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Departments of
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Oral Pathobiological Science and
Oral Health Science, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan;
First Department of Biochemistry, Sapporo Medical University, Sapporo, Japan; and
Department of Oral Health, National Institute of Public Health, Tokyo, Japan
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Abstract |
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B reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2
S40-I64 (a TLR2 mutant with a deletion of the region of Ser40 to Ile64) failed to activate NF-B in response to FSL-1. The deletion mutant TLR2C30-S39 induced NF-B reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu178 to Ala (TLR2E178A), TLR2E180A, TLR2E190A, and TLR2L132E induced NF-B activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2L107E, TLR2L112E (a TLR2 point mutant with a substitution of Leu112 to Glu), and TLR2L115E failed to induce NF-B activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2L115E, TLR2L112E, and TLR2S40-I64 were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2E190A were. In addition, these mutants, except for TLR2E180A, functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser40-Ile64 and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans. |
Introduction |
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Recent evidence has accumulated that microbial LP play pathological roles in bacterial infection (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Escherichia coli LP were first characterized and sequenced by Braun et al. (6), and they have been demonstrated to be biologically active (7, 8, 9, 10, 11). The part of lipoprotein responsible for biological activities has been demonstrated to be the N-terminal lipopeptide moiety, the structure of which is S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-Ser-Asn-Asn-Ala- (7). Mycoplasmas, wall-less microorganisms, also possess LP capable of activating macrophages or fibroblasts (12, 13, 14, 15, 16). Mühlradt et al. (13, 14) identified a 2-kDa lipopeptide called MALP-2 from Mycoplasma fermentans that is capable of activating monocytes/macrophages, and they determined the structure to be S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys. We have also found that Mycoplasma salivarium LP activate normal human gingival fibroblasts to induce the production of inflammatory cytokines and the surface expression of ICAM-1, and we have purified a 44-kDa lipoprotein (LP44) responsible for the activity (16). In addition, the structure of the N-terminal lipopeptide moiety of LP44 has been determined to be S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe-Thr-Gly-Trp-Val-Ala- (16). The lipopeptide S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1), synthesized on the basis of the N-terminal structure of LP44, showed the same activity as LP44 (16). The framework structure of FSL-1 is the same as that of MALP-2, but they differ in amino acid sequences and lengths of the peptide portion.
It has already been demonstrated that TLR2 functions as a receptor for microbial LP and lipopeptides (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35), PGN (36, 37, 38, 39, 40, 41, 42, 43, 44), and glycolipids (45, 46, 47, 48). Signaling by MALP-2, similar to FSL-1, has been demonstrated to be mediated by TLR2 (17, 24, 27, 28). Recently, it has been demonstrated that TLR2 requires TLR6 as a coreceptor for recognition of diacylated lipopeptides (24). We also found that FSL-1 is recognized by TLR2 (unpublished observations). Thus, numerous lines of evidence have been accumulated that PAMPs are recognized by TLRs, but it is not clear how TLRs recognize their ligands. We have a great interest in the mechanism by which TLRs recognize PAMPs because we think that elucidation of the mechanisms will provide insight into how microbial infections are controlled.
In this study, therefore, attempts were made to determine which region of TLR2 recognizes mycoplasmal diacylated LP and lipopeptides as well as Staphylococcus aureus PGN for comparative purposes.
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Materials and Methods |
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Human embryonic kidney (HEK) 293 cells obtained from American Type Culture Collection (CRL-1573; Manassas, VA) were maintained in DMEM (Life Technologies, Gaithersburg, MD) containing 10% FBS. A human acute monocytic leukemia cell line, THP-1 (49), obtained from Health Science Research Resources Bank (Osaka, Japan), was grown at 37°C in a humidified atmosphere of 5% CO2 in RPMI 1640 medium supplemented with 10% (vol/vol) FBS, penicillin G (100 U/ml), and streptomycin (100 µg/ml). E. coli LPS were obtained from Sigma-Aldrich (St. Louis, MO). S. aureus PGN were obtained from Fluka Chemie (Steinheim, Switzerland). FITC-conjugated goat anti-mouse IgG and peroxidase-conjugated anti-rabbit IgG Abs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Mouse IgG1 as an isotype control were purchased from BD Biosciences (San Jose, CA). Anti-TLR2 mAb (IMG-319) were purchased from Biocarta (San Diego, CA). All other chemicals were obtained from commercial sources and were of analytical or reagent grade.
