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Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse and hamster cells respond to the LPS partial structure lipid IVa, whereas human cells do not. TLR4 is involved in this ligand-specific recognition of LPS (17, 18). Recently, the involvement of MD-2 in this ligand-specific recognition of LPS was also demonstrated (19). Similar to lipid IVa, Taxol mimics the action of LPS on mouse cells but not on human cells. We have shown that MD-2 is responsible for this species-specific action of Taxol (10, 14). Furthermore, a single mutation of Gln22 of mouse MD-2 reduces its ability to confer Taxol responsiveness on mouse TLR4, but does not affect its ability to confer LPS responsiveness (10). These findings suggest that both LPS and Taxol physically associate with the TLR4-MD-2 complex. Furthermore, using LPS conjugated to a photo-activated cross-linker, Ulevitch and coworkers (20) observed LPS in close proximity to both TLR4 and MD-2 in the presence of CD14, which is an initial cell surface receptor for LPS. These findings, taken together, suggest that TLR4 and MD-2 constitute the central part of the LPS receptor complex.
Resolving how MD-2 contributes to formation of the LPS receptor complex is an important issue. The ability of MD-2 to form a complex with TLR4 on the cell surface is believed to be important for its ability to confer ligand responsiveness on TLR4. However, the amino acid residues of MD-2 that are important for cell surface complex formation with TLR4 and those that are important for ligand responsiveness of the TLR4-MD-2 complex have not been identified systematically. In this study, we addressed these issues by means of a strategy called alanine-scanning mutagenesis (21).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Taxol from Taxus brevifolia was purchased from Sigma-Aldrich (St. Louis, MO). LPS prepared from Escherichia coli 0111:B4 was purchased from List Biological Laboratories (Campbell, CA). Dishes, 100 mm in diameter, 24- and 6-well, were purchased from Corning Japan (Tokyo, Japan). Oligonucleotides were prepared commercially by Qiagen (Valencia, CA). MTS510 mAbs (11) were purchased from Medical and Biological Laboratories (Nagoya, Japan). FBS was purchased from Atlanta Biologicals (Norcross, GA). DMEM and PBS (pH 7.4, without CaCl2 and MgCl2) were purchased from Invitrogen (San Diego, CA). Prestained molecular mass standards and polyacrylamide gel were purchased from Bio-Rad (Hercules, CA). All other chemicals used were of reagent grade or better.
Stable transfectants and cell culture
HEK 293 cells introduced with an NF-
B-dependent luciferase
reporter construct (named 293/luc), and HEK 293 cells stably expressing
a recombinant mouse TLR4 bearing a flag and a 6 x His tag at
its C-terminal (named 293/mTLR4/luc) were generated previously
(10). The HEK 293 cell lines were maintained in DMEM
supplemented with 10% heat-inactivated FBS, penicillin G (100 U/ml),
and streptomycin sulfate (100 µg/ml) under a 5%
CO2 atmosphere at 100% humidity and 37°C.
Expression constructs
pEFBOS (22) vector-based expression constructs, which encode a recombinant mouse TLR4 or mouse MD-2 cDNA bearing a flag tag followed by a 6 x His tag at the C-terminal (11), were provided by Dr. K. Miyake (University of Tokyo, Tokyo, Japan), and were named in this study mTLR4fh and mMD-2fh, respectively. Mouse MD-2 mutant cDNAs were generated by PCR-based overlap extension (23) with Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). The sequences of the PCR primers are available upon request. Expression construct mMD-2fh was used as a PCR template, and every generated mutant MD-2 cDNA was designed to bear a flag tag followed by a 6x His tag at its C-terminal. The generated mutant MD-2 cDNAs were cloned into the XhoI and NotI sites of pEFBOS, and each mutant MD-2 expression construct is abbreviated as the wild-type residue (single letter amino acid designation) followed by the codon number and mutant residue (typically alanine). The cDNA inserts in the expression constructs were verified by sequencing with an ABI PRISM Genetic Analyzer (Applied Biosystems, Foster City, CA). Control expression vector pEFBOS- was generated previously (10).
Transient transfection
Cells (3 x 104/ml) were seeded into the wells of 24-well (1 ml/well) or 6-well (5 ml/well) dishes. After cultivation overnight, the cells were transfected with plasmids (0.3 µg/well of a 24-well dish, or 1.5 µg/well of a 6-well dish) using FuGENE 6 transfection reagent (Boehringer Mannheim, Indianapolis, IN). After a 24- to 48-h transfection, the culture medium was replaced with fresh medium. The plasmids used for the transfection were purified with a Wizard PureFection Plasmid DNA Purification system (Promega, Madison, WI).
