Molecular cloning and functional characterization of chicken toll-like receptors. A single chicken toll covers multiple molecular patterns.

Toll-like receptors (TLR) in the innate immune system have not been identified in non-mammalian vertebrates. Two types of TLR were cloned from a chicken bursa cDNA library using degenerate primers based on the consensus sequences of mouse and Drosophila Toll and designated as chicken TLR (chTLR) type 1 and type 2. Of the nine human TLRs reported to date, these chTLRs showed the highest homology to human TLR2. The extracellular regions of type 1 and type 2 contained a distinct approximately 200-amino acid stretch and were 45.3 and 46.3% homologous to that of human TLR2. The intracellular Toll/interleukin-1R homology domain of type 1 and type 2 was perfectly identical to each other and highly homologous (80.7%) to that of human TLR2. Both types were widely detected by reverse transcriptase-polymerase chain reaction and immunoblotting in various chicken organs, especially those rich in connective tissue. Both genes were mapped to chromosome 4q1.1, suggesting that they arose by gene duplication. By reporter gene assay, type 2 and to a lesser extent type 1, selectively signaled the presence of mycoplasma macrophage-activating lipopeptide-2/M161Ag in the human embryonic kidney 293 cell system. Cotransfection of type 2 and human CD14 or MD-2 into human embryonic kidney 293 cells allowed the response to Escherichia coli lipopolysaccharide (LPS), whereas type 1 did not signal LPS or any other microbial components tested. These results indicated that chTLR type 2 covers two major microbe patterns, lipoproteins and LPS, which are regulated by TLR2 and TLR4 in mammals. In oviparous animals, the duplicated TLRs in the pattern-recognition system may function for host-pathogen discrimination in a manner that is distinct from that in mammals.

The system responsible for sensing unique constituents of microorganisms has been identified as the innate immune sys-tem. It is generally accepted that the typical immune system consisting of both innate and acquired immunity emerged in the bony fish, the jaw-less fish, which are vertebrates, and all invertebrates survive without lymphocytes (1). The mammalian immune system is more complicated than those of invertebrates because of a conjunction of the innate immune system with the acquired immune system, which has been characterized as a mechanism for the recognition of microbe antigens by huge clonal variations and proliferation of lymphocytes. Lymphocyte activation tends to be augmented by the addition of innate immune stimuli. Thus, after connection of the innate and acquired systems, these two systems have become interdependent.
In human and mouse, the dysfunction of the lymphocyte system has very severe consequences (2). Hence, the lymphocyte system has been the main research target in the field of immunology for Ͼ50 years, but the essential requirement of innate immune responses by microbial stimuli for activation of antigen-presenting cells was proposed only a few years ago. Accordingly, insufficient information is available on the innate immune system of vertebrates to determine how the immune system was established. Phylogenic comparison of the innate immune system may further our understanding of this issue.
A major participant in the innate system was recently discovered in Drosophila melanogaster as the Toll receptor (3). In D. melanogaster, Toll and another Toll homologue, 18-Wheeler, participate in discrimination of microbial substances of fungi and bacteria, respectively (3,4). The major output of their signaling pathways is the secretion of anti-microbial peptides, which effectively eliminate invading microorganisms (3)(4)(5). This system should be representative of ancient host defense mechanisms.
The Toll-mediated host defense system has been considered to be conserved across a wide range of species from drosophila to humans (6), and in mammals it is defined as essential for the activation of antigen-presenting cells in the immune system (7,8). The current concept is that Toll-like receptors (TLR) 1 expressed on human or mouse macrophages and dendritic cells * This work was supported in part by grant-in-aids from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare of Japan and Organization for Pharmaceutical Safety and Research. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB050005 and AB046533.
