and diverse polygenic family of putative inhib-itory and stimulatory receptors (Hawke et al., 2001; Piyaviriyakulet al., 2007; Strong et al., 1999; Yoder et al., 2001, 2002). They be-long to a subset of the IgSF and do not undergo rearrangement orsomatic mutation (Litman et al., 2001; Yoder and Litman, 2000).In common with other immune genes, both natural killer receptors(NKRs) and NITRs appear to follow a ‘‘birth-and-death’’ model ofevolution (Nei and Rooney, 2005).

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The prototypic NITR cDNA was cloned more than many yearsago from the Southern pufferfish (Spheroides nephelus) (Rastet al., 1995) and then named as ‘‘Sn193’’. This NITR cDNA encodesan N-terminal extracellular variable (V) domain adjacent to a join-ing (J) domain, a C-terminal extracellular intermediate (I) domainwhich previously termed V-like C2 or V/C2 domain, a TM regionand a Cyt possessing an ITIM and a second ITIM-like (itim) se-quence. Subsequent studies have identified additional NITR genesfrom several fish species. Twenty-six genes were first characterizedin the Southern pufferfish (Strong et al., 1999), then with the isola-tion and description of four NITRs in the rainbow trout (Oncorhyn-chus mykiss) (Yoder et al., 2002), eighteen genes in the channelcatfish (Ictalurus punctatus) (Evenhuis et al., 2007; Hawke et al.,2001), one transcript in the Japanese flounder (Paralichthysolivaceus) (Piyaviriyakul et al., 2007) and the identification of 24families in medaka (Oryzias latipes) (Desai et al., 2008). Transcriptsencoding NITR proteins have also been found in the Atlantic sal-mon, Atlantic halibut, stickleback, Atlantic cod and lake whitefish(Yoder, 2009). Interestingly, no such homologs have yet been iden-tified in mammals (Litman et al., 2001). Several forms of NITRswere identified according to: (1) number of extracellular Ig do-mains (V-type and I-type) and exons encoding the ectodomains,(2) proteins that only possess a V-domain but lack an I domaincan be classified as NITRs if their genes are present within an NITRgene cluster, (3) presence and location of J or J-like sequences and(4) presence of ITIM or itim motifs in the Cyt region. All NITRs pos-sess one extracellular Ig domain of the V type, and most of thempossess a second extracellular Ig domain of the I type. Recent crys-tal structures of NITR V domains demonstrate that they are highlysimilar to the V domains of antigen receptors (Yoder, 2009). I do-main helps differentiate NITRs from other V domain receptors. NIT-Rs that encoded a Cyt region which typically possesses activatingor inhibitory signaling motifs. A smaller number of NITRs are con-sidered to be activating receptors, cause they possess a positivelycharged residue within the TM region and possesses a cytoplasmicimmunoreceptor tyrosine-based activation motif (ITAM) (Weiet al., 2007; Yoder et al., 2007). Most NITRs possess cytoplasmicITIMs and are classified as inhibitory forms. Moreover, like NKRs,the crystal structure of NITR Ig domains not only demonstrate theirpotential interaction with a polymorphic set of ligands, but alsoprovide evidence that binding specificity for the allogeneic deter-minant resides in the V domain CDR1-analogous loop (Cannonet al., 2008). NITR transcripts are detected in diverse hematopoieticlineages supporting the assumption that NITRs function in immu-nity (Hawke et al., 2001; Evenhuis et al., 2007).

Between mammalian leukocyte receptor complex (LRC) and theNITR gene clusters, comparative genomic analysis showed only aweakly conserved synteny. This can be included that the presenceof NITRs may be restricted to bony fish (Yoder, 2009). So far, thecollected data demonstrate that NITRs are representative of a rap-idly evolving and extraordinarily divergent gene family.

