Full-length sequence and expression analysis of a myeloid differentiation factor 88 (MyD88) in half-smooth tongue sole
Cynoglossus semilaevis
Y. Yu, Q. W. Zhong, Q. Q. Zhang, Z. G. Wang, C. M. Li, F. S. Yan & L. M. Jiang
Summary
Myeloid differentiation factor 88 (MyD88) is a universaland crucial adaptor protein, which plays an essential rolein the intracellular signalling elicited by IL-1R/TLR super-family. In the present study, we report the full-lengthsequence of MyD88 gene in half-smooth tongue sole(
Cynoglossus semilaevis
). In the 2855 bp genomic sequence,five exons and four introns were identified. The clonedcDNA exhibited 110 bp of 5
′
UTR, 576 bp of 3
′
UTR and858 bp of the entire open-reading frame encoding apolypeptide of 285 amino acids. The protein sequenceincluded a typical conserved cytosolic Toll/interleukin-1receptor (TIR) domain, an intermediate domain (ID) anda death domain (DD), and shared greater than 70% identitywith Japanese flounder
Paralichthys olivaceu
ortholog.Real-time polymerase chain reaction (RT-PCR) analysisindicated a broad expression of csMyD88, especially inovary and spleen. Quantitative RT-PCR analysis indicatedthat the csMyD88 mRNA levels were significantlyincreased in the spleen and head kidney after inactive
Vibrio anguillarum
challenge and the expression ofcsMyD88 appeared to be developmentally regulatedduring
C. semilaevis
ontogeny. Although, species-specificdifferences were present, the similarity between mammalianand piscine MyD88s suggested that the main function ofMyD88 might be conserved across vertebrates.
Introduction
The immune system of teleost fish, like that of higher verte-brates, has two components to fight against the invasionof pathogens, innate immunity and acquired immunity.Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) family members play crucial roles in host innateimmunity and acquired immunity by sensing invadingantigens (TLRs) (Medzhitov & Janeway, 1997; Kirk &
Bazan, 2005) and promoting their combat (IL-1R family)during inflammatory reaction (Dinarello, 1996). Althoughthe TLR and IL-1R family have complete differences intheir extracellular domains, but all the members in the IL-1R/TLR superfamily share a highly homologous domainin their intracellular part, the Toll-like/IL-1 receptor (TIR)domain, which plays an essential role in signalling down-stream of these receptors by recruiting several effectorsand favouring their functional interaction (Aderem &Ulevitch, 2000; Watters
et al
., 2007). Central to thissignalling pathway is the role of adaptor molecules, whichbridge the receptors to the protein kinases required topropagate the signal inside the cells (McGettrick &O’Neill, 2004). To date, five adaptors have been identi-fied, MyD88, MyD88-adaptor like (Mal, also known asTIRAP), TIR-domain containing adaptor protein induc-ing interferon beta (IFN-
β
) (TRIF, also known asTICAM1), TRIF-related adaptor molecule (TRAM, alsoknown as TICAM2) and sterile
α
- and armadillo-motifcontaining protein (SARM). MyD88 is utilized by thewhole IL-1R family members and all of the TLRs exceptTLR3, whereas TRIF signals downstream of TLR3 andTLR4 (Watters
et al
., 2007). These adaptors (with theexception of SARM, a negative regulator of signalling)recruit downstream signalling molecules which lead to theactivation of nuclear factor-
κ
B (NF-
κ
B) and members ofinterferon-regulated factor (IRF) family (Watters
et al
.,2007). This ultimately results in the production of proin-flammatory cytokines and type-1 IFNs (O’Neill & Bowie,2007), which are important for defence against infectionand subsequent tissue repair.
As an important adaptor protein, which functionsto couple activation of the receptor to downstream signal-ling components, MyD88 has a modular structure,comprising an N-terminal death domain (DD) and a C-terminal TIR domain, separated by a small intermediatedomain (ID). The TIR domain serves as a scaffold tomediate the binding between MyD88 and IL-1R/TLRsuperfamily by homotypic protein–protein interaction (Li
et al
., 2005). The entire non-TIR region is responsible forthe distinct subcellular localization of MyD88, and thecorrect cellular targeting of MyD88 is critical to its signal-ling function (Nishiya
et al
., 2007). The first step ofsignalling transduction is the recruitment of the adapterMyD88. Then, MyD88 recruits IL-1R-associated kinase-4(IRAK4) via homophilic DD interactions. Activated
Authors Yu and Zhong contributed equally to this work.