Cloning of human TLR2, TLR2 mutants, and TLR6
The cDNAs of human TLR2 (2.35 kb) and TLR6 (2.4 kb) were obtained by RT-PCR of RNA isolated from THP-1 cells. The cDNAs of TLR2 and TLR6 were cloned into a pEF6/V5-His TOPO vector (hereafter referred to as TLR2-TOPO and TLR6-TOPO; Invitrogen, Carlsbad, CA). The mutant TLR2 proteins used in this study are schematically presented in Fig. 1. The deletion mutants (TLR2S40-I64 (a TLR2 mutant with a deletion of the region of Ser40 to Ile64) and TLR2C30-S39) and the point mutants of the human TLR2 gene (TLR2E178A, TLR2E180A, TLR2E190A, TLR2L107E, TLR2L107G, TLR2L112E (a TLR2 point mutant with a substitution of leucine at a position 112 to glutamic acid), TLR2L112G, TLR2L115E, TLR2L115G, TLR2L123E, and TLR2L132E) were produced by a QuickChange XL site-directed mutagenesis kit (Stratagene, San Diego, CA) according to the manufacturer’s instructions with the TLR2-TOPO construct.
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LP were prepared from M. salivarium American Type Culture Collection 23064 by Triton X-114 phase separation according to the method described previously (50). Briefly, mycoplasmal cells were treated with Triton X-114 to extract LP. LP in the Triton X-114 phase were precipitated by methanol and used for stimulation after being suspended in sterile PBS by light sonication. The protein concentration was determined by the method described previously (50).
Synthesis of the diacylated lipopeptide FSL-1
FSL-1 was synthesized as follows. The side chain-protected Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe was built up with an automated peptide synthesizer (model 433; PE Applied Biosystems, Foster City, CA). F-moc-S-(2,3-bispalmitoyloxypropyl)-cysteine (Novabiochem, Laeufelfingen, Switzerland) was manually coupled to the peptide-resin using a solvent system of 1-hydroxy-7-azabenzotriazole-1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/CH2Cl2-DMF. The F-moc and resin were removed from the lipopeptide by trifluoroacetic acid. The lipopeptides were purified by preparative HPLC with a reverse phase C18 column (30 x 250 mm). The purity of FSL-1 was confirmed by analytical HPLC with a reverse phase C18 column (4.6 x 150 mm) to be 98%. The lipopeptide FSL-1 was dissolved in PBS.
NF-B reporter assay
HEK293 cells were plated at 1 x 105 cells/well in 24-well plates on the day before transfection. The cells were transiently transfected by FuGene 6 Transfection Reagent (Roche, Indianapolis, IN) with 30 ng of an NF-B reporter plasmid (pNF-B-Luc; Stratagene) and 3.5 ng of a construct directing expression of Renilla luciferase under the control of a constitutively active thymidine kinase promoter (pRL-TK; Promega, Madison, WI) together with 166.5 ng of each transfectant gene of TLR2-TOPO, TLR2 point mutants-TOPO, and TLR6-TOPO. Twenty-four hours after transfection, the cells were stimulated for 6 h with FSL-1, LP, and PGN in the absence of FBS, and luciferase activity was measured using a Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s instructions.