Miiuy croaker, Miichthys miiuy is not only an high-valued mar-ine aquaculture fish but also has more in-depth research inimmune and genetic background, some important immune genesand molecular markers have been reported (Cheng et al., 2010;Xu et al., 2010, 2011b). Nowadays, as lower vertebrates, miiuycroaker and other important cultured fish are afflicted by variousdiseases. It is very important to research miiuy croaker’s own in-nate immune mechanism, because innate immunity plays a signif-icant role in protecting the body from antigens infection (Xu et al.,2011a). In order to further elucidate the molecular immune mech-anisms in miiuy croaker, series of immune response and evolutionmechanism studies of the immune-related genes been carried outand reported (Cheng et al., 2011; Meng et al., 2012; Sun et al., 2011,2012). In this paper, in order to understand the immune signifi-cance of NITR gene in this species, we report the identification of

total 21 NITR genes which including inhibitory and activatingforms in miiuy croaker. Based on NITR nomenclature, we namedthese NITRs as MmNITR1 to MmNITR21, miiuy croaker NITRs gen-erated by repeated tandem duplication event, so they represent ahighly diversified gene complex. Furthermore, three typical inhib-itory NITRs (MmNITR1, MmNITR2 and MmNITR3) have been com-prehensive analyzed and examined its expression pattern invarious tissues in response to infection with pathogenic bacteria.

2. Materials and methods

2.1. Samples

Ten tissues (liver, spleen, kidney, intestines, heart, muscle, gill,brain, eye, and fin) of uninfected miiuy croaker and three immunetissues (liver, spleen and kidney) of challenged miiuy croaker in-jected with Vibrio anguillarum were sampled as described by Zhuet al. (2013). Total RNA was extracted from diverse tissues of indi-viduals using RNAiso Reagent (Takara) according to the manufac-turer’s instructions and the cDNA template was transcribed byreverse transcriptase M-MLV (Takara).

2.2. Database mining and Primer design

To identify all the NITR genes from miiuy croaker, all annotationNITRs from GenBank were used to construct a query set, local BLASTpand tBLASTn programs were conducted to search for all NITR genesfrom miiuy croaker whole-genome scaffold sequences (unpub-lished). In addition, three partial NITR cDNA sequences (Xu et al.,2010) were used to construct a query set, local BLASTN programwere used to search for full-length cDNA sequences and gene se-quences (MmNITR1, MmNITR2 and MmNITR3) from transcriptomeassembled sequences (Che et al., 2014) and whole-genome scaffoldsequences. Three pairs of primers (HM-NITR1-RT-F/R, HM-NITR2-RT-F/R and HM-NITR3-RT-F/R) were designed to detect the expres-sion level of NITR genes, primers b-actin-F/R were used as internalcontrol for NITRs expression analysis (Supplementary Table 1).

2.3. Tissue specific expressions of NITR gene

Ten uninfected tissues were used to determine the tissueexpression pattern of three MmNITRs. And, three immune tissuesafter V. anguillarum infection were used to determine the mRNAexpression profile. Real-time quantitative PCR was conducted ona 7300 real time PCR system (Applied Biosystems, USA) using aRealMaster Mix kit (TIANGEN). The PCR was carried out in a totalvolume of 20 ll, including 9 ll SYBR Green Real-time PCR mastermixtures, 1 ll cDNA sample, 1 ll sense primer, 1 ll anti-sense pri-mer and 8 ll ddH2O. Cycling conditions were as follows: 10 s at95 �C, followed by 40 cycles consisting of 5 s at 95 �C and 34 s at60 �C. Dissociation curve analysis was performed after each assayto determine target specificity. The data were analyzed using SPSSV13.0 software and 7300 System SDS Software v1.3.0 (Applied Bio-systems, USA). The base line was set automatically by the softwareand the expression levels were determined by the 2-DDCT method(Livak and Schmittgen, 2001).

2.4. Phylogenetic analyses

Predicted leader and TM regions of protein sequences wereidentified with SMART software (http://smart.embl-heidelberg.de/;) (Letunic et al., 2004). Homology models were viewed anddecorated using the SWISS-Pdb Viewer (http://swissmodel.expasy.org/spdbv/). The NITR V and I domains were aligned byClustal W in MEGALIGN software. Neighbor-joining tree were

F. Meng et al. / Developmental and Comparative Immunology 45 (2014) 269–277 271

constructed from pairwise Poisson correction distances with 2000bootstrap replication by MEGA 5.2 software (Tamura et al., 2011).