Department of Marine Biology, Ocean University of China, Qingdao, China
Received 8 February 2009; accepted 23 March 2009
Correspondence: Quanqi Zhang, Department of Marine Biology, Ocean University of China, 5 Yushan Road, Qingdao 266003, China., Tel/Fax: 0086-532-82031931; E-mail: [email protected]
IRAK4 phosphorylates and activates IL-1R-associated-kinase-1 (IRAK1) causing the oligomerization and activationof TNF receptor-associated factor-6 (TRAF6) (Wesche
et al
., 1997; Qian
et al
., 2001; Li
et al
., 2002; Burns
et al
.,2003). TRAF6 is a ubiquitin E3 ligase and functions withthe ubiquitin-conjugating enzyme Ubc13 and the Ubc-likeprotein Uev1a to catalyse the synthesis of Lys63-linkedpolyubiquitin chains on target proteins including TRAF6itself (Deng
et al
., 2000; Chen, 2005). UbiquitinatedTRAF6 then recruits TAK-1-binding protein-2 (TAB2)and activates the TAB2-associated transforming growthfactor-
β
-activated kinase-1 (TAK1) (Deng
et al
., 2000;Takaesu
et al
., 2000; Chen, 2005). Activated TAK1 thenactivates the I
κ
B kinase complex (IKK) complex whichphosphorylates I
κ
B resulting in its ubiquitination andsubsequent proteosome-mediated degradation. Thisleaves NF-
κ
B free to translocate to the nucleus and initiategene transcription (Wang
et al
., 2001; Hayden & Ghosh,2004). TAK1 is also responsible for the activation ofmitogen-activated protein kinase kinase 6 (MKK6) whichphosphorylates and activates Jun N-terminal kinase(JNK) and P38 and thereby mediates AP-1 activation(Wang
et al
., 2001). MyD88 also participates in theactivation of several members of the IRF family of tran-scription factors, namely, IRF1, IRF7 and IRF5 (Hochrein
et al
., 2004; Kawai
et al
., 2004; Honda
et al
., 2004, 2005;Uematsu
et al
., 2005).MyD88 was originally identified as one of the myeloid
., 1990).Subsequently, MyD88 orthologs have been described inmany species including human
Homo sapiens
(Hultmark,1994), rat
Rattus norvegicus
(Janssens & Beyaert, 2002),western clawed frog
Xenopus tropicalis
(Prothmann
et al
., 2000), fruit fly
Drosophila melanogaster
(Tauszig-Delamasure
et al
., 2002), etc. However, the informationabout MyD88 in teleost fish is limited. Completesequences and expression data of MyD88 have beenreported only in several teleost fish. Although piscineMyD88s shared high identity in amino acid sequence,diverse responses of MyD88 to experimental challengewere observed in different fish species. In Japanese floun-der
Paralichthys olivaceu
, the exposure to
Edwardsiellatarda
significantly elevated MyD88 mRNA copy numberin the blood, kidney and spleen (Takano
et al
., 2007).However, in Atlantic salmon
Salmo salar
L. and zebrafish
Danio rerio
, MyD88 showed only minor alterations in theexpression levels after experimental challenges (Meijer
et al
., 2004; Strandskog
et al
., 2008). Thus, isolation andanalysis of MyD88 gene from other teleost fish will behelpful to explain that discrepant responses of MyD88 indifferent fish species, to understand the mechanisms ofimmune recognition in teleost fish and to further evaluatethe evolutionary relationships of vertebrate MyD88s.
We reported here the full-length sequence of MyD88cDNA of half-smooth tongue sole
Cynoglossus semilaevis
,expression analysis of csMyD88 in different tissues anddifferent developmental stages. In addition, we also inves-tigated the expression of csMyD88 in spleen and head
kidney after the experimental challenge with inactive
Vibrio anguillarum
.