** To whom correspondence should be addressed. Tel./Fax: 81-6-6973-1209; E-mail: seya-tu@mc.pref.osaka.jp. 1 The abbreviations used are: TLR, Toll-like receptor; LPS, lipopolysaccharide; RACE, rapid amplification of cDNA ends; chTLR, chicken TLR; anti-chTLR Ab, anti-chicken TLR polyclonal antibody; MALP-2, macrophage-activating lipopeptide-2; TIR, Toll/interleukin-1 receptor; LRR, leucine-rich repeat; RT, reverse transcriptase; PCR, polymerase chain reaction; ORF, open reading frame; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; HEK, human embryonic kidney; NF-B, nuclear factor B. are involved in recognition and discrimination of microbial components. For example, TLR4 recognizes lipopolysaccharide (LPS), the major component of Gram-negative bacteria outer membrane (9 -11). TLR2, on the other hand, recognizes peptidoglycans and lipoproteins from Gram-positive bacteria, mycoplasmas, mycobacteria, and spirochetes (12). The TLR signaling in mouse macrophage dendritic cells results in induction or up-regulation of initial cytokines and costimulatory molecules, thereby augmenting the lymphocyte-activation ability of antigen-presenting cells (6 -8). In mammals, Toll signaling is evolved as an inducer of innate immune responses to complete activation of the acquired immune system. However, it is notable that in any lower vertebrates other than mammals, the TLR host defense system has not yet been systematically characterized.
In this study, we identified and characterized two chicken TLRs mapped to chromosome 4, a considerably conserved region among species from chickens to humans (13). One of these receptors governs both lipopeptide-and LPS-mediated signals in a distinct manner in terms of molecular combinations. In oviparous animals, the pattern recognition system may function in a unique fashion that is distinct from those of mammals as well as invertebrates.

MATERIALS AND METHODS
Cells and Reagents-Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin. RK13 cells (derived from rabbit kidney) were obtained from RIKEN Cell Bank (Wako, Saitama, Japan) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 60 g/ml kanamycin. LPS from Escherichia coli serotype 0127:B8 and Salmonella minnesota Re595 were purchased from Life Technologies, Inc., and Sigma. Staphylococcus aureus (pansorbin cells) was obtained from Calbiochem. Mycobacterium bovis Bacillus Calmette-Guerin Mycobacterium avium and Streptococcus pyogenes J17A4 were kindly provided by Dr. Y. Suzuki (Osaka Prefectural Institute of Public Health, Osaka, Japan). Inactivated Schizosaccharomyces cerevisiae L40 were prepared in our laboratory. E. coli DH5␣ was purchased from Life Technologies, Inc. Polymyxin B was obtained from Sigma. A synthetic lipopeptide based on the full-length MALP-2 was prepared using dipalmitoyl-S-glyceryl cysteine as described previously (14). Vectors containing cDNAs of human MD-2 (FLAG and His-tagged) or human CD14 were kind gifts from Dr. K. Miyake (Saga Medical School, Saga, Japan) (15) and Dr. H. Nishimura (Tsukuba University, Ibaraki, Japan) (16), respectively. Chicken MD-1 (17) was cloned in our laboratory and ligated into the pME18s mammalian expression vector. Anti-human CD14 monoclonal antibody and anti-FLAG M2 monoclonal antibody were purchased from Immunotech (Marseille, France) and Sigma, respectively.
Molecular Cloning of Chicken TLR (chTLR) cDNAs-The chicken bursa of Fabricius cDNA library was a kind gift from Dr. R. Goitsuka (Tokyo Scientific University, Tokyo, Japan) (18). A partial chTLR cDNA fragment was obtained by nested PCR with a degenerate primer and vector-specific primers. Degenerate reverse primer was designed based on the consensus C-terminal sequences of mouse TLR2, TLR4, and Drosophila melanogaster Toll (Fig. 1). Amplification was conducted by initial denaturation at 94°C for 2 min followed by 20 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 90 s. The first PCR products were used as the template in the next PCR performed under the same conditions for 35 cycles. The amplified products were cloned into the vector pCR2.1 (TA Cloning Kit, Invitrogen, San Diego, CA) and subjected to DNA sequencing using an ABI 377 sequencer. The complete chTLRs sequences were determined by sequencing liver mRNA of excel link species of chicken.
