トラフグ腫瘍壊死因子α誘導タンパク質遺伝子 (TNFAIP3/A20)の …

トラフグ腫瘍壊死因子α誘導タンパク質遺伝子(TNFAIP3/A

20)の発現解析

誌名魚病研究ISSN 0388788X著者名引間,順一

森田,美咲木下,峻介Basu,M.Biswas,G.河野,智哉酒井,正博

発行元 [出版者不明]巻/号 52巻1号掲載ページ p. 15-22発行年月 2017年3月

農林水産省農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

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158

論文修正

魚、病研究52巻 1号（2017年3月発行）， 15-22頁に掲

載された引間らの論文について訂正がありますのでお知

らせします。 Fig.1～Fig. 7の説明文において，「Hikima

et al., submittedJの記述は誤りでしたので，以下の Fig.

1～Fig. 7の説明文に差替えをお願いいたします。なお，

訂正済み論文の PDFは，学会ホームページからダウン

ロードできます（URL: http://www.fish-pathology.com/

鳩山yu／）。編集事務局は，本誤記載についてお詫びしま

す。

Erratu町E

In the published article, Hikima et al.，月shPathology

52(1), 15-22 (2017), there were several errors in the Fig閉

ure captions (Figs. 1-7) to be corrected. Therefore,

“Hikima et al., submitted" in Figs. 1-7 should read follow開

ing captions. Corrected article is available in the JSFS

homepage (URL: http://www.fish-pathology.com/en/

kenkyu乃. Editorial office sincerely apologizes for these

errors.

Corrected captions:

円g.1. Genomic structure of Fugu A20 gene (GenBank

accession no. LC076438). Exons are marked with gray

background. Capital letters indicated the exon region

and small letters showed the intron region. Deduced

amino acids indicated as one letter under the codon

sequence. Motifs of “gt”and“ag”in the intron were

underlined (i.e., GT-AG rule).

Fig. 2. Genomic organization of Fugu and human A20

genes. Exons and intrans were represented as closed

boxes and lines, respectively. The striped box in the

Fugu A20 indicated the Zf domains that were not

detected by PROSITE database analysis.

Fig. 3. Multiple alignments of the predicted Fugu A20

amino acid sequence with that of other teleosts and ver-

tebrates. DDBJ, EMBL, GenBank accession numbers of

sequences used in this analysis were: Fugu (GenBank

Accession number: LC076438), Nile tilapia, Oreochromis

niloticus (XM_005474330), zebrafish, Dania rerio

(XM 687830), coelacanth, Latimeria chalumnae

(XM 006000463), American Alligator, Alligator

mississippiensis (XM_014605568), chicken, Gallus

gal/us (XM 003640919), platypus, Ornithorhynchus

anatinus (XM一001509118),mouse, Mus musculus

(BC060221) and human, Homo sapiens (BC114480).

OTU domain and Zf domains were marked with gray

boxes. Identical amino acid residues were indicated by

dots (.), and dashes (-)were introduced to fill the gaps

for optimal alignment. The domains detected by

PROSITE database analysis were underlined, and four

cysteine residues in each Zf motif were highlighted by

light gray color. The catalytic sites (i.e., 0104, C107 and

H258) forming the ubiquitin-binding site in Fugu A20 were

shown with black triangle.

Fig. 4. Phylogenetic analysis of Fugu A20 with amino

acid sequences that of teleostean and tetrapoda. Num-

ber indicates bootstrap confidence values obtained for

each node after 1,000 replications.

Fig‘5. Quantitative real-time PCR analysis of Fugu

A20 gene in various tissues. Data were analyzed by the

2 ""cT method and normalized against f3-actin mRNA

levels. Data were presented as mean ± S.D. in trip！ト

cates. The fold induction values were calculated relative

to mRNA level in muscle.

Fig. 6. Relative expression (mean± S.D.; n = 3) of A20

(A) and TNF－α（B) genes at different time points in Fugu

head kidney cells stimulated with LPS, Poly l:C (Poly)

and imiquimod (IMQ). Asterisk above bar indicates a sig-

nificant difference (P < 0.01) in expression levels

between stimulated cells and unstimulated control cells

at a time point. The expression values were normalized

against f3聞 actinmRNA levels and fold induction values

were calculated relative to the average value of the non-

stimulated negative control at each time point (4, 12, 24,

and 48 h).

