The Archaic Roles of the Amphioxus NF-κB/I
κB Complex in Innate Immune Responses
This information is current as
of June 15, 2017.
Shaochun Yuan, Jie Zhang, Lingling Zhang, Ling Huang,
Jian Peng, Shengfeng Huang, Shangwu Chen and Anlong
Xu
J Immunol 2013; 191:1220-1230; Prepublished online 1 July
2013;
doi: 10.4049/jimmunol.1203527
http://www.jimmunol.org/content/191/3/1220
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Supplementary
Material
The Journal of Immunology
The Archaic Roles of the Amphioxus NF-kB/IkB Complex in
Innate Immune Responses
Shaochun Yuan,*,1 Jie Zhang,*,1 Lingling Zhang,* Ling Huang,* Jian Peng,*
Shengfeng Huang,* Shangwu Chen,* and Anlong Xu*,†
uclear factor kB belongs to a family of inducible dimeric
transcription factors that activate the expression of genes
involved in normal development, the immune response,
inflammation, and apoptosis (1). To date, NF-kB homologs have
been found in species ranging from single-cell organisms to
mammals, including coral, sea anemone, hydra, horseshoe crab,
fruit fly, zebrafish, Xenopus laevis, and human (reviewed in Ref. 2).
However, the functional mechanisms of the NF-kB homologs are
not consistent between invertebrates and vertebrates. In Drosophila,
three NF-kB molecules (Dorsal, Dif, and Relish) and an IkB homolog (Cactus) are responsible for regulating several biological
roles (3). Dorsal and Dif are involved in the Drosophila Toll signaling pathway, which recognizes Gram-positive bacteria and fungi,
N
*Department of Biochemistry, College of Life Sciences, Sun Yat-Sen (Zhongshan)
University, Guangzhou 510275, People’s Republic of China; and †Center of Scientific Research, Beijing University of Chinese Medicine, Beijing 100029, People’s
Republic of China
1
S.Y. and J.Z. contributed equally to this study.
Received for publication January 2, 2013. Accepted for publication May 24, 2013.
This work was supported by Project 2011CB946101 of the National Basic Research
Program (973), Project 2012AA092281 of the State High-Tech Development Project
(863), and Key Project 30730089 and Project 31270018 of the National Nature
Science Foundation of China.
The data sequences presented in this article have been submitted to the National
Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/) under
accession numbers KF006939, KF006940, and KF006941.
Address correspondence and reprint requests to Dr. Anlong Xu, College of Life
Sciences, Sun Yat-Sen University, 135 Xingangxi Road, Guangzhou, Guangdong
510275, People’s Republic of China. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: bbtRHD, recombinant bbtRel RHD; co-IP, coimmunoprecipitation; DD, death domain; GRR, glycine-rich region; NLS, nuclear
localization signal; RHD, Rel homology domain; TAD, transcription-activating
domain.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203527
and in the regulation of normal dorsal-ventral patterning during
Drosophila embryogenesis (4, 5). The third Drosophila NF-kB–
related protein, Relish, is reported to be involved in the immune
deficiency (IMD) pathway, which recognizes Gram-negative bacteria (6). However, owing to gene duplication, the mammalian NFkB family of proteins comprises five members (7). The p65, RelB,
and c-Rel proteins constitute the class II proteins, which positively
regulate target gene expression through the transcription-activating
domain (TAD). The class I proteins, p100 and p105, are precursors
of the mature forms, p50 and p52, which lack TADs and therefore
form heterodimers with TAD-containing family members to alter
their specificities for the kB site (1, 8). Although studies of geneknockout mice indicate that a lack of the p65 subunit leads to lethal
liver degeneration in embryos, roles for NF-kB proteins in dorsalventral patterning in mammalian embryos have not been found (9,
10). In contrast, mammalian RelB and c-Rel do play key roles in the
development of lymphoid organs, chiefly through response to
stimuli in lymphoid cells (9, 11–13).
Regardless of functional differences, the endoproteolytic mechanisms of class I members also differ between flies and humans. The
signal-induced endoproteolysis of Drosophila Relish requires the
activity of several gene products, including the IkB kinase complex
and the caspase 8 homolog Dredd (14). Activated Dredd cleaves off
an inhibitory C-terminal ankyrin repeat (ANK) domain from Relish, thereby allowing the translocation of the N-terminal portion to
the nucleus, where it induces the expression of AMP (antimicrobial
peptide) genes (15–18). In contrast to Relish, the proteasomemediated processing of p105 and p100 requires a 23-aa glycine-rich
region (GRR) (19–21). The GRR-dependent endoproteolytic
cleavage does not require specific downstream sequences, whereas
Relish requires a downstream sequence in the C-terminal for processing (17, 21).
Moreover, in contrast to Drosophila, which has only one IkB
protein, the mammalian IkB family commonly comprises IkBa,
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NF-kB transcription factors play important roles in immune responses and the development of the immune system. Many aspects
of NF-kB signaling differ significantly among distinct species, although many similarities in signaling exist in flies and humans.
Thus, to understand the functional refinement of the NF-kB cascade from invertebrates to vertebrates, the Rel and NF-kB
proteins, identified as bbtRel and bbtp105, were characterized in a basal chordate amphioxus. Consistent with the sequence
similarities, bbtRel was found to interact with a mammalian kB response element, to move into the nucleus when activated, and to
be inhibited by the NF-kB–specific inhibitor helenalin. Similar to the other class I members, bbtp105 could be cleaved into the
mature form p58. Such endoproteolysis depends on the GRR sequence and requires both protease degradation and caspase 8
cleavage. Furthermore, we found that bbtIkB and the unprocessed bbtp105 can inhibit the transcriptional activity of bbtRel,
whereas bbtp58 forms homodimers or heterodimers with bbtRel to create a mature NF-kB complex. Finally, we found that the
survival rate and the expression of bbtIkB and TNF-a–like genes were decreased when adult amphioxus were treated with
helanalin before immune challenge, suggesting the archaic roles for NF-kB signaling in innate immune responses in a basal
chordate. The presence of the NF-kB–IkB cascade in amphioxus indicates that it is a significant feature linking invertebrates to
vertebrates and is refined in vertebrates through the expansion and divergence of genes involved in the cascade. The Journal of
Immunology, 2013, 191: 1220–1230.
The Journal of Immunology
IkBb, IkBε, and Bcl-3, which exhibit structural and biochemical
similarities but play unique and nonredundant roles in regulating
NF-kB activation (1). For example, IkBa contains a functional
nuclear export signal, whereas IkBb does not, which may result in
differences in their subcellular locations and functions (22). IkBa
exhibits a rapid response to stimuli and is quickly resynthesized to
regulate transient NF-kB activation, whereas IkBb is less sensitive
to stimulus-induced degradation (22). Therefore, the roles of NFkB signaling in development, the endoproteolytic mechanisms,
and transcriptional regulation differ between flies and humans.