RT-PCR Analysis-Total RNAs were isolated from various chicken tissues as described previously (19) and were used as templates. RT-PCR was performed with primers specific for each type of chTLR under the following conditions: initial denaturation at 94°C for 1 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 59°C for 1 min, and extension at 72°C for 1 min. The PCR products were separated in a 1% (w/v) agarose gel.
Chromosome Preparation and in Situ Hybridization-Fluorescence in situ hybridization was used for chromosomal assignment of chTLR genes. The preparation of R-banded chromosomes and fluorescence in situ hybridization were performed as described by Matsuda and Chapman (20) and Suzuki et al. (21). Two probes with sequences specific for each type of chTLR were used for hybridization.
Construction of Expression Plasmid-C-terminal His-tagged type 1 and type 2 chTLRs containing the ORFs and signal peptides were ligated into the mammalian expression vector pME18 s. The plasmids were prepared with an endotoxin-free Plasmid Maxi kit (Qiagen, Valencia, CA).
Preparation of Rabbit Anti-chicken TLR Polyclonal Antibody-Rabbit anti-chicken TLR polyclonal antibody (anti-chTLR Ab) was produced by the method established in our laboratory (19). RK13 cells transiently expressing His-tagged type 2 chTLR were used as immunogens. IgG was purified from rabbit serum as described previously (19).
chTLR Protein Expression-Various chicken tissues were solubilized with lysis buffer containing 150 mM NaCl, 1% Nonidet P-40, 10 mM EDTA, 1 mg/ml iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, and 20 mM Tris-HCl, pH 7.4, using a potter-type homogenizer. After incubation at 4°C for 1 h, each lysate was centrifuged at 15,000 rpm at 4°C for 30 min. The supernatant was collected, and protein concentration was measured using a protein assay kit (Bio-Rad). Protein extracts (500 g) were separated by SDS-PAGE (6% acrylamide) under reducing conditions and transferred onto polyvinylidene difluoride membrane by the method described previously (22). After blocking with skimmed milk, blots were incubated with anti-chTLR Ab (5 g/ml) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1:10000) and detected using an ECL kit (Amersham Pharmacia Biotech).
Flow Cytometry-Transfected HEK293 cells were incubated with 30 l of 50 g/ml anti-chTLR Ab or 50 g/ml pre-immune rabbit IgG at 4°C for 1 h. After washing, cells were treated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG F(abЈ) 2 (American Qualex Antibodies, San Clemente, CA). The stained cells were analyzed using FACSCalibur.
Luciferase Reporter Gene Assay-HEK293 cells (3 ϫ 10 5 cells/well) were plated in 6-well plates, and the following day they were transiently transfected by calcium phosphate (Mammalian Transfection kit, Stratagene, La Jolla, CA) with 2.5 g of expression plasmids, 1.0 g of NF-B reporter plasmid-encoding luciferase (pNFB-Luc, Stratagene), and 2.5 g of pSV-␤-galactosidase control vector (Promega, Madison, WI) for normalization of transfection efficiency. The total expression plasmid concentration was made up to 5 g with empty vector (pME18 s). After 40 h, cells were harvested, seeded into five wells of 24-well plates, and treated with 10 g of LPS, 1 M MALP-2, or various bacterial suspensions at 37°C for 6 h. The cells were lysed, and luciferase activity was measured with the reagents and protocols from PicaGene (Toyo Ink, Tokyo, Japan). The specific activities were calculated as light intensity with stimulation/light intensity without stimulation.

Molecular Cloning and Properties of Chicken TLR
cDNAs-TLR has not been identified in non-mammalian vertebrates. First, we attempted to isolate chicken TLR homologues to compare their structural/functional relationship with those of mammals reported to date, i.e. mice and humans. Several degenerate primers were designed based on sequences of the TIR domain conserved among mice TLR2, TLR4, and D. melanogaster Toll (Fig. 1). Nested PCR was performed with two specific primers for the vector (pME18s), each degenerate primer and template (chicken bursa of Fabricius cDNA library). After second PCR, the PCR fragments of various sizes were subcloned into the vector pCR2.1, and their sequences were determined. One of the clones was found to contain a sequence similar to the TIR domain of human and mouse TLR2s. Finally, the full coding regions of two TLRs were identified by 3Ј and 5Ј RACE. The presence of these two TLR sequences was confirmed by RT-PCR in six clones from chicken liver mRNA, and then they were designated type 1 and type 2 TLR.
