Evidence for Evolving Toll-IL-1
Receptor-Containing Adaptor Molecule
Function in Vertebrates
This information is current as
of June 17, 2017.
Con Sullivan, John H. Postlethwait, Christopher R. Lage,
Paul J. Millard and Carol H. Kim
J Immunol 2007; 178:4517-4527; ;
doi: 10.4049/jimmunol.178.7.4517
http://www.jimmunol.org/content/178/7/4517
Subscription
Permissions
Email Alerts
This article cites 60 articles, 35 of which you can access for free at:
http://www.jimmunol.org/content/178/7/4517.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
References
The Journal of Immunology
Evidence for Evolving Toll-IL-1 Receptor-Containing Adaptor
Molecule Function in Vertebrates1
Con Sullivan,* John H. Postlethwait,† Christopher R. Lage,* Paul J. Millard,‡
and Carol H. Kim2*
T
he recent discovery of a functional type I IFN in the zebrafish, Danio rerio, provided the first insight into how
these essential antiviral cytokines evolved (1). Fish type I
IFNs are unique in that they possess introns and, based upon phylogenetic analyses, cluster away from mammalian type I IFNs (1,
2). The mechanisms underlying activation of fish type I IFNs have
yet to be explored. In mammals, transcription of type I IFNs can be
initiated by ligand activation of TLR3, TLR4, TLR7, TLR8, and
TLR9 (3–9). TLRs transduce signals from extracellular stimuli
into the cell through interactions with adaptor molecules. Both
TLRs and adaptor proteins possess Toll/IL-1R (TIR)3 domains that
facilitate the protein-protein interactions responsible for triggering
*Department of Biochemistry, Microbiology, and Molecular Biology, University of
Maine, Orono, ME 04469; †Institute of Neuroscience, University of Oregon, Eugene,
OR 97403; and ‡Department of Chemical and Biological Engineering and The Laboratory for Surface Science and Technology, University of Maine, Orono, ME 04469
Received for publication September 6, 2006. Accepted for publication January
10, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by Grant R15AI049237-02 (to C.S., C.R.L., and
C.H.K.) from the National Institute for Allergy and Infectious Disease, Grant R01
RR10715 (to J.H.P.) from the National Center for Research Resources, and Grant
R15AI065509-01 (to P.J.M.) from National Institute for Allergy and Infectious Disease. All institutes are components of the National Institutes of Health and its contents
are solely the responsibility of the authors and do not necessarily represent the official
views of National Center for Research Resources or National Institutes of Health.
the signal cascade (reviewed in Refs. 10 and 11). One such adaptor
known as TIR domain-containing adaptor molecule 1 (TICAM1,
also known as TRIF) plays an essential role in TLR3 and TLR4
signaling, allowing for the up-regulation of IFN-␤ (12, 13).
Mammalian TICAM1s are comprised of proline-rich N- and
C-terminal domains and a central TIR domain essential for interactions with other TIR domains (12, 13). Each domain plays
an important role in signaling. The TIR domain is responsible
for interacting with TLR3 or TICAM2. The N-terminal domain
can interact with TNFR-associated factor (TRAF)6 or form a
complex consisting of TANK-binding kinase (TBK)-1, IFN regulatory factors (IRF)3 and 7 (IRF7), TRAF3, I␬B kinase-related
kinase ␧, and NAK-associated protein-1 (14 –21). The C-terminal domain interacts with receptor-interacting protein (RIP)1
and RIP3.
In this study, we report the identification of a novel zebrafish
TICAM1 ortholog that exhibits unique structural and functional
features. Our results provide a glimpse into the evolutionary history of TLR-mediated type I IFN induction in a basally diverging
vertebrate TICAM1-mediated pathway. Our findings suggest that
zebrafish TICAM1 activates NF-␬B and type I IFN through mechanisms not observed in mammalian TICAM1 orthologs and that
while zebrafish type I IFN does not group with other avian or
mammalian clades (1, 2), its TICAM1-dependent induction indicates the presence of this potent antiviral pathway in early
vertebrates.
2
Address correspondence and reprint requests to Dr. Carol H. Kim, Department of
Biochemistry, Microbiology, and Molecular Biology, 5735 Hitchner Hall, University
of Maine, Orono, ME 04469. E-mail address: [email protected]
Materials and Methods
3
Nomenclature
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
Nomenclature rules for zebrafish, mouse, and human genes and proteins
follow different conventions. To minimize confusion in presenting these
data, gene names will be presented in italicized capital letters (e.g.,
TICAM1) and protein names will be presented in standard capital letters
(e.g., TICAM1).
Abbreviations used in this paper: TIR, Toll/IL-1R; TICAM, TIR domain-containing
adaptor molecule; TRAF, TNFR-associated factor; TBK, TANK-binding kinase; IRF,
IFN regulatory factor; RIP, receptor-interacting protein; ISRE, IFN-stimulated regulatory element; ZFL, zebrafish liver cell; RHIM, RIP homotypic interaction motif;
HA, hemagglutinin; poly(I:C), polyinosinic-polycytidylic acid.
www.jimmunol.org
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
In mammals, Toll-IL-1R-containing adaptor molecule 1 (TICAM1)-dependent TLR pathways induce NF-␬B and IFN-␤ responses. TICAM1 activates NF-␬B through two different pathways involving its interactions with TNFR-associated factor 6 and
receptor-interacting protein 1. It also activates IFN regulatory factor 3/7 through its interaction with TANK-binding kinase-1,
leading to the robust up-regulation of IFN-␤. In this study, we describe the role of zebrafish (Danio rerio) TICAM1 in activating
NF-␬B and zebrafish type I IFN. Zebrafish IFN is unique in that it cannot be categorized as being ␣- or ␤-like. Through
comprehensive sequence, phylogenetic, and syntenic analyses, we fully describe the identification of a zebrafish TICAM1 ortholog.
Zebrafish TICAM1 exhibits sequence divergence from its mammalian orthologs and our data demonstrate that these sequence
differences have functional consequences. Zebrafish TICAM1 activates zebrafish IFN; however, it does so in an apparently IFN
regulatory factor 3/7-independent manner. Furthermore, zebrafish TICAM1 does not interact with zebrafish TNFR-associated
factor 6, thus NF-␬B activation is dependent upon its interaction with receptor-interacting protein 1. Comparative genome
analysis suggests that TICAM1 and TICAM2 evolved from a common vertebrate TICAM ancestor following a gene duplication
event and that TICAM2 was lost in teleosts following the divergence of the rayfin and lobefin fishes 450 million years ago. These
studies provide evidence, for the first time, of the evolving function of a vertebrate TLR pathway. The Journal of Immunology,
2007, 178: 4517– 4527.
4518
TICAM SIGNALING DIFFERS IN BASALLY DIVERGING VERTEBRATES
DNA constructs
Cell culture
Zebrafish orthologs of TICAM1, RIP1, and TBK1 were identified through basic
local alignment search tool sequence analyses of zebrafish genome and expressed sequence tag databases using human and mouse orthologs and
available zebrafish sequence data. Each zebrafish ortholog was cloned and
deposited in GenBank (zebrafish TICAM1, accession no. DQ848679; zebrafish RIP1, accession no. DQ848680; zebrafish TBK1, accession no.
DQ860098). Zebrafish IFN promoter was cloned from a DraI-digested,
zebrafish genomic DNA pool through nested PCR, according to the protocol outlined in the GenomeWalker Universal kit (BD Clontech). Firstround PCR used primers AP1 (5⬘-GTAATACGACTCACTATAGGGC3⬘) and IFN PRO (5⬘-GTTATTATCCTGTATCGGCCAAGC-3⬘); nested,
second-round PCR used primers AP2 (5⬘-ACTATAGGGCACGCGTGGT3⬘) and IFN PRO NESTED (5⬘-CATTCGCAAGTAGACGCAGAG-3⬘),
and the product from the first round. Subsequently, the IFN promoter
(GenBank accession no. DQ855952) was subcloned in pGL3-Basic (Promega) for use in luciferase reporter assays. Mouse TICAM1, TRAF6, and
RIP1, along with the NF-␬B-luciferase reporter vector pBIIx and the IFNstimulated regulatory element (ISRE)-luciferase reporter vector ISRE-luc,
were provided by R. Medzhitov (Yale University, New Haven, CT). The
Mx187-luc reporter (22) was used as a secondary means to measure ISRE
activity in zebrafish liver cells (ZFL). The zebrafish expression vector
frm2bl (23) was a gift from P. Gibbs (University of Miami, Miami, FL).
pcDNA3.1-p35, which encodes the antiapoptotic protein p35, was provided
by W. Kaiser and M. Offermann (Emory University, Atlanta, GA).
