Novel Toll/IL-1 Receptor Homologous Region
Adaptors Act as Negative Regulators in
Amphioxus TLR Signaling
This information is current as
of June 18, 2017.
Jian Peng, Xin Tao, Rui Li, Jingru Hu, Jie Ruan, Ruihua
Wang, Manyi Yang, Rirong Yang, Xiangru Dong, Shangwu
Chen, Anlong Xu and Shaochun Yuan
J Immunol published online 31 August 2015
http://www.jimmunol.org/content/early/2015/08/30/jimmun
ol.1403003
http://www.jimmunol.org/content/suppl/2015/08/30/jimmunol.140300
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Supplementary
Material
Published August 31, 2015, doi:10.4049/jimmunol.1403003
The Journal of Immunology
Novel Toll/IL-1 Receptor Homologous Region Adaptors Act
as Negative Regulators in Amphioxus TLR Signaling
Jian Peng,*,1 Xin Tao,* Rui Li,* Jingru Hu,* Jie Ruan,* Ruihua Wang,*
Manyi Yang,* Rirong Yang,* Xiangru Dong,* Shangwu Chen,* Anlong Xu,*,† and
Shaochun Yuan*
T
oll-like receptors (TLRs) are a class of proteins that
recognize structurally conserved molecules derived from
microbes collectively referred to as pathogen-associated
molecular patterns (1, 2). In mammals, upon ligand stimulation,
the MyD88-dependent and Toll/IL-1R homologous region (TIR)
domain-containing adaptor inducing IFN-b (TRIF)–dependent
pathways can be triggered, leading to activation of NF-kB and the
*State Key Laboratory of Biocontrol, Guangdong Key Laboratory of Pharmaceutical
Functional Genes, College of Life Sciences, Sun Yat-Sen University, Guangzhou
510275, People’s Republic of China; and †Beijing University of Chinese Medicine,
Beijing 100029, People’s Republic of China
1
Current address: Department of Biotechnology, Guizhou Medical University,
Guiyang, Guizhou Province, People’s Republic of China.
Received for publication December 2, 2014. Accepted for publication August 3,
2015.
This work was supported by National Basic Research Program (973) Project
2013CB835303, National Natural Science Foundation of China Projects 31270018,
31470846, and 30730089, and New Star of Pearl River on Science and Technology
of Guangzhou Project 2014J2200017.
The sequences presented in this article have been submitted to the National Center
for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/genbank) under accession numbers KM288437, KM288438, KM288439, and KM288440.
Address correspondence and reprint requests to Dr. Anlong Xu and Dr. Shaochun
Yuan, State Key Laboratory of Biocontrol, Department of Biochemistry, College of
Life Sciences, Sun Yat-Sen University, 135 Xingangxi Road, Guangzhou 510275,
People’s Republic of China. E-mail addresses: [email protected] (A.X.) and
[email protected] (S.Y.)
The online version of this article contains supplemental material.
Abbreviations used in this article: ABIN-1, A20 binding and inhibitor of NF-kB;
ABL2, abelson murine leukemia viral oncogene homolog 2; bbt, Branchiostoma
belcheri tsingtauense; bbtRIP1b, bbt receptor interacting protein 1b; CARD, caspase
recruitment domain; Co-IP, coimmunoprecipitation; ER, endoplasmic reticulum;
EST, expressed sequence tag; FPP, fluorescence protease protection; HEK, human
embryonic kidney; IKK, inhibitor of NF-kB kinase; IRAK, IL-1R–associated kinase;
LTA, lipoteichoic acid; MyD88s, splice variant of MyD88; TICAM, TIR domaincontaining adaptor molecule; TIR, Toll/IL-1R homologous region; TRAF6,
TNFR-associated factor 6; TRAM, TRIF-related adaptor molecule; TRIF, TIR
domain-containing adaptor inducing IFN-b.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403003
induction of type I IFN. The MyD88-dependent pathway is universal for all TLRs except TLR3, whereas the TRIF-dependent
pathway is specifically used by TLR3 and TLR4 (3). When recruited by TLR4, MyD88 requires MyD88 adaptor-like (MAL,
also known as TIRPA), whereas TRIF needs TRIF-related adaptor
molecule (TRAM) as a partner (2, 4). The fifth adaptor SARM
plays an inhibitory role in the TRIF-dependent pathway (5, 6).
As an important component of the innate immune system,
mammalian TLR signaling is tightly regulated to avoid extreme
immune responses and to maintain tissue homeostasis (7). At the
receptor level, the TLR4 homolog, radioprotective 105-kDa protein (RP105, also called CD180) inhibits LPS-TLR4 binding and
impairs LPS-mediated NF-kB activation. Suppression of tumorigenicity 2–related protein also inhibits TLR4 signaling by competing for MyD88 binding (8, 9). Dominant-negative proteins
generated by stimulus-dependent alternative splicing are also
regulators at the adaptor level. For example, an LPS-inducible
splice variant of MyD88, MyD88s, is a dominant negative inhibitor of TLR/IL-1R–induced NF-kB activation (10). The splice
variant IL-1R–associated kinase (IRAK)-1c and IRAK-M are
dominant-negative IRAK forms that play negative regulatory roles
in Toll/IL-1R–induced inflammatory signaling (11, 12). In addition, posttranslation modification is widely used in negative
feedback regulation of TLR signal (13, 14). For instance, activation of the MyD88-dependent pathway leads to K63-linked
polyubiquitination of TNFR-associated factor 6 (TRAF6) and
the subsequent activation of the inhibitor of NF-kB kinase (IKK)
complex (15), whereas the K48-linked polyubiquitination of
TRAF6 results in proteasomal degradation of TRAF6 and inhibition of NF-kB (16). Because ubiquitination is a reversible process, deubiquitination of TRAF6 is another effective feedback
regulation of TLR signaling.