From the nucleotide sequences of the two chTLRs, typespecific sequences were identified at their 5Ј proximal sites, ϳ600 bp in their putative extracellular domains, and 3Ј proximal regions (Fig. 2). The former two sites led to differences in amino acid sequence between type 1 and type 2, whereas the latter did not. These two types of chTLRs were 88.5% homologous over the whole sequence and 57.0% homologous in the 600-bp type-specific sequences. The coding regions of both types consisted of a single exon. Furthermore, single introns of ϳ500 bp and 1.5-1.8 kilobase pairs were found immediately upstream of the coding regions of type 1 and type 2, respectively. Thus, the two independent genes encoded the two chTLR types, suggestive of no participation of alternative splicing in generating the two similar types of chTLRs.
The deduced amino acid sequences of type 1 and type 2 chTLR proteins are shown in Fig. 3. Both type 1 and type 2 possessed Ͼ12 LRRs and one C-terminal-flanking region in the putative ectodomains. Their TIR domains were completely identical. The type-specific amino acid sequences were found in the middle of the LRR domain and the N-terminal regions of the two types, reflecting the distinct nucleotide sequences. Of the nine human TLRs sequenced to date, both of the chTLR proteins exhibited the highest degrees of homology to human TLR2 (Table I). In particular, the TIR domain of chTLRs was ϳ80% identical to that of human TLR2.
Chromosomal Localization of chTLRs-To determine the chromosomal localization of type 1 and type 2 chTLRs, fluorescence in situ hybridization analysis was performed. Probes of ϳ800 bp containing the type-specific region of ϳ600 bp were used to map type 1 and type 2 individually. Because each probe detected the indistinguishable region, 4q1.1 proximal (Fig. 4), the two chTLRs genes were both localized to the q1.1 proximal region of chromosome 4 (ChickGBASE, www.ri.bbsrc.ac.uk/chickmap/).
Expression of chTLRs-To determine the tissue distributions of chTLR mRNAs, we first conducted RNA-blotting analysis. Total RNAs from various chicken tissues were blotted onto membrane, and type 2 chTLR ORF (2345 bp) was used as a probe. A single band was detected at ϳ3.4 kb in all tissues (data not shown). Because the probe contained the region common to both types of chTLR, we could not discriminate one from the other. We also confirmed the expression of chTLR mRNAs by RT-PCR. (Fig. 5A). Type 2 chTLR was detected in all tissues tested. Type 1, on the other hand, was hardly detected in the brain, spleen, and skeletal muscle. In heart, lung, liver, and ovary, both types of chTLR were abundantly expressed.
We next determined molecular masses and tissue distributions of the two chTLRs by immunoblotting. Various chicken tissues were homogenized, and aliquots of 500 g of each ex-tract were analyzed by SDS-PAGE followed by immunoblotting. ChTLRs were detected by anti-chTLR Ab established for this study. As controls, the soluble fraction of RK13 cells expressing each type of recombinant chTLR (His-tagged) were simultaneously analyzed on the same blots. Type 1 and 2 chTLR were detected at 102 kDa, and 97 and 99 kDa as a single band and double bands, respectively (Fig. 5B). These bands were also detected using anti-His-tag antibody (data not shown). ChTLRs were expressed in almost all tissues, although in the lung, kidney, testis, and ovary, the signals were marginal. Strong signals were detected in the heart, liver, gizzard, and muscle, which suggested that chTLRs are preferentially expressed in the connective tissues. We could not determine which type of chTLR was dominant in each tissue, because our polyclonal antibody recognized both type 1 and type 2.