Materials and methods
Fish and sampling
All experimental fish were collected from the commercialhatchery (Haiyang 863 High-tech base, Haiyang City,China). Tissues (brain, muscle, gonads, fin, blood, headkidney, intestine, gill, spleen, liver and heart) were col-lected from healthy individuals to investigate the tissue-specificity expression of csMyD88. Fertilized eggs of
C.semilaevis
were obtained by artificial fertilization andincubated at 22
°
C in clean sea water with aeration. Theembryonic stage for each sample was determined viamicroscopy. Ten embryonic stages (unfertilized egg, ferti-lized egg, multicell, blastula, gastrula, eye-bud, tail-budforming, tail-bud, heart-beating and hatching stage) andnine larva and juvenile stages (5, 10, 13, 15, 18, 19, 22,35 and 50 days after hatching) were selected, and sampleswere collected with a nylon net (100 mesh). In the chal-lenge experiment, 35
C. semilaevis
were injected withinactive
V. anguillarum
resuspended in 0.7% NaCl.Injected individuals were replaced into seawater tanksand five individuals were randomly sampled at 1, 4, 8, 16,24, 48 and 72 h. Thirty-five 0.7% NaCl-injected fishwere used as control group and five untreated individualswere used as blank control group (at 0 h). Spleen andhead kidney were taken from the fish used in the challengeexperiment. All samples were immediately frozen in liquidnitrogen and stored at –80
°
C.
RNA and genomic DNA extraction
Total RNA was extracted using the RNAprep AnimalRNA Purification Kit (Tiangen, Beijing, China), and thentreated with RNase-free DNase I (Tiangen) according tothe manufacturer’s protocol.
Genomic DNA was isolated from the blood. Simply,0.1 mL blood was collected from caudal vein using 1 mLsyringes, dissolved in 5 mL DNA extraction buffer con-taining 10 m
m
Tris-HCl (pH 8.0), 125 m
m
NaCl, 10 m
m
EDTA (pH 8.0), 0.5% SDS and 4
m
urea (TNES) andstored at room temperature until used. Fifteen microlitresof preserved sample was diluted by 600
μ
L TNES buffer.After overnight digestion with proteinase K (100
μ
g/mL,at 50
°
C), and extraction with phenol and chloroform/iso-amyl alcohol, DNA was precipitated with isopropanol,washed with 70% ethanol and resuspended in ddH
2
O.
Cloning and sequencing of
C. semilaevis
MyD88 gene
One microgram total RNA from
C. semilaevis
spleen wasreverse transcribed using RTase M-MLV (RNase H¯)(TaKaRa, Shiga, Japan) with Oligo-dT
15
primer (Tian-gen) to obtain the first-strand cDNA. The cDNA was thenused as a template for the subsequent polymerase chainreactions (PCRs). Degenerate primers for
MyD88 (MyD88-Fw1/MyD88-Rv1) were designed fromconserved regions of published nucleotide sequences ofMyD88 cDNAs (
D. rerio
: DQ100359,
Oncorhynchusmykiss
: AJ878918,
P. olivaceus
: AB241074). PCR condi-tions were: 94
°
C 5 min (initial denaturation), 30 cycleswith 94
°
C 30 s, annealing temperature 30 s (see below)and 72
°
C 30 s, 72
°
C 7 min (final extension). The anneal-ing temperature was decreased from 60
°
C to 50
°
C in thefirst 10 cycles (–1
°
C per cycle), then set at 50
°
C for theremaining 20 cycles. The SMART RACE cDNA Amplifi-cation Kit (BD Biosciences, Palo Alto, CA, USA) was usedto obtain 5
′
and 3
′
unknown cDNA regions according tothe manufacturer’s instructions. Specific primers wereused for 3
′
(MyD88–3
′
R) and 5
′
(MyD88–5
′
R) unknowncDNA regions. PCR conditions were: 94
°
C 5 min (initialdenaturation), 40 cycles with 94
°
C 30 s, annealing temper-ature 30 s (see below) and 72
°
C 1.5 min, 72
°
C 7 min (finalextension). The annealing temperature was decreasedfrom 65
°
C to 55
°
C in the first 10 cycles (–1
°
C percycle), then set at 55
°
C for the remaining 30 cycles. Thefull-length coding sequence and genomic sequence ofcsMyD88 were amplified with two gene-specific primers(MyD88-Fw2/MyD88-Rv2). PCR conditions were: 94
°
C5 min (initial denaturation), 40 cycles with 94
°
C 30 s,68
°
C 3 min. All PCRs were performed using a GeneAmpPCR System 9700 (Applied Biosystems, Forster City, CA,USA) and all the PCR products were separated on agarosegels, purified, cloned into pMD-18T vector (TaKaRa) andsequenced. All used primers were listed in Table 1.