Compared with vertebrates, basal deuterostomes possess greatly
expanded TLR repertoires. For example, the echinoderm sea urchin
has 222 TLRs and up to 26 potential TIR adaptors, whereas the
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Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and
at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor molecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how
these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA,
bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role
in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas
bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the
polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR
domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome,
suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play
nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of
innate immunity at basal chordate. The Journal of Immunology, 2015, 195: 000–000.
2
TIR ADAPTORS NEGATIVELY REGULATE TLR PATHWAY IN AMPHIOXUS
Materials and Methods
Animals and cells
Wild adult Chinese amphioxus Branchiostoma belcheri tsingtauense (bbt)
were obtained from Qingdao, China. Human embryonic kidney (HEK)
293T and Hela cells were grown in DMEM supplemented with 10% FCS
and antibiotics.
Cloning of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD cDNAs
TIR adaptor orthologs were identified in the B. floridae genome. Based on
these sequences, partial sequences of bbtTIRA, bbtTIRB, bbtTIRC, and
bbtTIRD were cloned from bbt intestinal cDNA by specific primer pairs
derived from bfTIRA (gene ID in B. floridae is 78250), bfTIRB (gene ID
in B. floridae is 89683), bfTIRC (gene ID in B. floridae is 89399), and
bfTIRD (gene ID in B. floridae is 69123). Subsequently, 59-RACE and
39-RACE were performed according to the manufacturer’s protocol using
a GeneRACE Kit (Invitrogen) for full-length sequence cloning. These
sequences have been submitted to the National Center for Biotechnology
Information database (http://www.ncbi.nlm.nih.gov/genbank/) and the
accession numbers are KM288437 (bbtTIRA), KM288440 (bbtTIRB),
KM288438 (bbtTIRC), and KM288439 (bbtTIRD), respectively.
Identification and sequence analysis of TIR adaptor-like genes
in Chinese amphioxus (B. belcheri)
The draft genome of B. belcheri and the related analysis tools can be
accessed at: http://mosas.sysu.edu.cn/genome. The following Pfam accession numbers were obtained from http://pfam.sanger.ac.uk/Software/
Pfam: Ankyrin repeat, PF00023; Arm, PF00514; CARD, PF00619;
Death, PF00531; DED, PF01335; Glycos_transf_1, PF00534; HEAT,
PF02985; Helicase_C, PF00271; LRR_1, PF00560; MBT, PF02820; NBARC, PF00931; OAS1_C, PF10421; Pkinase_Tyr, PF07714; Pro_isomerase, PF00160; Ras, PF00071; ResIII, PF04851; RIG-I_C-RD, PF11648;
SAM_1, PF00536; SEFIR, PF08357; TIR, PF01582; TPR_1, PF00515;
TSP_1, PF00090; WD40, PF00400. Domain and gene identification were
mainly performed with the HMMER2.0 plus SMART dataset (http://smart.
embl-heidelberg.de). Previously unidentified protein architectures were
validated by searching the expressed sequence tag (EST) and cDNA evidence from the EST dataset of B. belcheri, as well as the EST dataset and
cDNA sets of B. floridae (Supplemental Table I).
Acute immune challenges of adult amphioxus and real-time
PCR
A total of 15 ml per animal of LPS and lipoteichoic acid (LTA; 1 mg/ml)
mixture in PBS was injected into the amphioxus coelom. The challenged
and unchallenged amphioxus were cultured in separate tanks as described
in our previous study (20). Intestines from five individuals were collected
at 2, 4, 8, 12, 24, 36, 48, and 72 h postinjection as a single sample.
Intestines from five PBS-injected animals (15 ml per animal) were collected concurrently as nonchallenged controls.
For the NF-kB–specific inhibitor helenalin-treated experiments, the
adult amphioxus were separated into three groups and treated with: 1)
DMSO as a negative control, 2) the LPS and LTA (1 mg/ml) mixtures, or
3) helenalin administered before inoculation with the LPS and LTA mixtures (15 ml per animal). The animals were cultured in separate tanks, and
the intestines from five individuals were collected at 3 h postinjection as
a single sample.