Luciferase assay
Cells were lysed with cell culture lysis reagent (Promega), and luciferase activity in the cell lysates was measured with a Luciferase assay system (Promega), as described previously (14).
Cell surface staining
Unless indicated otherwise, all procedures were performed at 4°C or on ice. Three days after transfection, the cells in a 6-well dish were washed with 2 ml of buffer A (PBS containing 3% (v/v) FBS and 0.5 mg/ml NaN3), and then collected. The cells were incubated in buffer A containing 20 µg/ml MTS510 mAbs for 30 min. After incubation, the cells were washed with 1 ml of buffer A twice, and then incubated with buffer A containing 30 µg/ml dichlorotriazinyl amino fluorescein-conjugated anti-rat IgG (Immunotech, Luminy, France) for 30 min. Then the cells were washed with 1 ml of buffer A twice and collected. The collected cells were suspended in 500 µl of buffer A containing propidium iodide (10 µg/ml), and then analyzed with a FACSCalibur (BD Biosciences, Mountain View, CA). Alternatively, the cell surface was stained with an Enzymatic Amplification Staining kit (Flow-Amp Systems, Cleveland, OH) according to the manufacturer’s instructions with MTS510 (10 µg/ml) as the primary Ab.
SDS-PAGE and Western blotting
Each cell lysate was incubated with Ni-NTA agarose (Qiagen) under denaturing conditions according to the manufacturer’s instructions. The proteins absorbed to the resin were eluted with 0.1 M sodium-phosphate buffer (pH 8.0) containing 8 M urea and 250 mM imidazole. The eluted proteins were fractionated by SDS-PAGE (5–20% gradient) under reducing conditions (24). For Western blot analysis, proteins separated by SDS-PAGE were electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH) in 25 mM Tris/192 mM glycine/0.02% SDS/20% methanol at 6.6 V/cm for 18 h. Then each blot was incubated with Tetra-His Abs (Qiagen), and subsequently with anti-mouse IgG linked to HRP (Amersham Pharmacia Biotech, Piscataway, NJ). Cross-reactive proteins were detected with ECL Western blotting detection reagents (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell surface mouse TLR4-MD-2 complex formation has been
detected on a Ba/F3 stable transfectant expressing both mouse TLR4 and
mouse MD-2 by flow cytometry with an anti-mouse TLR4 mAb, MTS510,
which preferentially reacts with mouse TLR4, which is associated
with mouse MD-2 (11). Similarly, on the HEK 293 cell
surface, MTS510 reacts with the mouse TLR4-MD-2 complex more than mouse
TLR4 alone (Fig. 1
A). The
average signal intensity apparently increases (10.01 U) on expression
of mouse MD-2 in 293/mTLR4/luc cells, a HEK 293 cell line stably
expressing mouse TLR4, indicating that the TLR4-MD-2 complex is formed
on the surface of 293/mTLR4/luc cells (Fig. 1B). To identify
the important amino acid residues of mouse MD-2 for complex formation
with TLR4 on the cell surface, we generated expression constructs of
mouse MD-2 mutants in which amino acid residues from
Glu17 to C-terminal Asn160
were individually replaced by alanine. Alanine is usually chosen for
replacement because it eliminates the side chain beyond the 
carbon
yet does not alter the main-chain conformation or have an extreme
electrostatic or steric effect (21, 25). Because MD-2 is a
secretory protein (26), and the site between
Thr16 and Glu17 of mouse MD-2 was predicted to
be a possible cleavage site of the leader sequence with the PSORT II
program (http://psort.ims.u-tokyo.ac.jp/), we analyzed the amino acid
residues from Glu17 to C-terminal
Asn160. The expression constructs were introduced
into 293/mTLR4/luc cells, and cell surface TLR4-MD-2 complex formation
was measured by flow cytometry using MTS510 (Fig. 2). Expression of mouse MD-2 confers LPS
and Taxol responsiveness on 293/mTLR4/luc cells (Ref. 10
and Fig. 1C), and this responsiveness depends on the
expression of mouse TLR4 (10). The LPS and Taxol
responsiveness of 293/mTLR4/luc cells with the mutant MD-2 expression
constructs introduced was examined by measuring LPS or Taxol-induced
NF-B-dependent reporter (luciferase) activation (Fig. 2). Many MD-2
mutants showed reduced ability to confer LPS and/or Taxol
responsiveness on TLR4, and to form the TLR4-MD-2 complex that was
detected by MTS510, and the critical residues for the responses to LPS
and/or Taxol, and for the formation of the TLR4-MD-2 complex recognized
by MTS510 were not clustered in a particular region of mouse MD-2 (Fig. 2B).