Deglycosylation Analysis of chTLR-Both types of chTLR were predicted to have eight N-linked glycosylation sites from their primary sequences, and the molecular masses estimated from immunoblotting were larger than those expected. To determine the amount of N-linked sugar, we performed deglycosylation analysis of chTLRs. Lysates of RK13 cells transfected with vector only, type 1, or type 2 chTLR cDNA were treated with Nglycosidase followed by SDS-PAGE immunoblotting with anti-chTLR Ab. After glycosidase treatment, molecular sizes of both types were shifted down to the sizes predicted from the primary sequences (Fig. 5C). The signals were intensified through deglycosylation, suggesting that the epitopes may be disclosed by the removal of the sugars. Hence, type 1 and type 2 chTLRs were predicted to be type I transmembrane proteins with N-linked sugars corresponding to 19 kDa and 14 -17 kDa in their extracellular domain, respectively. Type 1 is more heavily glycosylated than type 2.
Functional Analysis of chTLRs-Both types of chTLR proteins may be analogues of human TLR2 (Table I). Human and mouse TLR2 act as pattern-recognition receptors and signal the presence of various bacteria through the activation of NF-B leading to immune responses. Here, we tested whether bacterial components activate NF-B via stimulation of chTLRs.
Human and mouse TLR2 activate NF-B in the HEK293 cell line. The TIR domain of chTLR was highly homologous to those of human TLR2, and the amino acid residues responsible for signaling were seemingly conserved in chTLR. Hence, the cDNA/plasmid of each chTLR was transfected into the HEK293 cell line. In some cases, other cDNAs were cotransfected together with chTLRs.
First, we checked the surface expression of type 1 and type 2 chTLR in transiently transfected HEK293 cells by flow cytometric analysis and using anti-chTLR Ab. Some shifts specific for chTLR were detected in type 1 and type 2 HEK293 cells (Fig. 6A), however, type 2 expression was lower than that of type 1. By immunoblotting analysis, the expression of type 1 and type 2 in HEK293 cells was clearly confirmed (Fig. 6A). Next, ligand-mediated activation of NF-B was analyzed using these type 1 and type 2 transfectants. LPS and MALP-2 were used as representative ligands for TLR4 and TLR2 in mammals, respectively (9 -11, 23, 24). MALP-2 served as a ligand for NF-B activation in HEK293 cells expressing type 1 or type 2 chTLR (Fig. 6D). Despite its low surface expression compared with type 1, type 2 can signal the presence of human TLR2 ligand mycoplasma lipoprotein, suggesting that even minimal expression of type 2 is sufficient for signaling. In fact, although we adjusted the expression levels of type 1 and type 2 in HEK293 transfectants, specific activity was not changed (data not shown). NF-B was more effectively activated by MALP-2 in the cells cotransfected with type 1 and type 2 chTLR than in those transfected with either type alone.