3. Results

3.1. cDNA and characterization of Miiuy croaker NITR genes

The BLAST programs were used to search for proteins similar toconfirmed NITRs in different teleost fishes, a total of twenty-oneNITR genes were found from miiuy croaker genome. Due to NITRsseem to have undergone species-specific gene birth and deathevents, there are few true orthologs between different fish species.Traditionally, NITR genes have been named based on the order oftheir identification in each species, so we named these NITRs inmiiuy croaker as NITR1 to NITR21 (GenBank accession number:KF264458–KF264460 and KJ575084–KJ575101). Among thesemiiuy croaker NITRs (MmNITRs), partial sequences of MmNITR4to MmNITR21 contain Ig domain and TM region. Depending ontheir TM region and Cyt region, three NITRs (MmNITR8,MmNITR15 and MmNITR16) are classified as activating receptorsand other NITRs are classified as inhibitory receptors. MmNITR8,MmNITR15 and MmNITR16 possess a conserved feature of activat-ing receptors, a charged residue (Arg) within their TM region. It iscorresponding to activating NITRs in other species such as zabra-fish NITR9 (Wei et al., 2007) and medaka NITR9 (Desai et al., 2008).

As the typical inhibitory receptors, the full-length cDNA ofMmNITR1, MmNITR2, MmNITR3 were 1767 bp, 1260 bp, 1678 bp,respectively (Fig. 1A). The genomic structure of MmNITR1 andMmNITR2 contain five exons and four introns. And MmNITR3 geneis composed of three exons and two introns. It is worth mentioningthat both MmNITR1 and MmNITR2 display an additional exon. ForMmNITR1, the additional exon exists between TM region and theCyt. But for MmNITR2, the additional exon exists between V do-main and TM region (Fig. 1B). It is important to note that there isanother intron about 578 bp between TM region and Cyt inMmNITR2 gene. The MmNITRs structure are similar to T-cell anti-gen receptor (TCR) and KIR (Fig. 1C), they all have Ig domain, TMregion, J or J-like sequence and ITIM motif.

3.2. Miiuy croaker NITR gene share conserved features

As reported in other teleost species, MmNITRs are related to Igand TCR. Common to Ig and TCR genes, conserved amino acid posi-tions are found in either NITR V domain and/or I domain. The initialsearch criteria for identifying MmNITRs was directed to the I do-main, which possess 6 highly conserved cysteines. But MmNITR8and MmNITR10 lack the forth cysteines. A joining (J; FXGXTXLXV)or J-like peptide sequence at the carboxyl-terminus of either the Vand I domain is a conserved feature of almost all miiuy croakerNITRs (Fig. 2).

Comparison of MmNITR1 and MmNITR3 to other miiuy croakerNITRs revealed two Ig domains, a V-type and an I region (Fig. 2).Eight conserved cysteine (Cys) residues (Piyaviriyakul et al.,2007) are found in MmNITRs which contain V and I domains, thesame with each of four rainbow trout NITRs (Yoder et al., 2002)and each of 15 zebrafish NITRs (Yoder et al., 2001). All Cys residuesare located within the extracellular regions, suggesting that theyform disulfide bonds as they do in other Igs. Each Ig domain (Vand I) of catfish NITRs appear to be formed by disulfide bond be-tween the 1st and 2nd Cys and the 3rd and 7th Cys, respectively(Hawke et al., 2001). Similarly in MmNITRs, disulfide bond of eachIg domain could be formed in a similar manner. Conserved domainsearch was performed using the SMART database (http://smart.embl-heidelberg.de/smart/). Two conserved domains weredetected, an IGv subfamily (SMART accession number: SM00406)

and an IG subfamily (SMART accession number: SM00409), whichappears to correspond to the V and I domains, respectively.