For luciferase assays, mouse TICAM1, zebrafish TICAM1, and deletion
constructs of zebrafish TICAM1 were amplified by PCR using DeepVent
polymerase (New England Biolabs) and subcloned in the zebrafish expression vector frm2bl at the KpnI and SpeI sites, resulting in the removal of
a GFP stuffer fragment and the insertion of the desired TICAM1 fragment.
Mouse TICAM1 possesses a KpnI restriction site in its coding sequence. To
facilitate its subcloning in frm2bl, mouse TICAM1’s native KpnI site was
destroyed without changing the amino acids it encodes via the QuikChange
XL Site-Directed Mutagenesis kit (Stratagene). The N-terminal deletion
(⌬N) of zebrafish TICAM1 lacks the sequence encoding the first 311 aa.
The C-terminal deletion (⌬C) lacks the sequence encoding the final 81 aa.
A construct consisting of the TIR domain alone (⌬N⌬C) was created by
amplification of a product that lacked the nucleotide sequence encoding the
N-terminal 311 aa and C-terminal 81 aa. A zebrafish TICAM1 with a TIR
domain deletion (⌬TIR) was created by PCR sewing. An N-terminal
TICAM1 with a C-terminal overlap was amplified along with a C-terminal
TICAM1 with an N-terminal overlap. Amplicons were combined, denatured, and spliced in the presence of DeepVent polymerase, which filled
in the overlapping PCR product. Following overlap extension, the spliced
product was amplified, and the product was subcloned in frm2bl. An RIP
homotypic interaction motif (RHIM) domain mutant was created by sitedirected mutagenesis. The resulting TICAM1 product contained changes of
amino acids 547–550 from Met-Ile-Gly-Asn to Ala-Ala-Ala-Ala (Fig. 1A).
For coimmunoprecipitation assays, full-length mouse and zebrafish
TICAM1 were subcloned in p3⫻FLAG-CMV-14 (Sigma-Aldrich) following
amplification with Phusion polymerase (New England Biolabs). An N-terminal zebrafish TICAM1 construct lacking the first 311 aa was subcloned in
p3⫻FLAG-CMV-14. Except for zebrafish TLR3, potential mouse and/or zebrafish TICAM1 interactors (zebrafish RIP1, zebrafish TBK1, mouse TRAF6,
and zebrafish TRAF6) were subcloned in pCMV3Tag9 (Stratagene). Zebrafish
TLR3 was subcloned in pcDNA3.1D/V5-His-TOPO with a hemagglutinin
(HA) tag and a stop codon introduced at the 3⬘ end by PCR. pcDNA3.1-p35
was transfected into cells used in coimmunoprecipitation experiments to
counter the apoptotic effects of TICAM1. pAdVAntage plasmid (Promega)
was included in each coimmunoprecipitation to bolster protein translation.
293H cells (Invitrogen Life Technologies) were cultured at 37°C, 6% CO2
in DMEM (high glucose) supplemented with 10% heat-inactivated FBS.
ZFL cells were grown in sealed vessels at 28°C in LDF medium, which
consists of 50% Leibovitz’s L-15 medium, 35% DMEM (high glucose),
and 15% F-12 nutrient mixture (Ham) supplemented with 5% heat-inactivated FBS, 0.5% heat-inactivated SeaGrow trout serum (East Coast Biologics), 50 ng ml⫺1 mouse epidermal growth factor, and 1⫻ insulin-transferrin-selenium-X. Unless otherwise noted, all culture products were
purchased from Invitrogen Life Technologies.
Representatives from a broad range of TIR domain-containing proteins
were aligned with DIALIGN (24), a multiple sequence alignment program
that compares amino acid sequences via a segment-to-segment approach
with no gap penalties imposed. This alignment method is useful in comparing proteins with similar domains that are otherwise unrelated. Molecular phylogenetic analyses were performed using PHYLIP, software version
3.6b (distributed by the author, J. Felsenstein, University of Washington, Seattle, WA at http://evolution.gs.washington.edu/phylip.html) (25). Sequences
were bootstrapped 1000 times with the program SEQBOOT and these
bootstrapped amino acid sequences were used to compute distance matrices with the program PROTDIST, according to the Jones-Taylor-Thornton
(26) model of amino acid replacement. Phylogenetic trees based upon these
data were generated by the neighbor-joining method (27), using the program NEIGHBOR, and from these trees, an extended majority rule consensus tree was created with the program CONSENSE.
293H and ZFL cells were plated in 24-well plates (Corning) so that they
were 90 –95% confluent on the day of transfection. Using Lipofectamine
2000 (Invitrogen Life Technologies), 400 ng of TICAM1 construct (mouse
TICAM1, zebrafish TICAM1, or indicated deletion construct), 400 ng of
reporter construct (NF-␬B: pBIIx-luc; ISRE: ISRE-luc; zebrafish minimal
Mx (ISRE) promoter: Mx187-luc; or zebrafish IFN promoter: pGL3-IFN
Pro), and 10 ng of pRL-CMV, which served as a Renilla luciferase internal
control to normalize data, were used to transfect 293H or ZFL cells. At
24 h posttransfection, 293H cells were lysed and luciferase activities were
measured using the Dual-Luciferase Reporter Assay system (Promega). At
48 h posttransfection, ZFL cells were lysed and luciferase activities were
measured using the Dual-Luciferase Reporter Assay system. All firefly
luciferase light outputs were normalized to Renilla luciferase activities.
Data are represented as fold induction over empty vector control. Error bars
indicate the SEM for ⱖ3 replicates in a representative experiment.
Coimmunoprecipitation
293H cells were plated in 25-cm2 flasks (Corning) so that they were 90 –
95% confluent on the day of transfection. Using Lipofectamine 2000, cells
were transfected with indicated amounts of plasmids totaling 8 ␮g (3 ␮g of
zebrafish or mouse TICAM1 construct, 3 ␮g of interactor construct, 1 ␮g
of pAdVAntage, and 1 ␮g of pcDNA3.1-p35). At 48 h posttransfection,
cells were washed twice with PBS (Invitrogen Life Technologies) and
lysed for 20 min in ice-cold buffer containing 50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 into which a general
use protease-inhibitor mixture containing 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, aprotinin, bestatin HCl, E-64, EDTA, and leupeptin hemisulfate (Sigma-Aldrich) was added to 10% of total volume.
Cell lysates were centrifuged at 16,000 ⫻ g for 15 min and soluble fractions were collected. Soluble lysates were transferred to plugged Handee
MiniSpin columns (Pierce) containing 10 ␮l of packed gel volume of
washed anti-FLAG M2 or anti-HA agarose affinity gel resin (SigmaAldrich). Samples were incubated on a rotating platform overnight at 4°C.
Following overnight incubation, samples were washed eight times in 25
mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.05% Tween 20, and eluted in 40
␮l of 2⫻ lane marker nonreducing sample buffer (Pierce) during a 5-min
incubation at 100°C. Samples were reduced in 2-ME, separated by PAGE,
and transferred to BioTrace nitrocellulose membranes (Pall Corporation)
using the Mini Trans-Blot cell (Bio-Rad). Membranes were blocked in
TBS (pH 7.5) with 0.1% Tween 20 containing 5% nonfat dried milk and
exposed to rabbit anti-FLAG polyclonal (Affinity Bioreagents), anti-HA
polyclonal (Invitrogen Life Technologies), or anti-myc polyclonal Ab (Affinity Bioreagents) overnight at 4°C. Following overnight incubation,
membranes were washed three times in TBST and incubated in goat antirabbit secondary Ab conjugated to alkaline phosphatase (Bio-Rad) for 1 h
at room temperature. Membranes were washed three times in TBST. CDPStar AP substrate (Novagen) was applied to membranes according to the
manufacturer’s recommendations and the membranes were exposed
to film.