Generation of polyclonal Ab to human TLR2
An extracellular domain of TLR2 protein (Met1-Arg508) was subcloned into the pGEX-2T (Amersham Pharmacia Biotech, Tokyo, Japan) vector by the PCR method to generate a GST-TLR2 fusion protein and was transformed into E. coli DH5
. The fusion protein formed inclusion bodies in the cells. Therefore, they were collected from the cells lysed in 20 mM Tris-HCl (pH 8.0) containing 0.5% Nonidet P-40 after treatment with lysozyme by centrifugation at 8000 x g for 30 min. The purified inclusion bodies were subjected to SDS-PAGE, and the band corresponding to the GST-TLR2 fusion protein was excised from the SDS-PAGE gels and then homogenized. The homogenate was suspended in PBS and used for immunogen after being mixed at a ratio of 1:1 with CFA. The polyclonal anti-TLR2 Ab was generated by immunizing Japanese White rabbits with the mixture. On days 1, 14, and 28, rabbits were injected into five s.c. and i.m. sites. Sera were drawn 1 wk after the final immunization and were used as polyclonal anti-TLR2 Ab.
Expression of TLR2 and TLR6 in THP-1 and HEK293 transfectants by RT-PCR
RNAs were prepared from THP-1 and HEK293 cells using an RNeasy kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions and were dissolved in 50 µl of RNase-free water. By using an RT-PCR kit (Takara Shuzo, Shiga, Japan), the RNAs (0.1 µg) were transcribed with avian myeloblastosis virus reverse transcriptase using the antisense primer of 
-actin (15) and those of human TLR2 and TLR6. The forward and reverse primers for TLR2 or TLR6, which were used for their gene cloning, were designed to contain their start and stop codons on the basis of DNA sequences of TLR2 (GenBank accession no. XP_003304) and TLR6 (GenBank accession no. NM_006068) as follows: TLR2, 5'-CACTGGACAATGCCACATAC-3' (forward) and 5'-CCCAACTAGACAAAGACTGG-3' (reverse); and TLR6, 5'-CAACATCATGACCAAAGA-3' (forward) and 5'-CACCATCATCCAAGTAAATA-3' (reverse). The specificities of the primers for TLR2 and TLR6 were confirmed by Southern hybridization with a probe coding the internal sequence. The RT reaction was performed in an automated DNA thermal cycler according to the manufacturer’s instructions. Briefly, a 1-µl volume (0.1 µg) of RNA was adjusted to a total volume of 20 µl in 10 mM Tris-hydrochloride (pH 8.3) containing 1 µM each of dATP, dCTP, dTTP, and dGTP; 1 µM of the antisense primer for the cytokine; 5 mM MgCl2; 50 mM KCl; 20 U of an RNase inhibitor; and 5 U of avian myeloblastosis virus reverse transcriptase. The RNA was transcribed at 55°C for 30 min after incubation at 30°C for 10 min and was then denatured at 99°C for 5 min and cooled at 5°C for 5 min. The resulting mixture containing cDNA was added to 80 µl of a mixture containing 10 mM Tris-hydrochloride (pH 8.3), 0.25 µM of each of the sense primers for -actin and TLR2, 1.8 mM MgCl2, and 1 U of Taq polymerase (Takara) and was amplified by PCR as follows: after 2 min of denaturation at 94°C, 28 amplification cycles (30 s of denaturation at 94°C, 30 s of annealing at 60°C, and 1.5 min of extension at 72°C) were performed. The PCR products were separated on 2% gel of NuSieve 3/1 agarose (FMC, Rockland, ME) in 0.5x TBE buffer containing ethidium bromide (5 µg/ml).
Western blotting
The transfected HEK293 cells grown in a six-well plate were washed twice with ice-cold PBS; lysed with 62.5 mM Tris-HCl (pH 6.8) containing 2% SDS, 10% glycerol, and 50 mM DTT (an SDS sample buffer) in the presence of inhibitor cocktails of proteases (Sigma-Aldrich); and boiled for 10 min. The lysates were centrifuged at 14,000 rpm for 10 min, and the resulting supernatants containing cytosolic and membrane proteins were collected. Proteins in the supernatant were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. The membrane was incubated at 4°C overnight with polyclonal anti-TLR2 Ab as described above and then with peroxidase-conjugated anti-rabbit IgG Ab. Immunoreactive proteins were detected using ECL detection reagents (Amersham Pharmacia Biotech).