3.3. Expression of NITR mRNAs in miiuy croaker

NITR transcripts can be detected in multiple immune relatedtissues including the hematopoietic kidney, spleen, liver and intes-tine (Evenhuis et al., 2007). In this study, three typical inhibitoryreceptor genes (MmNITR1, MmNITR2 and MmNITR3) could bedetected in ten normal tissues. As shown in Fig. 3, MmNITRs wereexpressed strongly in eye, muscle and fin and expressed weakly inheart (p < 0.05). The expression of MmNITRs were detected inimmune-related organs in agreement with observations in otherreported species. However, the expression level in organs wasdifferent in each NITRs family (Hawke et al., 2001; Yoder et al.,2002).

In liver, the expression level of MmNITR mRNAs decreased from6 h to 36 h after infection, but significantly increased and reachedits peak level in 48 h. A very low levels of MmNITRs were observedin 72 h for bacterial infection (p < 0.05). In contrast, expression ofthe three MmNITRs in spleen at high level could be detected on72 h. In kidney, highest expression was detected on 72 h whilelowest expression was detected on 6 h. The trend of the relativeexpression of the three inhibitory MmNITRs are consistent in liver,spleen and kidney (Fig. 3).

3.4. The immunoglobulin domains of MmNITRs

Since the ‘‘Sn193’’ NITR cDNA was initial discovered, numerousNITR cDNAs have been predicted or identified from the fish gen-omes of Tetraodon, zebrafish, catfish, flounder, fugu, sea bass andmedaka. The majority of all characterized NITRs possess a V andan I Ig domains, while both Ig domains may possess a J or J-like se-quence. All current data suggest that with no evidence for recom-bination, V-J or I-J sequences are always encoded within a singleexon. MmNITR3, MmNITR6, MmNITR7, MmNITR13, MmNITR15,MmNITR16, MmNITR17 and MmNITR19 were vivid cases in point(striking example). However, in MmNITR1, MmNITR4, MmNITR8,MmNITR9, MmNITR10, MmNITR11 and MmNITR14 genes, their Vand I domains are encoded by two separate exons, cause of theyhave a I-J-like sequence. All NITR genes have one unifying featurethat they all encode an authentic V domain (Litman et al., 2001).NITR V domains include three hypervariable (HV) regions whichcorrespond to the complementarity determining regions (CDRs)in TCRs (Fig. 4) (Yoder et al., 2004a). Alignment of the NITR V do-mains sequences defines three general HV regions. High variationis observed between Cys-23 (C23) and Trp-41 (W41), Gly-47 (G47)and Leu-89 (L89), and between Cys-104 (C104) and the J-like se-quence, which correspond to HV region 1 (HV1), HV2, and HV3,respectively (Fig. 2). Compared NITR V domains to TCR V domains,the positioning of the HV1 and HV3 regions corresponds to thepositions of complementarity-determining region 1 (CDR1) andCDR3. However, the positioning of the HV2 region overlaps CDR2positioning. In addition, recent crystal structures of NITR V do-mains indicate that they have highly similarity to the antigenreceptor V domains (Cannon et al., 2008). Overall, these data indi-cate that NITR V domains (Fig. 4A) are similar to TCR V domains(Fig. 4B).

3.5. Comparative models suggest structural differences between NITRsand V-type Ig domains

The V domain of miiuy croaker NITRs are highly divergent(13.1–86.9% identical) with much of the diversity (Table 1). Basedon secondary structure analysis, similarity of MmNITRs with otherteleosts was also confirmed. MmNITRs 3-D structures were

Fig. 1. Nucleotide and deduced amino acid sequences of MmNITR genes. Sequences of translated regions are shown in capital letters. The deduced amino acid residues areshown in capital letters. Non-coding regions are shown in small letters. (A) Stand for MmNITR genes. N-link giycosylation sites are indicated by red lines. J or J-like motifs areshown in blue boxes. ITIM motifs are shown in green boxes. (B) Stand for the domains of MmNITR genes. Leader peptide (L), variable domain (V), intermediate domain (I),transmembrane (TM), cytoplasmic tail immunoreceptor tyrosine-based inhibitor motif (ITIM), J-like motifs, additional exon and are indicated by different colours. (C)Compare protein structures of MmNITRs to T-cell antigen receptor (TCR) and killer Ig-like receptors (KIR). (For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)