Because amphioxus occupies the crucial evolutionary position
linking invertebrates to vertebrates, characterization of the NFkB–directed transcription process in this basal chordate will help
to define the endoproteolytic mechanism of the class I NF-kB
subgroup in the transition from invertebrates to vertebrates as well
as demonstrate how NF-kB and IkB have coevolved to participate
in embryogenesis and immunity.
Materials and Methods
Adult Chinese amphioxus Branchiostoma belcheri tsingtauense were obtained from Qingdao, China. HEK293T (human embryonic kidney) and
HeLa cells were grown in DMEM supplemented with 10% FCS and
antibiotics. The following inhibitors were used: helenalin (Alexis Biochemicals), Bay 11-7082 (Enzo Life Sciences), evodiamine (Enzo Life
Sciences), and MG132 (Sigma-Aldrich).
Cloning of the bbtRel, bbtp105, and bbtIkB cDNAs
Initially, we examined the gene family that contained Rel homology
domains (RHDs) and identified two partial sequences in the Branchiostoma
floridae genome. Based on these two sequences, gene-specific primers
were designed, and partial bbtRel and bbtp105 sequences (bbtRel and
bbtp105 from B. belcheri tsingtauense) were cloned from Chinese amphioxus intestinal cDNA. Next, 59-RACE and 39-RACE were performed
using the GeneRACE Kit (Invitrogen), in accordance with the manufacturer’s protocol, for full-length sequence cloning. The bbtIkB cDNA
was identified from the amphioxus neurula stage embryo cDNA library.
National Center for Biotechnology Information accession numbers
KF006939, KF006940, and KF006941 are assigned for bbtRel, bbtp105,
and bbtIkB, respectively (http://www.ncbi.nlm.nih.gov/).
Plasmid construction
For the expression of bbtRel, bbtp105, bbtIkB, and their truncated mutants
in HEK293T cells, PCR fragments encoding amino acids 1-755, 1-350,
351-452, 453-755, and 1-170+453-755 of bbtRel; 1-1024, 1-250, 1-581,
251-580, 581-792, 793-1024, and 1-580+793-1024 of bbtp105; and 1-350
of bbtIkB were fused with the Flag or HA tag, and inserted into the expression plasmid pcDNA3.1 (Invitrogen)(Figs. 3A, Supplemental Fig. 2A).
For the study of subcellular localization, full-length bbtRel, bbtp105,
bbtIkB, and the indicated fragments described above were inserted into
pEGFP-N1 (Clontech).
cell suspension was subjected to ultrasonication for 5 periods, each consisting of 90 cycles of 5 s at 5-s intervals. The final product was centrifuged at 12,000 3 g for 30 min, and the supernatant was harvested and
filtered. The filtered supernatant was applied to an IMAC (Sepharose Fast
Flow; GE Healthcare) column that was chelated with Ni2+ and equilibrated
with solution A. The column was rinsed with solution A at a flow rate of
2 ml/min to baseline level. Solution B (20 mM Tris-HCl, pH 8.0; 150 mM
NaCl; and 100 mM imidazole) was used to wash away the non–specifically
bound proteins. The target protein was rinsed with solution C (20 mM TrisHCl, pH 8.0; 150 mM NaCl; and 200 mM imidazole). The fraction corresponding to the protein peak was collected and applied to a hydrophobic
column (G25; GE Healthcare). The column was pretreated with solution D
(50 mM NH4HCO3) and was washed and used to establish the baseline
value. The corresponding protein fractions were collected and lyophilized
for further use (Supplemental Fig. 3A). A New Zealand rabbit was immunized with 500 mg purified bbtRel-RHD protein in combination with
Freund’s adjuvant (Sigma-Aldrich). For the booster immunization, 200 mg
recombinant proteins was mixed with an equal volume of Freund’s incomplete adjuvant and injected s.c. into the rabbit’s back. A booster immunization was performed 30 d after the first immunization, with two
further injections administered at intervals of 14 d. Whole-blood antiserum
was obtained by cardiac puncture. Anti-RHD poly-Ab was purified and
determined by Western blots (Supplemental Fig. 2F).
Nuclear extraction and EMSA
A biotin-labeled 22-bp duplex bearing the NF-kB binding sequence was
synthesized by Invitrogen [NF-kB wt (sense): 59-AGT TGA GGG GAC
TTT CC CAG GC-39; NF-kB wt (anti-sense): 59-GCC TGG GAA AGT
CCC CTC AAC T-39; NF-kB mutant (sense): 59-AGT TGA GGC GAC
TTT CCC AGG C-39; and NF-kB mutant (anti-sense): 59-GCC TGG GAA
AGT CGC CTC AAC T-39]. The biotinylated complementary oligonucleotide pairs were annealed to generate double-stranded and biotinlabeled probes (10 pmol/ml) by combination in buffer (10 mM Tris and 1
mM EDTA) at 95˚C for 5 min, followed by gradual cooling to room
temperature. The unlabeled complementary oligonucleotide pairs were
annealed to generate double-stranded competitor probes (10 pmol/ml) and
were used at a 200-fold concentration excess over the labeled probes
during the experiments.
Nuclear extracts from the bbtRel overexpressing cells were obtained
using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit
(Thermo Fisher Scientific). Protein concentrations were measured using the
Pierce BCA Protein Assay Kit. The EMSA reaction solutions were prepared
in accordance with the manufacturer’s protocol (LightShift Chemiluminescent EMSA Kit; Thermo Fisher Scientific). For binding reactions,
amounts equivalent to 10 fmol of the duplex were incubated with 200 ng
bbtRel-RHD purified protein or 5 mg nuclear extract. The protein–probe
mixture was separated using a 10% polyacrylamide 1 native gel and was
transferred to a Biodyne B Nylon Membrane (Thermo Fisher Scientific).
Migration of the biotin-labeled probes was detected on x-ray film, using
streptavidin–HRP conjugates to bind biotinylated probes and a chemiluminescent substrate. Both were used in accordance with the manufacturer’s
protocol.
Embryo collection, section in situ hybridization, Southern blot hybridization, RT-PCR analysis, immunofluorescence imaging, transient transfection, luciferase reporter assay, and coimmunoprecipitation (co-IP) were
performed as described previously (23–25). Primers for hybridization probe
preparation and RT-PCR are listed in Supplemental Table I.
Acute immune challenge in adult amphioxus
Heated bacterial mixtures (105 CFU) of V. vulnificus and S. aureus were
prepared for injection as described in our previous study (23). The adult
amphioxus was separated into three groups and was treated with 1) DMSO
as a negative control, 2) the bacterial mixtures, or 3) helenalin administered prior to inoculation with the bacterial mixtures. The animals were
cultured in separate tanks, and the intestines from five individuals were
collected at 0, 2, 4, 6, 8, and 12 h postinjection as a single sample.