The following primer pairs were used: BbtTIRA-F: 59-GGCTATCAGGCTCTACGGACTTC-39, BbtTIRA-R: 59-CGCTTGACTTGCTGTTGCTTGT-39; BbtTIRB-F: 59-TGGTCTACTTCGGTGGCTTGGA-39, BbtTIRB-R:
59-CGGCTCATCGCTCTGTTCATCTTG-39; BbtTIRC-F: 59-GTCATCGGTGTCCAAGTT-39, BbtTIRC-R: 59-TCTCCTCCAGCATGTCAT-39;
BbtTIRD-F: 59-AGCCTTCTACTGGACATACCTGAG-39, BbtTIRD-R: 59-CACGCCACCGACAAGACATATC-39; Bbtb-actin-F: 59-CCCTGTGCTGCTGACTGAG-39, Bbtb-actin-R: 59-ACACGCCATCTCCAGAATCC-39.
Expression plasmids
For the expression of bbtTIRA in HEK293T cells, PCR fragments
encoding for aa 1–160, 161–620, 621–889, 890–1142, and 1–1142 of
bbtTIRA were inserted into pCMV-Flag, pCMV-Myc, and pCMV-HA
(Clontech) and designated bbtTIRA1, bbtTIRA4, bbtTIRA7, bbtTIRA9,
and bbtTIRA-FL, respectively (Fig. 3B). For the expression of bbtTIRC in
HEK293T cells, PCR fragments encoding for aa 1–161, 162–302, and
1–302 of bbtTIRC were inserted into pCMV-Flag, pCMV-Myc, and
pCMV-HA (Clontech) and designated bbtTIRC1, bbtTIRC2, and bbtTIRCFL, respectively (Fig. 4D). For the expression of bbtTIRB and bbtTIRD in
HEK293T cells, PCR fragments encoding for aa 1–493 of bbtTIRB and 1–
429 of bbtTIRD were inserted into pCMV-Flag, pCMV-Myc, and pCMVHA (Clontech) and designated bbtTIRB-FL and bbtTIRD-FL, respectively.
For the study of subcellular localization, full-length bbtMyD88, bbtTICAM,
bbt receptor interacting protein 1b (bbtRIP1b), and bbtTRAFs were
inserted into pEGFP-N1 (Clontech). To determine topologies of membrane
proteins, we inserted full-length bbtTIRD into pEGFP-N1 and inserted
full-length bbtTIRB into pEGFP-C1 (Clontech) (Fig. 2B). PCR fragment
encoding for mCherry was inserted into pcDNA3.1 (Invitrogen). PCR
fragments encoding for aa 1–864 of bbtTICAM fused with 39Flag tag was
inserted into a pcDNA3.0 vector (Invitrogen) and designated bbtTICAMFL. Full-length bbtMyD88, bbtTRAF6, and bbtRIP1b were inserted into
a pcDNA3.0 vector (Invitrogen) fused with 39Flag and 39HA tag. For the
endosomal and mitochondrial markers, the PCR fragment encoding for the
endosomal marker protein CD63 and the mitochondrial marker PHB1
were inserted into pEGFP-N1 vectors fused with GFP-tag, respectively.
For the endoplasmic reticulum (ER), the PCR fragment encoding for the ER
marker ERM was inserted into a pEGFP-N1 vector fused with RFP-tag.
Fluorescence protease protection
Protein localization could be determined by fluorescence protease protection (FPP) assay (24) and passage of the Hela cells into cell chambers
(Lab-Tek chambered cover glass). At 16 h postpassage, cells were transfected with plasmid coding for the bbtTIRB or bbtTIRD tagged with GFP
and soluble fluorescent protein mcherry (Red) expression plasmid. After
20 h posttransfection, cell culture medium was removed and cells were
washed three times for 1 min each in KHM (110 mM potassium acetate,
2 mM MgCl2, 20 mM HEPES, pH 7.2) buffer at 25˚C. Then 20 mM
digitonin (Sigma-Aldrich) in KHM buffer was added to the cells to
permeabilize the plasma membrane. To determine the permeabilization
situation after digitonin application, the red fluorescent of mcherry (Red)
was tested with microscopy. When the red fluorescent disappeared, the
KHM buffer with digitonin was removed and cells were washed quickly
but thoroughly with KHM buffer. Then 4 mM of the protease trypsin (in
KHM buffer) was added directly onto the cells, and images were immediately taken in the fluorescence microscope to record whether GFP
fluorescent signals persist or disappear.
Coimmunoprecipitation (Co-IP), immunofluorescence imaging, transient transfection, and luciferase reporter assay were performed as previously described (21, 25).
Results
Sequences and structure analyses of bbtTIRA, bbtTIRB,
bbtTIRC, and bbtTIRD
Annotation of immune-related molecules in amphioxus B. floridae
has identified .40 TIR adaptors and ongoing domain shuffling
among these adaptors (17). Using another recently completed
genome of amphioxus B. belcheri, we conducted a genomic
survey of the TIR adaptor in B. belcheri and compared with
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cephalochordate amphioxus possesses at least 39 TLRs and .40
TIR adaptors (17). Among these adaptors, amphioxus MyD88,
TRAM-like [also known as amphioxus TIR domain-containing
adaptor molecule (TICAM)], and SARM have been well studied.