Results
Identification of a full-length zebrafish TICAM1 ortholog
exhibiting considerable sequence divergence from mammalian
orthologs
The TIR domain of the zebrafish TICAM1 was originally described by Meijer et al. (28). The TICAM1 TIR domain cDNA
sequence was used to search zebrafish expressed sequence tag and
genome resources. A predicted full-length TICAM1 sequence was
identified using the online resources of the Zebrafish Genome
Project and zebrafish TICAM1 was cloned. The open reading frame
of zebrafish TICAM1 is 1701 bp. Comparisons of syntenic regions
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Phylogenetic reconstruction
Luciferase reporter assays
The Journal of Immunology
4519
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 1. TICAM1 alignment and domain architecture. A, Alignment of human, mouse, and zebrafish TICAM1 orthologs using AlignX (Vector NTI;
Invitrogen Life Technologies), a ClustalW-based algorithm. Dashes indicate gaps in amino acid sequence. The TRAF6-binding motif, TIR domain, and
RHIM domain are identified by underlines and annotations. The arrow above the sequence Met-Ile-Gly-Asn at positions 547–550 identifies the amino acid
residues targeted by QuikChange mutagenesis to make the Ala-Ala-Ala-Ala RHIM domain mutant. Zebrafish TICAM1 is 566 aa; human TICAM1 is 712
aa; and mouse TICAM1 is 732 aa. B, Both the N- (1–318 aa) and C-terminal (456 –566 aa) domains of zebrafish TICAM1 are truncated, calling into question
their capacity to interact with homologs of proteins known to interact in mammals.
among zebrafish, mouse, and human genomes indicate that zebrafish TICAM1 is orthologous to mammalian TICAM1s. Despite
this conserved synteny, the zebrafish TICAM1 gene encodes a
smaller protein (566 aa) than does the mouse (732 aa) or human
ortholog (712 aa). A ClustalW-based alignment (AlignX, Vector
NTI; Invitrogen Life Technologies) identified significant gaps in
the N and C termini of zebrafish TICAM1 amino acid sequence
when compared with human and mouse, raising questions about
zebrafish TICAM1’s capacity to transduce downstream signals
(Fig. 1A). Furthermore, no identifiable TRAF6-binding motif at
consensus positions 265–270 was identified in zebrafish TICAM1.
Some sequence divergence was also noted in the BB loop of TIR
domain and the RHIM domain (consensus positions 682–720),
raising questions about zebrafish TICAM1’s capacity to interact
with TLR3 and RHIM domain-containing proteins (RIP1, RIP3),
respectively. Zebrafish TICAM1 shares 26% sequence identity
and 46% sequence similarity with human and mouse across
aligned residues, excluding gaps. Zebrafish TICAM1 is comprised of 318-aa N terminus, a 137-aa TIR domain, and a 110-aa
C terminus (Fig. 1B).
4520
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 2. TICAM1 and TICAM2
share a close evolutionary relationship.
A, Zebrafish TICAM1 forms a monophyletic group with mouse and human
TICAM1 and mouse and human
TICAM2, exhibiting 100% bootstrap
support. Representatives from a broad
range of TIR domain-containing proteins were aligned with DIALIGN
(55), a multiple sequence alignment
program that compares amino acid sequences via a segment-to-segment approach with no gap penalties imposed. All bootstrap values ⬍91% are
shown while those ⱖ91% are not
shown. B, The TICAM paralogon
from the human genome. Segments of
chromosomes 1, 5, 15, and 19 are
paralogous. Data from National Center for Biotechnology Information
Gene Database: (www.ncbi.nlm.nih.
gov/entrez/query.fcgi?db ⫽ gene).
TICAM SIGNALING DIFFERS IN BASALLY DIVERGING VERTEBRATES
The Journal of Immunology
4521
TICAM1 and TICAM2 evolved in a gene duplication event
Mammalian TLRs use four different TIR domain-containing adaptor proteins to transduce signals: MyD88, Mal, TICAM1, and
TICAM2 (TRAM) (28, 29). All but TICAM2 have been identified in
fish, and accumulating evidence indicates that TICAM2 does not exist
in fishes (30). Phylogenetic analysis demonstrates that TICAM1 and
TICAM2 form a monophyletic group with 100% bootstrap support
(Fig. 2A). These findings are supported by Iliev et al. (30) and suggest
that TICAM1 and TICAM2 are sisters from a gene duplication event.
The TICAM gene pair is part of a paralogon, or a group of paralogous
regions within the same species (31, 32), in the human genome (Fig.
2B). The paralogon includes Hsa19p13 with FEM1A, TICAM1,
TMED1, and SEMA6B; Hsa5q21-q23 with FEM1C, TICAM2,
TMED7, and SEMA6A; Hsa15q21-q25 with FEM1B, TMED3, and
SEMA6D; and Hsa1q21-q22 with SEMA6C and other genes. These
four paralogous chromosome segments probably originated in the two
rounds of genome duplication that likely preceded the vertebrate radiation (33).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 3. Comparative genomics
of TICAM regions. Comparisons
between human and zebrafish genomes show regions in each containing TICAM1 (A) or TICAM2 (B)
and the near-neighbors. Zebrafish
data are from the Zv6 version of the
zebrafish genome (www.ensembl.
org/Danio_rerio/index.html).
The zebrafish TICAM1 gene on Chr:22 (ENSDART
00000062685) is embedded in one of two zebrafish duplicates of
the region of Hsa19p13 that surrounds TICAM1, with orthologous
near-neighbors in both genomes (Fig. 3A, bottom panel). The other
copy of the human region lies on zebrafish chromosome 2 in the
Zv6 version of the zebrafish genome (Fig. 3A, top panel). Both
zebrafish duplicate regions include genes slightly further away on
Hsa19p13.3 (Fig. 3A, middle panel). The genomic neighborhood
of TICAM2 is better conserved in zebrafish than is the TICAM1containing region, despite the lack of a TICAM2 ortholog (Fig.
3B). Genes that immediately neighbor TICAM2 in the human genome have orthologs that are neighbors in the zebrafish genome
(FEM1C and TMED7), but without an ortholog of TICAM2 between them (Fig. 3B). In humans, a sequence appropriate to encode
a human membrane-bound isoform of TICAM2 has been identified
but not described (GenBank accession no. AY304581). It apparently occurs through an alternative splicing event that combines
the open reading frame of TMED7 with TICAM2, its 3⬘ neighbor.
4522
TICAM SIGNALING DIFFERS IN BASALLY DIVERGING VERTEBRATES
FIGURE 4. A hypothesis for the history of TICAM
genes. R1, First round of gene duplication; R2, second
round of gene duplication. A, Predicted portion of a
chromosome from the last pregenome duplication common ancestor of vertebrates possessed genes in following order: FEM1, TICAM, TMED, SEMA6, and KCNN.
B, Following the first round of genome duplication (Post
R1), one of the TICAM genes was lost. C, After the
second round of genome duplication (Post R2), the
genes that became TICAM1 and TICAM2 appeared. In
the preduplication teleost (D), the TICAM2 gene was
lost, but the TICAM1 gene was retained. In humans (E),
TICAM1 and TICAM2 were both retained and evolved
distinct functions in TLR signaling.
and TMED7 are adjacent in the frog would suggest that the gene
has been lost from the frog genome in an event independent from
the loss from the fish genomes. Given that rayfin fish genomes lack
TICAM2 (30), this gene may have been lost early after the divergence of rayfin and lobefin fishes 450 million years ago.
Zebrafish TICAM1 fails to interact with TRAF6 but can
associate with TLR3, RIP1, and TBK1
Mammalian TICAM1 interacts with TLR3, TRAF6, TBK1, and
RIP1 (15, 17, 40, 41) and these interactions potentiate downstream
antiviral signaling, leading to the activation of NF-␬B and IFN-␤.
Zebrafish TICAM1’s unique structural features, particularly the
presence of a hydrophilic glutamine residue in the typically hydrophobic ␪1 position, the absence of an obvious TRAF6-binding
motif, the truncation of its N- and C-terminal domains, and the
sequence differences observed in the RHIM domain (Fig. 1), call
into question its capacity to interact with TLR3, TRAF6, TBK1,
and RIP1 and transduce vital immune signals. To address zebrafish
TICAM1’s capacity to associate with these known interactors, coimmunoprecipitation experiments were performed (Fig. 5).
