Lysines 128 and 132 Enable Lipopolysaccharide Binding to MD-2, Leading to Toll-like Receptor-4 Aggregation and Signal Transduction
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m306802200
ISSN1083-351X
AutoresAlberto Visintin, Eicke Latz, Brian G. Monks, Terje Espevik, Douglas T. Golenbock,
Tópico(s)Immunotherapy and Immune Responses
ResumoThree cell-surface proteins have been recognized as components of the mammalian signaling receptor for bacterial lipopolysaccharide (LPS): CD14, Toll-like receptor-4 (TLR4), and MD-2. Biochemical and visual studies shown here demonstrate that the role of CD14 in signal transduction is to enhance LPS binding to MD-2, although its expression is not essential for cellular activation. These studies clarify how MD-2 functions: we found that MD-2 enables TLR4 binding to LPS and allows the formation of stable receptor complexes. MD-2 must be bound to TLR4 on the cell surface before binding can occur. Consequently, TLR4 clusters into receptosomes (many of which are massive) that recruit intracellular toll/IL-1/resistance domain-containing adapter proteins within minutes, thus initiating signal transduction. TLR4 activation correlates with the ability of MD-2 to bind LPS, as MD-2 mutants that still bind TLR4, but are impaired in the ability to bind LPS, conferred a greatly blunted LPS response. These findings help clarify the earliest events of TLR4 triggering by LPS and identify MD-2 as an attractive target for pharmacological intervention in endotoxin-mediated diseases. Three cell-surface proteins have been recognized as components of the mammalian signaling receptor for bacterial lipopolysaccharide (LPS): CD14, Toll-like receptor-4 (TLR4), and MD-2. Biochemical and visual studies shown here demonstrate that the role of CD14 in signal transduction is to enhance LPS binding to MD-2, although its expression is not essential for cellular activation. These studies clarify how MD-2 functions: we found that MD-2 enables TLR4 binding to LPS and allows the formation of stable receptor complexes. MD-2 must be bound to TLR4 on the cell surface before binding can occur. Consequently, TLR4 clusters into receptosomes (many of which are massive) that recruit intracellular toll/IL-1/resistance domain-containing adapter proteins within minutes, thus initiating signal transduction. TLR4 activation correlates with the ability of MD-2 to bind LPS, as MD-2 mutants that still bind TLR4, but are impaired in the ability to bind LPS, conferred a greatly blunted LPS response. These findings help clarify the earliest events of TLR4 triggering by LPS and identify MD-2 as an attractive target for pharmacological intervention in endotoxin-mediated diseases. Invasive Gram-negative infection is a life-threatening medical disorder that accounts for >300,000 cases of sepsis in the United States annually (1Angus D.C. Linde-Zwirble W.T. Lidicker J. Clermont G. Carcillo J. Pinsky M.R. Crit. Care Med. 2001; 29: 1303-1310Crossref PubMed Scopus (6531) Google Scholar). One-quarter to one-third of afflicted individuals will die. 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There is now abundant evidence that TLR4 functions as the mammalian signal transducer for lipopolysaccharides (LPS) from Enterobacteriaceae (9Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6349) Google Scholar, 10Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1592) Google Scholar, 11Lien E. Means T.K. Heine H. Yoshimura A. Kusumoto S. Fukase K. Fenton M.J. Oikawa M. Qureshi N. Monks B. Finberg R.W. Ingalls R.R. Golenbock D.T. J. Clin. Invest. 2000; 105: 497-504Crossref PubMed Scopus (678) Google Scholar), the family of Gram-negative bacteria that includes many of the most common human pathogenic bacteria. Activation of TLR4 by LPS is absolutely dependent upon the presence of MD-2 (12Schromm A.B. Lien E. Henneke P. Chow J.C. 