Protein expression, purification and rabbit antiserum
preparation
The expression vector was constructed by inserting the bbtRel RHD into the
expression vector pET-28a (Novagen), and the construct was transformed
into the expression host BL21 (Novagen). The selected transformant was
inoculated in 23 YT medium that was supplemented with kanamycin
(50 mg/ml) and was stimulated with isopropyl b-D-thiogalactoside at
a concentration of 0.5 mM to induce expression at 25˚C. The induced
culture was centrifuged, and the cell pellets were resuspended in solution
A (20 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 20 mM imidazole). The
Results
Structural and phylogenetic analyses of bbtRel, bbtp105, and
bbtIkB
To characterize the roles of amphioxus NF-kB cascade in innate
immune responses, two genes contained RHDs (named bbtRel and
bbtp105) were obtained by RT-PCR and RACE from the Chinese
amphioxus (B. belcheri tsingtauense, bbt). BbtRel contains an
open reading frame of 2265 bp and encodes a 755-aa protein,
which includes an N-terminal RHD and a C-terminal TAD. The
two conserved motifs, the DNA binding motif (RXXRXRXXC)
and the nuclear localization signal [(NLS): RKRQK], are in the
RHD of bbtRel (Fig. 1A). Genomic organization analysis demonstrated that bbtRel is encoded by 9 exons, of which exons 2–4
are responsible for the RHD (Supplemental Fig. 1A). Southern
blot analysis demonstrated that bbtRel is a single-copy gene in the
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Animals, cells, and reagents
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CHARACTERIZATION OF AMPHIOXUS NF-kB/IkB COMPLEX
amphioxus genome (Supplemental Fig. 1C). A neighbor-joining
tree based on the protein sequences of the conserved RHD
showed that bbtRel is clustered with the vertebrate class II NF-kB
proteins, which include RelA, RelB, and c-Rel, whereas the dorsallike proteins that belong to invertebrate class II NF-kB are clustered as a separate invertebrate group (Fig. 1B). These results suggest that bbtRel is the ancestor of the vertebrate class II NF-kB
group of proteins.
Another NF-kB family member, bbtp105, encodes a 1024-aa
protein, which includes a conserved GRR motif (Supplemental
Fig. 1B), an N-terminal RHD, and a C-terminal IkB-like domain
containing six ANK repeats (Fig. 1A). A death domain (DD) is
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FIGURE 1. Sequence analysis of bbtRel, bbtp105,
and bbtIkB. (A) Domain topology of bbtRel,
bbtp105, and bbtIkB. The domains were predicted
using the SMART database (http://smart.emblheidelberg.de/smart/set_mode.cgi?NORMAL=1),
whereas the NLS presented in bbtRel (RKRQK)
and bbtp105 (KKRKK), the caspase 8 recognition
site (PxExx[Ac/Ar], x represents any residue) at
position 579–582 in bbtp105, and the C-terminal
phosphorylation sites of bbtp105 (DSGLEQ) and
bbtIkB (TSGLEDS) were manually predicted using sequence alignment in accordance with published sequences. (B) The bbtRel and bbtp105
neighbor-joining trees were constructed using protein sequences from the RHD region. Clustal W
1.83 software was used for multiple alignments.
The phylogenetic tree was built using Mega v3.1
software. The neighbor-joining method was used
to calculate the trees with 1000 bootstrap tests and
handling gaps with pairwise deletion. Aa, Aedes
aegypti; Ag, Anopheles gambiae; Cr, Carcinoscorpius rotundicauda; Dm, Drosophila melanogaster; Dr, Danio rerio; Gg, Gallus gallus; Hs,
Homo sapiens; Mm, Mus musculus; Sp, Strongylocentrotus purpuratus; Xl, Xenopus laevis. The
numbers at the nodes indicate bootstrap values.
located at the C terminus, and an NLS is in the IPT domain
(Fig. 1A). IPT is a domain that contains an Ig-like fold and is found
in cell surface receptors as well as in intracellular transcription factors. A potential PEST domain in amino acids 467–490
(APPDDDDLITDSAPTEDTMAMSQA) was predicted by the
PEST find Web service (http://emboss.bioinformatics.nl/cgi-bin/
emboss/pestfind). Phylogenetic analysis places bbtp105 at the
base of the NF-kB clade, indicating that bbtp105 is more homologous to the ancestral NF-kB proteins (p100 and p105) than
to the Rel proteins in vertebrates, whereas the Drosophila Relishlike sequence clustered as an invertebrate-specific group (Fig.
1B). Therefore, we propose that all Rel proteins evolved from
The Journal of Immunology
FIGURE 2. In vitro characterization of
bbtRel. (A) The NF-kB–specific inhibitor
helenalin attenuates the transcriptional activity of bbtRel in a dose-dependent manner.
At 24 h post transfection, the indicated
amounts of helenalin were added to the cell
culture medium, the cells were incubated for
12 h, and the reporter expression was measured. Data are shown as the mean 6 SD of
three samples per treatment. Values were
considered significant when p , 0.05. The
results were confirmed by at least three separate experiments. (B) Helenalin specifically
inhibits the binding of bbtRel to the kB element. At 24 h post transfection, the nuclear
extracts were prepared, and binding with kB
probes was determined by gel shift assays.
(C) Binding activity of the bbtRel protein to
the human kB probe in a dose-dependent
manner with increasing amounts of bbtRelRHD protein. (D) EMSA showed that helenalin and MG132 can inhibit the binding
activity of bbtRel protein with human kB
probe. (E) Increased protein expression and
nuclear translocation of bbtRel after bacterial
challenge in amphioxus gut tissues. (F) Increased binding activity of bbtRel to the kB
probe in cells derived from amphioxus gut
tissue after bacterial challenge was measured
by EMSA.
Functional characterization of bbtRel
To define the transcriptional activity of bbtRel, NF-kB reporter
assays were performed using the transient expression of bbtRel in
293T cells. In the presence of bbtRel, the transcription of the
reporter gene was significantly induced (Fig. 2A). Further reporter
assays showed that the truncated bbtRel mutants did not exhibit
transcriptional activity (Supplemental Fig. 2A, 2B), indicating that
the transcriptional activity of bbtRel depends both on the atypical
C-terminal TAD and on the N-terminal RHD. Investigation of the
subcellular location demonstrated that the bbtRel-RHD was distributed evenly in the nucleus, whereas the truncated mutants
missing the RHD were distributed evenly in the cytoplasm, indicating that the NLS in the RHD region is essential for the nuclear
location of bbtRel (Supplemental Fig. 2C).
Next, we assessed whether the NF-kB inhibitors BAY11-7082,
evodiamine, and helenalin could block the transcriptional activity
of bbtRel. Increased concentrations of helenalin resulted in a dosedependent reduction in reporter expression; however, this was not
true for BAY11-7082 or evodiamine (Figs. 2A, Supplemental Fig.
2D). Then, nuclear extracts of 293T cells in which bbtRel or
bbtp105 was overexpressed were used for gel shift assays. The
results showed that helanalin can impair the formation of the
bbtRel–DNA complex, but not the bbtp105–DNA complex, indicating that helanalin is a specific inhibitor of bbtRel (Fig. 2B).
