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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/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
18 J. Hikima, M. Morita, S. Kinoshita, M. Basu, G. Biswas, T. Kono and M. Sakai
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
20 J. Hikima, M. Morita, S. Kinoshita, M. Basu, G. Biswas, T. Kono and M. Sakai
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
I I 0 05
Fig. 4. Hikima et al., submitted.
ぐ炉
t
ペ
ぐふ
・*ふ‘coooeo
令◇‘
5
>
7
、ふ9
‘
0ぐら
0
参‘9
令ぐト
゜e
令> 0
、◇ 4̀
§、ふ
ペ◇`
*ぐ
令ぶ
岳怨
8
7
6
5
4
3
2
1
0
7
s1a > a1 VNtJW a >lle-aa
Fig. 5. Hikima et al., submitted.
(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
Fig. 6. Hikima et al., submitted.
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.
22 J. Hikima, M. Morita, S. Kinoshita, M. Basu, G. Biswas, T. Kono and M. Sakai
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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