Sequence analysis
The protein domains of the putative amino acid sequenceof csMyD88 were analysed by SMART (http://smart.embl-heidelberg.de/). Multiple alignments of aminoacid sequences were obtained by the software
clustalx
1.81 (Thompson
et al
., 1997). Phylogeneticanalysis was conducted using
mega
3.0 (Kumar
et al
.,2004). A phylogenetic tree was constructed using theneighbour-joining method (Saitou & Nei, 1987) based onthe Poisson-corrected distances. Node robustness wasevaluated by the bootstrap method (
N
= 1000 replica-tions). MyD88 of
D. melanogaster
,
Aedes aegypti
and
Culex quinquefasciatus
were used as outgroups.
RT-PCR analysis
Total RNA extracted from different tissues were treatedand purified as described above. For each sample, 1
μ
g totalRNA was reverse transcribed using RTase M-MLV (RNaseH¯) (TaKaRa) with random hexamers (Tiangen) in a 20-
μLfinal volume to obtain the first-strand cDNA. RT products(1 μL) were amplified with two gene-specific primersMyD88-Fw3/MyD88-Rv3 (Table 1). PCR conditions wereas follows: 94 °C 5 min (initial denaturation), 35 cycleswith 94 °C 30 s, 64.5 °C 30 s, 72 °C 30 s, and 72 °C 5 min(final extension). PCR products (3.5 μL) were analysed ona 2% agarose gel and confirmed by sequencing. The qualityof extracted RNA and the efficiency of reverse transcriptionwere tested using 18S rRNA as a reference gene.
Preparation of inactive V. anguillarum and challenge
experiment
Vibrio anguillarum was cultured in marine broth at 17 °Cfor 48 h and then fixed in 0.5% formalin for 24 h. Thebacterial cells were centrifuged at 5000 g at 4 °C for10 min, and then resuspended in 0.7% NaCl to an OD600
of 1.0 for injection.The 72 h experiment was carried out on C. semilaevis
of 200–250 g held in circular 500 L tanks at 16 °C. All fishwere anaesthetized with benzocaine (0.5 mg/L) prior tointraperitoneal injection. Thirty-five individuals wereinjected with formalin-killed V. anguillarum bacteria(100 μL/100 g body weight, OD600 = 1.0), and 0.7%NaCl was injected into another 35 fish (100 μL/100 gbody weight). Five individuals without experimentalinjection were used as blank control group.
Quantitative real-time PCR
Total RNA were isolated from samples at different develop-mental stages and tissues of the fish used in the challengetrial. The cDNA were generated with Quantscript RT Kit(Tiangen), random hexamers (Tiangen) and 1.0 μg ofRNA. Amplifications were performed in a 25 μL finalvolume containing 1 × SYBR® Premix Ex Taq™ II (PerfectReal Time) (TaKaRa), 0.2 μm each of specific forwardand reverse primer, and 1.0 μL diluted cDNA (50 ng/μL).18S rRNA was selected as a normalizer to control for dif-ferences in RNA and cDNA loading (Zhong et al., 2008).Negative control (no-template reaction) was alwaysincluded. Specific primers MyD88-Fw3/MyD88-Rv3(Table 1) were used in quantitative real-time (RT) PCRanalysis. An ABI Prism 7500 Sequence Detection System(Applied Biosystems) was programmed for an initial stepof 95 °C 15 s, followed by 45 thermal cycles of 95 °C 5 sand 64.5 °C 40 s. Fluorescent detection was performedafter each extension step. A dissociation protocol was addedafter thermocycling to verify that only a single productwas amplified. A serial dilution of plasmids containingeither 18S rRNA or MyD88 gene fragments were used tocompute the amounts of template. All samples were ampli-fied in triplicate and means were used for further analyses.