To eliminate interference from endo-human NF-kB and to investigate whether bbtRel recognizes the kB response element
through its RHD in vitro, an EMSA was performed using a
recombinant bbtRel RHD (bbtRHD) and the kB element. The
results demonstrated that the bbtRHD recognized the mammalian
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a common ancestral RHD-ankyrin structure within a unique superfamily and diverged into the two subgroups that expanded in
vertebrates as the result of two whole-genome duplications.
The 1,029-bp open reading frame in bbtIkB encodes a protein
with 343 aa residues and contains several features found in IkB
members, such as six ANK repeats at the C terminus and two
serine residues (Fig. 1A). The two serine residues in the N-terminal
serine-rich region are critical for IkB degradation (Supplemental
Fig. 1D). In contrast to human IkBa and IkBb, a potential PEST
domain in amino acids 54–76 (KSCQDDTEDDCIESDEDPNEEQ)
is located at the N terminus. Because the PEST region is important
for its constitutive phosphorylation and intrinsic stability (26), the
different location of the PEST domain suggests that bbtIkB may
be regulated by different kinetics of degradation, which need to be
further discussed. Southern blot analysis clearly demonstrated that
bbtIkB is a single-copy gene in the amphioxus genome (Supplemental Fig. 1C). The genomic sequence of bbtIkB is relatively
small, spanning 3.2 kb, and contains six exons ranging in size from
87 to 276 bp and five introns ranging from 208 to 875 bp (Supplemental Fig. 1A). The genomic sequence of human IkBa also
contains six exons and is similar in size to bbtIkB, indicating the
evolutionarily conserved genomic structure of the IkB family in
invertebrates and vertebrates. In addition, the intron/exon boundaries in human IkBb and Bcl3 are similar to those in human IkBa,
and individual IkB family members are distributed on different
chromosomes (27). Thus, we conclude that IkBa and other family
members have not arisen recently through simple gene duplication
events but have gradually evolved from an ancestral IkB molecule
(Supplemental Fig. 1E).
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kB element, which was also recognized by human NF-kB and
Drosophila Dorsal (Fig. 2C). This interaction was specific because
bbtRHD did not bind the mutant kB probes and the competitor
(unlabeled kB probes) blocked such binding (Fig. 2C). The gel
shift complexes were partially reduced by increasing the dose of
helenalin or MG132, further suggesting that these two inhibitors
impair the interaction of bbtRel with the target sequence (Fig. 2D).
To test whether bbtRel can translocate into the nucleus in response
to activation, we raised the poly-Ab of bbtRel-RHD, and the
subcellular location of bbtRel was tested (Supplemental Fig. 2F).
Results showed that following a bacterial challenge, the amount of
bbtRel in the nucleus and the binding activity of the bbtRel with the
kB probe increased in the cells derived from amphioxus gut tissue,
suggesting that nuclear localization of bbtRel after a bacterial challenge is due to its protein activity, but not the cell type (Fig. 2E–F).
These data indicate that the specific recognition sequence in NF-kB
was acquired early and was maintained during evolution.
CHARACTERIZATION OF AMPHIOXUS NF-kB/IkB COMPLEX
The NLS and DD are important for the localization of bbtp105
For functional characterization of bbtp105, several truncated mutants of bbtp105 were constructed and overexpressed in 293T
cells (Fig. 3A). Reporter assay showed that only RHD-IPT and
RHD-DD induced reporter gene expression, indicating that the
ANK region may play an inhibitory role (Fig. 3A, 3B). To determine the subcellular location of bbtp105, the truncated forms of
bbtp105 were transfected into HeLa cells and the locations of the
expressed proteins were investigated. The results show that the
proteins that contained an NLS and were missing the DD were
located around the nucleus, whereas proteins containing the DD
were distributed throughout the cytoplasm (Fig. 3C). The Western
blots demonstrated that, as with human NF-kB1/2 and Drosophila
Relish, bbtp105 was processed spontaneously into the mature form,
which was measured at ∼ 58 kDa (named bbtp58) (Supplemental
Fig. 2E). The Western blot results also suggested that the truncated
mutants containing amino acids 260–582 mediated the processing
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FIGURE 3. The NLS and DD are important for the localization of bbtp105. (A) Description of the bbtp105-truncated mutants used in this study. (B)
Reporter assays show that, compared with the wild-type bbtp105, proteins without the ANK region enhanced the transcriptional activity of bbtp105. (C)
Subcellular localization of the full-length bbtRel, bbtp105, and bbtIkB, indicating that the proteins are largely restored to the cytoplasm when inactivated.
Overexpression of the bbtp105-truncated mutants indicates that the proteins that contain the NLS and are missing the DD are located around the nucleus,
whereas the proteins that contain a DD are distributed throughout the cytoplasm. (D) Amphioxus caspase 8 promotes the endoproteolytic cleavage of
bbtp105, whereas the caspase 8 inhibitor zVAD-FMK inhibits the transcriptional activity of bbtp105 and reduces production of the mature bbtp58. (E)
Deletion of the GRR sequence at 390–442 aa led to the incorrect processing of bbtp105 and inhibited the translocation of bbtp58 from the cytosol to the
nucleus, suggesting that the GRR motif provides a recognition and termination signal for proteolysis. (F) Western blotting shows the inhibition of bbtp105
processing by the protease inhibitor MG132 in 293T cells. (G) Ubiquitination assays show that K63-linked ubiquitination is important for the processing of
bbtp105 and that ubiquitination is dependent on the K48 site.
The Journal of Immunology
The relationship among bbtRel, bbtIkB, and bbtp105
To determine the relationship among bbtRel, bbtIkB, and bbtp105,
reporter assays were performed and results showed that the transcription activity of bbtRel was severely inhibited by bbtpl05 and
abolished when bbtIkB was coexpressed (Fig. 4A). ELISA assays
further revealed that when bbtRel was overexpressed in 293T cells,
the secretion of human TNF-a was induced in a dose-dependent
manner, and such induction could be inhibited when bbtIkB was
coexpressed with bbtRel (Fig. 4B). Co-IP assays demonstrated
that bbtRel interacts directly with bbtIkB and bbtp105, and that
bbtp105 formed homodimers (Fig. 4C). We also demonstrated that,
in cells overexpressing bbtRel or in cells treated with MG132, the
proteolytic degradation of bbtIkB was inhibited (Fig. 4D). In addition, both K48 and K63 mutants were ubiquitinated in bbtIkB
(data not shown). Given the distinct location of the PEST motif of
bbtIkB, our results not only indicate that a similar feedback regulation exists between bbtRel and bbtIkB but also suggests that
bbtIkB may depend on another unidentified ubiquitination mechanism.
Because co-IP assays demonstrated that bbtRel interacts directly
with bbtp105 (Fig. 4C), to determine which domain was responsible for the bbtp105–bbtRel interaction, further co-IP assays with
truncated bbtp105 were performed. The data suggest that, in
contrast to interaction through the ANK repeats that is observed in
mammals, bbtRel interacts with bbtp105 through the IPT region
in amphioxus (Fig. 4E). Next, bbtRel was coexpressed with different truncated bbtp105 mutants, and reporter assays demonstrated that the IPT region blocked the activity of bbtRel (Fig. 4F).