272 F. Meng et al. / Developmental and Comparative Immunology 45 (2014) 269–277

predicted by amino acid homology modeling using the SWISS-MODEL alignment interface mode (http://swissmodel.expasy.org/)by submitting a CLUSTALW alignment containing representativeMmNITRs (MmNITR1, MmNITR2 and MmNITR3) as targetsequences and IpNITR11 (or a bispecific alpha/beta TCR) as thetemplate. The highest similarity of MmNITR1 and MmNITR2 werefound with the I. punctatus NITR11 (Evenhuis et al., 2007) (Fig. 4A),which in turn demonstrated to be structurally similar to TCR(Fig. 4B) in chain topology and dimerisation mode. However, thehighest similarity of MmNITR3 was found with a bispecificalpha/beta TCR (Fig. 4C).

3.6. Phylogenetic relationships of miiuy croaker NITRs within teleosts

In an effort to ensure MmNITR genes patterns of divergence andto characterize possible orthologous relationships between NITRsin different species, phylogenetic comparisons were performed.In general, the phylogenetic tree of miiuy croaker NITR V domain(Fig. 5A) can be divided into three groups. Seven MmNITRs(MmNITR1, MmNITR4, MmNITR8, MmNITR9, MmNITR10,MmNITR11 and MmNITR14) are included in group 1. They have acommon feature that V and I domain are encoded in two separateexons. Group 2 contain eight MmNITRs (MmNITR3, MmNITR6,

Fig. 2. Peptide sequence alignment of miiuy croaker NITR V and NITR I regions. Highly conserved Ig domain residues are designated in one-letter code and highlighted inpink. The location of sequence regions corresponding to Hypervariable1 (HV1), HV2 and HV3 as well as J-related domains are indicated by blue shaded. The putativeglycosylation sites in miiuy croaker NITRs are shown in yellow boxes. In I domain, the rest of conserved cysteines were indicated by asterisks. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)

F. Meng et al. / Developmental and Comparative Immunology 45 (2014) 269–277 273

MmNITR7, MmNITR13, MmNITR15, MmNITR16, MmNITR17 andMmNITR19), because their V and I domain are encoded in a singleexon. All NITRs in group 1 and group 2 have two extracellular Igdomains, maybe that is the reason why the two groups get to-gether first. In group 3, six NITR genes (MmNITR2, MmNITR5,MmNITR12, MmNITR18, MmNITR20 and MmNITR21) only en-coded V domain.

The phylogenetic tree of NITR V domain (Fig. 5B) can be dividedinto three groups. Group1 contains NITR genes which only encodeda V domain. In group2, NITR genes have a common that V and I do-main in two separate exons. And NITR genes in group3 have a fea-ture that V and I domain in a single exon. As a few catfish NITRs (IpNITR5–14), sea bass NITRs (Ol NITR3, 11a/12), zebrafish NITRs(DrNITR7, 10/11 and 13/14a) and MmNITR2 possess a V regionbut not an I region, phylogenetic trees were separately estimatedfor V (Fig. 5B) and I regions (Fig. 5C). As shown in Fig. 5B, NITRscan be classified into a few distinct groups for a given species.For example, pufferfish can be divided into two parts. One part(pufferfish 1) contains the largest numbers of NITRs (Sn NITR1–4,7, 9 and 11–26), the second part (pufferfish 2) contains threereceptors (Sn193, Sn NITR5 and 10) and cannot (yet) be groupedwith other NITRs.

Meanwhile, the phylogenetic tree of NITR I domain was dividedinto two groups (Fig. 5C), NITR genes which encoded V and I

domain in two separate exons form a cluster in group1. Anothergroup contain the NITR genes which V and I domain in a singleexon. In rainbow trout, the I regions of three out of four NITRsare closely related, whereas the V regions are dissimilar to eachother. No matter in the phylogenetic tree of NITR V or I domain,MmNITRs are in different groups. The phylogenetic trees show thatNITR genes which form a cluster, because of their similaritycommon genomic structure.