Surface expression of TLR2 and its point mutants in HEK293 transfectants by flow cytometry
To assess the surface expression of TLR2 and its point mutants in HEK293 cells by flow cytometry, HEK293 cells were removed from the plastic dishes 72 h after transient transfection with the same amount of TLR2 or its point mutant genes. The cells were incubated at 4°C for 1 h with or without isotype-matched mouse IgG or anti-TLR2 mAb (IMG-319) and then with FITC-conjugated anti-mouse IgG. Surface expression was measured using a flow cytometer FACSCalibur (BD Biosciences).
Laser scanning confocal microscopic analysis of the expression of TLR2 on the surfaces of HEK293 cells
HEK293 cells were grown on poly-L-lysine-coated coverglasses on the day before transfection. The cells were transiently transfected by FuGene 6 transfection reagent with a TLR2 gene. After a 24-h incubation, the medium was removed, and the cells were incubated with serum-free DMEM containing rhodamine-conjugated Con A (5 µg/ml; Molecular Probes, Eugene, OR), followed by methanol fixation (4 min at -20°C). The cells were washed with PBS and then with 1 µg/ml rabbit polyclonal anti-TLR2 Ab generated in our laboratory diluted with PBS and were further incubated with Alexa-anti-rabbit IgG Ab (Molecular Probes) at room temperature for 45 min. The cells were finally washed three times with PBS, sealed in the presence of 90% glycerol, and observed using a laser microscope (LSM510; Carl Zeiss, Tokyo, Japan). Digital images were acquired and processed using Adobe Photoshop (version 5.0; Mountain View, CA).
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Results |
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First an experiment was conducted to determine whether endogenous TLR2 and TLR6 are expressed in HEK293 cells. RT-PCR analysis indicated that mRNAs of endogenous TLR2 and TLR6 were not expressed in nontransfected HEK293 cells (Fig. 2A), although Kirschning et al. (51) demonstrated that endogenous TLR2 mRNA is not expressed in HEK293 cells. HEK293 cells were transiently transfected with TLR2 and/or TLR6 genes and examined for the expression of mRNAs by RT-PCR and for the expression of proteins of wild-type TLR2 (TLR2wt) and its mutants by Western blotting. RT-PCR analysis indicated that mRNA of TLR2 and/or TLR6 was expressed in each of their transfectants (Fig. 2A). Analysis by Western blotting revealed that proteins of TLR2wt and its mutants were expressed in the transfectants (Fig. 2B).
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S40-I64) and TLR2wt (Fig. 3).
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S40-I64as well as TLR2wt were expressed on the cell surface of their transient transfectants (Fig. 4). Ratios of mean fluorescence intensities of samples stained with anti-TLR2 mAb to that of an isotype control are shown in each histogram of Fig. 4. The ratio of TLR2wt was the highest of all tested, and the ratios of TLR2L115E, TLR2L112E, TLR2E190A, and TLR2S40-I64 decreased in this order (Fig. 4).
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Takeuchi et al. (24) suggest that TLR2 requires TLR6 as a coreceptor for recognition of diacylated lipopeptides such as mycoplasmal lipopeptides. Experiments were therefore conducted to determine whether FSL-1 is recognized by HEK293 cells transiently transfected with TLR2 and/or TLR6 gene(s). FSL-1 stimulated NF-B reporter activity in HEK293 cells transfected with a TLR2 gene and in the cells transfected with both TLR2 and TLR6 genes, but not in the cells transfected with only a TLR6 gene, in a dose-dependent manner (Fig. 5). The level of activity in transfectants with both TLR2 and TLR6 genes was higher than that in transfectants with only a TLR2 gene, supporting the finding of Takeuchi et al. (24). However, FSL-1 failed to induce NF-B activation in the TLR6 transfectant (Fig. 5), indicating that HEK293 cells do not possess a functional endogenous TLR2. Therefore, the following experiments were conducted in HEK293 cells transiently transfected with TLR2wt or TLR2 mutant genes together with a TLR6 gene.