Fig. 7. Relative expression (mean土S.D.;n = 3) of A20 (A) and TNF－α（B) genes at different time points in Fugu

spleen cells stimulated with LPS, Poly l:C (Poly) and

imiquimod (IMO). Asterisks above bars indicate a signifi-

cant difference in expression levels between stimulated

cells and unstimulated control cells at a time point (*P <

0.05; **p < 0.01 ). The expression values were norma卜

ized against f3-actin mRNA levels and fold induction va卜

ues were calculated relative to the average value of the

non-stimulated negative control at each time point (4,

12, 24, and 48 h).

魚病研究 Fish Pathology, 52 (1), 15-22, 2017. 3

Research article

c2017 The Japanese Society of Fish Pathology

Molecular Characterization and Expression Analysis of Tumor Necrosis Factor Alpha-induced Protein 3 (TNFAIP3/A20)

Gene from Japanese Pufferfish Takifugu rubripes

Jun-ichi Hikima 1 *, Misaki Morita 1, Shunsuke Kinoshita 1, Madhubanti Basu町

Gouranga Biswas3, Tomoya Kono1 and Masahiro Sakai1

1 Department of Biochemistry and Applied Biosciences, Faculty of Agriculture,

University of Miyazaki, Miyazaki 889-2192, Japan 2Fish Health Management Division, ICAR-Central Institute of Freshwater

Aquaculture, Odisha 751002, India 3Kakdwip Research Centre of ICAR-Central Institute of Brackishwater

Aquaculture, West Bengal 7 43347, India

(Received January 17, 2016)

ABSTRACT-Tumor necrosis factor alpha (TNF-a)-induced protein 3 (TNFAIP3/A20) is an important deubiquitinating enzyme that takes part in homeostasis of immunity induced by TNF-a. During viral and bacterial infection, it plays a crucial role in negative regulation of innate immune responses in mammals. However, this molecule in fish is still poorly understood. In this study, we identified full-length A20 gene of Japanese pufferfish (Fugu) Takifugu rubripes and performed its expression analyses in various tis-sues and stimulated cells. Total length of the determined Fugu A20 gene spaned 6,229 bp and con-sisted of 9 exons and 8 introns. The Fugu A20 gene contained a 2,394 bp open reading frame (ORF) that encoded a 797-amino acid residue. In the exon-intron structure of Fugu A20 gene, the intron-boundary positions in the region having 7 zinc finger (Zf) domains were different from those of human. Expression analysis of Fugu A20 gene exhibited a higher transcription in thymus, head kidney (HK) and spleen tissues than the others. In HK and spleen cells stimulated with LPS, polyl:C and imiquimod, A20 gene was expressed after up-regulation of TNF-a gene expression. These results together suggest that expression of Fugu A20 gene is associated with bacterial and viral infections.

Key words: Tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20), TNF-a, Zinc finger domain, Takifugu rubripes, Japanese pufferfish, Fugu

Tumor necrosis factor alpha (TNF-a)-induced pro-

tein 3 (TNFAIP3) is a deubiquitinating enzyme, which is

also called A20, belonging to the ovarian tumor family

(Parvatiyar and Harhaj, 2011). A20 has an amino-ter-

minal ovarian tumor ubiquitin (OTU) domain to cleave

K63-linked ubiquitin residues and seven carboxy

(C)-terminal Zinc finger (Zf) domains to conjugate K48-

linked ubiquitin chains, and thus de-ubiquitination occurs

through attenuating activation of specific E3-ubuqutin

ligase such as the TNF receptor-associated factor 6

(TRAF6) (Frias-Staheli et al., 2007; Parvatiyar and

Harhaj, 2011; Wertz et al., 2015). In human, A20 is

induced by TNF-a to play a key role in the homeostasis

of immune system (Dorronsoro and Lang, 2013). A20

can also suppress nuclear factor (NF)-KB activity.

Bacterial or viral simulations are transferred to TRAF6

* Corresponding author E-mail: [email protected]

through Toll like receptor (TLR) 4 and TRAF6 activates

NF-KB by phosphorylation (Kawai and Akira, 2009;

Parvatiyar and Harhaj, 2011; Harhaj and Dixit, 2012).

Activated NF-KB migrates into the cell nucleus and con-

sequent inflammatory response occurs with the tran-

scription of pro-inflammatory cytokines and A20 genes

(Parvatiyar and Harhaj, 2011; Harhaj and Dixit, 2012).

Due to the denaturation of TRAF6, inflammatory

response is suppressed resulting in no activation of

NF-KB (Parvatiyar and Harhaj, 2011; Harhaj and Dixit,

2012). Thus, A20 plays a role as negative feedback

agent in the NF-KB pathway (Parvatiyar and Harhaj,

2011).