4. Discussion

It is a challenge to deciphering NITR genetic origins, since theNITRs may be restricted to bony fish species and are extraordi-narily diverged. In this review, we summarize the current knowl-edge concerning these genes, identify twenty-one NITR genesfrom miiuy croaker, define the conserved features of MmNITRs,and investigate the phylogenetic diversity of NITRs within teleostspecies.

MmNITRs share common features within Ig domains with otherreported teleost species. A glycine bulge motif (e.g. MmNITR1FGEG232,MmNITR3FGEG249) in the end of I domain indicates a core feature of Jregions (FGXG) of other Ig domains (Chothia et al., 1985). Theconsensus joining (J) sequence is FGXGTXLX (V/L). A J-like sequencepossesses either motif FGXG (GXG) or TXLX (V/L/I) or partial

Fig. 3. Expression of NITR genes in various tissues (brain (B), eye (E), fin (F), gill (G), heart (H), intestine (I), kidney (K), liver (L), muscle (M) and spleen (S)) of uninfected miiuycroaker. Expression of NITR genes in three tissues after injection with V. anguillarum sampled at six time point (p < 0.05). (A) Expression of MmNITR genes in ten normaltissues. The expression data to heart have a value of 1 as control. (B–D) Expression of MmNITR genes in liver, spleen and kidney after V. anguillarum infection, respectively.Above these data, same lowercase letters stand for that they are not significantly different, whilst different lowercase letters stand for that they are significantly different.

Fig. 4. Homology model of miiuy croaker NITR V domain. The crystal structure of the channel catfish NITR11 (IpNITR11) V domain (A), TCR Va (B) and bispecific alpha/betaTCR V domain (C). MmNITR1 and MmNITR2 V domains have the highest similarity with IpNITR11 V domain. And MmNITR3 V domain is similar to bispecific alpha/beta TCR Vdomain. Hypervariable1 (HV1)/complementarity-determining region 1 (CDR1) is highlighted in yellow, HV2/CDR2 is highlighted in green and HV3/CDR3 in blue. IpNITR11 Vdomain is shown in aquamarine, TCR Va is shown in gold and bispecific alpha/beta TCR V domain is shown in purple. The V domains used in the comparison are PDB code3BDB, 2ICW and 2P1Y. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

274 F. Meng et al. / Developmental and Comparative Immunology 45 (2014) 269–277

sequence of both motifs. Besides the MmNITRs which only encodedV domain and MmNITR4, other MmNITRs contain a J or J-like motifin their I domain. But MmNITR8, MmNITR9, MmNITR10,MmNITR13 and MmNITR14 have two J-like motifs, one located inV domain, while another one located in I domain. All MmNITRs con-tain potential N-linked glycosylation sites (N-X-S/T), but the quan-tity are different. For example, MmNITR1 and MmNITR3extracellular portion contain two potential N-linked glycosylationsites and MmNITR2 contains four potential N-linked glycosylationsites (Fig. 1A), while the extracellular portions of NITRs observedin other fish contain 2–7 sites. The glycosylation sites of other IgSFreceptors are also located within their Ig domains (Barclay, 1999). A

few of specific glycoforms have been concerned in recognitionevents, although sugars in glycoproteins have a main function tostabilize the attached protein (Rudd et al., 2001). These events in-creased the possibility that NITRs function in cell–cell adhesionthrough these Ig domains. If so, whether the V domains recognizeforeign proteins or self-proteins located on the cell surface or othersoluble proteins are needed to make a decisions. MmNITR TM re-gions are all composed of 23 residues. As activating receptors, theTM sequences of MmNITR8 (VWLSIIRTGIILFFVTVCLLVYT),MmNITR15 (YFWRGFSAFTTILVVLLAFSVCM) and MmNITR16(YFWRGFSAFTTILVVLLAFSVCM) are different from inhibitoryreceptors. The three TM sequences all contain a charged residue

Tabl

e1

The

NIT

RV

dom

ain

sequ

ence

iden

tity

ofm

iiuy

croa

ker

com

pare

dw

ith

each

othe

r.