Statistical analysis was performed with spss13.0 software(SPSS Inc., Chicago, IL, USA). Significant differencesbetween samples were analysed via one-way anova (analysisof variance) using Duncan’s test. Differences of P < 0.05were considered significant.
Results
Cloning and characteristics of MyD88 gene from
C. semilaevis
The adopted RACE approach yielded a full length cDNA(accession number: FJ641054) with 110 bp of 5′ UTR,576 bp of 3′ UTR and 858 bp of the entire open readingframe (Fig. 1). The predicted amino acid sequence ofMyD88 was 285 residues long with an estimated Mw of33 kDa. Typical MyD88 domains including a DD, an IDand a TIR domain were identified in csMyD88 deducedamino acid sequence using SMART program (Fig. 1). Theamino acid sequence of csMyD88 shared high identitywith Japanese flounder MyD88 (BAE75959.1), zebrafishMyD88 (AAZ16494.1), human MyD88 (AAC50954) andmouse MyD88 (AAC53013) at 75.4%, 66.8%, 62.0%and 57.9%, respectively. The alignment analysis of theamino acid sequence of csMyD88 with other MyD88srevealed that the sequences of TIR domains were muchmore conserved than those of DDs and IDs. The TIRdomain, DD and ID of csMyD88 shared sequence homologywith that of Japanese flounder MyD88 (BAE75959.1) at84.6%, 68.5% and 69.6%, respectively. Three typicalconserved boxes in TIR domains were identified incsMyD88, the first one (F150DAFICY156), the second one(R185DVLPG190) and the last one (F174WDRL278).
The genomic sequence of csMyD88 spanned 2855 bpcontaining five exons and four introns (Fig. 1). The lengthof the five exons in csMyD88 was exactly like Japaneseflounder’s, but different from that of zebrafish and humanMyD88. The position of four introns in csMyD88 wassimilar to that of Japanese flounder, zebrafish and humanMyD88. In accordance with other MyD88 genes,csMyD88 gene showed the conserved intron–exonboundaries conforming to the GT-AG rule (Fig. 1).
To evaluate the evolutionary relationships of csMyD88in the context of vertebrate MyD88s, a phylogenetic treewas constructed (Fig. 2). All vertebrate MyD88s formed amonophyletic group, and the observed relationshipswithin this cluster agreed with the taxonomic classifica-tion of these species. The mammalian and fish MyD88were grouped, respectively, with above 98% bootstrapsupport. The putative amino acid sequence of csMyD88was obviously located within the fish MyD88 group with75% bootstrap support (Fig. 2).
Expression of MyD88 mRNAs in tissues
RT-PCR analysis was used to determine the expression ofcsMyD88 in various tissues. csMyD88 had a wide distri-
bution of expression in all 12 tested tissues. High expres-sion levels of csMyD88 were detected in ovary and spleen;moderate in testis, heart, head kidney, blood, fin, liver,brain, gill and intestine; but weak in muscle (Fig. 3).
Expression of MyD88 mRNAs in developmental stages
The expression of MyD88 seemed to be developmentallyregulated (Fig. 4). In the embryonic development of C.semilaevis, the highest expression level of MyD88 mRNAwas detected in unfertilized eggs. After fertilization, therelative level of MyD88 mRNA continually decreaseduntil blastula stage, about nine times lower than that atunfertilized-egg stage. After that, the expression level ofMyD88 elevated steeply, about two-fold, and was main-tained until eye-bud stage. At the later stages of embry-onic development, the expression level of MyD88 kept anapproximately constant level with only slight fluctuations.Then it elevated steeply, about two-fold, from heart-beatingstage to hatching stage (Fig. 4a). After hatching, the levelof MyD88 mRNA changed only slightly from days 5to 18. Then it sharply decreased on day 19, and reachedthe minimum level on day 22 (metamorphic stage). Sub-sequently, the transcription level recovered on day 35 andkept the increase until the 50-day stage, the last stage westudied (Fig. 4b).