Amphioxus MyD88 was shown to mediate the activation of NFkB through its middle and death domains, whereas TICAM was
shown to mediate the most primitive TRIF-dependent activation
of NF-kB (18–20). Amphioxus SARM plays inhibitory roles in
both MyD88- and TICAM-dependent pathways by interacting
with MyD88, TRAF6, and TICAM (19, 21). Recently, ubiquitination
was shown to be essential in regulating the activation of amphioxus NF-kB (22, 23). In addition to these TIR adaptors having
homologies with their vertebrate counterparts, most amphioxus
TIR adaptors exhibit novel protein domain architectures, such as
the TIR domain linked with the kinase domain and the TIR domain linked with the caspase recruitment domain (CARD). To
reveal how these novel TIR adaptors function in amphioxus TLR
signaling, we selected four representatives for functional analyses.
The study will not only aid in defining the relationship among
amphioxus TIR adaptors, but also will provide further knowledge
on the regulation of TLR signaling in vertebrates.
The Journal of Immunology
3
B. floridae. We found a similar number of TIR adaptors in the two
species. In addition to several TIR adaptors that showed homol-
ogies with MyD88, TICAM, and SARM, some adaptors were
shown to be similar to orphan vertebrate TIR genes, and most of
FIGURE 2. Inhibitory efforts of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD on the NF-kB activation mediated by bbtMyD88 or bbtTICAM and their
distributions. (A) bbtTIRB and bbtTIRD colocalized with the endosome marker CD63. Immunofluorescence microscopy images taken from HeLa cells
cotransfected with GFP-fused CD63, together with HA-tagged pCMV-bbtTIRB, bbtTIRD, and stained with anti-HA and Alexa Fluor 532 Ab. (B) The FPP
results showed that bbtTIRB anchored to the endosome with its TIR domain degraded by protease trypsin, whereas the TIR domain of bbtTIRD was not
affected. The HeLa cells were transfected with mCherry (Red) inserted into pcDNA3.1 and bbtTIRB inserted into pEGFP-C1 or bbtTIRD inserted into
pEGFP-N1. Fluorescence microscopy images were taken before or after digitonin and trypsin application. Fluorescence microscopy images are representative of at least three independent experiments in which .80% of the Hela cells showed similar patterns. (C–E) HEK293T cells were cotransfected with
NF-kB transcriptional luciferase reporter constructs, together with bbtMyD88 or bbtTICAM and (increasing amounts of) bbtTIRA, bbtTIRB, and bbtTIRC
vectors as indicated. All reporter assays performed in HEK293T cells are shown as mean 6 SD of three samples per treatment, and values were considered
significant when p , 0.05. Results were confirmed by at least three separate experiments. *p , 0.05.
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FIGURE 1. Genomic sequence analysis of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD and their domain topology. (A–D) The TIR domain, STYKc
domain, and transmembrane regions were predicted by the SMART Web site (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1). The
TRAF6-binding motif [PxExx(Ac/Ar): Ar for aromatic residues, Ac for acidic residues, x for any residue]. Sequence analyses indicated that all bbtTIRA,
bbtTIRB, bbtTIRC, and bbtTIRD possess several conserved kB-binding motifs in the promoter region, and the reporter assays confirmed that the region
containing some kB-binding sites upstream of the ATG of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD is essential for the binding to bbtRel. For the reporter
assays, the increasing amounts of pGL3 basic vectors containing 2-kb genomic sequences upstream of the ATG of bbtTIRA, bbtTIRB, bbtTIRC, and
bbtTIRD were transfected into HEK293T cells in the presence of 50 ng HsP65 or bbtRel expression vector. Hsp65 indicates Homo sapiens p65. Data are
shown as mean 6 SD of three samples per treatment, and values were considered significant when p , 0.05. Results were confirmed by at least three
separate experiments. *p , 0.05, **p , 0.01.
4
TIR ADAPTORS NEGATIVELY REGULATE TLR PATHWAY IN AMPHIOXUS
conserved protein structures: the TIR domain and the STYKc
domain. The STYKc domain of bbtTIRA showed 30% amino
acid identity with mammalian Tyrosine-protein kinase abelson
murine leukemia viral oncogene homolog 2 (ABL2) (Fig. 1A,
Supplemental Fig. 1A). BbtTIRC encodes a polypeptide of 302 aa
with a conserved orphan TIR domain and two TRAF6-binding
motifs (PxExx) in the C terminus (Fig. 1C) (26, 27). Bedsides,
bbtTIRB encodes a polypeptide of 493 aa, and bbtTIRD encodes
a polypeptide of 430 aa. Although both bbtTIRB and bbtTIRD
contain two transmembrane regions and a TIR domain, the TIR
domain of bbtTIRB is located at the N terminus, whereas that of
bbtTIRD is located at the C terminus (Fig. 1B, 1D). Further sequence alignment showed that the characteristic sequence Box1,
Box2, Box3, and the BB loop are well conserved in the TIR
domains of these four adaptors (Supplemental Fig. 1B).
bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD are the target genes
of NF-kB
RT-PCR was performed to determine the tissue distribution of
bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, and results showed
that transcripts of all the tested genes are abundant in amphioxus
FIGURE 3. bbtTIRA interacted with bbtTICAM and bbtRIP1b. (A) The full-length of bbtTIRA colocalized with bbtTICAM in Hela cells. (B) Truncated
and site-directed mutants of bbtTIRA inserted into pCMV-Myc vector and named as indicated. Lys635 was substituted for Arg635. (C) bbtTIRA1, bbtTIRA4,
and bbtTIRA7, but not bbtTIRA9, depressed the activation of the NF-kB by bbtTICAM. (D) Co-IP results showed that bbtTIRA1, bbtTIRA4, and
bbtTIRA7, but not bbtTIRA9, could interact directly with bbtTICAM. (E) bbtTIRA inhibited NF-kB activation mediated by bbtRIP1b in HEK293T cells.