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A. 2001; 98: 12156-12161Crossref PubMed Scopus (197) Google Scholar, and 19Latz E. Visintin A. Lien E. Fitzgerald K.A. Monks B.G. Kurt-Jones E.A. Golenbock D.T. Espevik T. J. Biol. Chem. 2002; 277: 47834-47843Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). In addition, sensitive responses to LPS depend on two additional proteins, lipopolysaccharide-binding protein (LBP) and CD14. Both of these molecules play a pivotal role in providing LPS to the signal transduction machinery (20Ingalls R.R. Heine H. Lien E. Yoshimura A. Golenbock D. Infect. Dis. Clin. North Am. 1999; 13: 341-353Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Using forward genetic screening, we identified an LPS non-responder cell line derived from CD14-transfected Chinese hamster ovary cells. These cells expressed a point mutation in MD-2 that resulted in the conversion of a conserved cysteine to tyrosine (12Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Crossref PubMed Scopus (237) Google Scholar). Human MD-2 bearing the same amino acid substitution was unable to confer LPS responsiveness to TLR4-expressing cells. These findings implied an absolute requirement for MD-2 in LPS receptor complex function. This conclusion has been confirmed both in vitro (e.g. Refs. 21Re F. Strominger J.L. J. Biol. Chem. 2002; 277: 23427-23432Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar and 22Kawasaki K. Nogawa H. Nishijima M. J. Immunol. 2003; 170: 413-420Crossref PubMed Scopus (72) Google Scholar) and in vivo (13Nagai Y. Akashi S. Nagafuku M. Ogata M. Iwakura Y. Akira S. Kitamura T. Kosugi A. Kimoto M. Miyake K. Nat. Immunol. 2002; 3: 667-672Crossref PubMed Scopus (827) Google Scholar), as animals with a targeted disruption of the MD-2 gene exhibit the same LPS non-responder phenotype. To gain insights into the mechanisms by which the LPS receptor complex activates cells, we analyzed the molecular basis for the impaired function of MD-2C95Y and found that it lacks both the ability to bind TLR4 on the cell surface and to interact with LPS. Based on the lipid-binding motifs of other LPS-interacting proteins (23Ferguson A.D. Welte W. Hofmann E. Lindner B. Holst O. Coulton J.W. Diederichs K. Struct. Fold Des. 2000; 8: 585-592Abstract Full Text Full Text PDF Scopus (180) Google Scholar, 24Mancek M. Pristovsek P. Jerala R. Biochem. Biophys. Res. Commun. 2002; 292: 880-885Crossref PubMed Scopus (66) Google Scholar, 25Schumann R.R. Lamping N. Hoess A. J. Immunol. 1997; 159: 5599-5605PubMed Google Scholar, 26Lamping N. Hoess A. Yu B. Park T.C. Kirschning C.J. Pfeil D. Reuter D. Wright S.D. Herrmann F. Schumann R.R. J. Immunol. 1996; 157: 4648-4656PubMed Google Scholar), we hypothesized that a highly positively charged region in human MD-2 (amino acids 122–132) might be involved in LPS recognition. Using targeted point mutagenesis, we discovered that lysines 128 and 132 are critical for binding to LPS, but not to TLR4. As no previous work had demonstrated how CD14 and LBP relate to MD-2, we sought to characterize this interaction. We found that MD-2 binding to LPS is greatly enhanced by both CD14 and LBP, thus explaining their LPS-sensitizing effect. In addition, we found evidence that TLR4 and LPS physically interact, consistent with a previous study from our group concerning the pharmacology of TLR4 and lipid A (27Golenbock D.T. Hampton R.Y. Qureshi N. Takayama K. Raetz C.R. J. Biol. Chem. 1991; 266: 19490-19498Abstract Full Text PDF PubMed Google Scholar). The formation of these stable complexes requires the presence of MD-2. The functional outcome of this interaction is the formation of surface aggregates of TLR4 that can be visually observed on the cell surface following LPS binding to TLR4 on transfected HEK293 cells. We found that the resultant cytoplasmic surface formed by this cluster rapidly recruits toll/IL-1/resistance domain-containing adapter molecules via homophilic interactions and propagates the activation signal to downstream effectors, culminating in the activation of NF-κB-dependent gene expression. We propose that the induction of large aggregates of TLR4 promoted by MD-2 is the critical event that initiates LPS signal transduction. HEK293 cell lines stably expressing fluorescent protein-tagged TLR4 have been described previously (19Latz E. Visintin A. Lien E. Fitzgerald K.A. Monks B.G. Kurt-Jones E.A. Golenbock D.T. Espevik T. J. Biol. Chem. 2002; 277: 47834-47843Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). HEK293 cells stably expressing MD-2 or MD-2C95Y were generated by retroviral transduction (18Visintin A. Mazzoni A. Spitzer J.A. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12156-12161Crossref PubMed Scopus (197) Google Scholar). Escherichia coli 0111:B4 LPS was purchased from Sigma and repurified to remove contaminating TLR2 ligands as described (28Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (957) Google Scholar). Monoclonal antibody (mAb) HTA125 (IgG2a), which recognizes TLR4, was a gift from Dr. Kensuke Miyake (29Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1717) Google Scholar). The mAb and polyclonal antiserum against green fluorescent protein (GFP) were from Clontech and Molecular Probes, Inc. (Eugene, OR), respectively. The horseradish peroxidase-conjugated anti-biotin polyclonal antibody was from New England Biolabs Inc. (Beverly, MA). Unless otherwise stated, all other reagents were purchased from Sigma. Adherent cells from a confluent 10-cm dish were labeled with biotin (Pierce) on ice, solubilized in detergent (1% Triton X-100, 10 mm Tris-Cl (pH 7.4), 137 mm NaCl, 10% glycerol, 2 mm EDTA, and protease inhibitors), subjected to immunoprecipitation with the indicated antibodies (2 μg/ml) in 20 μl of packed protein A-Sepharose (Amersham Biosciences, Uppsala, Sweden) for 16 h at 4 °C, resolved by SDS-PAGE, and electrotransferred onto Hybond-C nitrocellulose membranes (Amersham Biosciences). The membranes were blocked in 5% dry milk in phosphate-buffered saline (PBS) and 0.1% Tween 20 for 30 min at 37 °C and probed for an additional 30 min at 37 °C with horseradish peroxidase-conjugated anti-biotin polyclonal antibody (1 μg/ml). Biotinylated proteins were revealed by enhanced chemiluminescence using a commercial kit for this purpose (Amersham Biosciences). Identical conditions for immunoprecipitation and immunoblotting were used for all of the blots presented in this work. FLAG-tagged MD-2 was revealed by probing the membranes with horseradish peroxidase-conjugated mAb M2, whereas GFP-tagged TLR4 was detected by blotting with anti-GFP mAb, followed by a horseradish peroxidase-conjugated anti-mouse secondary reagent (Bio-Rad). When necessary, membranes were stripped for 30 min in 0.1 m glycine (pH 2.2), 1% Tween 20, and 0.1% SDS and reprobed. The polysaccharide moiety of repurified LPS was biotin-labeled using biotin hydrazide (Pierce) according to the manufacturer's instructions. 1 mg of LPS was treated with 10 mm of sodium metaperiodate on ice for 30 min in coupling buffer (0.1 m sodium acetate at pH 5.5). After quenching in 50 mm glycerol for 5 min, excess oxidant and glycerol were removed by gel filtration using a Sephadex G-25 PD-10 desalting column (Amersham Biosciences), and LPS was eluted in coupling buffer. Biotinylation was performed by adding biotin hydrazide to a 5 mm final concentration for 2 h at room temperature. Unreacted biotin hydrazide was removed by a second purification step with a PD-10 column, and biotinylated LPS was finally eluted in Hanks' balanced saline solution. Biotinyated LPS was subjected to SDS-PAGE and detected by Western blotting using horseradish peroxidase-conjugated anti-biotin polyclonal antibody (see above), which revealed a smeared band ranging from 100 to 20 kDa, as would have been predicted because of the heterogeneous nature of the polysaccharide portion of LPS and the likelihood that the number of biotins per LPS would not be constant (30Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar). Furthermore, the bioactivity of the LPS was retained as assessed by comparing the ability of non-biotinylated LPS with that of biotinylated LPS in an NF-κB activation assay of TLR4-expressing HEK293 cells. The binding of LPS to FLAG-tagged soluble MD-2 was examined by incubating 10 ml of supernatant from HEK293 cells that had been transfected with MD-2 with 0.5–1 μg of biotinylated LPS/ml either overnight at 4 °C or for 1 h at room temperature in the