Zebrafish TLR3-HA and zebrafish TICAM1–3⫻FLAG were
overexpressed in 293H cells for 48 h, and following a coimmunoprecipitation experiment, were shown to associate with one another (Fig. 5A, middle panel, lane 1). Empty vector controls were
included to show that the interactions were not the result of nonspecific interactions (Fig. 5A, middle panel, lanes 2 and 3).
Through coimmunoprecipitation, it was shown that zebrafish
TICAM1 associated with zebrafish RIP1 (Fig. 5B, middle panel,
lane 1) when overexpressed in 293H cells. Despite its truncated N
terminus, zebrafish TICAM1 also is capable of interacting with
zebrafish TBK1 (Fig. 5C, lower middle panel, lane 1). Like mammals, this interaction relies on the N terminus of TICAM1, as a
TICAM1 protein lacking the N terminus failed to associate with
TBK1 (Fig. 5C, lower middle panel, lane 3). Despite the similarity,
the functional significance of the TICAM1-TBK1 interaction in
zebrafish, based upon the observations described later, is in question. In contrast, following overexpression in 293H cells, zebrafish
TICAM1 failed to coimmunoprecipitate zebrafish TRAF6 or
mouse TRAF6 (Fig. 5D, middle panel, lanes 1 and 2). Analysis of
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
To further test for the presence of a zebrafish ortholog of TICAM2,
we performed 3⬘ RACE to determine whether such a membranebound form of TICAM2 could be identified in zebrafish. A single
fragment was amplified, but analysis showed it to consist solely of
sequences orthologous to TMED7 (data not shown).
Comparative genomic analysis of available sequence data reveals that in the opossum (Monodelphis domesticus), a marsupial
diverging from the placental lineage ⬃175 million years ago (34),
part of ENSMODT0000001740 on chromosome 6 is orthologous
to TICAM2 and part to TMED7; the opossum FEM1C ortholog
(ENSMODT00000019962) is unlinked, appearing on chromosome
3. In Xenopus, as in zebrafish, FEM1C (ENSXETT00000040604)
and TMED7 (ENSXETT00000040594) genes are adjacent to each
other, but TICAM2 does not appear between them. In chicken
(Gallus gallus), TMED7 (ENSGALT00000034557) is on chromosome Z, but FEM1C (ENSGALT00000013361) is unassigned
making it difficult to draw conclusions. The nonvertebrate chordate
Ciona intestinalis has an ortholog of TICAM genes (ENSCINT
00000022578), of TMED7/3/1 (ENSCINT00000003864), and of
FEM1 genes (ENSCINT00000027118). These three genes are not
adjacent in the Ciona genome, but the FEM1 ortholog has loci that
are close neighbors with loci that are close neighbors of FEM1C
and FEM1B in human.
These genomic data suggest the following history for TICAM1
and TICAM2 (Fig. 4). A chromosome in the last pregenome
duplication common ancestor of vertebrates possessed FEM1,
TICAM, and TMED genes in that order, in addition to SEMA6,
KCNN, and other genes (Fig. 4A). After the first round of genome
duplication (R1), the TICAM gene on one of the duplicate chromosomes was lost (Fig. 4B), and after the second round of genome
duplication (R2), produced what became TICAM1 and TICAM2
(Fig. 4, C and E). In the teleost lineage, the TICAM2 gene was
eventually lost, but TICAM1 was retained (Fig. 4D). Some of the
other genes in the paralogon are present in duplicate copy in teleosts today, due to a genome duplication event in the teleost ancestry ⬃300 million years ago (35–39) (data not shown in the
figure). According to this proposed history, the current absence of
TICAM2 from the genome databases of chicken and Xenopus
could be due either to its absence from the animals’ genomes or to
a hole in the genome libraries, although the finding that FEM1C
The Journal of Immunology
4523
zebrafish genome databases fails to uncover a sequence for a second TRAF6 gene, and thus the potential for another TRAF6 coevolving with TICAM1 in a way that would allow for their proteinprotein interaction to be maintained appears extremely unlikely.
Zebrafish TRAF6 exhibits 58% identity and 73% conservation
with mouse TRAF6. Mouse TICAM1 associated both with zebrafish and with mouse TRAF6 (Fig. 5D, middle panel, lanes 4 and
5). These data, coupled with the alignment data, imply that zebrafish TICAM1 does not interact with zebrafish TRAF6 and that
NF-␬B activation occurs through an alternative mechanism. Based
upon the coimmunoprecipitation data collected, the evidence indicates that NF-␬B activation may occur through a TICAM1-RIP1
association.
Zebrafish TICAM1 activates NF-␬B in human 293H and
zebrafish ZFL cells
Alignment of zebrafish TICAM1 with mammalian orthologs reveals clear sequence differences that may confound its capacity to
transduce signals (Fig. 1A). Through overexpression of TICAM1
and various deletion constructs, it has been shown that full-length
human TICAM1 can activate NF-␬B, IFN-␤ promoter, and ISRE
luciferase reporters and that individual TICAM1 domains (N terminus, C terminus, and/or TIR domains) contribute differentially
to this signaling capacity (13, 40, 42). To examine zebrafish
TICAM1 and its domains’ signaling capacities, full-length zebrafish TICAM1, along with several zebrafish TICAM1 domain
deletion constructs, a zebrafish TICAM1 RHIM mutant construct,
and a mouse TICAM1-positive control, were overexpressed in hu-
man 293H and zebrafish ZFL cells to assay their effects on NF-␬B
activation, using the reporter construct pBIIx-luc (43) (Fig. 6, A
and B). In 293H cells (Fig. 6A), full-length zebrafish TICAM1
retained a capacity to activate NF-␬B (9.3-fold induction), albeit at
levels below that of full-length mouse TICAM1 (21.6-fold induction). Similarly in ZFL cells (Fig. 6B), full-length TICAM1 activated NF-␬B (3.3-fold induction), but in this instance, mouse
TICAM1 exhibited diminished NF-␬B activation (1.2-fold induction). Overexpression of a zebrafish TICAM1 N-terminal deletion
construct enhanced NF-␬B activation over zebrafish full-length
TICAM1 in both 293H and ZFL cells, with 24.2-fold (Fig. 6A) and
4.2-fold (Fig. 6B) activations noted, respectively. These data indicate that the TIR and C-terminal domains of zebrafish TICAM1
are responsible for NF-␬B activation and are corroborated by the
diminished activities noted upon overexpression of deletion constructs missing both the N and C termini (ZFL: 1.4-fold induction;
293H: 1.1-fold induction) or the C-terminal domain (ZFL: 2.0-fold
induction; 293H: 2.1-fold induction) (Fig. 6, A and B). Deletion of
the TIR domain also diminished NF-␬B activation in ZFL cells
(1.4-fold induction) (Fig. 6B), but appeared not to disrupt NF-␬B
signaling in 293H cells (11.2-fold induction), when compared with
the full-length TICAM1 result (9.3-fold induction) (Fig. 6A). Furthermore, site-directed mutagenesis of residues 547–550 (MetIle-Gly-Asn) in the RHIM domain of zebrafish TICAM1, which
correspond to residues 687– 690 in human TICAM1 and 688 –
691 in mouse TICAM1 (Val-Gln-Leu-Gly), to Ala-Ala-AlaAla, as previously demonstrated by Meylan et al. (40), led to a
diminished capacity for zebrafish TICAM1 to activate NF-␬B
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 5. Zebrafish TICAM1 associates with TLR3, RIP1, and TBK1, but not TRAF6. A, Zebrafish TICAM1 (middle panel, lane 1) coimmunoprecipitates with zebrafish TLR3 (top panel, lane 1). B, Zebrafish RIP1 (middle panel, lane 1) coimmunoprecipitates with zebrafish TICAM1 (top panel, lane
1). C, Zebrafish TBK1 (lower middle panel, lane 1) coimmunoprecipitates with zebrafish TICAM1 (top panel, lane 1). Zebrafish TICAM1 lacking the
N-terminal residues (top middle panel, lane 3) fails to associate with zebrafish TBK1 (lower middle panel, lane 3), despite evidence of strong expression
in whole cell lysates. D, Zebrafish TRAF6 (middle panel, lanes 1) fails to coimmunoprecipitate with zebrafish TICAM1 (top panel, lane 1), despite strong
expression levels in the whole cell lysate (bottom panel, lane1). Similarly, mouse TRAF6 fails to coimmunoprecipitate with zebrafish TICAM1 (top panel,
lane 2), despite strong expression levels in the whole cell lysate (bottom panel, lane 2). Mouse TICAM1 coimmunoprecipitates with both zebrafish TRAF6
(middle panel, lane 4) and mouse TRAF6 (middle panel, lane 5).