(F) Confocal microscopy showed that bbtTIRA colocalized with bbtRIP1b. (G) The Co-IP results showed that bbtTIRA1, bbtTIRA4, and bbtTIRA7, but not
bbtTIRA9, interacted directly with bbtRIP1b. (H) Compared with wild type bbtTIRA, the bbtTIRA-mutant could depress the activation of the NF-kB by
bbtTICAM in a low level. Immunofluorescence microscopy images are representative of at least three independent experiments, in which .80% of the Hela
cells showed similar staining patterns. All reporter assays performed in HEK293T cells are shown as mean 6 SD of three samples per treatment, and values
were considered significant when p , 0.05. Results were confirmed by at least three separate experiments. *p , 0.05.
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them contain novel domain recombination, such as TIR+PkinaseTyr, CARD+TIR, and death effector domain (DED)+CARD+TIR
(Supplemental Table I). To reveal how the novel TIR adaptors
function in amphioxus TLR signaling, we chose eight TIR novel
adaptors (CARD-TIR, B. floridae gene ID: 89399, 96757; TIRHEAT, B. floridae gene ID: 93247; TIR-Pkinase_Tyr, B. floridae
gene ID: 78250; TPR-TIR, B. floridae gene ID: 63858 and 68270;
TIR, B. floridae gene ID: 69123 and 89683) with the following
two characteristics for further cloning. First, they should have
novel domain architectures. Second, they should exist in both
B. belcheri and B. floridae simultaneously and not experience gene
duplication. Adaptors with these two features should be more stabilized and have more specialized functions.
Unfortunately, just four of them with differential characteristics
could be isolated from the B. belcheri tsingtauense intestine cDNA
library and designated as bbtTIRA (corresponding to B. floridae
gene ID: 78250), bbtTIRB (B. floridae gene ID: 89683), bbtTIRC
(B. floridae gene ID: 89399), and bbtTIRD (B. floridae gene ID:
69123), respectively, suggesting that the other four TIR adaptors
may be pseudogenes or genes with inducible transcription.
BbtTIRA encodes a polypeptide of 1143 aa with two highly
The Journal of Immunology
To reveal whether bbtTIRA, bbtTIRB, and bbtTIRC are critical
for the activation of NF-kB or other immune-related transcription
factors, we conducted luciferase assays. Because of the lack of
amphioxus cell lines at present, mammal cell lines were chosen.
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digestive system (Supplemental Fig. 1C), which is considered to
be the first defense line of amphioxus (25, 28). To further study
the immunological significance of bbtTIRA, bbtTIRB, bbtTIRC,
and bbtTIRD in adults, we performed real-time PCR analyses, and
results showed the transcriptions of these TIR adaptors in amphioxus intestines to be upregulated after challenge with LPS and
LTA mixture (Supplemental Fig. 1D). In our previous study, we
have demonstrated that bbtRel is the ancestor of the vertebrate
class II NF-kB proteins (25) and can interact with kB motif.
Moreover, the interaction between kB motif and bbtRel can be
blocked by the NF-kB–specific inhibitor helenalin (25, 29). In this
study, we further showed that the upregulation of bbtTIRA,
bbtTIRB, bbtTIRC, and bbtTIRD were inhibited in adult amphioxus, which are treated with helenalin before challenge with LPS
and LTA mixture (Supplemental Fig. 1E). These results suggested
that the transcription of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD
are tightly regulated by amphioxus NF-kB.
To further investigate whether bbtTIRA, bbtTIRB, bbtTIRC,
and bbtTIRD are classical NF-kB target genes, we obtained the
2-kb genomic sequences upstream of the ATG of bbtTIRA,
bbtTIRB, bbtTIRC, and bbtTIRD and subjected them to the
Transcription Element Search System prediction program (www.
cbil.upenn.edu/cgi-bin/tess/tess) to determine whether these
regions contain conserved NF-kB–binding motifs. All promoter
regions of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD were
found to contain several conserved kB-binding sites (Fig. 1).
Then the sequence including kB-binding sites of bbtTIRA,
bbtTIRB, bbtTIRC, and bbtTIRD was inserted into pGL3 basic
reporter vector and cotransfected with bbtRel or human p65 expression plasmid in HEK293T to reveal whether p65 can recognize these promoter sequences. Reporter assays showed that
hsp65 or bbtRel can recognize the sequence including kB-binding
sites of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, suggesting
that these TIR adaptors are classical targets of amphioxus NF-kB
signaling (Fig. 1).