Therefore, the unprocessed bbtp105 interacts with bbtRel through
its IPT region and to inhibit its transcription activity by its ANK
domain. Because the processed mature bbtp58 may form a hetero
dimer with bbtRel to create a mature NF-kB complex, and co-IP
assay demonstrated that bbtp105 can form homodimers (Fig. 4C),
for a more detailed analysis of the p58–p58 homodimer and the
p58–bbtRel heterodimer, we generated constructs that expressed
two covalently linked proteins and a flexible peptide linker (Fig.
4G). The 20-aa peptide linker in these constructs was used previously to link multimeric complexes in in vivo and in vitro assays
(30). The reporter assays demonstrated that the bbtRel–bbtp58
heterodimer is a more effective transcription factor than is the
wild-type bbtRel or the linked bbtRel homodimer. In addition, the
bbtp58–bbtp58 homodimer induced higher levels of transcription
than did the wild-type bbtp105 or bbtp58 (Fig. 4G). Therefore, we
can assume that the functional NF-kB complex contains the
bbtRel–bbtp58 heterodimer or the bbtp58–bbp58 homodimer.
The temporal expression and tissue distribution of bbtRel,
bbtp105, and bbtIkB in the adult amphioxus
Because NF-kB plays important roles in dorsal-ventral patterning
in Drosophila, we used whole-mount in situ hybridization to investigate the temporal expression of bbtRel during amphioxus
embryogenesis. The bbtRel transcripts were abundant from the
blastula stage of embryogenesis and were distributed to the dorsal
and ventral regions at the neurula stage (Supplemental Fig. 3A).
At the larval stage, although still primarily located in the dorsalventral region, the bbtRel transcripts were abundant in the anterior
region, suggesting that bbtRel may be involved in the dorsalventral and anterior-posterior patterning of amphioxus embryos
(Supplemental Fig. 3A). Moreover, RT-PCR showed that bbtp105
and bbtIkB were also abundant during embryogenesis (Supplemental Fig. 3B).
To determine the tissue distribution of bbtRel, bbtp105, and
bbtIkB, RT-PCR was performed and results showed that transcripts of all the tested genes are abundant in amphioxus digested
tissues (Fig. 5B). Further section in situ hybridization was performed, and results showed that bbtRel transcripts were extensively distributed in the gill slits, hepatic cecum, intestine, and
gonads, whereas bbtp105 and bbtIkB transcripts were mainly
found in the hepatic cecum and intestine in adult amphioxus that
was immune challenged with the Gram-negative bacteria V. vulnificus (Fig. 5A). Because the digestive system is considered the
primary line of defense in amphioxus, to confirm the immune
significance, real-time RT-PCR analysis was performed to monitor
the expression of bbtRel, bbtp105 transcripts in adults challenged
with V. vulnificus. Results showed that transcripts of bbtRel and
bbtp105 were upregulated in 2 h and reached the highest level 6–8 h
post infection. The upregulation of bbtRel is consistent with the
result that the amount of bbtRel is greatly increased after bacterial
challenge (Fig. 5C), indicating that bbtRel may be regulated by
other immune-related transcription factors, such as IFN regulatory
factors, in amphioxus.
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of bbtp105, whereas the truncations containing the DD region were
inhibitory (Supplemental Fig. 2E), indicating that the DD is important for stability of the bbtp105 in the cytoplasm. These
observations, along with the reporter expression data, suggest that
the NLS in the IPT region is essential for nuclear localization of
p105; however, when the protein is in the precursor form, the DD
is important for its cytoplasmic distribution.
To investigate the processing mechanism of bbtp105, we analyzed the bbtp105 sequence and determined that it contains a
GRR at position 391–442 in the N terminus and a conserved
caspase 8 recognition motif, DEGD, at position 579–582 (Fig.
1A). Considering that the mature bbtp105 protein is ∼ 58 kDa, it
is reasonable to assume that a caspase mediates the cleavage
process. Therefore, reporter assays were performed, which demonstrated that, when coexpressed with increasing amounts of
bbtcaspase 8 protein [characterized in our previous study (28,
29)], the transcription activity of bbtp105 was enhanced, and the
mature bbtp58 was increased. In addition, when the cells were
treated with the caspase 8–specific inhibitor zVAD-FMK, the
transcriptional activity of bbtp105 and the mature bbtp58 were
reduced (Fig. 3D). To determine whether the GRR region is indispensable for the processing of bbtp105, a deletion mutant in
which amino acids 391–442 were removed was constructed and
expressed in 293T cells. Incorrect processing of bbtp105 occurred, and the translocation of bbtp58 from the cytosol to the
nucleus was inhibited, indicating that GRR sequence provides
the correct termination signal for the caspase 8–dependent cleavage (Fig. 3E). The proteasome complex and the ubiquitination
of bbtp105 are implicated in the degradation of the C-terminal
region of bbtp105, as treatment with the proteasome inhibitor
MG132 blocked the processing of bbtp105 in vitro (Fig. 3F).
Next, we showed that neither K48 nor K3 mutants could affect
the ubiquitination of bbtRel. However, when the K48 and K63
were double mutated, ubiquitination of bbtRel could not be observed, indicating that both K48- and K63-linked ubiquitination
are important for the degradation of bbtRel. Because K63-linked
ubiquitination is used to mediate the regulation of gene functions, whereas K48-linked ubiquitination leads to protein degradation, we concluded that the K63-linked ubiquitination may
assist the K48-linked ubiquitination of bbtp105 during its processing (Fig. 3G). Taken together, these ideas suggest that the
bbtp105 processing mechanism required both caspase 8 cleavage and proteasome degradation. The caspase 8 recognition
motif and the GRR sequence may provide the processing signal, whereas the C-terminal DD blocks the processing of this
protein.
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CHARACTERIZATION OF AMPHIOXUS NF-kB/IkB COMPLEX
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FIGURE 4. The relationships among bbtp105, bbtRel, and bbtIkB. (A) The transcriptional activity of bbtRel is inhibited by bbtp105 and bbtIkB in
a dose-dependent manner. (B) ELISA assay showed that the overexpressed bbtRel can induce the secretion of human TNF-a in 293T cells, and such
induction could be inhibited by bbtIkB in a dose-dependent manner. (C) Co-IP assays indicate that bbtRel interacts directly with bbtp105 and bbtIkB, and
that the bbtp105 protein forms homodimers. (D) A co-IP assay showed that in cells that overexpressed bbtRel or in cells treated with MG132, the proteolytic degradation of bbtIkB was inhibited. (E) Co-IP assays indicate that bbtRel interacts with the bbtp105-truncated mutants that contain the IPT region,
indicating that amino acids 251–580 contain the motif responsible for the interaction of bbtp105 with bbtRel. (F) The coexpression of bbtRel with the IPT
region of bbtp105 inhibits the transcriptional activity of bbtRel. (G) Reporter assays show that the bbtp58–bbp58 homodimer and the bbtRel–bbtp58
heterodimer are more effective than the wild-type proteins. All data from reporter assays are shown as the means 6 SD of three samples per treatment, and
values were considered significant when p , 0.05. The results were confirmed by at least three separate experiments.