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The reactivity of MTS510 mAbs, which specifically react with the
cell surface TLR4-MD-2 complex, with 293/mTLR4/luc cells with C37A,
L71A, L78A, C95A, Y102A, C105A, E111A, V113A, I117A, P118A, F119A,
E136A, I138A, L146A, C148A, and T152A introduced was <5% of that on
cells expressing mouse MD-2 (Figs. 2
and 3, C and
D). As shown in Fig. 3E, the expression levels of
these mutant MD-2 proteins in 293/mTLR4/luc cells were similar to that
of the mouse MD-2 protein, indicating that these mutations did not
affect the protein expression levels. It is noteworthy that the LPS-
and Taxol-induced luciferase activity in 293/mTLR4/luc cells with the
MD-2 mutants introduced, the expression of which did not lead to
Ab-detectable TLR4-MD-2 complex formation on 293/mTLR4/luc cells, was
<9 and 2%, respectively, of that in cells expressing mouse MD-2
(Figs. 2 and 3, C and D). These results suggest
that the ability of MD-2 to form a complex with TLR4 on the cell
surface is essential for its ability to confer LPS and Taxol
responsiveness on TLR4.
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The reactivity of MTS510 mAbs with 293/mTLR4/luc cells expressing
the Y34A, Y36A, G59A, V82A, I85A, F126A, P127A, G129A, I153A, I154A,
and H155A mutants was >55% of that of cells expressing mouse MD-2
(Figs. 2 and 3, A and B). Although the abilities
of these mutants to form a complex with TLR4 on the cell surface were
similar to or somewhat lower than that of mouse MD-2, their abilities
to confer LPS and Taxol responsiveness on 293/mTLR4/luc cells were
apparently lower than that of mouse MD-2 (Fig. 3, A and
B). These results suggest that cell surface complex
formation with TLR4 is not sufficient for MD-2 to confer LPS or Taxol
responsiveness on TLR4.
Furthermore, we examined the amino acid residues replaced by alanine
with which the ability to form a complex with mouse TLR4 was retained,
but with which the ability to confer both LPS and Taxol responsiveness
on mouse TLR4 was apparently reduced. Ile85,
Ile153, and Tyr34 of mouse
MD-2 were individually replaced by Met, Leu, and Trp, respectively, and
then the ability to confer LPS and Taxol responsiveness on
293/mTLR4/luc cells was examined. As shown in Fig. 4A, 293/mTLR4/luc cells
expressing the I85 M mutant showed similar Taxol sensitivity to that of
cells expressing mouse MD-2. But cells expressing the I85 M mutant
showed apparent lower LPS sensitivity than cells expressing mouse MD-2.
These results show that replacement of Ile85 by
Met specifically reduces the ability of mouse MD-2 to confer LPS
responsiveness on mouse TLR4, and that replacement of
Ile85 by Ala reduces the ability of mouse MD-2 to
confer both LPS and Taxol responsiveness on mouse TLR4. In contrast,
293/mTLR4/luc cells expressing the I153L mutant showed similar LPS
sensitivity to that of cells expressing mouse
MD-2, but cells expressing the I153L mutant did
not respond to Taxol stimulation (Fig. 4B). Similarly,
293/mTLR4/luc cells expressing the Y34W mutant showed similar LPS
sensitivity to that of cells expressing mouse MD-2, but cells
expressing the Y34W mutant showed apparent lower Taxol sensitivity than
ones expressing mouse MD-2 (Fig. 4C). These results show
that replacement of Ile153 and
Tyr34 by Leu and Trp, respectively, specifically
reduces the ability of mouse MD-2 to confer Taxol responsiveness on
mouse TLR4, and that replacement of Ile153 and
Tyr34 by Ala reduces the ability of mouse MD-2 to
confer both LPS and Taxol responsiveness on mouse TLR4. These results,
taken together, suggest that the amino acid residues at codon numbers
34, 85, and 153 of mouse MD-2 are important for conferring both LPS and
Taxol responsiveness on mouse TLR4, and that the required amino acid
residues at these positions for conferring LPS responsiveness are
partly different from those for Taxol responsiveness.