The function of chMD-1 has not been determined (17), although its human homologue was shown to be a cofactor for RP-105, which is an LRR protein and enhances LPS signaling on B cells (25,26). In this study, chMD-1 was found to subtly up-regulate MALP-2-dependent chTLR type 2 activation by NF-B reporter gene assay (Fig. 6D). On the other hand, MALP-2dependent type 1 activation was not potentiated by cotransfection with chMD-1. Hatched and stippled bars, specific regions for type 1 and type 2, respectively. Homologies between two types of chTLRs in the whole sequence and the specific sequences are indicated. The Kozak sequence was conserved in both types. The introns, indicated by closed bars, were shown to be 500 bp and 1.5-1.8 kilobase pairs for type 1 and type 2, respectively, by PCR analysis of a chicken genome library (GenBank TM accession numbers AB 050005 (for type 1 chTLR) and AB 046533 (for type 2 chTLR)).   (Fig. 6D). In mammals, CD14 and/or MD-2 have been identified as TLR cofactors for LPS signaling to facilitate NF-B activation (27)(28)(29). Therefore, we attempted to coexpress chTLRs in combination with these cofactors. Cotransfection with type 2 and either human MD-2 or human CD14 allowed the transfectants to respond to E. coli LPS. In particular, CD14 more efficiently enhanced type 2-mediated response to LPS than MD-2. These responses were inhibited by the treatment with polymyxin B (data not shown), indicating that the observed NF-B activation was attributable to a specific chTLR response by LPS. Salmonella LPS, on the other FIG. 6. Expression and signaling of chTLRs in HEK293 transfectants. A, the protein expression of chTLRs in HEK293 transfectants was assessed by flow cytometric analysis and Western blotting. Left, HEK293 cells were transiently transfected with pME18s (vector only) (thin lines) or those containing each chTLR cDNA (thick lines). After 24 h, the cells were collected and treated with anti-chTLR Ab or preimmune rabbit IgG (broken lines). The mean fluorescence intensities of the transfectants were as follows: vector only, 7.0; type 1-His chTLR, 14.1; and type 2-His chTLR, 8.3. More fluorescent shift was observed with type 2 transfectants when the amount of the vector was increased (data not shown).) Right, each HEK293 transfectant was lysed (1 ϫ 10 8 cells/ml), and 30 l of each lysate was subjected to SDS-PAGE followed by immunoblotting with anti-chTLR Ab. Types 1 and 2 chTLR were detected as single and double bands, respectively, as indicated by arrows. B, protein expression of human CD14 in HEK293 transfectants was assessed by flow cytometry. HEK293 cells transfected with human CD14 and type 1 or type 2 chTLR were collected and treated with anti-huCD14 monoclonal antibody (thick lines) or normal mouse IgG (thin lines). The levels of huCD14 were evaluated by flow cytometry. Untransfected cells are indicated by broken lines. C, protein expression of human MD-2 in HEK293 transfectants was assessed by immunoblotting. HEK293 cells were transfected with the FLAG-tagged huMD-2 and type 1 or type 2 chTLR. After 48 h, the supernatants of HEK293 transfectants were collected and subjected to SDS-PAGE followed by immunoblotting with anti-FLAG monoclonal antibody. D, luciferase reporter gene assay for measurement of NF-B activation in HEK293 cell chTLR transformants. HEK293 cells transfected with NF-B-luciferase reporter plasmid were cotransfected with empty vector (vector control is shown on the left), chTLR type 1-His (hatched bars), or type 2-His (closed bars) in addition to the other expression plasmids as indicated. chMD-1, chicken MD-1; huMD-2, human MD-2; huCD14, human CD14. After 40 h, cells were stimulated with MALP-2 (treated with polymyxin B) or LPS for 6 h, and NF-B activation was determined by luciferase activity in cell lysates. Specific activity was obtained by the formula described under "Materials and Methods." Luciferase activity of the HEK293 transfectants with one of the cofactors (open bars) or with type 1 and type 2 chTLRs (stippled bars) was measured along as controls. Experiments were performed two times in triplicate, and the results are expressed as means Ϯ S.D. Statistical significance was confirmed by InStat program. A two-tailed p value of Ͻ0.05 was taken to significant differences indicated by asterisks .   FIG. 4. Chromosomal localization of chTLRs. The specific sequences for each type of chTLR were used as biotinylated probes. Both types of chTLR gene were localized to chromosome 4q1.1 proximal.

FIG. 5. Characterization of chTLR mRNAs and proteins. A,
RT-PCR analysis of two chTLRs in various chicken tissues. Specific primers for each type of chTLR were used. Unsaturated cycles of PCR conditions were adopted for these analyses. PCR amplification of ␤-actin was performed for the control. B, protein-blotting analysis of chTLRs in chicken tissues. Various chicken tissue extracts were blotted onto a membrane and probed with anti-chTLR Ab (type 2 chTLR for antigen), which cross-reacted with type 1 as well as type 2 chTLR as described under "Materials and Methods." The anti-chTLR Ab recognized type 1-His and type 2-His chTLR in cell lysates of RK13 transfectants. The molecular masses of chTLRs were calculated from standard curve of molecular markers. C, N-glycosylation of type 1 and type 2 chTLR produced in RK13 cells. The lysates of the RK13 cells expressing either type 1 or type 2 chTLR were treated with N-glycosidase. The samples were subjected to SDS-PAGE and blotted onto a membrane. ChTLR proteins were detected with anti-chTLR Ab.