In rainbow trout, a partial nucleotide sequence of

A20 cDNA has been identified as a transcript up-regu-

lated by LPS-stimulation, and this gene was significantly

induced by LPS and zymosan, but not by polyl:C in the

macrophages (lliev et al., 2006). In zebrafish, a full-

length nucleotide sequence of A20 cDNA encoding OTU

16 J. Hikima, M. Morita, S. Kinoshita, M. Basu, G. Biswas, T. Kono and M. Sakai

and Zf domains has been identified (Oehlers et al.,

2011). However, information on A20 gene in fish is

still insufficient and it should additionally be discussed

for expression pattern. In the present study, we have

cloned the entire nucleotide sequence of the A20 gene

from the Japanese pufferfish, Fugu, Takifugu rubripes,

predicted the structure and examined a response to bac-

terial and viral mimic stimulations in the head kidney and

spleen cells.

Materials and Methods

Fish maintenance and handling

The Japanese pufferfish, Fugu, Takifugu rubripes

(mean mass: 100土 8g) was obtained from Matsumoto

Fisheries Farm in Miyazaki, Japan. The fish were firstly

acclimatized in an aerated seawater tank at 20°C and

fed with a commercial diet (Sango, Higashimaru Co.

Ltd.) at 1 % body weight per day for two weeks under a

natural photoperiod prior to their use in the study. All

animal experiments were conducted according to the rel-

evant national and international guidelines,'Act on

Welfare and Management of Animals'(Ministry of the

Environment, Japan). Ethics approval from the local

IACUC was not sought since this law does not mandate

protection of fish.

cDNA synthesis

Fish was anesthetized with 2-phenoxyethanol

(0.05%, Sigma-Aldrich) before being killed for tissue col-

lection. Tissues from healthy Fugu [thymus, head kid-

ney (HK), trunk kidney (TK), spleen, liver, intestine,

brain, heart, gill, muscle and skin] were isolated under

sterile condition from six individual fish for total RNA

extraction.

Total RNA was isolated using ISOGEN (Nippon

Gene) following the manufacturer's instructions and

poly(A) mRNA was purified using a quick prep micro

mRNA kit (Amersham Pharmacia Biotech). cDNA was

synthesized from 2μg of mRNA using ReverTra Ace RT

kit (Toyobo) and used as a template for PCR.

Molecular cloning and sequencing

Initially, PCR was performed using the HK cDNA

prepared above, with primers Fw/A20 F3 and Rv/A20 R1

(Table 1) specific to the region including open reading

frame (ORF) and untranslated regions, to allow ampli-

fication of A20 gene. After amplification of this partial

Fugu A20 sequence, the 5'and 3'end were obtained by

RACE-PCR using SMARTer RACE cDNA amplification

kit (Clontech Laboratory Inc.) according to the manufac-

turer's instruction. RACE PCR was performed with Rv/

A20 GSP2, Fw/A20 GSP2 or Fw/A20 NGSP2 (Table 1).

For sequencing of Fugu A20 gene, amplification was

performed in a 50μL reaction volume containing 5μL of

dNTP mixture (2.5 mm each dATP, dCTP, dGTP, dTTP}

and 10x Gene Taq Universal buffer, 0.5μL of Taq poly-

merase (5 unitsμL―1; Nippon Gene), 5μL of each primer

set (F and R; 2.5μM), 28.5μL of distilled water and

1μL of Fugu thymus cDNA. The cycling conditions for

all conventional PCRs were: 1 cycle of 95°C for 1 min,

35 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for

3 min, followed by 1 cycle of 72°C for 5 min. Genomic

DNA was isolated from the head kidney of Fugu with a

blood and cell culture DNA mini kit (Qiagen) according

to the manufacturer's instructions. The Fugu A20 gene

was PCR-amplified using the Taq polymerase with the

specific primers in Table 1. The PCR profile was same

Table 1. Oligonucleotide primers used in this study

Primers Sequences (5'→ 3') bp Application

Rv/A20 GSP2 CAGCATGTATTGAGAGGTGGCATGAAGC 28 5'-RACE PCR

Fw/A20 GSP2 GAGGGCTACTGTGACAAGTGCTATGTC 227 7 3'-RACE PCR

Fw/ A20 NGSP2 TT AACCAGGTGGCACATCGCTCGACAC

Fw/A20 F1 ACGTTGGAGCAGGAGAAGAA 20

Fw/A20 F3 ATTTCTCCCCACAGTCTCCAC 21

Fw/A20 F5 TATCCTGGACAGAGCCATGC 20

Fw/A20 F6 ACATTCTTCGCAGACCCATC 20

Fw/A20 F7 AGCTCGTCAACCACGAGTTC 20 Fw/A20 FB CTTTGTGAGCGCTGTTTCAA 20 Conventional PCR for gene sequencing Rv/A20 R1 A TTGAGAGGTGGCA TGAAGC 20