4524
TICAM SIGNALING DIFFERS IN BASALLY DIVERGING VERTEBRATES
(2.4-fold activation over empty vector control and 3.9-fold reduction compared with wild-type full-length TICAM1) (Fig.
6A). Collectively, these data demonstrate an essential role for
the C-terminal RHIM domain in zebrafish TICAM1-mediated
NF-␬B activation.
Zebrafish TICAM1 activation of type I IFN occurs through
N-terminal-independent and ISRE-independent mechanisms
Mammalian TICAM1 has been shown to play an essential role in
TLR3- and TLR4-mediated IFN-␤ activation through an IRF3/
IRF7-dependent mechanism (12, 16, 44, 45). TBK1 is brought into
a complex with TICAM1 at its N terminus via TRAF3 and phosphorylates IRF3 and IRF7, allowing for its eventual nuclear
translocation and up-regulation of IFN-␤ (15, 17, 19 –21).
Overexpression of zebrafish TICAM1 led to minimal activation
of an ISRE-luciferase reporter in 293H cells (2.6-fold above
empty vector) (Fig. 6C) and no activation in ZFL cells (Fig.
6D). Similarly, using the zebrafish-derived Mx187-luc construct,
which is a minimal IFN-inducible Mx promoter construct possessing two ISREs, a 3.4-fold induction over empty vector control was
observed in ZFL cells (Fig. 6E). In contrast, mouse TICAM1 overexpression robustly activated ISRE-luciferase reporter in 293H
cells (136-fold above empty vector) (Fig. 6C); however, when
compared with the 293H data, it exhibits minimal activation of
ISRE in ZFL cells (ISRE-luc: 1.2-fold above empty vector;
Mx187-luc: 4.9-fold above empty vector) (Fig. 6, D and E). Despite the unexpected absence of robust ISRE activation in ZFL
cells, as seen in 293H cells, zebrafish type I IFN was activated by
zebrafish TICAM1 (9.5-fold induction) (Fig. 6F). Mouse TICAM1
strongly activated the zebrafish type I IFN promoter (22.9-fold
induction) (Fig. 6F). Overexpression of the zebrafish TICAM1 Nterminal deletion construct led to an even more enhanced type I
IFN activation (16.9-fold induction) than with full-length zebrafish
TICAM1 (Fig. 6F). These results correlated with the observed
NF-␬B activation, in which the N-terminal deletion construct exhibited stronger activation (Fig. 6, A and B). The C-terminal deletion retained a capacity to activate IFN promoter (Fig. 6F) (10.8fold induction), but it appeared not to be strongly ISRE-induced in
293H cells (2.4-fold induction) or ZFL cells (ISRE-luc: 1.7-fold
induction; Mx187-luc: 3.4-fold induction) (Fig. 6, D and E). Interestingly, site-directed mutagenesis of the C-terminal RHIM domain at residues 547–550 from Met-Ile-Gly-Asn to Ala-Ala-AlaAla disrupted IFN promoter activation (4.0-fold induction over
empty vector control), leading to a 2.4-fold reduction from the
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 6. Overexpression of zebrafish TICAM1 activates NF-␬B and type I IFN promoter, but in an ISRE-independent manner. Luciferase assays
demonstrate the effect of mouse TICAM1, zebrafish TICAM1, and zebrafish TICAM1 mutants on NF-␬B activation in 293H (A) and ZFL (B) cells. A RHIM
domain mutation causes a decrease in NF-␬B-luciferase activation relative to the control zebrafish TICAM1 in 293H cells (A). C, Unlike mouse TICAM1,
zebrafish TICAM1, and deletion mutants demonstrate limited ISRE-luciferase activation in 293H cells. D and E, In ZFL cells, minimal ISRE-luc and
Mx187-luc activation is observed upon overexpression of mouse TICAM1, zebrafish TICAM1, and zebrafish TICAM1 deletion constructs. F, Despite the
absence of strong ISRE-luciferase activity, type I IFN promoter-luciferase activation is observed upon overexpression of mouse TICAM1, zebrafish
TICAM1, and zebrafish TICAM1 deletion constructs. All firefly luciferase activities are normalized to constitutively active Renilla luciferase levels. Data
are presented as fold induction over empty vector. Error bars represent one SEM. Empty, empty vector control; Mouse, full-length mouse TICAM1, Danio,
full-length zebrafish TICAM1; ⌬N, N-terminal deletion (1–311 aa) of zebrafish TICAM1; ⌬C, C-terminal deletion (485–566 aa) of zebrafish TICAM1;
⌬TIR, deletion of TIR domain (312– 482 aa) of zebrafish TICAM1; ⌬N⌬C, deletion of N- and C-termini of zebrafish TICAM1 leaving TIR domain
(312– 482 aa); RHIM Mutant, site-directed mutagenesis of amino acids 547–550 from Met-Ile-Gly-Asn to Ala-Ala-Ala-Ala.
The Journal of Immunology
Discussion
Phylogenetic analyses imply that fish type I IFNs form clades distinct from those of other mammalian and avian type I IFNs and
thus cannot be classified as being ␣- or ␤-like, except by function
(1, 2). These findings, coupled with the unique gene structure of
fish type I IFNs, raise interesting questions about how they are
triggered to signal and whether TLR-mediated pathways are responsible for their induction. In describing the first fish type I IFN,
which we cloned from the zebrafish, our laboratory noted its antiviral potential, as it was inducible, in ZFL cells, by the TLR3
agonist polyinosinic-polycytidylic acid (poly(I:C)) and was protective of cells infected with snakehead rhabdovirus (1). Subsequently, we noted that zebrafish type I IFN exerts its antiviral
effects through the induction of antiviral proteins like Mx (22) and
can itself be induced in vivo by snakehead rhabdovirus (46).
The findings described herein illustrate a TICAM1-dependent
mechanism by which fish type I IFN expression can be induced. In
mammals, TLR3 and TLR4 signaling through TICAM1 leads to
the specific up-regulation of IFN-␤. Direct IFN-␣ up-regulation
through TICAM1-dependent pathways has yet to be described and
indeed may not occur. Furthermore, based upon the current literature, direct IFN-␣ induction appears restricted to MyD88-dependent signaling pathways (6). These findings are interesting in light
of the fact that zebrafish type I IFN, as already discussed, cannot
be defined as ␣- or ␤-like in terms of phylogeny but may be considered ␤-like in terms of its induction by TICAM1 (Fig. 6F).
Comprehensive analyses of syntenies and phylogenies from representative organisms of each vertebrate class provide excellent
insight into how TLR signaling has evolved (Figs. 2 and 3). For
example, our data confirm a recent hypothesis proposed by Iliev
et al. (30) about TICAM2’s absence in fish and goes further by
indicating that TICAM2 most likely arose from an early gene duplication (Fig. 4) and then was lost after the divergence of rayfin
and lobefin fishes 450 million years ago. Our inability to identify
TICAM2 in the chicken and Xenopus leaves open the possibility
that TICAM2 was lost from each of these genomes in events independent of TICAM2’s loss in the fish genomes. Indeed, in Xenopus, the maintained arrangement of the flanking genes FEM1C
and TMED7 and the absence of TICAM2 makes this a likely scenario. Unfortunately, similar evidence could not be derived from
the chicken genome due to sequence gaps. Although our hypothesis is not the most parsimonious, it is clear, based upon the history
of the TICAM genes (Fig. 4), that this explanation is the most
likely in describing what has happened to TICAM2 at each vertebrate class, if they indeed have been lost. Thus, it is a strong possibility that fishes, and perhaps amphibians and birds, rely solely
upon TICAM1, MyD88, and Mal to transduce signals from TLRs.