The potential roles of the amphioxus NF-kB cascade in
immune defense
For further characterization of the roles of amphioxus NF-kB
cascade in immune defense, we treated adult amphioxus with 4
mM helenalin before bacterial challenge. Blocking NF-kB by dietary supplementation of 4 mM helenalin decreased the survival
rate of amphioxus following oral infection with V. vulnificus (Fig.
6A). Because IkB and TNF-a are two classical NF-kB target
genes, we first cloned the genomic sequences of bbtIkB and
bbtTNFa-like. Then the genomic sequences were uploaded to the
TESS online prediction program (www.cbil.upenn.edu/cgi-bin/
tess/tess) to search whether these regions contain the conserved
NF-kB binding motif. The genomic sequence of bbtIkB, including
the 2-kb sequence upstream of the ATG and the coding region,
The Journal of Immunology
1227
contains three conserved kB binding sites in the first intron of
bbtIkB. Furthermore, reporter assays indicated that the 700-bp
sequence upstream of the second CDS in bbtIkB responded in
a dose-dependent manner, suggesting that this region is indispensable for the interaction with bbtRel (Fig. 6B). Then the expression of bbtIkB following bacterial challenge was determined
by RT-PCR, and a significant induction was observed, consistent
with studies in Drosophila and humans, in which activation of the
NF-kB pathway increased the expression of IkB (Fig. 6C). Similar
to the vertebrate TNF-a homolog, the bbtTNFa-like gene contains
an NF-kB binding site in its promoter region that responds to the
overexpression of bbtRel in a dose-dependent manner (Fig. 6D).
Over the same period, bbtTNFa-like, another classical NF-kB
target gene required for the innate immune response in vertebrates,
was upregulated slightly (Fig. 6E). Moreover, treatment with the
helenalin and MG-132 prior to infection consistently suppressed
the upregulation of bbtTNFa-like and bbtIkB (Fig. 6C, 6E). Because the TLR signaling pathway have been well characterized in
amphioxus, the bbtIkB protein was also coexpressed with amphioxus MyD88 or TICAM (31, 32), and the activation of NF-kB
mediated by bbtMyD88 or bbtTICAM was inhibited by bbtIkB in
a dose-dependent manner (Fig. 6F). Thus, we proposed that the
rapid activation of NF-kB in the digestive system of amphioxus
provides prompt protection against pathogenic infection, likely by
modulating amphioxus TLR signaling to target many immunerelated effector genes.
Discussion
The evolution and functional conservation of the NF-kB–IkB
cascade
Despite the evolutionary distance between amphioxus and vertebrates, we determined that bbtRel, bbtp105, and bbtIkB contain
signature motifs similar to those found in the vertebrate orthologs,
notably the DNA binding motif and the NLS in bbtRel, as well as
the six ANK repeats and the N-terminal potential phosphorylation
sites in bbtIkB. Like human p100 and p105 and insect Relish,
bbtp105 is a mosaic protein that contains both the RHD and the
inhibitory IkB domain. Despite the sequence similarities among
species, the overexpression of bbtIkB and bbtp105 specifically
inhibits the translocation of bbtRel to the nucleus and reduces its
transcriptional activity, suggesting that bbtIkB and bbtp105 are
natural inhibitors of bbtRel. Moreover, the genomic sequence of
bbtIkB contains several kB-binding motifs, and the ANK repeats
are the site of the bbtIkB–bbtRel interaction, suggesting that the
roles of NF-kB and IkB have coevolved and remained conserved
throughout the evolution of chordates.
When the Toll pathway is activated, Drosophila Dorsal and/
or DIF proteins are released from inhibition and translocate to
the nucleus, where they bind to kB response elements and transactivate a specific set of genes, such as the drosomycin gene (33).
In this study, we determined that in the presence of helenalin,
the survival rate of amphioxus was decreased after an infection
challenge. Moreover, we showed that bbtRel recognizes the kB
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FIGURE 5. The tissue distribution of bbtRel, bbtp105, and bbtIkB. (A) Section in situ hybridization analyses of bbtRel, bbtp105, and bbtIkB expression
in adult amphioxus. g, gill slits; hc, hepatic cecum; i, intestine; m, muscle; n, notochord; nc, nerve cord; o, ovary; s, spermary. Blue indicates strong
hybridization; dark brown indicates a weak signal. (B) RT-PCR showed that transcripts of bbtRel, bbtp105, and bbtIkB are abundant in the digestive tissues
of normal amphioxus. The results are presented as expression relative to that in muscles. (C) RT-PCR analyses of the expression patterns of bbtRel, bbtp105,
and bbtIkB after a challenge with Gram-negative V. vulnificus. The results are presented as fold induction in the abundance of mRNA relative to that in
samples injected with PBS and were determined using the 22OOCt method from two parallel experiments performed in triplicate. The endogenous control
for normalization is cytoplasmic bbtb-actin.
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CHARACTERIZATION OF AMPHIOXUS NF-kB/IkB COMPLEX
element and induces the expression of immune-related genes, such
as bbtIkB and bbtTNFa-like. Sequence analysis showed that the
promoter region of the amphioxus TNFa-like and IkB genes
contains conserved kB binding sites that can respond to the activation of bbtRel, further suggesting a role for NF-kB in the amphioxus innate immune response. Because the phylogenetic
analysis indicated that bbtp105 and bbtRel are the ancestors of the
vertebrate class I and II subgroups, respectively, this study not
only demonstrates the archaic roles of the amphioxus NF-kB–IkB
cascade in immunity but also provides crucial information regarding the functional refinement of NF-kB molecules during the
evolution from invertebrates to vertebrates.
The mature NF-kB complex in amphioxus exhibits both
similarities and differences
Our study demonstrated that bbtp105 inhibits bbtRel, suggesting
that, in activated cells, bbtp105 is processed into p58, which results
in the coactivation of bbtp105 and bbtRel. We also determined
amphioxus caspase 8 as the bbtp105 endoprotease, suggesting that
this caspase-mediated endoproteolytic process is similar to the
Dredd-mediated process in Drosophila. However, unlike Drosophila Relish, a direct interaction between bbtp105 and bbtcaspase
8 was not observed. It is reasonable to presume that this interaction
is instantaneous and difficult to detect by overexpression. Alternatively, the interaction may require other unidentified proteins for
endoproteolysis to proceed. When the GRR was deleted, endoproteolysis did not occur, regardless of the presence of bbtcaspase
8, suggesting that the GRR motif is a separate recognition and
termination signal for proteolysis. We also demonstrated that, as in
vertebrate class I NF-kBs, the endoproteolytic processing required
the K63-linked ubiquitination of bbtp105 and the degradation by
a protease. Therefore, we can assume that the protease-dependent
mechanism was established before the evolution of chordates, and,
because of improvements to the process, the caspase 8–dependent
processing was ultimately lost in vertebrates, although it is still
conserved in protochordates.