Rv/A20 R6 GCCAGTGGAGTGGCAGATAA 20

Rv/A20 R7 CCACGGAAACGTAAAAGGTG 20

Rv/A20 RS TTCTGGTTCACGACCAGTTG 20

Rv/A20 R9 T AAGCCTCGCACTTTGCTC 19

Fw/A20-qF2 CGTAAAGATGGCCTCTCCAG 20 Rv/A20-qR2 GATGGGTCTGCGAAGAATGT 20 Fw/TNFa-qF CAGGCTTCTTTCCGAGTGAC 20

Rv/TNFa—qR TTCTGACACGCTGACCTCAC 20 qPCR

Fw//3-actin Fw2 CGTGCGTGACATCAAGGA 18

Rv//3-actin Rv2 GCAGCGGTGCCCATCTC 17

Characterization and expression of Fugu A20 gene 17

as above-mentioned.

Obtained PCR products were ligated into the

pGEM-T Easy vector (Promega) and transfected into

competent Escherichia coli TAM competent cells (Active

Motif), where recombinants were identified through red-

white color selection when grown on MacConkey agar

(Sigma-Aldrich). Plasmid DNA from at least three

clones was extracted using a QIAprep Spin Miniprep Kit

(Qiagen) and sequenced using a CEQ8000 Automated

Sequencer (Beckman Coulter). The sequences gener-

ated were analyzed for similarity with other known

sequences using the FASTA (Pearson and Lipman,

1988) and BLAST (Altschul et al., 1990) programs.

Sequence and structural analysis

Multiple sequence alignments were generated using

ClustalX v1 .81 (Thompson et al., 1997) and homology

analysis was performed using the MatGat software

v2.02 (Campanella et al., 2003). Finally, the protein

family signature was analyzed using the ExPASy

PROSITE database of protein families and domains

(http://prosite.expasy.org/; Falquet et al., 2002), and

phylogenetic analysis was performed on the full-length

amino acid sequences of the known A20 molecules

using the neighbor-joining method (Saitou and Nei,

1987). MEGA6 (Tamura et al., 2013) was used to con-

struct the tree with confidence limits (Felsenstein, 1985).

Quantification of Fugu A20 gene expression

HK and spleen cells were collected under sterile

condition from freshly killed Fugu (three individuals) and

gently pushed through a 100μm nylon mesh (John

Staniar and Co.) with RPMI 1,640 medium (Gibco BRL)

supplemented with 5% fetal bovine serum (FBS; Gibco)

and 1 % Streptomycin/Penicillin (Gibco). After washing

with the above medium, HK and spleen cells were stimu-

lated with 20μg mlー1of lipopolysaccharide (LPS;

Sigma-Aldrich), the viral mimic, polyinosinic acid-poly-

cytidylic acid (polyl:C; Sigma-Aldrich) and lmiquimod

(Wako Pure Chemical Industries, Ltd.) for 0, 4, 12, 24

and 48 h at 22°c (0 h was a non-treated control).

cDNA was synthesized from stimulated HK and spleen

cells, and unstimulated tissues (muscle, brain, TK, heart,

skin, gill, intestine, spleen, HK, thymus and liver) as

described above and diluted to 20 times with 10 mM Tris

buffer (pH 8.0).

The qPCR reaction was conducted using

THUNDERBIRD™ SYBRRqPCR Mix (Toyobo Co., Ltd.) for each sample run in triplicate as per the protocol

described earlier (Kinoshita et al., 2014). The compar-

ative threshold cycle (CT) method (2―△△ CT method)

(Schmittgen and Livak, 2008) was used to analyze the

expression levels of A20 and TNF-a genes using Fugu

/3-actin (GenBank Accn no. U37499) as an internal control

gene. One-way analysis of variance was performed to

find out any difference in quantified relative expression

of a particular gene under different stimulations at a

given time point using SPSS for Windows v. i 7.0 pro-

gram (SPSS Inc.).