5
bbtTIRA inhibits the bbtTICAM-dependent pathway, whereas
bbtTIRB and bbtTIRC affect the bbtMyD88-dependent pathway
Because bbtTIRB and bbtTIRD are transmembrane proteins with
their TIR domain locating at a distinct terminus, to investigate
whether they have a specific subcellular location, we cotransfected them with three organelle markers. Results showed
bbtTIRB and bbtTIRD to be colocalized with endosomes, but not
with mitochondrial and ER when overexpressed in Hela cells
(Fig. 2A, Supplemental Fig. 2A, 2B). To investigate whether TIR
domains of bbtTIRB and bbtTIRD faced to the cytosol, or
enclosed in the endosome, we used further FPP experiment. In
the FPP procedures, the bbtTIRB or bbtTIRD expressed cells
were first treated with cholesterol-binding digitonin and then
with trypsin. Because the plasma membrane contains more
cholesterol than intracellular organelles, the low concentration of
cholesterol-binding digitonin can increase the permeability of
cell membrane and allow trypsin get into cytosol, but not intracellular organelles. Results showed that, when treated with
trypsin, the fluorescence of GFP disappeared in GFP-tagged
bbtTIRB-expressed cells, but was retained in GFP-tagged
bbtTIRD-expressed cells. These results suggested that bbtTIRB
anchored to the endosome with its TIR domain faced to the cytosol, whereas the TIR domain of bbtTIRD enclosed in the
endosome or ER. When the TIR domain enclosed in the endosome or ER, it may have no chance to interact with other
TIR-containing molecules, suggesting that bbtTIRD may be a
redundant protein without signaling transduction activities in
amphioxus TLR signaling (Fig. 2B).
FIGURE 4. bbtTIRB and bbtTIRC interacted with bbtMyD88 and
inhibited its transcription stimulatory activity. (A) The full length of
bbtTIRB and bbtTIRC colocalized with bbtMyD88. Immunofluorescence
microscopy images conducted in Hela cells are representative of at least
three independent experiments, in which .80% of the cells showed similar
staining patterns. (B and C) The Co-IP results showed that bbtTIRB and
bbtTIRC interacted directly with bbtMyD88, but not with bbtTICAM. (D)
Truncated mutants used in this study. (E) bbtTIRC2 inhibited NF-kB activation to the same extent as seen with the full length, but bbtTIRC1 had
no effect. (F) The Co-IP results showed that the mutant bbtTIRC1 and
bbtTIRC2 interacted directly with bbtMyD88. All reporter assays performed in HEK293T cells, and data are shown as mean 6 SD of three
samples per treatment. Values were considered significant when p , 0.05.
Results were confirmed by at least three separate experiments. *p , 0.05.
6
TIR ADAPTORS NEGATIVELY REGULATE TLR PATHWAY IN AMPHIOXUS
Results showed none to have an effect on the induction of type I
IFN or on the activation of AP1 and NF-kB in HEK293T cells
(Supplemental Fig. 2C–E). Because MyD88-dependent and
TICAM-dependent pathways have been demonstrated in amphioxus (18, 19), we further used luciferase assays to test whether
bbtTIRA, bbtTIRB, and bbtTIRC are involved in amphioxus
MyD88- and TICAM-dependent signaling. Results showed that
bbtTIRA specifically inhibited the bbtTICAM-dependent activation of NF-kB (Fig. 2C), whereas bbtTIRB and bbtTIRC could
attenuate the bbtMyD88-mediated activation of NF-kB in a dosedependent manner (Fig. 2D, 2E).
BbtTIRA interacts with bbtTICAM and bbtRIP1b to inhibit the
bbtTICAM-mediated NF-kB activation
To further assess how bbtTIRA affects the bbtTICAM-mediated
activation of NF-kB, we first performed confocal microscopy
to show the colocalization of bbtTIRA with bbtTICAM, but not
with bbtMyD88 in Hela cells (Fig. 3A, Supplemental Fig. 2F).
Then four truncated mutants of bbtTIRA were constructed (Fig.