Expression of MyD88 mRNAs after challenged with
inactive V. anguillarum
In teleost fish, spleen and head kidney are very importantlymphoid organs just as bone marrow and lymph nodes inmammals (Solem & Stenvik, 2006). Spleen and headkidney of C. semilaevis were selected to investigate theresponse of MyD88 to the injection with inactive V.anguillarum. Compared with its control group, the tran-scription level of csMyD88 in head kidney of test groupkept an approximately constant level with only slightfluctuations until 4 h after intraperitoneal injection, andthen it sharply increased and reached the maximum levelat 8 h (P < 0.05), about three times higher than its controlgroup. After that the mRNA expression recovered at 16 hpostinjection (Fig. 5a). However, the response profile ofcsMyD88 in spleen presented a different shape to that inhead kidney. Quantitative RT-PCR analysis indicated thatno obvious change was observed in spleen of tested groupuntil 48 h after injection, and then it was up-regulated at72 h (P < 0.05), about two-fold relative to its controlgroup (Fig. 5b). No significant differences were observedbetween the level of MyD88 mRNA of all control groups(injected with 0.7% NaCl) and blank group in headkidney and spleen, indicating the exclusion of the influenceinduced by intraperitoneal injection in the challengeexperiment (Fig. 5).
Discussion
Since the first discovery of the MyD88 gene in M1 myelo-leukaemic cells (Lord et al., 1990), MyD88 orthologs
Characterization of MyD88 from Cynoglossus semilaevis 177
have been described in many species (Hultmark, 1994;Prothmann et al., 2000; Janssens & Beyaert, 2002;Tauszig-Delamasure et al., 2002; McGettrick & O’Neill,2004; Takano et al., 2006; Qiu et al., 2007). In this study,
we isolated MyD88 cDNA from a kind of flatfish, C. semi-laevis, predicted the amino acid sequence and character-ized its potential functional domains. Aligned with otherMyD88 proteins, the highest identity was found in its fish
Figure 1. Genomic sequence of csMyD88 (FJ641054). CDS (coding sequence) are shown in capital letters, whereas the 5′ untranslated region, the 3′ untranslated region and the introns are shown in lower case letters. The deduced amino acid sequence is shown by single letter code of amino acids below the CDS. DD is underlined, ID is noted with gray highlighting and TIR domain is represented with double line. The three conserved region (box 1, box 2 and box 3) of TIR domain are boxed.
Figure 2. Phylogenetic analysis of MyD88 amino acid sequences in representative vertebrates. MyD88 of Drosophila melanogaster, Aedes aegypti and Culex quinquefasciatus were used as outgroups. Numbers at tree nodes refer to percentage bootstrap values after 1000 replicates; the scale bar refers to a phylogenetic distance of 0.1 amino acid substitutions per site. The accession numbers for MyD88 sequences are as follows: Homo sapiens, AAC50954; Pongo pygmaeus, BAG55251; Pan troglodytes, NP001123935; Macaca mulatta, NP001124153; Sus scrofa, NP001093393; Mus musculus, AAC53013; Rattus norvegicus, EDL76917; Gallus gallus, NP001026133; Xenopus tropicalis, NP001016837; Danio rerio, DQ100359; Ictalurus punctatus, ACD81929; Paralichthys olivaceus, AB241074; Oncorhynchus mykiss, AJ878918; Salmo salar, ABV59003; Chlamys farreri, ABB76627; Drosophila melanogaster, NP610479; Aedes aegypti, XP001658635; Culex quinquefasciatus, XP001868621 and Cynoglossus semilaevis, FJ641054.
Figure 3. Tissue distribution of MyD88 gene expression. Real-time polymerase chain reaction (RT-PCR) was conducted for 35 cycles (a) using mRNA isolated from various tissues of healthy adult fish; PCR products were analysed by electrophoresis. A fragment of 18S rRNA was amplified in all samples using RT-PCR (b). Negative control without template was included.
Figure 4. Quantitative analyses of the expression profiles of MyD88 gene in Cynoglossus semilaevis embryos and larvae. (a) The expression level of MyD88 mRNA in the embryos from unfertilized egg to hatching stage. (b) The expression level of MyD88 mRNA in larvae from days 5 to 50. The relative expression variance is shown as ratio (the amounts of MyD88 mRNA normalized to the corresponding 18S rRNA values). Data are shown as mean ± standard error of the mean (n = 3) (P < 0.05). Groups marked with the same letters are not statistically different.