Results

Structural analysis of Fugu A20 gene

Fugu A20 gene consists of 6,229 bp containing nine

exons and eight introns, and the coding 2,394 bp mRNA

(deposited in GenBank under the accession number

LC076438) translates a protein of 797 amino acids (aa)

in length (Fig. 1), which was located at Fugu genome

scaffold_124 (791,414-797,300 bp) in Ensemble

Genome Browser (FUGU 4.0) (www.asia.ensembl.org/

Takifugu_rubripes/). Although, the splice donor/ accep-

tor sequence (GT/AG) was conserved at the 5'and 3'

ends of the introns (Fig. 1), between exon 6 and exon 7

it was CT/TG. The exon-intron structure of Fugu A20

gene was very similar to that in human although the

divided positions of exons in the region having Zf

domains when compared to those of humans were dif-

ferent in number (Figs. 2 and 3). The results of

PROSITE database analysis showed that Fugu A20 pro-

tein contains only five Zf DNA binding domains (i.e., Zf1: E3as_E410; Zf2: A4a1ーFs11;Zf4: Aso4—ys31; ZfS: Nsss -Ksa3;

Zf7: K765-H790) (the regions underlined in Fig. 3), com-

paring with the human A20 protein possessing seven Zf domains such as S380-N416 (Zf1), S471-S507 (Zf2), D514-S548

(Zf3), 0soo_Hs3s (Zf4), Rsso_Fsas (Zf5), S1os_p745 (Zf6) and

D755-G790 (Zf7) (the regions in the gray boxes in Fig. 3;

Wertz et al., 2015). However, four cysteines (i.e.,

Cx4Cx11Cx2C and Cx2Cx11Cx2C) in seven regions similar

to the human Zf domains were well-conserved in fish.

Interestingly, one common aa deletion among teleosts

was observed in Zf6 (Fig. 3). This deletion is probably

one of the features in the fish A20 aa sequences. One

Fugu OTU domain (N102—Q257) from the result of PROSITE database analysis was also shortened from

the OTU domain (M1ーS360)known in human (the regions

in the gray boxes; Wertz et al., 2015). The catalytic site

forming the ubiquitin-binding site in the human OTU

domain (D100, c103 and H256) (Accession no. P21580 in

UniProt database: http://www.uniprot.org/uniprot/

P21580) is highly conserved in the Fugu OTU domain

(D104, c107 and H258). It was suggested that the aa

sequence of Zf and OTU domain were conserved among

vertebrates.

Multiple alignments and phylogenetic analysis of A20

sequence

Multiple alignments with known A20 molecules in

other vertebrates revealed a high conseNation between

the mammalian, bird, reptile and fish aa sequences (Fig.

3). The high identities of A20 were shown between Fugu

and zebrafish (75.0%) or tilapia (87.3%), but the low

identities were shown between Fugu and coelacanth


5400

6229

Fig. 1. Hikima et al., submitted

Fugu (9 exons & 8 intrans): 6,229 bp; 797 aa

............、、..........

’ I OTU domain I ,・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ .、

一椙=r.町一',,. ¥',, ＼

、、',,_ ¥_・-,,_ -,,、',,.',ヽ'•zr1 Zf2 Zf3 Zf4 Zf5 Zf6辺f7

',, __ ¥、、--,,_',,_'ヽ、ふ、

1 2 3 45 6 7

Human (9 exons & 8 intrans): 19,810 bp; 790 aa

Fig. 2. Hikima et al., submitted

Fugu Ti lapw Zebrafish Coelacanth Alligator Chicken Platypus Mouse Human

Fugu Tilopw Zebrafish Coelacanth Alligator Chicken Platypus Mouse Human

Fugu Tl lapta Zebrahsh Caelacanth All 1gator Chicken Platypus Mouse Human

Characterization and expression of Fugu A20 gene

I OTU domain I

『1~i-~~-,~1;,1i:tilu:!! , ...』.I ..... I . . . . . .. 20 .... I .... I .... I ... 40 .... I .... I .... I ... 60 .... I .... I .... I ... 80 .... I .... I ~SQGQNFLPKFLFVSNLLKAVKIRQRVPNOWKPANGSGLIHHLRGMHRYTLEMIAMNHFPQAFR印'VQAAILORAMQATLEQE CRE 100

198 • • W• ••• ••• A ...•• .. . ........ A . ••. .. .••.... K •.....• L . .•..•.... •• ... ••• D .• E ••••..••••. --.... .. . . . .•. . 198 •• 恥・'"••• A ... L. ....•...... A • . A ... •. •. TD. LT ... T .• LH ...••....•• S. L ••. . .. V . . • 0 .. . • • •. --• . . . . . . . • . • . . 198 1:: 屈;~J,:. ・・:・... W. I. •...•••... YRA • ...•• RN.KF .W. LASV ••.. . VE ••.. . N.R •. DD •• E. L .E. ... TTAKGQ ...• YS ..• E.. 200