hand, failed to activate NF-B through type 2 signaling. Unlike type 2, type 1 barely signaled the presence of either LPS. The expression levels of CD14 or MD-2 were not affected by the cotransfection of either of the chTLRs (Fig. 6, B and C). Moreover, cotransfection of chMD-1 with chTLRs showed no effect on NF-B activation in response to LPS. Hence, type 2 cannot solely act as a receptor for LPS but can do so for E. coli LPS in combination with CD14 or MD-2.
Responses to whole inactivated bacteria were also analyzed using the same experimental protocol (Table II). Only type 2-bearing cells responded to whole inactivated E. coli, S. aureus, and M. bovis. Unlike human TLR2 (30) type 2 chTLR failed to respond to S. cerevisiae as well as zymosan even with human CD14 (data not shown). Thus, type 2 chTLR acts solely as a pattern-recognition receptor that is almost similar to human or mouse TLR2 (12,(31)(32)(33) and in combination with MD-2 and/or CD14 acts as a TLR4-like receptor. The role of type 1 chTLR remains to be determined. DISCUSSION Molecular cloning and functional characterization of human and mouse TLRs have been reported. This is the first report on the structure and function of TLR in a non-mammalian vertebrate.
Here, we successfully cloned two chicken TLRs, named type 1 and type 2 chTLR. The nucleotide sequence of type 1 was 88.5% identical to that of type 2, both of which exhibited the highest degrees of similarity to human TLR2 among the human TLRs reported to date. There are three inconsistent regions in the ORF of type 1 and type 2, 5Ј end of 30 -60 bp, 3Ј end of 10 bp, and the middle region of 600 bp, making a clear distinction between type 1 and type 2. Type 1 and type 2 are not alternatively spliced products, but they are generated from distinct genes because single exons with different nucleotide sequences encode the ORF of type 1 and type 2. These two genes were mapped in close proximity to chromosome 4q1.1, suggesting that duplication followed by homologous recombination of an ancestral TLR2 gene gave rise to two distinct TLR2-like chicken genes. Duplicated TLR2 genes have not been reported in other species. The whole genome size of chickens is one third of that of humans. The chicken may have adopted a strategy of gene duplication to yield multiple TLRs to cover a variety of microbial repertoires. From an evolutionary view point, the TLR2 prototype should exist in an ancestor of the birds, and gene duplication should have occurred exclusively in birds.
Based on the predicted amino acid sequences of type 1 and type 2, chTLRs consist of an extracellular scaffold of LRR domains and the intracellular TIR domain. The chTLR proteins share the same TIR domain, which is 80% identical to that of human TLR2. Different amino acid sequences of type 1 and type 2 are localized in the N-terminal region and the extracellular 200-amino acid stretch, resulting in 12 LRR domains of type 1 and 13 LRR domains of type 2. Major structural differences between type 1 and type 2 should be attributable to the functional divergence of type 1 and type 2.
RT-PCR and Northern blotting analysis indicated that in humans TLR2 expression is limited to the brain, heart, lung, spleen, and muscle (27,34). In contrast, chTLR mRNAs are ubiquitously expressed in all organs tested (Fig. 5A). Either type 1 or type 2 chTLR protein could be detected in all organs tested, however the protein signals were weak in the lung, kidney, testis, and ovary, some of which contain abundant chTLR mRNAs. The level of human TLR2 mRNA is often higher than its protein level (35), suggesting that the messageto-protein discrepancy may commonly occur in TLRs. Otherwise, it may predict the presence of other TLRs with similar nucleotide sequence regions but distinct protein sequences. Alternatively, chTLR may form a complex with other molecules, which causes a lesser detectable amount of the expected size of the TLR proteins. Strong protein signals were detected in the heart, muscle, and gizzard in which myoblasts and connective tissue are differentiated. This finding was reminiscent of the function of D. melanogaster Toll which participates in myogenesis (36). The distribution profile of chTLRs may predict additional functions.