It is possible that additional TIR domain adaptor proteins may
exist, but this appears unlikely based upon the plethora of genome
data available for representatives of each vertebrate class. The significance of TICAM2’s absence in basally diverging vertebrates
clearly needs to be explored, as it has been shown to be essential
to TLR4-mediated IFN-␤ induction in mammals. It is also clear
that the role of MyD88, Mal, and TICAM1 in lower vertebrate
TLR signaling also needs to be fully characterized. The data presented here attempt to describe TICAM1’s function in zebrafish
TLR signaling, beginning with an experiment designed to test
whether zebrafish TICAM1 can interact with zebrafish TLR3, as it
does in mammals.
TLR3 recognizes dsRNA (3) and endogenous cellular RNA (47)
and unleashes a potent MyD88-independent antiviral pathway (12,
13). Human TLR3 is a 904-aa long protein expressed on the cell
surface of human fibroblast epithelial cells (48) and within dendritic cells (49, 50). It possesses a horseshoe-shaped ectodomain
consisting of 23 leucine-rich repeats (51, 52), a transmembrane
domain, and a TIR domain required for downstream signaling (3).
TLR3 orthologs have been cloned from a broad range of vertebrates, from zebrafish to humans (53). The actual role of TLR3 in
mediated antiviral responses is under scrutiny (54); however, specific evidence does indicate a real immunological function. Specifically, TLR3 is believed to interact with dsRNA intermediates of
single-stranded RNA viruses like respiratory syncytial virus (55)
or West Nile virus (56). Interactions were also noted between
TLR3 and mouse CMV, a DNA virus (57), and TLR3 and reovirus, a dsRNA virus (3, 54). Interestingly, TLR3 was shown to be
activated in response to nonviral dsRNAs as well. dsRNAs from
eggs of the helminth parasite Schistosoma mansoni were shown to
trigger an immune response through the TLR3 signal transduction
cascade (58). TLR3 activation by ligands appears to occur in the
acidic compartments of early phagolysosomes or endosomes, as an
acid pH is required (59). Disruption of acidic pH blocked TLR3
activation by poly(I:C). Additionally, TLR3 must form multimers
to be active (59). Upon activation, the antiviral signal cascades
through the adaptor molecule TICAM1 (12, 13). The TLR3TICAM1 interaction requires their respective BB loops. The consensus sequence for the BB loop, a conserved sequence contained
within the TIR domain and shown to be important for TLR and
TICAM1 signaling, is RDx␾1␾2G, where x represents any residue
and ␾ represents any hydrophobic residue. The mutation of the ␾2
residue from proline to histidine results in the inability of the human TICAM1 to interact with human TLR3 and thereby up-regulate human IFN-␤ (12). Similarly, when the ␾2 position of human
TLR3 was mutated from alanine to histidine, TLR3 failed to interact with human TICAM1 and therefore failed to up-regulate
IFN-␤ (12). The BB loop of the putative zebrafish TICAM1 differs
from the consensus sequence and from the mammalian TICAM1s
(Fig. 1A). The proline at ␾2 is conserved in known mammalian
TICAM1s. In the putative zebrafish TICAM1, this position contains an alanine instead. In addition, in zebrafish, ␾1 is occupied by
a glutamine, and the x position contains an alanine. We believed
that the differences noted in these positions may be important to
TLR3-TICAM1 signaling in zebrafish; however, our coimmunoprecipitation data indicate that TLR3 and TICAM1 can associate
and thus this interaction appears conserved despite sequence differences (Fig. 5A). It is noteworthy that human and mouse TLR3
contain alanines in the ␾2 positions, while human and mouse
TICAM1 contain prolines in their ␾2 positions. These occurrences
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
wild-type TICAM1 and 2.7-fold reduction from the C-terminal
deletion TICAM1 construct (Fig. 6F). The apparent difference in
IFN promoter activation capacity between the C-terminal deletion
TICAM1 construct and the RHIM mutant indicates not only an
activating role for the RHIM domain in IFN activity, but also a
possible inhibitory role for other portions of the C terminus. Constructs lacking the TIR domain retained a capacity to activate the
IFN promoter construct (6.7-fold induction) (Fig. 6F), but again
exhibited minimal ISRE activation in 293H (1.9-fold induction)
(Fig. 6C) or ZFL (ISRE-luc: 1.5-fold induction; Mx197-luc: 2.0fold induction) cells (Fig. 6, D and E). Overexpression of the combined N-terminal, C-terminal deletion, which consists largely of
the TIR domain alone, resemble the data presented by Yamamoto
et al. (13). The IFN promoter construct was activated 2.4-fold in
ZFL cells (Fig. 6F); the ISRE-luc reporter was induced 1.5-fold in
293H cells (Fig. 6C) and 1.3-fold in ZFL cells (Fig. 6D); the
Mx187-luc reporter was activated 1.1-fold above empty vector
control (Fig. 6E). These data show that the TIR domain of zebrafish TICAM1, when expressed alone, has a dominant-negative
effect.
4525
4526
TICAM SIGNALING DIFFERS IN BASALLY DIVERGING VERTEBRATES
deletion of the C terminus of TICAM1 did not alter IFN promoter activation in ZFL cells, mutation of the C-terminal RHIM
domain at residues 547–550 from Met-Ile-Gly-Asn to Ala-AlaAla-Ala resulted in a diminished capacity for IFN activation
(Fig. 6F). These findings show a clear role for the RHIM domain in IFN activation but also indicate that other portions of
the C terminus may negatively regulate IFN activation. Further
investigation of the zebrafish pathway may provide the context
necessary to resolve some of the discrepancies noted in characterizing the role and importance of TICAM1’s domains in
tetrapods.
Acknowledgments
We thank Brian Niland, Akshata Nayak, and Jeremy Charette for their
technical assistance. This is Maine Agricultural and Forest Experiment
Station publication 2923.
Disclosures
The authors have no financial conflict of interest.
References
1. Altmann, S. M., M. T. Mellon, D. L. Distel, and C. H. Kim. 2003. Molecular and
functional analysis of an interferon gene from the zebrafish, Danio rerio. J. Virol.
77: 1992–2002.
2. Robertsen, B. 2006. The interferon system of teleost fish. Fish Shellfish Immunol.
20: 172–191.
3. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition
of double-stranded RNA and activation of NF-␬B by Toll-like receptor 3. Nature
413: 732–738.
4. Diebold, S. S., T. Kaisho, H. Hemmi, S. Akira, and C. Reis e Sousa. 2004. Innate
antiviral responses by means of TLR7-mediated recognition of single-stranded
RNA. Science 303: 1529 –1531.
5. Heil, F., H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning, S. Akira,
G. Lipford, H. Wagner, and S. Bauer. 2004. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 303: 1526 –1529.
6. Kawai, T., S. Sato, K. J. Ishii, C. Coban, H. Hemmi, M. Yamamoto, K. Terai,
M. Matsuda, J. Inoue, S. Uematsu, et al. 2004. Interferon-␣ induction through
Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6.
Nat. Immunol. 5: 1061–1068.
7. Lund, J. M., L. Alexopoulou, A. Sato, M. Karow, N. C. Adams, N. W. Gale,
A. Iwasaki, and R. A. Flavell. 2004. Recognition of single-stranded RNA viruses
by Toll-like receptor 7. Proc. Natl. Acad. Sci. USA 101: 5598 –5603.
8. Hoshino, K., T. Kaisho, T. Iwabe, O. Takeuchi, and S. Akira. 2002. Differential
involvement of IFN-␤ in Toll-like receptor-stimulated dendritic cell activation.
Int. Immunol. 14: 1225–1231.
9. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto,
K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor
recognizes bacterial DNA. Nature 408: 740 –745.
10. Kopp, E., and R. Medzhitov. 2003. Recognition of microbial infection by Tolllike receptors. Curr. Opin. Immunol. 15: 396 – 401.
11. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol.
4: 499 –511.
12. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, and T. Seya. 2003. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated
interferon-␤ induction. Nat. Immunol. 4: 161–167.
13. Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Takeuchi, K. Takeda, and
S. Akira. 2002. Cutting edge: a novel Toll/IL-1 receptor domain-containing
adapter that preferentially activates the IFN-␤ promoter in the Toll-like receptor
signaling. J. Immunol. 169: 6668 – 6672.