Characterization of MyD88 from Cynoglossus semilaevis 179
orthologs. Phylogenetic analysis also showed that theputative csMyD88 clustered within the fish MyD88 groupwith 75% bootstrap support. All observed relationshipswithin this cluster were coincident with the taxonomicpositions of the species. Predicted amino acid sequence ofcsMyD88 had a modular structure, comprising a C-terminalTIR domain and an N-terminal DD, separated by ID. TIRdomain of csMyD88 shared a high identity with humanand other piscine MyD88s, which suggested the similarbinding mechanism between MyD88 and its receptors.There were three important regions in the TIR domain ofMyD88, named box 1, box 2 and box 3. Box 1 seemed toconserve the described (F/Y)DAΦΦXΦ consensus motif(Φ indicates hydrophobic residues and X represents anyamino acid). The alanine residue of the (F/Y)DAΦΦXΦmotif was reported to be the first residue of a β-sheet (Xuet al., 2000). Box 2 contained three conserved aa residues,R, D and G, reported to constitute a loop in the TIRdomain (Xu et al., 2000). Box 3 was characterized by aFWXXΦ motif, which was found in the last α helix of TIRdomain (Xu et al., 2000; Jault et al., 2004). Mutagenesisstudies showed that box 1 and box 2 were essential forsignalling (Xu et al., 2000; Watters et al., 2007). Box 3was involved in signalling, although it might not have asimportant a role as box 1 and box 2 (Slack et al., 2000).Compared with the TIR domain, the DD of csMyD88showed less identity with that of other MyD88s. DD wasinitially defined as the region of similarity between thecytoplasmic tails of the FAS/Apo-1/CD95 and necrosisfactor (TNF) receptors required for their induction of
cytotoxic signalling (Itoh & Nagata, 1993; Tartagliaet al., 1993), and then was found in many additional pro-teins (Feinstein et al., 1995; Weber & Vincenz, 2001). Byprotein–protein interactions with other DD sequencesforming either homo- or heterodimers (Boldin et al.,1995), DD was utilized by many members of the TNFsuperfamily for building signalling complexes that caninduce responses such as cytotoxicity, activation of JNK/stress-activated protein kinases (SAPKs), and/or activationof NF-κB (Nagata, 1997; Burns et al., 1998). The similarsequences and location of DD in MyD88s suggested thatit should have a similar function in signalling transduction.
In mammals, MyD88 was observed in many tissues andin B and T cells. High level MyD88 mRNA transcript wasdetected in spleen, lung and liver of adult healthy mouse,as well as in B and T cells (Hardiman et al., 1997). It wasalso detected in many adult human tissues, in addition tohuman monocyte, T, B, natural killer (NK) and dendriticcell lines (Hardiman et al., 1996). Like mammals, MyD88was found in a broad array of tissues in teleost fish(Takano et al., 2006). In this study, csMyD88 mRNA wasdetected in all tested tissues, the strongest expressions inovary and spleen, while the least expression in muscle.The high expression of MyD88 in spleen was also foundin mouse (Hardiman et al., 1997) and Japanese flounder(Takano et al., 2006). Spleen is considered as a veryimportant immune organ. High expression of MyD88 inspleen indicated its important function in immune system,and that was consistent with the role of MyD88, which isknown to function as a common adaptor protein in the
Figure 5. Quantitative analyses of the changes of csMyD88 expression in head kidney (a) and spleen (b) after challenged with inactive Vibrio anguillarum. The relative expression variance is showed as ratio (the amounts of MyD88 mRNA normalized to the corresponding 18S rRNA values). Data are shown as mean ± standard error of the mean (n = 5) (P < 0.05). BC, blank control. Groups marked with the same letters are not statistically different.
downstream signalling pathways of all the IL-1R/TLRsuperfamily members except TLR3. The strong expressionof csMyD88 in gonads was coincident with the findings inmammals. The abundant expression of a majority of TLRfamily members (TLR1-9), its adaptors (MyD88 andTRIF) and activation targets (NF-κB) were demonstratedin rat gonads (Palladino et al., 2007). The presences ofthese immune-related molecules in gonads suggested thatthey provided broad-spectrum detection of bacteria andviruses that might enter the tract to protect the process ofgametogenesis and to maintain the homeostasis of gonadsin mammals and teleost fish.