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B activation in HEK293 cells transiently transfected with TLR2 deletion mutants
To determine which region of the N-terminal extracellular domain of TLR2 is responsible for FSL-1 signaling, we examined NF-B reporter activity in HEK293 cells transiently transfected with each of two TLR2 deletion mutant genes together with a TLR6 gene. Two deletion mutants (TLR2S40-I64 and TLR2C30-S39) were constructed, because the region Ser40-Ile64, but not Cys30-Ser39, of TLR2 was demonstrated to be essential for recognition of S. aureus PGN by TLR2 (38). Although TLR2S40-I64 was expressed on the cell surface of the transfectant (Fig. 4), it failed to activate NF-B when stimulated with FSL-1 (0.1, 1.0, and 10 nM; Fig. 6). On the other hand, TLR2C30-S39 induced NF-B reporter activity, but the level of activity was significantly reduced compared with that induced by TLR2wt (Fig. 6). The same results were obtained with HEK293 cells transiently transfected with each of TLR2 mutant genes, but not with a TLR6 gene, although the level of activity was lower (data not shown). These results suggest that TLR2S40-I64 and, to a lesser extent, TLR2C30-S39 are involved in the recognition of FSL-1 as well as S. aureus PGN.
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B activation in HEK293 cells transiently transfected with TLR2 point mutants
Point mutants with a substitution of Glu178, Glu180, and Glu190 to alanine and a substitution of Leu115 and Leu132 located in a leucine-rich repeat (LRR) motif to glutamic acid were made as described in Materials and Methods. TLR2E178A, TLR2E180A, TLR2E190A, and TLR2L132E induced NF-B activation, but TLR2L115E failed to induce NF-B activation when stimulated with FSL-1 (Fig. 7A). In addition, TLR2L115E and TLR2E190A were expressed on the cell surface of the transfectants, and the level of expression of the former was slightly higher than that of the latter (Fig. 4). These results suggest that Leu115 is essential for recognition of FSL-1 by TLR2. Experiments were therefore conducted to determine whether Leu115 is involved in recognition of other TLR2 ligands, M. salivarium LP and S. aureus PGN. In the case of PGN stimulation, HEK293 cells were transiently transfected with each of the TLR2wt and mutant genes, but not with a TLR6 gene, because PGN is recognized by TLR2 in the absence of TLR6 (38, 43, 44). Neither LP nor PGN was recognized by TLR2L115E in the same way as FSL-1 (Fig. 7, B and C), suggesting that Leu115 is a critical residue in recognition of these TLR2 ligands. A substitution of Glu190 to alanine reduced the level of NF-B activation in response to PGN stimulation, but not to FSL-1 and LP (Fig. 7, A–C), suggesting that the recognition site for PGN is slightly different from that for LP or FSL-1.
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B activation when stimulated with FSL-1 (Fig. 8A). In addition, TLR2L112E as well as TLR2L115E were expressed on the cell surface of the transfectants (Fig. 4). However, TLR2L123E induced NF-B activation, but the level of activation was significantly reduced compared with that induced by TLR2wt (Fig. 8A). The same results were obtained when LP and PGN were used as stimulants (data not shown). Thus, it was found that Leu107, Leu112, Leu115, and, to a lesser extent, Leu123 in the LRR motif are involved in the recognition of LP and PGN as well as FSL-1 by TLR2. This finding encouraged us to investigate whether substitutions of each of these leucine residues to a hydrophobic glycine, not to a hydrophilic glutamic acid, affected NF-B activation. Neither TLR2L112G nor TLR2L115G induced the activation (Fig. 8B). However, substitution of TLR2L107G induced the activation, but the level of activation was significantly reduced (Fig. 8B). These results strongly suggest that Leu107, Leu112, and Leu115 in an LRR motif of human TLR2 play important roles in the recognition of M. salivarium LP, S. aureus PGN, and the diacylated lipopeptide FSL-1.