14. Doyle, S., S. Vaidya, R. O’Connell, H. Dadgostar, P. Dempsey, T. Wu, G. Rao,
R. Sun, M. Haberland, R. Modlin, and G. Cheng. 2002. IRF3 mediates a TLR3/
TLR4-specific antiviral gene program. Immunity 17: 251–263.
15. Sato, S., M. Sugiyama, M. Yamamoto, Y. Watanabe, T. Kawai, K. Takeda, and
S. Akira. 2003. Toll/IL-1 receptor domain-containing adaptor inducing IFN-␤
(TRIF) associates with TNF receptor-associated factor 6 and TANK-binding kinase 1, and activates two distinct transcription factors, NF-␬B and IFN-regulatory
factor-3, in the Toll-like receptor signaling. J. Immunol. 171: 4304 – 4310.
16. Fitzgerald, K. A., D. C. Rowe, B. J. Barnes, D. R. Caffrey, A. Visintin, E. Latz,
B. Monks, P. M. Pitha, and D. T. Golenbock. 2003. LPS-TLR4 signaling to
IRF-3/7 and NF-␬B involves the toll adapters TRAM and TRIF. J. Exp. Med.
198: 1043–1055.
17. Han, K. J., X. Su, L. G. Xu, L. H. Bin, J. Zhang, and H. B. Shu. 2004. Mechanisms of the TRIF-induced interferon-stimulated response element and NF-␬B
activation and apoptosis pathways. J. Biol. Chem. 279: 15652–15661.
18. McWhirter, S. M., K. A. Fitzgerald, J. Rosains, D. C. Rowe, D. T. Golenbock,
and T. Maniatis. 2004. IFN-regulatory factor 3-dependent gene expression is
defective in Tbk1-deficient mouse embryonic fibroblasts. Proc. Natl. Acad. Sci.
USA 101: 233–238.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
are reversed in zebrafish, where TLR3 possesses a proline in the ␾2
position and putative TICAM1 possesses an alanine in its ␾2 position. This “complementary switch” may be noteworthy from an
evolutionary perspective, but may also have practical impacts on
TLR3-TICAM1 interactions and signaling.
In mammals, TBK1 interacts with the N terminus of TICAM1
and then phosphorylates IRF-3 and IRF-7, leading to up-regulation
of IFN-␤ (15–18). Although zebrafish TICAM1 retains the capacity to coimmunoprecipitate TBK1 through its N terminus (Fig.
5C), indicating this interaction can occur, it is unclear what role the
TBK1 interaction plays in the up-regulation of zebrafish type I
IFN. In fact, upon deletion of the N terminus responsible for the
TICAM1-TBK1 interaction in mammals, enhanced IFN activation
was noted (Fig. 6F), indicating that the N terminus, and thus potentially the TBK1 interaction, has one or both of the following
effects: it may facilitate an inhibitory portion of the zebrafish
TICAM1 pathway, or it may sterically hinder TICAM1’s interaction with other protein partners that are yet to be described.
It was noteworthy that zebrafish TICAM1, lacking an apparent
TRAF6-binding motif with the consensus PxExx[Ac/Ar] (P represents proline, x represents any amino acid, and [Ac/Ar] represents
an acidic or aromatic amino acid), failed to coimmunoprecipitate
zebrafish TRAF6 (Fig. 5D). This finding confounds TRAF6’s role
in mediating a NF-␬B response, at least in the zebrafish, and supports the assertion by Gohda et al. (60) that the TICAM1-TRAF6
interaction may be nonessential in the mammalian TLR3 pathway.
Our finding is in contrast to reports by Sato et al. (15) and Jiang
et al. (41), who showed that TICAM1-TRAF6 interaction is important for NF-␬B activation in human 293 cells. Furthermore,
overexpression of an N-terminal deletion zebrafish TICAM1 in
293H and ZFL cells, causing enhanced NF-␬B activation relative
to full-length zebrafish, was noted (Fig. 6, A and B). These data
lend additional credence to an aforementioned potential inhibitory
role the N terminus plays in zebrafish TICAM1. Indeed, our data
indicate that TICAM1 signaling is driven by the TIR domain and
C terminus, with the RHIM domain playing an important role. Our
finding that zebrafish TICAM1 and RIP1 can interact with one
another (Fig. 5B), and that mutation of the TICAM1 RHIM domain at residues 547–550 from Met-Ile-Gly-Asn to Ala-Ala-AlaAla disrupts NF-␬B activation in 293H cells (Fig. 5A), suggests
that this activation occurs because of this association, as is the case
in mammals (40, 42).
The evolutionary history of TLR-mediated induction of type I
IFN responses provides essential perspective into the origins and
complexities of antiviral immunity. The data presented herein represent the first insights into the underlying mechanisms basally
diverging vertebrates use to counter pathogens through TICAM1dependent signaling. Our findings also demonstrate that TICAM1dependent induction of type I IFNs is an ancient mechanism, although the means by which TICAM1 induces this particular
zebrafish type I IFN is through an apparently alternative signaling
pathway. Although the complete details of how TICAM1 induces
this zebrafish type I IFN are yet to be determined, our findings may
be predictive in defining undiscovered TICAM1-dependent, IRF3/
7-independent type I IFN induction schemes in mammals, perhaps
through a RHIM-dependent mechanism. In mammals, the TICAM1TBK1 interaction is essential to type I IFN activation (15). Our data
show that while this interaction also occurs in zebrafish (Fig. 5C), its
importance in mounting the TLR-mediated antiviral response is
unclear. In particular, our surprising finding that a TICAM1
construct lacking the N-terminal domain necessary for interaction with TBK1 exhibited an enhanced activation of the zebrafish type I IFN promoter (Fig. 6F) indicated that other parts
of the TICAM1 protein play a role in this activation. Although
The Journal of Immunology
42. Kaiser, W. J., and M. K. Offermann. 2005. Apoptosis induced by the Toll-like
receptor adaptor TRIF is dependent on its receptor interacting protein homotypic
interaction motif. J. Immunol. 174: 4942– 4952.
43. Saksela, K., and D. Baltimore. 1993. Negative regulation of immunoglobulin ␬
light-chain gene transcription by a short sequence homologous to the murine B1
repetitive element. Mol. Cell Biol. 13: 3698 –3705.
44. Oshiumi, H., M. Sasai, K. Shida, T. Fujita, M. Matsumoto, and T. Seya. 2003.
TIR-containing adapter molecule (TICAM)-2, a bridging adapter recruiting to
Toll-like receptor 4 TICAM-1 that induces interferon-␤. J. Biol. Chem. 278:
49751– 49762.
45. Doyle, S. E., R. O’Connell, S. A. Vaidya, E. K. Chow, K. Yee, and G. Cheng.
2003. Toll-like receptor 3 mediates a more potent antiviral response than Tolllike receptor 4. J. Immunol. 170: 3565–3571.
46. Phelan, P. E., M. E. Pressley, P. E. Witten, M. T. Mellon, S. Blake, and
C. H. Kim. 2005. Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio). J. Virol. 79: 1842–1852.
47. Kariko, K., H. Ni, J. Capodici, M. Lamphier, and D. Weissman. 2004. mRNA is
an endogenous ligand for Toll-like receptor 3. J. Biol. Chem. 279: 12542–12550.
48. Matsumoto, M., S. Kikkawa, M. Kohase, K. Miyake, and T. Seya. 2002. Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks
double-stranded RNA-mediated signaling. Biochem. Biophys. Res. Commun.
293: 1364 –1369.
49. Matsumoto, M., K. Funami, M. Tanabe, H. Oshiumi, M. Shingai, Y. Seto,
A. Yamamoto, and T. Seya. 2003. Subcellular localization of Toll-like receptor
3 in human dendritic cells. J. Immunol. 171: 3154 –3162.
50. Muzio, M., D. Bosisio, N. Polentarutti, G. D’Amico, A. Stoppacciaro,
R. Mancinelli, C. van’t Veer, G. Penton-Rol, L. P. Ruco, P. Allavena, and
A. Mantovani. 2000. Differential expression and regulation of Toll-like receptors
(TLR) in human leukocytes: selective expression of TLR3 in dendritic cells.