The functional profile of chTLRs was examined in this study. Type 2 per se served as a receptor for the mycoplasma lipopeptide MALP-2 and in conjunction with human CD14 or MD-2 served as a receptor for LPS. Thus, type 2 encompasses the function of human TLR2 and TLR4, thereby covering two major signals induced by lipoprotein and LPS. After completing this study, the same was found true in humans (28). Human TLR2 solely recognizes lipoproteins and together with CD14 and/or MD-2 recognizes LPS, although human TLR4 acts as the main receptor for LPS. Thus, the TLR2-CD14/MD-2-dependent LPS recognition system would be conserved across mammals and chickens.
Type 1 possessed less ability to act as a receptor for MALP-2 than type 2 and did not respond to LPS. The different region of type 1 versus type 2 in chTLRs may provide a hint to determine the portion responsible for ligand recognition or molecular complex formation. Indeed, type 1 and type 2 showed structural diversity, particularly in the ϳ200-amino acid LRR region. In light of the properties of human/mouse TLR2, which recognize bacterial components by forming a molecular complex with CD14, MD-2 (28), TLR1, or TLR6 (37)(38)(39), coupling components to type 1 on the cell surface may differ from those of type 2. Identification of such components will facilitate further functional characterization of type 1. In summary, the functional properties of type 2 chTLR are similar to those of mouse and human TLR2, but type 1 should confer distinct or unique properties on chTLR.
MD-1 was first reported as a Myb-inducible gene in chickens (17). Later, mammalian MD-1 was identified as a stabilizer of RP-105. MD-1⅐RP-105 complex is required for LPS recognition and signaling by TLR4 on the B cell (25,26). In this study, we confirmed that chicken MD-1 did not serve as a cofactor for TLR2-like chTLRs. Although it has not been tested for its TLR cofactor function, chicken MD-1 probably serves as a cofactor for unidentified LRR proteins. This point should be clarified after identification of chicken MD-1 partners. At present, we have no evidence for the presence of a TLR4 analogue in the chicken. Although human TLR4 can respond to a variety of LPS species, chicken TLR4, if any, will not cover a wide variety of LPS, because only limited species of bacteria bearing LPS can induce cellular responses (40,41). In contrast, type 2 chTLR message is expressed ubiquitously, hence, it is likely that type 2 chTLR covers the function of TLR4. The overlapping coverage of two human TLRs 2 and 4 with one chicken TLR is an attractive hypothesis, because the genome size of the chicken is relatively small compared with those of mammals. Their genome size is about one third that of humans, and thus chromosomal recombination is likely to occur with high frequency. With regard to the origin of the two chTLRs, we favor the interpretation that there was a single ancestor TLR gene that underwent gene duplication, shuffling, and insertion diverging into the two chTLRs. However, there are no introns in the ORFs of the two chTLR genes, suggesting that the parent genes with introns existed in another region of the chicken genome. In fact, in humans, TLR3 and others are present in the form of genes with introns. If this is the case in chickens, the number of chTLRs should be more than that identified here. Moreover, there is a number of microchromosomes in which other innate immune-or complement-related genes (regulator of complement activation) are clustered (42). The genome size of chicken MHC is one tenth that of humans (43). These properties of immune-competent genes are not common in those of mammals. We should compare the chicken TLR system with those of fish as well as lampreys to test what properties are common or general in lower vertebrates, which will be determined when the fish genome project is complete.
A characteristic point in oviparous animals is the duplicated TLRs involved in the pattern-recognition system, which is distinct from that in mammals. The bird-specific immune facility should be highlighted in the innate system beside the well known bursa of Fabricius, a high frequency of gene conversion in B cells and the small MHC alleles (44).