I ( . . • ... W. I.. V •..•.... FSA. R. I. .RN. KL .W.R. T .K .... VE. .. C. D.R •.. . .• ENLI.. T .. DT .MAR .... YNA .. E. . 197

I .. ~ . 1, '.M •• A . ..• W.IE. V ........ FSA.R.I. .RN.KL.W.R.A.K ..•• V ...• HFD.R ••.... EYLIE. T .. ET .GAR.R.PYNA .. E.. 197

,:: 通？知M .. A •• . . W .. .•. .. .•• •.. F .A •...•• RN.KF .W.N.S.K .... VE ..... D.R .. D ..• ENL . ••.. ADT. VSRSA .• YN ... E.. 197 .. AC •.. W .. ••....••• A. CST. ... •• RN. KF .W. L. SLK .... VE ... C. D.R .. ND .•• NL ..... ADTPAARS ... YN ... E. . 196

I ,. ・・ ;1.;・; 益.M ....... W •..••• . ...• A. FST ...... RN. KF. W. L. SLK .... VE. .• C. D.R •• ND ... NL!. ... TDTPMARS • .. YN •.• E. . 196

＇ APLITVKDSGPEIRAVPLINPGRGGFEELKVHFLMEKE 298

V I ••• •• ••• • •••••• ••• ••• • T. • • ... .•.••. .. •• Q •• ••.• ..••....• 贔.....I. ....•. .... • ... R.N.............. 298 • . . .•... .. . ••... . L. . ... ..• F ... . ••....••.... G ••....••....• "ll・..... I. .. ... ....••....•• ... .• R ••.. T. • • . 298 I ••• A . ..•• T •...•. NML .. LE .... FA ... I. .... ••• .•• Q ...•........ T、,... V.L .•....•..... MYNSM.RA. 叩.....T.T. 300 . V. V . ...•. V .. L. .KM ... LE. ... F •.. HI ....... L.. TEQ .. R •...•••• NM . T •. V. L •• •... •• .... VSQE .. K •• D ... •• . TOA. 297 • V .A ••••. .. V. L. .K •••• LE. .•• FA . . •••. V . ••• L. .GE ••. R .. . •.... NMJ. T .• V. L ... . .......• VTSE •. R .. D.S .... TDA. 297 I. V .A ........ I .S.KML .. LE .... F •.. K .••.. .. •. . . AH .•. R ..••••••..• II V •. V. L ......••...• V .RE •. R .• D •...•. T .P. 297 I. V ••. . . . ••• . . . S.KML .. LE ••. NFA •• K ........... AQ ... R ......... jl V .. V. L . •.... L. .... V .RD .. R . • 0 ...... TDP .N 296 V .C. ......... S.KML.. LE. .. NFA .. K . . •.. •.• •.. AQ ••. R ..•• • ... -~,~ V .• y.L . •• 弁 - ~ · ・・・・ . 'J .~D • . R •. D. ~ ・・・ .:fD~. 296

@TI Fugu 』 KERLLNOYLILIEIPVIGLGYOTIQII氾ARLOEGNLPEOMNLMEOYLQLVNHEFQRWQEOKEQAIVAAQPQR-PPPFSVS SLI -- ---- -- 397

>!!::"i. 旦~ニ!!~~喜~im·~~!!!Coelacanth All 1gator Chicken Platypus Mouse Human

i:!!:;:," 冒ui[~l~{!;l~li\~;;~!i!fr'"'m~I'"I"'Illi胃

dl冒!~;ごここQごご!I三~I!~ff~I』［言I~Fugu Tilopw Zebrafish Coelacanth Alligator Chicken Platypus Mouse Human

Fugu Ti laprn Zebrahsh Coelacanth All igatar Ch1cken Platypus Mouse Human

Fugu Ti laprn Zebrafish Coelacanth Alligator Chicken Platypus Mouse Human

Fugu Ti lapta Zebra fish Coelacanth Alligator Chicken Platypus Mouse Human

>~lillll!~l~1:1ij;~II~·~~TI!;;: 贔晶霊IPAPIQNRHGLE F E AR QVAHRSTHSPPLVVRDRAVKPRSSQQ TQNQT (WR C N-VSPGCT 739

A S ..... ............... V .. V ...................... N. --T ... --L. E .. A .......... . -. IV. P .. TS ...... CP •........ E ......... N .. F .......... A. S. GSQ ... --LVT .. S .A. PPPP .. --A .. R .... k. -L. .... . SP. SG. LVAGT. . P .MAP. l. ... S. Y .NPLF .... Q .. FLEA. OH .. EARWSGEQLARHYE. APHLR. MQHNV S-Q. LK. A .. H. N. F. PSR-OE 770 SPASGQTGTSA .. H.1VS. L .. E .. TL.ST ...... Q

言［言胃□打『!I且！言！［『"盲言直三

Fig. 3. Hikima et al., submitted.