3B) and luciferase assays showed that the TIR domain, middle
region, and STYKc domains of bbtTIRA attenuated the activation of NF-kB mediated by bbtTICAM (Fig. 3C). Co-IP assays
confirmed that TIR domain, middle region, and STYKc domain
of bbtTIRA, but not the C-terminal region, can interact with
bbtTICAM (Fig. 3D). Because our previous study demonstrated
that the association of bbtRIP1b with bbtTICAM is a crucial
step in the bbtTICAM-mediated activation of NF-kB (19), to
investigate whether bbtTIRA would interrupt the interaction
between bbtTICAM and bbtRIP1b, we performed luciferase
assays. Results showed that bbtTIRA attenuated the activation
of NF-kB mediated by bbtRIP1b (Fig. 3E). Signal analysis by
confocal microscopy indicated that bbtRIP1b colocalized with
bbtTIRA when overexpressed in Hela cells (Fig. 3F). Furthermore, Co-IP assays showed that bbtTIRA could interact with
bbtRIP1b through its N-terminal, middle region, and STYKc
domain (Fig. 3G).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 5. bbtTIRC inhibited the NF-kB activation induced by bbtTRAF6. (A) HEK293T cells were cotransfected with NF-kB transcriptional luciferase reporter constructs, together with bbtTRAF6 and increasing amounts of bbtTIRC vectors as indicated. (B) The full length of bbtTIRC colocalized with
bbtTRAF6. Immunofluorescence microscopy images are representative of at least three independent experiments, in which .80% of the Hela cells showed
similar staining patterns. (C) The Co-IP results showed bbtTIRC to interact directly with bbtTRAF6. (D) bbtTIRC2 inhibited NF-kB activation to the same
extent as seen with the full length, but mutant bbtTIRC1 had no effect. (E) Co-IP results showed that bbtTIRC2 interacted directly with bbtTRAF6. (F) Sitedirected mutants used. The TRAF6-binding motif (PxExx(Ac/Ar): Ar for aromatic residues, Ac for acidic residues, x for any residue). (G) bbtTIRC-mutant
did not interact with bbtTRAF6. (H) Compared with the wild-type bbtTIRC, activity of bbtTIRC-mutant was attenuated to 40%. (I) Compared with the
wild-type bbtTIRC, bbtTIRC mutant almost did not depress the polyubiquitination of bbtTRAF6. All reporter assays performed in HEK293T cells, and data
are shown as mean 6 SD of three samples per treatment. Values were considered significant when p , 0.05. Results were confirmed by at least three
separate experiments. *p , 0.05.
The Journal of Immunology
Sequence analysis of the STYKc domain of bbtTIRA and
mammalian ABL2 showed ∼30% identity (Supplemental Fig. 1A),
leading to the question of whether this domain can mediate tyrosine phosphorylation to affect NF-kB activation. The inactive
mutation of Abl2-like kinase into c-Abl1, in which the Lys290
critical for ATP binding was mutated to arginine, resulted in
a complete loss of kinase effect (30). Thus, site-directed mutagenesis of bbtTIRA was conducted by replacing the conserved
residues 635K within the STYKc domain with 635R (Fig. 3B).
Compared with wild type bbtTIRA, reporter assays showed that
the K635R mutant can suppress the NF-kB activation mediated by
bbtTICAM in a low level (Fig. 3H). Thus, it appears that the TIR
domain, middle region, and STYKc domain of bbtTIRA participated in inhibiting the activation of NF-kB in the MyD88-independent signal pathway.
BbtTIRC interacts with bbtMyD88 and bbtTRAF6 and
depresses polyubiquitination of bbtTRAF6
TRAF6, but not bbt NF-kB essential modulator and human NF-kB
essential modulator (Supplemental Fig. 3A–D). Compared with
wild-type bbtTIRC, the ability of the bbtTIRC-mutant to depress
polyubiquitination of TRAF6 was also attenuated (Fig. 5I). Thus,
we suggest that bbtTIRC inhibited the TRAF6-mediated signaling
by depressing the polyubiquitination of TRAF6 (Fig. 6).
Discussion
Rudiment of amphioxus TLR signaling
Basal deuterostomes possess many more TIR adaptors than the
five seen in vertebrates. For example, the sea urchin possesses 26
potential TIR adaptors, including 4 MyD88-like, 15 SARM1-like,
and 7 orphan TIR genes (33). In comparison of the genomes between B. floridae and B. belcheri, we found that the basal chordate amphioxus possesses .40 TIR adaptor proteins, including
homologs of MyD88, TICAM, and SARM; adaptors similar to
orphan vertebrate TIR genes; and adaptors with novel domain
recombination (17). The identification of both TLR and MyD88
in various evolutionary stages, and the functional interaction
of amphioxus TLR with MyD88, suggests that the MyD88dependent pathway is conserved and is crucial for invertebrate
TLR signaling (18, 33–35). Because no homolog of vertebrate TICAM1 and TICAM2 was identified in invertebrates, the
MyD88-independent pathway was long believed to be a vertebrate
FIGURE 6. Novel TIR adaptors play nonredundant roles in both
MyD88-dependent and -independent signal pathways in amphioxus.
bbtTIRA shows a unique inhibitory role in amphioxus MyD88-independent pathway by interacting with bbtTICAM and bbtRIP1b, whereas
bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway
by interacting with bbtMyD88 and depressing the polyubiquitination of
bbtTRAF6. At the same time, their expressions are controlled by NF-kB,
causing an effective feedback regulation of amphioxus NF-kB signaling.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Because bbtTIRB and bbtTIRC were shown to attenuate the
NF-kB activation mediated by bbtMyD88, confocal microscopy
was performed. Results showed both bbtTIRB and bbtTIRC to
colocalize with bbtMyD88, but not with bbtTICAM when overexpressed in Hela cells (Fig. 4A, Supplemental Fig. 2G). Since
bbtTIRB contains two transmembrane regions, it is easily
gathered and shows significant organelle location (Fig. 2A,
Supplemental Fig. 2A, 2B). However, bbtTIRC may be widely
distributed in the cytosol. Thus, when bbtMyD88 was co-transfected with bbtTIRB or bbtTIRC, it showed distinct distribution.