presence of 25 μl of packed streptavidin-Sepharose CL-4B. Beads were collected by centrifugation, washed three times with Hanks' balanced saline solution, and resuspended in reducing SDS sample buffer, and anti-FLAG Western analysis was performed as described above. Washing with lysis buffer did not dissociate MD-2·LPS complexes. To study LPS interactions with TLR4, adherent cells from 10-cm confluent dishes were washed extensively with PBS and incubated for 30 min with 5 ml of prewarmed complete medium containing 1 μg/ml biotinylated LPS. Cells were then washed with Hanks' balanced saline solution and lysed as described above for immunoprecipitations. Biotinylated LPS·protein complexes were collected by addition of 20 μl of packed streptavidin beads and processed exactly as described for the immunoprecipitation protocol. The presence of TLR4 in the LPS complexes was assessed by Western blotting with anti-GFP antibody (which also recognizes the spectral variants of GFP such as yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)). Confocal Microscopy—To visualize the surface distribution of TLR4 and MD-2, HEK293 cells stably expressing YFP-TLR4 were seeded on 35-mm glass-bottom tissue culture dishes (Mattek Corp., Ashland, MA) and transfected with MD-2 or MD-2C95Y using GeneJuice (Novagen, La Jolla, CA). The following day, cells were washed twice with PBS supplemented with 1% fetal bovine serum and incubated for 30 min on ice with mouse anti-FLAG mAb M2 (10 μg/ml). After two washes with PBS/fetal bovine serum, the cells were incubated with Alexa 647-conjugated anti-mouse IgG (2 μg/ml) for 15 min on ice. Confocal images were collected from living cells incubated on a warm-stage apparatus at 37 °C for 15 min with a Zeiss Axiovert 100-M inverted microscope using an LSM 510 laser scanning unit. YFP was excited using the 514-nm line of a 25-milliwatt argon laser, and Alexa 647 was excited with a helium/neon laser emitting at 633 nm. Band-pass or long-pass filters were chosen to optimally separate the fluorescence emissions between the different photomultipliers using single-labeled samples of the probes as controls. LPS-induced clustering of TLR4 and MyD88 recruitment was monitored as follows. HEK293 cells stably expressing YFP-TLR4 and MD-2 or YFP-TLR4 and MD-2C95Y were grown on glass-bottom tissue culture dishes and transiently transfected with CFP-tagged MyD88. The cells were then incubated with 250 ng of Cy5-labeled repurified E. coli 0111:B4 LPS/ml of culture medium. After 10 min, the cells were washed twice with prewarmed culture medium and imaged with a Zeiss confocal microscope using a warm-stage apparatus set at 37 °C. Sequential scans were taken for CFP, YFP, and Cy5 using excitation at 458, 514, and 633 nm, respectively. z-stacks were acquired by scanning the cells from top to bottom. Side views of the cells were generated by electronically overlaying the images in the xy axis and visualizing the z axis. Scanning Electron Microscopy—Cells were grown on poly-l-lysine-treated coverslips and treated with 1 μg/ml LPS for 5 min at 37 °C. Samples were chilled on ice, washed with ice-cold Hanks' balanced saline solution, fixed for 20 min in 4% glutaraldehyde, and stained for 30 min with mAb HTA125 (1 μg/sample) in 200 μl. Antigen·antibody complexes were revealed by a 30-min incubation with a Nanogold™-labeled anti-mouse F(ab′)2 polyclonal antibody, followed by silver enhancement (LIS silver enhancement kit, L-24919, Molecular Probes, Inc.) according to the manufacturer's protocol. The silver-enhanced coverslips were then washed twice for 5 min with ultrapure water, dehydrated through a graded series of ethanol soaks to 100%, and then critical point-dried in liquid CO2 (31Robards A.W. Wilson A.J. Procedures in Electron Microscopy, Current Core Edition & Updates. John Wiley & Sons, Inc., New York1999Google Scholar). The coverslips with the dried cells were mounted and gold-coated for scanning electron microscopy. Samples were examined on an Etec Autoscan electron microscope at 20 kV. Atomic contrast imaging was performed on the samples' backscatter to confirm the