19


Table 2. Identities of A20 amino acid sequence between Fugu and the other vertebrates

＼ Tilapia Zebrafish Coelacanth Alligator Chicken Platypus Mouse Human

Fugu 87.3% 75.0% 46.7% 45.1% 45.6% 46.2% 43.5% 44 7%

Tilapia 72.7% 46.1% 42.8% 431% 44.1% 41.8% 42.7%

Zebrafish 47.0% 45.0% 45.8% 46.9% 44.9% 449%

Coelacanth 54.9% 53.6% 55.5% 54.5% 54.8%

Alligator 78.0% 68 0% 65.1% 66.2%

Chicken 675% 64.9% 66.0%

Platypus 761% 770%

Mouse 879%

Teleosts

100

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(46.7%), chicken (45.6%), alligator (45.1 %) or mammals

(43.5-46.2%).

Phylogenetic analysis revealed that A20 identified

from Fugu formed a cluster with A20s identified from

other teleosts distinctly away from the clades of coel-

acanth, chicken, alligator and mammalian species

(Fig. 4).

Expression analysis of Fugu A20 gene

A constitutive expression of the A20 gene was

observed in all the tissues examined, with the highest

□ Control口LPS口PolylC ■ IMQ

8 (A)

*T l l A20

6

4

2

(/) 恩

乏゜0:: 4h 12h 24h 48h E (B)

こ器a::Q) 3 5 l ＊

T TNFa 30

25

20

15

10

5

゜ 4h 12h 24h 48h

Time post stimulation


expression seen in spleen, HK and thymus of healthy

Fugu (Fig. 5).

LPS and polyl:C stimulations caused a significant

increase (P < 0.01) in A20 gene expression at 12 h after

treatment in HK cells (Fig. 6A). In the same samples,

expression of TNF-a was up-regulated (P < 0.01) at 4

and 12 h post LPS, polyl:C and imiquimod treatment

（日g.6B). In contrast, no change in the expression of

A20 gene was noticed in HK cells treated with imiquimod

(Fig. 6A). Furthermore, expression of A20 and TNF-a

genes were sufficiently down-regulated in HK cells at 24

h post-stimulation by LPS, polyl:C and imiquimod (Fig.

6).

In spleen cells, the expression of A20 gene was sig-

nificantly increased (P < 0.01; 0.05) at 4 and 12 h after

polyl:C and imiquimod treatments (Fig. 7A). An

increased expression of TNF-a was only (P < 0.01; 0.05)

at 4 h post LPS, polyl:C and imiquimod treatments (Fig.

Characterization and expression of Fugu A20 gene 21

(A) oControl □ LPS ロPolyl:C■IMQ

5

4

3

2

A20 ＊＊

s1a > a1 ¥iN<l Ea>―-e-ar

゜ 4h 12h 24h 48h

8

5

2

9

6

3

0

1

1

1

(B)

＊＊

4h 12h 24h 48h

Fig. 7.

7B).

Time post stimulation

Hikima et al., submitted

Discussion

In Fugu A20, this catalytic site forming the ubiquitin-

binding site was also well-conserved (D 100 103

, C and

H256). One common aa deletion in Zf6 was conserved

in teleosts and coelacanth (Fig. 3), suggesting that their

common ancestor molecules could be the one-aa-dele-

tion type and that one aa could be obtained during the

diversion from coelacanth to reptiles. In mammals,

14-3-3 protein binding sequence (BS) (i.e., RSKSDP)

between Zf3 and Zf4 of A20 was functionally important

to a number of signaling pathways and interaction of

A20 with 14-3-3 protein alters localization of the former

from a punctuated cytoplasmic staining to a more dif-

fused cytoplasmic pattern, which is also associated with

decreased amount of A20 in the insoluble cell fraction

(Vincenz and Dixit, 1996). Lademann et al. (2001)

reported that the binding to 14-3-3 protein protects A20

from ubiquitin degradation. The 14-3-3 BS was con-

served in the mammals and coelacanth, but not in tele-

ost fish species (Fig. 3). Therefore, teleost A20 may

not have the 14-3-3 protein binding function, suggesting

that teleost A20 may not conjugate K48-linked ubiquitin

chains, and also the amount of A20 in the cytoplasm

may not be affected by the interaction with 14-3-3 pro-

tein.