However, a number of obvious differences were also observed.
First, unlike Relish, in which the C-terminal 107 aa are required for
endoproteolysis and signal-dependent phosphorylation by Drosophila IkB kinase b (15), the C-terminal sequence of bbtp105 is
the inhibition signal for processing of the protein to mature p58.
Second, the N-terminal serine-rich region of Relish and the PEST
domain negatively regulated Relish activation, whereas the ANK
repeats were responsible for the negative regulation of bbtp105 in
amphioxus. Third, the IPT region is the foundation for the heterodimerization between bbtRel and the mature p58 form of the
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FIGURE 6. Potential roles of the amphioxus NF-kB in immune defense. (A) The survival rate of amphioxus after a challenge with Gram-negative
bacteria V. vulnificus in the absence or presence of 4 mM helenalin. The bacterial challenge was performed at 1 h post treatment with DMSO (gray curve)
and helenalin (black curve). The results are presented as the survival rates from two parallel experiments. (B) The reporter assays demonstrated that the 2-kb
sequence upstream of the ATG did not respond to the overexpression of bbtRel, whereas the 700-bp sequence upstream of the second CDS in bbtIkB
responded in a dose-dependent manner. (C) RT-PCR analyses of the expression patterns of bbtIkB after a challenge with Gram-negative V. vulnificus in the
presence or absence of helenalin. (D) Sequence analysis indicated that the 1-kb sequence upstream of ATG contains one kB binding site in the bbtTNFalike gene, and reporter assay confirmed that bbtRel can bind to this region. (E) RT-PCR analyses of the expression patterns of bbtTNFa-like after
a challenge with Gram-negative V. vulnificus in the presence or absence of helenalin. The bacterial challenge was performed at 1 h post treatment with
DMSO (blue curve), helenalin (green curve), or MG-132 (red curve). The results are presented as fold induction in the abundance of mRNA relative to that
in samples injected with DMSO and were determined using the 22OOCt method from two parallel experiments performed in triplicate. The endogenous
control for normalization is cytoplasmic bbtb-actin. (F) Reporter assays showed that bbtIkB inhibited the activation of NF-kB, as mediated by bbtMyD88
and bbtTICAM in a dose-dependent manner. All reporter assay data are shown as the means 6 SD of three samples per treatment, and values were
considered significant when p , 0.05. The results were confirmed by at least three separate experiments.
The Journal of Immunology
protein. Moreover, the IPT region significantly inhibits the transcriptional activity of bbtRel and controls the nuclear location of
bbtp105, suggesting that when bbtRel forms a heterodimer with
the mature p58, p58 not only assists the nuclear translocation of
bbtRel but is critical for its binding to the kB element. Therefore,
although not as many members of the NF-kB family are found in
amphioxus as are found in vertebrates and insects, the functional
bbtRel–bbtp58 heterodimer and the bbtp58–bbp58 homodimer in
amphioxus exert functions that are as powerful as those found in
other species.
Amphioxus NF-kB facilitates an understanding of the evolution
of the immune system
Disclosures
The authors have no financial conflicts of interests.
References
1. Ghosh, S., and M. S. Hayden. 2008. New regulators of NF-kappaB in inflammation. Nat. Rev. Immunol. 8: 837–848.
2. Gilmore, T. D., and F. S. Wolenski. 2012. NF-kB: where did it come from and
why? Immunol. Rev. 246: 14–35.
3. Khush, R. S., F. Leulier, and B. Lemaitre. 2001. Drosophila immunity: two paths
to NF-kappaB. Trends Immunol. 22: 260–264.
4. Govind, S. 1999. Control of development and immunity by rel transcription
factors in Drosophila. Oncogene 18: 6875–6887.
5. Gross, I., P. Georgel, C. Kappler, J. M. Reichhart, and J. A. Hoffmann. 1996.
Drosophila immunity: a comparative analysis of the Rel proteins dorsal and Dif
in the induction of the genes encoding diptericin and cecropin. Nucleic Acids
Res. 24: 1238–1245.
6. Hedengren-Olcott, M., M. C. Olcott, D. T. Mooney, S. Ekengren, B. L. Geller,
and B. J. Taylor. 2004. Differential activation of the NF-kappaB-like factors
Relish and Dif in Drosophila melanogaster by fungi and Gram-positive bacteria.
J. Biol. Chem. 279: 21121–21127.
7. Graef, I. A., J. M. Gastier, U. Francke, and G. R. Crabtree. 2001. Evolutionary
relationships among Rel domains indicate functional diversification by recombination. Proc. Natl. Acad. Sci. USA 98: 5740–5745.
8. Fusco, A. J., O. V. Savinova, R. Talwar, J. D. Kearns, A. Hoffmann, and
G. Ghosh. 2008. Stabilization of RelB requires multidomain interactions with
p100/p52. J. Biol. Chem. 283: 12324–12332.
9. Gerondakis, S., M. Grossmann, Y. Nakamura, T. Pohl, and R. Grumont. 1999.
Genetic approaches in mice to understand Rel/NF-kappaB and IkappaB function: transgenics and knockouts. Oncogene 18: 6888–6895.
10. Beg, A. A., W. C. Sha, R. T. Bronson, S. Ghosh, and D. Baltimore. 1995.
Embryonic lethality and liver degeneration in mice lacking the RelA component
of NF-kappa B. Nature 376: 167–170.
11. Carrasco, D., F. Weih, and R. Bravo. 1994. Developmental expression of the
mouse c-rel proto-oncogene in hematopoietic organs. Development 120: 2991–
3004.
12. Weih, D. S., Z. B. Yilmaz, and F. Weih. 2001. Essential role of RelB in germinal
center and marginal zone formation and proper expression of homing chemokines. J. Immunol. 167: 1909–1919.
13. Snapper, C. M., F. R. Rosas, P. Zelazowski, M. A. Moorman, M. R. Kehry,
R. Bravo, and F. Weih. 1996. B cells lacking RelB are defective in proliferative
responses, but undergo normal B cell maturation to Ig secretion and Ig class
switching. J. Exp. Med. 184: 1537–1541.
14. Ertürk-Hasdemir, D., M. Broemer, F. Leulier, W. S. Lane, N. Paquette,
D. Hwang, C. H. Kim, S. Stöven, P. Meier, and N. Silverman. 2009. Two roles
for the Drosophila IKK complex in the activation of Relish and the induction of
antimicrobial peptide genes. Proc. Natl. Acad. Sci. USA 106: 9779–9784.
15. Leulier, F., A. Rodriguez, R. S. Khush, J. M. Abrams, and B. Lemaitre. 2000.
The Drosophila caspase Dredd is required to resist gram-negative bacterial infection. EMBO Rep. 1: 353–358.
16. Stöven, S., I. Ando, L. Kadalayil, Y. Engström, and D. Hultmark. 2000. Activation of the Drosophila NF-kappaB factor Relish by rapid endoproteolytic
cleavage. EMBO Rep. 1: 347–352.