Co-IP assays further confirmed the direct interaction of bbtMyD88
with both bbtTIRB and bbtTIRC (Fig. 4B, 4C). To investigate
which domain of bbtTIRC is responsible for its function, two
truncated mutants, bbtTIRC1 (the N-terminal) and bbtTIRC2 (the
C-terminal TIR domain with TRAF6-binding motifs), were constructed for further analysis (Fig. 4D). Reporter and Co-IP assays
showed that although bbtTIRC1 and bbtTIRC2 were both shown
to interact with bbtMyD88, only bbtTIRC2 inhibited NF-kB activation mediated by bbtMyD88 in a dose-dependent manner (Fig.
4E, 4F). Because recruitment of TRAF6 to MyD88 complexes is
a key step in the activation of NF-kB (31, 32), we performed
reporter assays to investigate the effect of bbtTIRC on bbtTRAF6
activity. Results showed that bbtTIRC inhibits TRAF6-mediated
NF-kB activation in a dose-dependent manner (Fig. 5A). Moreover, bbtTIRC can interact and colocalize with bbtTRAF6 (Fig.
5B, 5C). As mentioned earlier, the analysis of the bbtTIRC sequence revealed two TRAF6-binding motifs (PxExx) in the C
terminus. To verify whether the activity of bbtTIRC depends
on these two motifs, two truncated mutants (bbtTIRC1 and
bbtTIRC2) were coexpressed with bbtTRAF6. Results showed
that only bbtTIRC2, which contains two TRAF6 binding motifs,
can interact with bbtTRAF6 and inhibit the TRAF6-mediated
activation of NF-kB in a dose-dependent manner (Fig. 5D, 5E).
To further reveal whether the inhibitory activity of bbtTIRC is due
to these two TRAF6 binding motifs, we conducted site-directed
mutagenesis by replacing the conserved residues 162PVE164 and
257PPE259 into 162AVA164 and 257APA259 (Fig. 5F). In contrast with wild-type bbtTIRC, the bbtTIRC-mutant did not interact
with bbtTRAF6 (Fig. 5G), and the capacity of the bbtTIRC-mutant
to attenuate the activation of NF-kB mediated by bbtTRAF6 was
reduced to 40% (Fig. 5H).
Because K63-linked autopolyubiquitination of TRAF6 is another crucial step in the activation of NF-kB, to investigate whether
bbtTIRC could affect the polyubiquitination of bbtTRAF6, we
performed ubiquitination assays. Results showed that bbtTIRC
can depress the polyubiquitination of bbtTRAF6 and human
7
8
TIR ADAPTORS NEGATIVELY REGULATE TLR PATHWAY IN AMPHIOXUS
Novel TIR adaptors negatively regulated amphioxus TLR
signaling at distinct aspects
In addition to the regulation at the adaptor level, posttranslation
modification of the key molecules in TLR signaling is a key
mechanism of feedback regulation, including ubiquitination and
phosphatation. K63 autopolyubiquitination of TRAF6 in conjunction with the E2 enzyme Uev1a/Ubc13 is essential to activate
NF-kB signaling, whereas the deubiquitinases A20 and CYLD
(38, 39), which remove the K63-linked polyubiquitin chains from
TRAF6, can suppress such activation (40). In addition, A20
binding and inhibitor of NF-kB (ABIN-1) and ABIN-3 contain
a ubiquitin binding domain and may contribute to the termination
of the NF-kB response downstream of TRAF6 by recruiting A20
to ubiquitylated IKKg (41). Our previous study showed that
bbtABIN2 can compete with bbtTRAF6 for the K63-linked
ubiquitin chains to negatively regulate NF-kB activation. In this
study, we observed that bbtTIRC binds human TRAF6 and
bbtTRAF6 to depress their polyubiquitination. In vertebrates,
phosphatation of a series of kinases is another crucial step in the
release and subsequent translocation of NF-kB into the nucleus. In
this study, when the 635K within the STYKc domain of bbtTIRA
was replaced with 635R, the suppression of bbtTICAM-mediated
activation of NF-kB was eliminated, indicating that the STYKc
domain of bbtTIRA is important for the regulation of NF-kB.
Studies in vertebrates have shown that Abl1, an Abl2-like tyrosine
kinase, can mediate the phosphorylation of inhibitor of NF-kB a
at Tyr305 and increase its stability (42), resulting in the inhibition
of NF-kB. Because the STYKc domain in bbtTIRA is homologous
with the tyrosine kinase of Abl2, further characterization of the
relationships of bbtTIRA with inhibitor of NF-kB a or other
substrates should help to define the distinct functions of bbtTIRA
in anti-inflammation.
In conclusion, to our knowledge, this study provides the first
evidence that the expanded TIR adaptors in basal chordate play
nonredundant roles in the activation of NF-kB, adding a further
layer of complexity to amphioxus innate immunity, not only with
respect to the diversity of receptor recognition, but also in the
cytoplasmic regulation (Fig. 6).
Disclosures
The authors have no financial conflicts of interest.
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9