The highest level of csMyD88 mRNA was detected atunfertilized-egg stage, and then decreased gradually afterfertilization. That suggested the presence and consumptionof maternal MyD88 mRNA. The existence of maternalMyD88 mRNA was also identified in zebrafish (van derSar et al., 2006). Except MyD88, other immune-relatedmaternal mRNAs have been found in teleost fish. In ourobservation, maternal TLR9 was also found in unferti-lized eggs of C. semilaevis (data not shown). Maternal IgmRNA was detected in the eggs of sea bass (Picchiettiet al., 2004) and maternal C3 mRNA was present inunfertilized eggs and young embryos of carp (Huttenhuiset al., 2006). The presence of those immune-relatedmaternal mRNAs indicated either involvement of thosefactors in development itself, or more probably involvedin defending the egg and made a preparation of theimmune system for the posthatching period (Huttenhuiset al., 2006). In the embryo of zebrafish, maternal MyD88transcript levels reduced after fertilization and returned tohigher levels after gastrula stage (van der Sar et al., 2006).Similar tendency of MyD88 expression was found in C.semilaevis. Unlike other teleost fish, flatfishes undergometamorphosis, which arises from a series of regulatedprocesses including tissue differentiation, eye migrationand other biochemical, molecular or physiologicalchanges (Okada et al., 2001; Bolker et al., 2005). Aftermetamorphosis, the symmetrical body of flatfish larvaedramatically changes into an asymmetrical form. In thisstudy, the expression of csMyD88 was steeply reduced atmetamorphic stage. We also found the dramatic decreaseof other immune-related molecules, such as, TLR9, MHCIIB during the metamorphosis of C. semilaevis (data notshown). The significant decease of the expression of theseimmune-related molecules at metamorphic stage indi-cated they might be involved in the complicated changesduring metamorphosis.
As a crucial adaptor, MyD88 is a pivotal componentof the innate immune system and plays a critical role ininitiating and activating the immune system, especiallyin MyD88-dependent TLR/IL-1R signalling pathway(Janssens & Beyaert, 2002; Goldstein, 2004). Mice lack-ing MyD88 displayed a high susceptibility to Leishmaniamajor infection (Medvedev et al., 2002), Listeria mono-cytogenes infection (De Muraille et al., 2003) and Toxo-plasma gondii infection (Scanga et al., 2002). In addition,cells from MyD88-deficient mice were totally unresponsiveto peptidoglycan, lipoprotein, CpG DNA and imidazo-
quinolines in terms of cytokine production (Hacker et al.,2000; Hemmi et al., 2002). In Japanese flounder, theMyD88 mRNA levels were increased in blood, gill, kidneyand spleen after E. tarda challenge (Takano et al., 2007)and the population of MyD88-positive cells increased inkidney and spleen on day 3 after infection (Takano et al.,2006). Agreeing with these evidences, the expression ofcsMyD88 in head kidney and spleen showed a significantincrease after intraperitoneal injection of inactive V.anguillarum. However, different responses of MyD88 toexperimental challenges were observed in teleost fish.MyD88 expression levels showed only minor alterationsin the head kidney and spleen of Atlantic salmon afterexperiment challenge (CpGs or poly I : C) (Strandskoget al., 2008) and no obvious changes were observed inzebrafish after Mycobacterium marinum infection (Meijeret al., 2004). That probably suggested that the expressionof MyD88 might be differently regulated lying on thestimuli and the species involved.
In conclusion, the present study reported the sequenceand expression data of csMyD88, indicating the con-served protein primary structure and interesting similarresponses in the immune stimulation across vertebrates.The expression of MyD88 during ontogeny suggested itspossible function in the development of C. semilaevis.
Acknowledgements
This work was supported by State 863 High TechnologyR&D Project of China (no. 2006AA10A404,2006AA10A414) and the National Natural ScienceFoundation of China (no. 3067162, 30600455).
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