17. Stoven, S., N. Silverman, A. Junell, M. Hedengren-Olcott, D. Erturk,
Y. Engstrom, T. Maniatis, and D. Hultmark. 2003. Caspase-mediated processing
of the Drosophila NF-kappaB factor Relish. Proc. Natl. Acad. Sci. USA 100:
5991–5996.
18. Meinander, A., C. Runchel, T. Tenev, L. Chen, C. H. Kim, P. S. Ribeiro,
M. Broemer, F. Leulier, M. Zvelebil, N. Silverman, and P. Meier. 2012. Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling. EMBO J. 31: 2770–2783.
19. Heusch, M., L. Lin, R. Geleziunas, and W. C. Greene. 1999. The generation of
nfkb2 p52: mechanism and efficiency. Oncogene 18: 6201–6208.
20. Blank, V., P. Kourilsky, and A. Israël. 1991. Cytoplasmic retention, DNA binding
and processing of the NF-kappa B p50 precursor are controlled by a small region
in its C-terminus. EMBO J. 10: 4159–4167.
21. Lin, L., and S. Ghosh. 1996. A glycine-rich region in NF-kappaB p105 functions
as a processing signal for the generation of the p50 subunit. Mol. Cell. Biol. 16:
2248–2254.
22. Cheng, J. D., R. P. Ryseck, R. M. Attar, D. Dambach, and R. Bravo. 1998.
Functional redundancy of the nuclear factor kappa B inhibitors I kappa B alpha
and I kappa B beta. J. Exp. Med. 188: 1055–1062.
23. Li, J., S. Yuan, L. Qi, S. Huang, G. Huang, M. Yang, L. Xu, Y. Li, R. Zhang,
Y. Yu, et al. 2011. Functional conservation and innovation of amphioxus RIP1mediated signaling in cell fate determination. J. Immunol. 187: 3962–3971.
24. Yuan, S., T. Liu, S. Huang, T. Wu, L. Huang, H. Liu, X. Tao, M. Yang, K. Wu,
Y. Yu, et al. 2009. Genomic and functional uniqueness of the TNF receptorassociated factor gene family in amphioxus, the basal chordate. J. Immunol. 183:
4560–4568.
25. Yuan, S., K. Wu, M. Yang, L. Xu, L. Huang, H. Liu, X. Tao, S. Huang, and
A. Xu. 2010. Amphioxus SARM involved in neural development may function
as a suppressor of TLR signaling. J. Immunol. 184: 6874–6881.
26. Rottjakob, E. M., S. Sachdev, C. A. Leanna, T. A. McKinsey, and M. Hannink.
1996. PEST-dependent cytoplasmic retention of v-Rel by I(kappa)B-alpha: evidence that I(kappa)B-alpha regulates cellular localization of c-Rel and v-Rel by
distinct mechanisms. J. Virol. 70: 3176–3188.
27. Huguet, C., P. Crepieux, and V. Laudet. 1997. Rel/NF-kappa B transcription
factors and I kappa B inhibitors: evolution from a unique common ancestor.
Oncogene 15: 2965–2974.
28. Xu, L., S. Yuan, J. Li, J. Ruan, S. Huang, M. Yang, H. Huang, S. Chen, Z. Ren,
and A. Xu. 2011. The conservation and uniqueness of the caspase family in the
basal chordate, amphioxus. BMC Biol. 9: 60.
29. Yuan, S., H. Liu, M. Gu, L. Xu, S. Huang, Z. Ren, and A. Xu. 2010. Characterization of the extrinsic apoptotic pathway in the basal chordate amphioxus.
Sci. Signal. 3: ra66.
30. Wang, X. W., N. S. Tan, B. Ho, and J. L. Ding. 2006. Evidence for the ancient
origin of the NF-kappaB/IkappaB cascade: its archaic role in pathogen infection
and immunity. Proc. Natl. Acad. Sci. USA 103: 4204–4209.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
In our previous studies of amphioxus TLRs and related signaling
molecules, we provided compelling evidence for a functional intracellular TLR–NF-kB signaling cascade (25, 31, 32, 34). Amphioxus expresses $48 TLRs and .40 TIR-containing adaptors,
which suggests an expanded repertoire of adaptors and intermediate signal transducers in its TLR signaling pathways. In the
current study, we determined that the amphioxus genome contains
only two NF-kB family members, bbtRel and bbtp105. Moreover,
the NF-kB complexes in amphioxus are simpler than those in
Drosophila, which further supports our previous observation
that ancestral chordates contain a highly elaborate innate recognition and signaling transduction system, but a much simplified terminal signal pathway (34). Therefore, the complexity
of the amphioxus innate immune system depends on a complex
recognition and cytoplasmic regulation system, but not on transcriptional effectors.
In vertebrate evolution, along with the innovation of adaptive
immunity, whole-genome duplication extended the number of NFkB family members, and their expression and function became
more specialized with evolution. Mammalian p50 and p65 expression varies widely among cell types, whereas the expression
of RelB is restricted to specific regions of the thymus, lymph
nodes, and Peyer’s patches. Because the expression of c-Rel is
confined to hematopoietic cells and lymphocytes, mammalian
classical NF-kB signaling functions chiefly in the acute immune
response and normal development, whereas in lymphoid cells, the
nonclassical pathway responds chiefly to stimuli through the activation of RelB homodimers and heterodimers with another Rel
protein (11–13). In the current study, we propose that the nonclassical pathway is not established in protochordates, which, together with the absence of classical MHC genes and other lymphoid-specific receptor genes in the amphioxus genome, provides
further evidence that vertebrate-specific adaptive immunity did not
exist in basal chordates.
In sum, our study not only provides novel evidence pertaining to
the evolution of NF-kB and IkB molecules but also describes the
archaic roles of the NF-kB and IkB cascade in amphioxus immunity, contributing to further understanding of the formation of
terminal amphioxus innate immune signaling.
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31. Yuan, S., S. Huang, W. Zhang, T. Wu, M. Dong, Y. Yu, T. Liu, K. Wu, H. Liu,
M. Yang, et al. 2009. An amphioxus TLR with dynamic embryonic expression
pattern responses to pathogens and activates NF-kappaB pathway via MyD88.
Mol. Immunol. 46: 2348–2356.
32. Yang, M., S. Yuan, S. Huang, J. Li, L. Xu, H. Huang, X. Tao, J. Peng, and A. Xu.
2011. Characterization of bbtTICAM from amphioxus suggests the emergence
of a MyD88-independent pathway in basal chordates. Cell Res. 21: 1410–1423.
CHARACTERIZATION OF AMPHIOXUS NF-kB/IkB COMPLEX
33. Matova, N., and K. V. Anderson. 2006. Rel/NF-kappaB double mutants reveal
that cellular immunity is central to Drosophila host defense. Proc. Natl. Acad.
Sci. USA 103: 16424–16429.
34. Huang, S., S. Yuan, L. Guo, Y. Yu, J. Li, T. Wu, T. Liu, M. Yang, K. Wu,
H. Liu, et al. 2008. Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity. Genome Res. 18:
1112–1126.
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