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B activation in HEK293 cells transiently cotransfected with TLR2wt and TLR2 mutants
In addition, experiments were conducted to determine whether these mutants functioned as a dominant negative form of TLR2. HEK293 cells were transfected with the same amounts of TLR2 and TLR2 mutant genes and then stimulated with PGN. Cotransfection with TLR2L107E, TLR2L112E, TLR2L115E, and TLR2S40-I64, which did not induce NF-B activation in response to PGN stimulation, reduced NF-B activation (Fig. 9). However, cotransfection with TLR2 E180A, which induced NF-B activation in response to PGN stimulation, enhanced NF-B activation (Fig. 9). These results suggest that TLR2L107E, TLR2L112E, TLR2L115E, and TLR2S40-I64, which did not recognize PGN, functioned as dominant negative forms of human TLR2.
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Discussion |
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B activation. The cytoplasmic domains of TLR homologues to IL-1R have been demonstrated to be essential for signaling leading to NF-B activation (1, 2, 3). Because each TLR is involved in signaling by different bacterial ligands, the extracellular domains of TLRs may define the specificities for various ligands. We therefore focused on the extracellular domain of TLR2 that is responsible for recognition of LP and lipopeptides, because it is not known how they are recognized by TLR2. Mitsuzawa et al. (38) recently found, using several deletion mutants of TLR2, that the extracellular region Ser40-Ile64 of TLR2 is critical for the recognition of S. aureus PGN. Furthermore, to answer the criticism that the conformational change due to the 25-aa deletion caused the receptor to be inactive, they confirmed that the synthetic peptide corresponding to the TLR2 region of Ser40-Ile64 competed with TLR2wt for PGN recognition.
On the basis of the report by Mitsuzawa et al. (38), two types of TLR2 deletion mutants (TLR2S40-I64 and TLR2C30-S39) were used to determine the region of the extracellular domain of TLR2 critical for recognition of the diacylated lipopeptide FSL-1. In addition, point mutants with a substitution of Glu178, Glu180, and Glu190 of TLR2 to alanine and a substitution of Leu115 and Leu132 to glutamic acid were made. This is because the amino acid residues Glu178, Glu180, and Glu190 were suggested by the results of computer analysis to be surface-exposed and because Leu115 and Leu132 are located at an LRR of TLR2.
The deletion mutant TLR2S40-I64 failed to activate NF-B when stimulated with FSL-1 (Fig. 6). On the other hand, TLR2C30-S39 induced NF-B reporter activity, but the level of activity was significantly reduced compared with that induced by TLR2wt (Fig. 6). In addition, TLR2S40-I64 revealed a similar localization pattern in the transfectants as TLR2wt (Fig. 3) and was indeed expressed on the cell surface of the transfectants, although the expression level was lower that that of TLR2wt (Fig. 4). That is, these results suggest that the conformational change due to the 25-aa deletion did not abrogate surface expression of the deletion mutant. Therefore, it is considered that the region of Ser40-Ile64 and, to a lesser extent, the region of Cys30-Ser39 are responsible for recognition of FSL-1 as well as S. aureus PGN.