Mm

1M

m2

Mm

3M

m4

Mm

5M

m6

Mm

7M

m8

Mm

9M

m10

Mm

11M

m12

Mm

13M

m14

Mm

15M

m16

Mm

17M

m18

Mm

19M

m20

Mm

21

Mm

1⁄⁄⁄

20.6

19.6

7167

.319

.619

.618

.731

.823

.464

.521

.523

.424

.320

.618

.717

.821

.518

.719

.621

.5M

m2

20.6

⁄⁄⁄

20.6

20.6

21.5

20.6

21.5

20.6

11.2

21.5

21.5

22.4

20.6

1419

.622

.418

.720

.618

.748

.650

.5M

m3

19.6

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F. Meng et al. / Developmental and Comparative Immunology 45 (2014) 269–277 275

(Arg), a conserved feature of activating receptor. On the contrary,the majority of NITRs in miiuy croaker can be classified as inhibitorybased on the presence of one or two ITIM or itim motifs. As well asby the absence of a charged residue in their TM region. For instance,the intracellular region of typical inhibitory MmNITR1 showed twoconsensus ITIM (VNYAAL316 and SVYSQV340) (Fig. 1A). Both of theITIMs of MmNITR1 shows conserved sequences as previously pro-posed (I/V/L/S)XYXX(V/L) (Ravetch and Lanier, 2000). The ITIM ofMmNITR1 is located at a distance of 41 amino acid residues fromthe TM region. The length between the positions of the two tyrosineresidues of ITIM region is 24 amino acid residues which is similar tothe length found in KIRs by Blery et al. (2000). ITIM plays an impor-tant role in mediating an inhibitory signal of KIRs, through somestudies on ITIM-bearing receptors and their mutation of the tyro-sine residue (Blery et al., 2000; Yusa et al., 2004). Currently reportedNITRs structure have some different in terms of the number ofextracellular domains, J or J-like sequences, ITIM and itim motifs(Hawke et al., 2001; Yoder et al., 2004).

Though various receptors possess V domains, as a receptor onlypossess a V domain does not define a NITR. In order to classify a Vdomain protein as a NITR, it also needs to possess an I domain withsix conserved cysteines (Fig. 2). NITR I domains contain many ofthe same conserved residues which existed in the NITR, TCRs andIgs V domains. Among these conserved residues, C23 and C104 arepredicted to form a disulfide bond for purpose of stabilizing theIg fold: as well as four additional conserved cysteines consist in Idomains of NITRs (Fig. 2) (Giudicelli et al., 2006); NITR I domainsconsensus sequence reveal extra conserved residues, for examplea tyrosine residue (Y) and an alanine–valine dipeptide sequence(A-V). MmNITR2, MmNITR5, MmNITR12, MmNITR18, MmNITR20and MmNITR21 only encoded V domain. For example, MmNITR2characterized by lack of the I domain-encoding exon, which is re-placed by a novel exon. Importantly, if proteins which possess aV domain alone and lack an I domain can be classified as NITRs,the precondition is that their genes are present within an NITRgene cluster. So MmNITR2 belongs to NITR family (Fig. 5B) andother five MmNITRs also belong to NITR family (Fig. 5A). WhilstV-only NITR genes have been reported in several other species(zebrafish, channel catfish, medaka and sea bass), the novel exonhas been observed so far in I. punctatus NITR5–11, sea bass NITR15and NITR28–30. Mentioning the evolution of novel exons, there aretwo mechanisms have been proposed. The first one, ‘‘exonization’’of an intronic region, needs the recruited genomic sequence wassurrounded by the evolution of a splice site (Sorek, 2007). Theother one, ‘‘exon shuffling’’, resulting in the insertion of an exonfrom a ‘‘donor’’ gene into a preexisting intron of a recipient gene,facilitating the gain of an exon through crossing over among non-homologous genes (Patthy, 2008). Under the first model, a se-quence which is similar to the extra exon should be retained inthe corresponding introns of related genes that did not obtain a no-vel exonic sequence. Therefore, just like already reported for KIRgenes, exon shuffling is the most likely mechanism for novel exonacquisition (Rajalingam et al., 2004). At present, the function of thepeptide which encoded in the additional exonic sequence is notclearly, because no functional domain or motif has been identified.NITR genes are predominantly expressed in tissues which in agree-ment with other teleost species (Ferraresso et al., 2009; Piyaviriya-kul et al., 2007; Yoder et al., 2002).