J. Immunol. 164: 5998 – 6004.
51. Choe, J., M. S. Kelker, and I. A. Wilson. 2005. Crystal structure of human
Toll-like receptor 3 (TLR3) ectodomain. Science 309: 581–585.
52. Bell, J. K., I. Botos, P. R. Hall, J. Askins, J. Shiloach, D. M. Segal, and
D. R. Davies. 2005. The molecular structure of the Toll-like receptor 3 ligandbinding domain. Proc. Natl. Acad. Sci. USA 102: 10976 –10980.
53. Phelan, P. E., M. T. Mellon, and C. H. Kim. 2005. Functional characterization of
full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio). Mol. Immunol. 42: 1057–1071.
54. Edelmann, K. H., S. Richardson-Burns, L. Alexopoulou, K. L. Tyler,
R. A. Flavell, and M. B. Oldstone. 2004. Does Toll-like receptor 3 play a biological role in virus infections? Virology 322: 231–238.
55. Rudd, B. D., E. Burstein, C. S. Duckett, X. Li, and N. W. Lukacs. 2005. Differential role for TLR3 in respiratory syncytial virus-induced chemokine expression.
J. Virol. 79: 3350 –3357.
56. Wang, T., T. Town, L. Alexopoulou, J. F. Anderson, E. Fikrig, and R. A. Flavell.
2004. Toll-like receptor 3 mediates West Nile virus entry into the brain causing
lethal encephalitis. Nat. Med. 10: 1366 –1373.
57. Tabeta, K., P. Georgel, E. Janssen, X. Du, K. Hoebe, K. Crozat, S. Mudd,
L. Shamel, S. Sovath, J. Goode, et al. 2004. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus
infection. Proc. Natl. Acad. Sci. USA 101: 3516 –3521.
58. Aksoy, E., C. S. Zouain, F. Vanhoutte, J. Fontaine, N. Pavelka, N. Thieblemont,
F. Willems, P. Ricciardi-Castagnoli, M. Goldman, M. Capron, et al. 2005. Double-stranded RNAs from the helminth parasite Schistosoma activate TLR3 in
dendritic cells. J. Biol. Chem. 280: 277–283.
59. de Bouteiller, O., E. Merck, U. A. Hasan, S. Hubac, B. Benguigui, G. Trinchieri,
E. E. Bates, and C. Caux. 2005. Recognition of double-stranded RNA by human
Toll-like receptor 3 and downstream receptor signaling requires multimerization
and an acidic pH. J. Biol. Chem. 280: 38133–38145.
60. Gohda, J., T. Matsumura, and J. Inoue. 2004. Cutting edge: TNFR-associated
factor (TRAF) 6 is essential for MyD88-dependent pathway but not toll/IL-1
receptor domain-containing adaptor-inducing IFN-␤ (TRIF)-dependent pathway
in TLR signaling. J. Immunol. 173: 2913–2917.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
19. Sasai, M., H. Oshiumi, M. Matsumoto, N. Inoue, F. Fujita, M. Nakanishi, and
T. Seya. 2005. Cutting edge: NF-␬B-activating kinase-associated protein 1 participates in TLR3/Toll-IL-1 homology domain-containing adapter molecule-1mediated IFN regulatory factor 3 activation. J. Immunol. 174: 27–30.
20. Oganesyan, G., S. K. Saha, B. Guo, J. Q. He, A. Shahangian, B. Zarnegar,
A. Perry, and G. Cheng. 2006. Critical role of TRAF3 in the Toll-like receptordependent and -independent antiviral response. Nature 439: 208 –211.
21. Hacker, H., V. Redecke, B. Blagoev, I. Kratchmarova, L. C. Hsu, G. G. Wang,
M. P. Kamps, E. Raz, H. Wagner, G. Hacker, et al. 2006. Specificity in Toll-like
receptor signalling through distinct effector functions of TRAF3 and TRAF6.
Nature 439: 204 –207.
22. Altmann, S. M., M. T. Mellon, M. C. Johnson, B. H. Paw, N. S. Trede, L. I. Zon,
and C. H. Kim. 2004. Cloning and characterization of an Mx gene and its corresponding promoter from the zebrafish, Danio rerio. Dev. Comp. Immunol. 28:
295–306.
23. Gibbs, P. D., and M. C. Schmale. 2000. GFP as a genetic marker scorable
throughout the life cycle of transgenic zebra fish. Mar. Biotechnol. 2: 107–125.
24. Morgenstern, B. 2004. DIALIGN: multiple DNA and protein sequence alignment
at BiBiServ. Nucleic Acids Res. 32: W33–W36.
25. Felsenstein, J. 1989. Phylip: phylogeny inference package (version 3.2). Cladistics 5: 164 –166.
26. Jones, D. T., W. R. Taylor, and J. M. Thornton. 1992. The rapid generation of
mutation data matrices from protein sequences. Comput. Appl. Biosci. 8:
275–282.
27. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406 – 425.
28. Meijer, A. H., S. F. Gabby Krens, I. A. Medina Rodriguez, S. He, W. Bitter,
B. Ewa Snaar-Jagalska, and H. P. Spaink. 2004. Expression analysis of the Tolllike receptor and TIR domain adaptor families of zebrafish. Mol. Immunol. 40:
773–783.
29. Jault, C., L. Pichon, and J. Chluba. 2004. Toll-like receptor gene family and
TIR-domain adapters in Danio rerio. Mol. Immunol. 40: 759 –771.
30. Iliev, D. B., J. C. Roach, S. Mackenzie, J. V. Planas, and F. W. Goetz. 2005.
Endotoxin recognition: in fish or not in fish? FEBS Lett. 579: 6519 – 6528.
31. Coulier, F., C. Popovici, R. Villet, and D. Birnbaum. 2000. MetaHox gene clusters. J. Exp. Zool. 288: 345–351.
32. Leveugle, M., K. Prat, N. Perrier, D. Birnbaum, and F. Coulier. 2003. ParaDB:
a tool for paralogy mapping in vertebrate genomes. Nucleic Acids Res. 31: 63– 67.
33. Dehal, P., and J. L. Boore. 2005. Two rounds of whole genome duplication in the
ancestral vertebrate. PLoS Biol. 3: e314.
34. Hedges, S. B., and S. Kumar. 2002. Genomics: vertebrate genomes compared.
Science 297: 1283–1285.
35. Amores, A., A. Force, Y. L. Yan, L. Joly, C. Amemiya, A. Fritz, R. K. Ho,
J. Langeland, V. Prince, Y. L. Wang, et al. 1998. Zebrafish hox clusters and
vertebrate genome evolution. Science 282: 1711–1714.
36. Amores, A., T. Suzuki, Y. L. Yan, J. Pomeroy, A. Singer, C. Amemiya, and
J. H. Postlethwait. 2004. Developmental roles of pufferfish Hox clusters and
genome evolution in ray-fin fish. Genome Res. 14: 1–10.
37. Naruse, K., M. Tanaka, K. Mita, A. Shima, J. Postlethwait, and H. Mitani. 2004.
A medaka gene map: the trace of ancestral vertebrate proto-chromosomes revealed by comparative gene mapping. Genome Res. 14: 820 – 828.
38. Postlethwait, J. H., Y. L. Yan, M. A. Gates, S. Horne, A. Amores, A. Brownlie,
A. Donovan, E. S. Egan, A. Force, Z. Gong, et al. 1998. Vertebrate genome
evolution and the zebrafish gene map. Nat. Genet. 18: 345–349.
39. Taylor, J. S., I. Braasch, T. Frickey, A. Meyer, and Y. Van de Peer. 2003. Genome duplication, a trait shared by 22000 species of ray-finned fish. Genome Res.
13: 382–390.
40. Meylan, E., K. Burns, K. Hofmann, V. Blancheteau, F. Martinon, M. Kelliher,
and J. Tschopp. 2004. RIP1 is an essential mediator of Toll-like receptor 3-induced NF-␬B activation. Nat. Immunol. 5: 503–507.
41. Jiang, Z., T. W. Mak, G. Sen, and X. Li. 2004. Toll-like receptor 3-mediated
activation of NF-␬B and IRF3 diverges at Toll-IL-1 receptor domain-containing
adapter inducing IFN-␤. Proc. Natl. Acad. Sci. USA 101: 3533–3538.
4527