It was found that TLR2E178A, TLR2E180A, TLR2E190A, and TLR2L132E induced NF-B activation, but TLR2L115E failed to induce the activation when stimulated with FSL-1 (Fig. 7A). In addition, it was found that TLR2L115E and TLR2E190A were expressed on the cell surface of the transfectants, and the level of the expression of the former was slightly higher than that of the latter (Fig. 4), suggesting that the conformational change due to the substitution of Leu115 to glutamic acid did not abrogate the surface expression. Therefore, it is considered that Leu115 plays an important role in recognition of FSL-1 by TLR2. The fact that Leu115 is located in a LRR of TLR2, and there are several leucine residues in the LRR encouraged us to examine whether these leucine residues are involved in the recognition. Indeed, it was found that TLR2L107E, TLR2L112E, and TLR2L115E failed to induce the NF-B activation when stimulated with LP and S. aureus PGN as well as FSL-1 (Fig. 7, B and C). The cellular localization patterns of TLR2L112E and TLR2L115E were similar to that of TLR2wt (Fig. 3), and they were expressed on the cell surface of the transfectants (Fig. 4). Similar results were obtained when TLR2L107G, TLR2L112G, and TLR2L115G were stimulated (Fig. 8B). However, TLR2L107E failed to induce NF-B activation, whereas a weaker, but significant, activation was observed in the case of TLR2L107G (Fig. 8B). These results suggest that the recognition of FSL-1 by TLR2 is based on a hydrophobic interaction. On the basis of these results, it was concluded that Leu107, Leu112, and Leu115 in an LRR motif of TLR2 is critical for recognition of LP and S. aureus PGN as well as FSL-1 by TLR2.
This is the first report to show that leucine residues in an LRR are critical for the recognition of PGN, diacylated LP, and diacylated lipopeptide by TLR2, although Mitsuzawa et al. (38) suggested, on the basis of results of experiments using deletion mutants of TLR2, that an LRR around the region of Ser40-Ile64 is responsible for S. aureus PGN signaling. The structural characteristic of TLR proteins is that they possess LRR motifs, which appear to be involved in protein-protein interaction (52, 53). LRR motifs were first discovered in 2-glycoprotein of human serum (54). LRRs are 20- to 29-residue sequence motifs present in a diverse group of molecules with differing functions (52). Individual repeats correspond to structural - units, consisting of a short -strand and -helix approximately parallel to each other (52). TLR2 possesses >10 LRRs. Alignment of the region around Leu107-Leu123 of TLR2 with an LRR consensus sequence (LEXLXLXXCXLTXXXCXXL) described by Kobe and Deisenhofer (52) suggests that Leu107 and Leu112 are located at a short -strand and that Leu115 is located at an -helix region (Fig. 10). Based on this speculation, a substitution of Leu107 and Leu112 located in the -strand region to a hydrophilic amino acid, glutamic acid, would cause a small conformation change in the surface-exposed -helix region, which, in turn, would abrogate the recognition by TLR2. This result suggests that each of these leucine residues is involved in recognition of diacylated LP, diacylated lipopeptide, and PGN.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ken-ichiro Shibata, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, Nishi 7, Kita 13, Kita-ku, Sapporo 060-8586, Japan. E-mail address: shibaken{at}den.hokudai.ac.jp
3 Abbreviations used in this paper: TLR, Toll-like receptor; FSL-1, S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe; LP, lipoproteins; LP44, a lipoprotein with a molecular mass of 44 kDa; LRR, leucine-rich repeat; MALP-2, S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys; PAMP, pathogen-associated molecular pattern; PGN, peptidoglycans; TLR2L112E, a TLR2 point mutant with a substitution of leucine at a position 112 to glutamic acid; TLR2S40-I64, a TLR2 mutant with a deletion of the region of Ser40 to Ile64; TLR2-TOPO, the cDNA of TLR2 cloned into a pEF6/V5-His TOPO plasmid; TLR6-TOPO, the cDNA of TLR6 cloned into a pEF6/V5-His TOPO plasmid; TLRwt, wild-type TLR.