Fugu A20 gene was expressed in all the tissues

examined with higher expression level observed in the

immune-competent organs such as spleen, HK and thy-

mus. In mice, A20 mRNAs were detected in lymphoid

tissues like thymus, spleen and gut-associated lymphoid

tissue (GALT), especially in thymus, meaning that A20

mRNAs are expressed in T-and B-cells and dendritic

cells (Tewari et al., 1995; Hong et al., 2011). It sug-

gests that Fugu A20 mRNAs are probably expressed in

various leukocyte types.

In this study, we conducted an expression analysis

of A20 gene using LPS as a representative ligand of

bacterial infection, polyl:C (double strand RNA virus) as

representatives of virus infection, and imiquimod as a

TLR7 ligand. In case of LPS and polyl:C stimulation,

an increased expression of A20 was observed in HK

cells after 12 h (Fig. 5), suggesting that at 12 h post-

stimulation, NF-KB or I RF3 activities could be reduced

by de-ubiqutination of TRAF6 or TRAF3 mediated

through the increased A20 (Kawai and Akira, 2009,

Parvatiyar and Harhaj, 2011). In rainbow trout macro-

phages, A20 gene is also up-regulated by LPS between

6 and 24 h of stimulation but not by polyl:C (lliev et al.,

2006). It indicates that other type of immune cells

excluding macrophages in Fugu HK cells might have

induced A20 gene expression by polyl:C. Fugu spleen

cells also revealed induction of A20 gene by polyl:C.

As described above, mammalian A20 is highly

expressed in lymphoid cells and dendritic cells, suggest-

ing that polyl:C could induce A20 gene expression in fish

lymphocytes and dendritic cells. In contrast, in stimula-

tion by imiquimod, an increased expression of A20 gene

was observed in spleen but not in HK cells. Furthermore,

since the expression of TLR7 gene in the carp HK cells

is also induced by imiquimod (Tanekhy et al., 2010), it

seems that the HK cells are stimulated by imiquimod.

These all suggest that the transcriptional regulation of

A20 gene could vary depending on the cell-type.

In mammals, TNF-a and IL-1 f3 proteins also induce

expression of A20 gene as like LPS (Dixit et al., 1990;

Dorronsoro and Lang, 2013) and in other words, A20

transcription is controlled by NF-KB (Dixit et al., 1990).

In Fugu HK cells, following LPS and polyl:C stimulations,

an elevated expression of TNF-a gene was observed at

4 h and expression of A20 gene was up-regulated at 12

h. The timing of A20 gene induction seems that TNF心

induced A20 expression after stimulation. On the other

hand, induction timing of A20 gene in the spleen by

polyl:C and imiquimod was earlier than that in HK cells.

In this study, the expression of A20 gene is increased in

Fugu organs stimulated by the LPS, polyl:C and imiqui-

mod, although there are differences in the expression

patterns between HK and spleen, suggesting that each

different cell types (i.e., lymphocytes and dendritic cells)

in these tissues would show different expression pattern

of A20 mRNAs after stimulation.


Acknowledgements

This research was supported by a Grant-in-Aid for

Scientific Research (C) (No. 23580257) from the Japan

Society for the Promotion of Science (JSPS).

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トラフグ腫瘍壊死因子 a誘導タンパク質遺伝子

(TNFAIP3/ A20)の発現解析

引間順一• 森田美咲• 木下峻介・M.Basu

G. Biswas・河野智哉・酒井正博

腫瘍壊死因子 a (TNF-a)誘導タンパク質 (TNFAIP3/

A20)はTNF-aにより誘導される免疫機構において重要

な脱ユビキチン化酵素であり，哺乳類ではウイルスや細

菌感染時の自然免疫応答を制御するために重要な因子で

ある。本研究でクローン化したトラフグ A20遺伝子の全

長は 6,229bpで9エキソン・ 8イントロンであった。ま

たORFは2,394bpで797アミノ酸残基をコードしてい

た。 7つの zinc-finger(Zf) ドメインを含む領域におけ

るイントロン分断箇所がヒトのものとは異なっていた。

A20遺伝子の発現は，胸腺，頭腎および牌臓において高

いことが確認された。また， LPS, polyl:Cおよびイミキ

モドで刺激した頭腎および牌臓細胞において， TNF-a遺

伝子発現の後で A20遺伝子の発現が誘導された。

魚病研究， 52(1), 15-22 (2017)

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トラフグ腫瘍壊死因子α誘導タンパク質遺伝子 (TNFAIP3/A20)の …