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Previously, we demonstrated that a single mutation at
Gln22 of mouse MD-2 reduces its ability to confer
Taxol responsiveness on mouse TLR4, but does not affect its ability to
confer LPS responsiveness (10). In addition to a single
alanine mutation at Gln22, single alanine
mutations at Asp101 and
Glu122 also reduced the ability of mouse MD-2 to
confer Taxol responsiveness, but not LPS responsiveness (Fig. 5A). Furthermore, we examined
its ability to confer LPS and Taxol responsiveness on TLR4 by measuring
dose responses against LPS or Taxol stimulation. Consistent with the
previous findings, 293/mTLR4/luc cells expressing the Q22A mutant
showed lower Taxol sensitivity than that of cells expressing mouse
MD-2, and cells expressing the Q22A mutant showed similar LPS
sensitivity to that of cells expressing mouse MD-2 (Fig. 5B). Cells expressing the D101A or E122A mutant showed
apparently lower Taxol sensitivity than ones expressing mouse MD-2, and
their LPS sensitivity was similar to that of cells expressing mouse
MD-2 (Fig. 5, C and D). In contrast, cells
expressing the R157A mutant showed lower sensitivity to LPS than ones
expressing mouse MD-2, and they showed similar Taxol sensitivity to
that of cells expressing mouse MD-2 (Fig. 5, A and
E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown that alanine substitution at Tyr34, Tyr36, Gly59, Val82, Ile85, Phe126, Pro127, Gly129, Ile153, Ile154, or His155 of mouse MD-2 apparently reduces its ability to confer LPS and Taxol responsiveness on TLR4, but does not affect its ability to form a complex with TLR4 so much. These amino acid alterations may affect the direct interaction between MD-2 and ligands, or may induce conformational changes of TLR4-MD-2 complex that are important for the direct interaction with ligands or for inducing intracellular signaling. The precise molecular mechanisms underlying the involvement of these amino acid residues conferring ligand responsiveness on TLR4 remain to be for further analysis.
In this study, we systemically analyzed mouse MD-2 residues that affect the formation of the TLR4-MD-2 complex using MTS510 Abs, which react more strongly with the mouse TLR4-MD-2 complex than TLR4 alone. We found that some mouse MD-2 mutants showed dramatically reduced ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510. The Ab was raised against mouse TLR4, was reported to react with TLR4 but not with MD-2, and was suggested to recognize the specific conformation of mouse TLR4 that is associated with mouse MD-2 (11). Therefore, 293/mTLR4/luc cells that express a mutant mouse MD2 but do not react with MTS510 might be defective in the TLR4-MD-2 complex formation on the cell surface because of a defect in the physical association of the mutant MD-2 with TLR4, and/or a defect in the translocation of the TLR4-MD-2 complex, which has been shown to be formed in the endoplasmic reticulum (26) to the cell surface. Alternatively, such cells might have lost the conformational epitope recognized by the Ab. In addition, we found that the mouse MD-2 mutants showed reduced ability to confer LPS and Taxol responsiveness. All things considered, we suggest that the ability of MD-2 to form the cell surface TLR4-MD-2 complex is essential for its ability to confer LPS and Taxol responsiveness on TLR4, but not sufficient.
Cysteine residues, which form intra and intermolecular disulfide bonds, are generally thought to be important for the molecular structure. Human MD-2 contains seven cysteine residues, which are conserved between mouse and man, and these residues are important for the formation of large disulfide-linked oligomers (26, 31). Our results showed that Cys residues, such as Cys37, Cys95, Cys105, and Cys148 of mouse MD-2 are important for conferring LPS and Taxol responsiveness on mouse TLR4 and for forming the cell surface TLR4-MD-2 complex recognized by MTS510. In contrast, substitution of Cys133 by Ala did not affect the ability to confer LPS and Taxol responsiveness on TLR4, or the ability to form the TLR4-MD-2 complex recognized by MTS510 so much. Structural analysis of MD-2 and the TLR4-MD-2 complex is essential for further understanding of the molecular mechanisms underlying the recognition of ligands and signal transduction. We believe that our functional analysis of MD-2 residues should be helpful for the modeling of the structure of MD-2.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Masahiro Nishijima, Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail address: nishim{at}nih.go.jp
3 Abbreviations used in this paper: TLR, Toll-like receptor; HEK, human embryonic kidney.
Received for publication June 4, 2002. Accepted for publication October 21, 2002.
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B translocation in murine macrophages. Infect. Immun. 64:878.[Abstract]
B by lipopolysaccharide. J. Immunol. 167:3354.This article has been cited by other articles:
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