silver nature of the white particles. TLR4-induced cell signal transduction was monitored by measuring either NF-κB activity or interleukin-8 (IL-8) secretion. To measure NF-κB activation, 2 μg of a reporter plasmid in which NF-κB drives the synthesis of luciferase was transiently cotransfected with 5 μg of the indicated cDNA by lipofection (GeneJuice) in 10-cm tissue culture dishes following the manufacturer's recommendations. The following morning, cells were seeded at a density of 50,000 cells/well in a 96-well plate, allowed to recover for 5 h, and stimulated as indicated between 6 and 18 h. Luciferase activity was measured with a plate reader luminometer (Victor2, PerkinElmer Life Sciences) using chemicals provided with the luciferase assay system (Promega, Madison, WI). All data are presented as the means ± S.D. of triplicate well readings, normalized to a value of 1.0 in comparison with an unstimulated negative control. Antibody cross-linking experiments were performed by coating the 96-well high protein-binding tissue culture plates (catalog no. 3361, Costar, Corning, NY) overnight with 50 μl of mAbs (or LPS) in PBS at the indicated concentrations. Excess antibody was removed by washing the plates with pyrogen-free PBS, and cells transfected with the NF-κB-luciferase reporter plasmid were seeded. At 6–18 h post-seeding, NF-κB activation was assessed as described above, and IL-8 levels in the supernatants were measured using a commercial IL-8 enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) following the manufacturer's recommendation. As a negative control, antibodies were boiled for 10 min before coating the plates. TLR4 and MD-2 Form Noncovalently Bound Complexes on the Cell Surface of HEK293 Cells—Substitution of cysteine 95 with a tyrosine residue generates a mutant form of human MD-2 (MD-2C95Y) that binds to TLR4 in the cytoplasm and partially immunoprecipitates with TLR4 (12Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Crossref PubMed Scopus (237) Google Scholar). MD-2C95Y is unable to confer LPS responsiveness to TLR4 (12Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Crossref PubMed Scopus (237) Google Scholar). However, substituting cysteine 95 with a serine residue, which is sterically closer to a cysteine, generates a mutant form of MD-2 (MD-2C95S) that has been shown to be slightly active when cotransfected with TLR4 (32Mullen G.E. Kennedy M.N. Visintin A. Mazzoni A. Leifer C.A. Davies D.R. Segal D.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3919-3924Crossref PubMed Scopus (64) Google Scholar). We hypothesized that the failure of MD-2C95Y to enable LPS signaling was due to its failure to properly bind to surface TLR4. Cells stably expressing YFP-tagged TLR4 were transfected with FLAG-tagged MD-2, MD-2C95Y, or MD-1. Cell-surface proteins were biotin-labeled, and whole cell lysates were subjected to immunoprecipitation with anti-GFP polyclonal antiserum, which recognizes epitopetagged TLR4. Anti-GFP precipitates were resolved by electrophoresis, blotted, and probed for surface biotin (Fig. 1). The same lysates were also precipitated with anti-FLAG mAb, subjected to SDS-PAGE, and transferred to nitrocellulose. Because of size differences in the proteins of interest, this blot was then bisected, and each portion was developed individually by Western blotting using anti-FLAG antibody for the lower portion or anti-GFP antibody for the upper portion of the blot. We found that TLR4 and MD-2 co-immunoprecipitated from the cell surface (Fig. 1, lane 1). In contrast, neither the MD-2C95Y mutant protein nor MD-1 associated with TLR4 on the cell surface (Fig. 1, lanes 2 and 3), despite comparable levels of these proteins in the lysates (lanes 4–9). MD-2C95Y Does Not Co-localize with TLR4 on the Cell Surface—Western blot analysis of transfected HEK293 cells and their supernatants established that MD-2C95Y is synthesized, glycosylated, and secreted (data not shown). HEK293 cells stably expressing YFP-tagged TLR4 were transiently transfected with MD-2 (Fig. 2, upper panels) or the MD-2C95Y mutant (lower panels) and stained with anti-FLAG