Received for publication March 20, 2003. Accepted for publication July 31, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  M. Iyori, H. Kataoka, H. M. Shamsul, K. Kiura, M. Yasuda, T. Nakata, A. Hasebe, and K.-i. Shibata Resveratrol Modulates Phagocytosis of Bacteria through an NF-{kappa}B-Dependent Gene Program Antimicrob. Agents Chemother., January 1, 2008; 52(1): 121 - 127. [Abstract] [Full Text] [PDF]
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 T. Into, Y. Kanno, J.-i. Dohkan, M. Nakashima, M. Inomata, K.-i. Shibata, C. J. Lowenstein, and K. Matsushita Pathogen Recognition by Toll-like Receptor 2 Activates Weibel-Palade Body Exocytosis in Human Aortic Endothelial Cells J. Biol. Chem., March 16, 2007; 282(11): 8134 - 8141. [Abstract] [Full Text] [PDF]
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 N. Satta, S. Dunoyer-Geindre, G. Reber, R. J. Fish, F. Boehlen, E. K. O. Kruithof, and P. de Moerloose The role of TLR2 in the inflammatory activation of mouse fibroblasts by human antiphospholipid antibodies Blood, February 15, 2007; 109(4): 1507 - 1514. [Abstract] [Full Text] [PDF]
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J. J. Senn Toll-like Receptor-2 Is Essential for the Development of Palmitate-induced Insulin Resistance in Myotubes J. Biol. Chem., September 15, 2006; 281(37): 26865 - 26875. [Abstract] [Full Text] [PDF]
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 P. Henneke and R. Berner Interaction of neonatal phagocytes with group B streptococcus: recognition and response. Infect. Immun., June 1, 2006; 74(6): 3085 - 3095. [Full Text] [PDF]
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 M. Hashimoto, K. Tawaratsumida, H. Kariya, K. Aoyama, T. Tamura, and Y. Suda Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall components Int. Immunol., February 1, 2006; 18(2): 355 - 362. [Abstract] [Full Text] [PDF]
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 B. Fournier and D. J. Philpott Recognition of Staphylococcus aureus by the Innate Immune System Clin. Microbiol. Rev., July 1, 2005; 18(3): 521 - 540. [Abstract] [Full Text] [PDF]
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 Y. Nakao, K. Funami, S. Kikkawa, M. Taniguchi, M. Nishiguchi, Y. Fukumori, T. Seya, and M. Matsumoto Surface-Expressed TLR6 Participates in the Recognition of Diacylated Lipopeptide and Peptidoglycan in Human Cells J. Immunol., February 1, 2005; 174(3): 1566 - 1573. [Abstract] [Full Text] [PDF]
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 C. E. Moore, S. Segal, A. R. Berendt, A. V. S. Hill, and N. P. J. Day Lack of Association between Toll-Like Receptor 2 Polymorphisms and Susceptibility to Severe Disease Caused by Staphylococcus aureus Clin. Vaccine Immunol., November 1, 2004; 11(6): 1194 - 1197. [Abstract] [Full Text] [PDF]
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 N. Sato, N. Takahashi, K. Suda, M. Nakamura, M. Yamaki, T. Ninomiya, Y. Kobayashi, H. Takada, K. Shibata, M. Yamamoto, et al. MyD88 But Not TRIF Is Essential for Osteoclastogenesis Induced by Lipopolysaccharide, Diacyl Lipopeptide, and IL-1{alpha} J. Exp. Med., September 7, 2004; 200(5): 601 - 611. [Abstract] [Full Text] [PDF]
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N. W. J. Schroder, H. Heine, C. Alexander, M. Manukyan, J. Eckert, L. Hamann, U. B. Gobel, and R. R. Schumann Lipopolysaccharide Binding Protein Binds to Triacylated and Diacylated Lipopeptides and Mediates Innate Immune Responses J. Immunol., August 15, 2004; 173(4): 2683 - 2691. [Abstract] [Full Text] [PDF]
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T. Okusawa, M. Fujita, J.-i. Nakamura, T. Into, M. Yasuda, A. Yoshimura, Y. Hara, A. Hasebe, D. T. Golenbock, M. Morita, et al. Relationship between Structures and Biological Activities of Mycoplasmal Diacylated Lipopeptides and Their Recognition by Toll-Like Receptors 2 and 6 Infect. Immun., March 1, 2004; 72(3): 1657 - 1665. [Abstract] [Full Text] [PDF]
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