In summary, this is the first time that 21 NITR genes were iden-tified from miiuy croaker. Not merely provide a new species forNITR gene, but also provide significant insights into the evolutionand function of NITR genes in bony fish. It is very important to dis-cern the relationships of NITR genes to the growing number ofinhibitory receptors which regulate immune recognition functions.Based on the results reported here, in addition to those collected sofar in other species, the following conclusions can be drawn: (1)

Fig. 5. Phylogenetic relationships between published NITR V and I domains. (A) Phylogenetic relationships between miiuy croaker NITR V domains. (B) V domains from miiuycroaker NITR1, 2 and 3 were compared with V domains of published NITRs. (C) Phylogenetic analysis of NITR I domains. Published NITR sequences are defined by the two-letter species abbreviation and described from Raja eglanteria (Re), Sciaenops ocellatus (So), Southern pufferfish (S. nephelus, Sn), zebrafish (D. rerio, Dr), rainbow trout(Oncorhynchus mykiss, Om), channel catfish (I. punctatus, Ip), Japanese flounder (Paralichthys olivaceus, Po), medaka (O. latipes, Ol), European sea bass (D. labrax, Dl) and miiuycroaker (Miichthys miiuy, Mm) were included in the alignment. NITR genes which encode only the V domain, V and I domain in two separate exons or V and I domain in asingle exon are highlighted with different colours. MmNITR genes are also indicated in red box or red letters. Bootstrap values less than 50 are not shown. The species namesand the GenBank accession numbers are as follows: IpNITR1–16, NM_001200280.1, NM_001200094.1, NM_001200095.1, NM_001200096.1, NM_001200097.1,NM_001200277.1, NM_001200098.1, AF397461.1, NM_001200332.1, NM_001200099.1, NM_001200333.1, NM_001200240.1, NM_001200241.1, NM_001200242.1,NM_001200243.1, EF490807.1; OmNITR1–4, AF345875–AF345878; DrNITR1–13, NM_001007219.1, NM_001018162.1, NM_131652.1, NM_131654.1, NM_001005768.1,NM_199130.3, NM_001115088.1, NM_001005577.1, AY570237.1, NM_001083034.1, NM_001025501.1, NM_001025496.1, EU170263.1; DlNITR1–6, DlNITR 8–17, DlNITR 19–26, DlNITR 28–30, GQ385039–GQ385065; OlNITR1a–5a, 7a–9a, 10b, 11a, 12a, 13–22, http://www.biomedcentral.com/content/supplementary/1471-2148-8-177-S1.doc;PoNITR, AB254823.1; MmNITR1, KF264458; MmNITR2, KF264459; MmNITR3, KF264460. MmNITR4–MmNITR21, KJ575084–KJ575101; SoIgSF, JQ031817.1; ReMDIR(modular domain immune-type receptor type II IgSF domain gene), DQ278458.1. (For interpretation of the references to color in this figure legend, the reader is referred tothe web version of this article.)

276 F. Meng et al. / Developmental and Comparative Immunology 45 (2014) 269–277

NITRs seem to have undergone species-specific gene birth anddeath events there are few true orthologs between fish species,(2) exon/intron acquisition/deletion contributed to the remarkablediversity of domain and genomic organization of fish NITRs, (3) dif-ferential expression might be a further dimension of NITR diver-sity, (4) NITR genes represent an extraordinarily divergent andrapidly evolving gene complex.

Acknowledgements

This study was supported by National Natural Science Founda-tion of China (31370049) and Important Science and ZhejiangProvince National Funds for Distinguished Young Scientists(R14C040001).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.dci.2014.03.016.

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