mAb. MD-2 was then cross-linked and visualized by incubating these cells at 37 °C with Alexa 647-conjugated anti-mouse IgG polyclonal antibody. Confocal microscopic scans of YFP fluorescence (TLR4) and Alexa 647 fluorescence (MD-2) were obtained. In the absence of antibody treatment, TLR4 is uniformly distributed on the cell surface, as well as in the Golgi apparatus (19Latz E. Visintin A. Lien E. Fitzgerald K.A. Monks B.G. Kurt-Jones E.A. Golenbock D.T. Espevik T. J. Biol. Chem. 2002; 277: 47834-47843Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). Additionally, quantitative analysis of TLR4 by flow cytometry demonstrated that, in transfected HEK293 cells, the level of TLR4 surface expression was independent of MD-2 or MD-2C95Y coexpression (data not shown). Treatment of wild-type MD-2 with anti-FLAG antibody resulted in the clustering of MD-2 on the cell surface. Because MD-2 is bound to TLR4 on the cell surface, we subsequently observed the reorganization of surface TLR4 in the exact same distribution as MD-2 (yellow signal). Surprisingly, although our immunoprecipitation analysis suggested that MD-2C95Y would not bind TLR4 (and it is not known to bind any other TLR), it was consistently found on the surface of HEK293 cells that had been transfected with the TLR4 and MD-2C95Y cDNAs. Antibody cross-linking aggregated the mutant form of MD-2, but no commensurate changes in the distribution of TLR4 were observed (Fig. 2, lower panels). These results indicate that the MD-2C95Y mutant is not capable of binding to TLR4. Antibody-mediated Aggregation of MD-2 Efficiently Triggers TLR4 —The co-localization of TLR4 with MD-2 correlates with LPS responsiveness and thus suggests that binding of MD-2 to TLR4 is required for the assembly of an active LPS receptor. It is reasonable to postulate that the MD-2C95Y mutant failed to function because of its inability to mediate the assembly of a signaling complex. If the assembly of a heteromeric complex were truly the initiating event in signal transduction, then cross-linking TLR4 and/or MD-2 with antibodies instead of a ligand should mimic receptor activation. To directly test this hypothesis, we coated the surface of plastic dishes with mAbs and plated transfected cells on the antibody-coated surface. mAb HTA125 (29Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1717) Google Scholar) recognizes TLR4, and anti-FLAG mAb M2 binds the epitope tag on MD-2 and MD-2C95Y. As shown in Fig. 3, anti-FLAG mAb stimulated IL-8 secretion when wild-type MD-2 was present, but not when the MD-2C95Y mutant was expressed. This indicates that TLR4 is present on the cell surface despite the presence of a mutant form of MD-2 (because the plated antibody can bind only to surface TLR4). Identical antibody-induced activation was obtained in cells that did not express any exogenous MD-2 (data not shown). Isotype-matched antibodies and boiled antibodies did not activate cells (see Fig. 5) (data not shown), indicating that activation was not caused by contaminating LPS. We conclude that aggregation is an effective triggering signal for TLR4-mediated cellular activation. The MD-2C95Y mutant is incapable of activating TLR4 when cross-linked with a mAb because it fails to co-aggregate TLR4.Fig. 5MD-2 has a TLR4-binding domain and a second positively charged domain that is necessary for full activation.A, shown is an alignment of a highly basic region of MD-2 orthologs. The asterisks indicate the lysine residues in human MD-2; the lysine residues that were used to prepare mutant MD-2 molecules (MD-2K128E and MD-2K132E) are shown in boldface. B, HEK293 cells stably expressing YFP-TLR4 were transiently transfected with wild-type MD-2 (MD-2wt) or mutant MD-2 constructs and an NF-κB/luciferase reporter plasmid. TLR4 and MD-2 were immunoprecipitated (IP) from cell lysates or supernatants using anti-GFP or anti-FLAG antibody as indicated. Immunoprecipitates were resolved by 4–15% reducing SDS-PAGE, transferred to a Hybond membrane, bisected with a razor, and probed individually for TLR4 or MD-2
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