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B by Lipopolysaccharide
Division of Microbiology, National Institute of Health Sciences, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B by LPS. LPS binding to
cells expressing CD14, TLR4, and MD-2 was unaffected by these
mutations. These observations demonstrate that hMD-2 undergoes
N-linked glycosylation at Asn26 and
Asn114, and that these glycosylations are crucial for
TLR4-mediated signal transduction of LPS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and plays a major role in
septic shock in humans (1). CD14 is believed to be a
recognition molecule of LPS and is thought to play a central role in
the activation of cells with the help of LPS-binding protein
(LBP)2 (3, 4). However, because CD14, a GPI-anchored protein, lacks a
transmembrane region (5), a search was made for
transmembrane molecules that transmit LPS signaling into the cytoplasm,
and Toll-like receptors (TLRs) were identified as candidates
(6, 7, 8, 9).
TLRs are mammalian homologues of the Drosophila Toll
protein, which plays critical roles in the establishment of
dorsoventral polarity and the antifungal response in adult flies
(10). In mammals, the TLR family consists of at least
eight members. Two of them, TLR2 and TLR4, have been found to confer
responsiveness to bacterial products (6, 7, 9, 11). TLR2
responds to peptidoglycans and lipoteichoic acids
(12), whereas TLR4 is involved in LPS signaling. A recent
study demonstrated that TLR4-mediated activation of NF-B by LPS
required MD-2, a novel accessory molecule expressed on the cell surface
and associated with TLR4 (13).
Human (h)MD-2 consists of 160 amino acids residues with a predicted
molecular mass of 18 kDa, and there are two potential
N-linked glycosylation sites in this amino acid sequence.
Although the general role of glycosylation of protein molecules has not
been completely established, many functions of particular
glycosylations have been reported. These functions include modulation
of enzyme and hormone activity, regulation of intracellular traffic,
control of protein folding, ligand recognition, and cell-cell
interaction (see Ref. 14 for review). However, the
structural and functional implications of glycosylation have not been
elucidated in regard to the hMD-2 molecule. In this study, we found
that hMD-2 undergoes N-linked glycosylation, and that the
glycosylation is essential for hTLR4-mediated activation of NF-B
by LPS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human embryonic kidney 293 cell line (obtained from the Human Science Research Resource Bank, Tokyo, Japan) was grown in DMEM (Life Technologies, Rockville, MD) supplemented with 10% (v/v) heat-inactivated FCS (Life Technologies), penicillin (100 U/ml), and streptomycin (100 µg/ml). The human monocytic cell line THP-1 was grown in the same way as the 293 cells except that RPMI 1640 (Life Technologies) was used instead of DMEM. LPS was prepared from Escherichia coli 03K2a2B:H2 (S type) and R3 F653 (R type) as described by Westphal et al. (15) and Galanos et al. (16), respectively, and was purified according to the methods of Hirschfeld et al. (17). An antiserum (1060) against the equine infectious anemia virus (EIAV)-tag epitope (amino acid sequence ADRRIPGTAEE) was a kind gift of Dr. N. Rice (National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD). A goat anti-human TLR4 Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Sulfosuccinimidyl-6-(bioitinamido)-6-hexanamido hexanoate (sulfo-NHS-LC-LC-biotin) and immobilized streptavidin agarose were from Pierce (Rockford, IL). PNGase F was purchased from New England Biolabs (Beverly, MA). Alexa fluor 594-conjugated LPS was purchased from Molecular Probes (Eugene, OR). Unless otherwise noted, all other chemicals were obtained from Wako Pure Chemical (Osaka, Japan).
Plasmid construction and site-directed mutagenesis
Plasmid containing human CD14 cDNA was provided by Dr. S. Yamamoto (Medical College of Oita, Oita, Japan). The coding regions of hMD-2 and hTLR4 were amplified by RT-PCR from total RNA prepared from THP-1 and human spleen total RNA (OriGene Technologies, Rockville, MD), respectively. The coding regions of hMD-2, CD14, and TLR4 minus their predicted signal peptide sequences were subcloned downstream of a mammalian expression vector in which the preprotrypsine signal peptide sequence precedes the NH2-terminal EIAV tag (these constructs will be referred to as pEIAV-hMD-2, pEIAV-hCD14, and pEIAV-hTLR4, respectively). Epitope tagging had no influence on the activity of hMD-2, CD14, and TLR4 in tissue culture. Luciferase reporter plasmid endothelial cell-leukocyte adhesion molecule (pELAM)-L, was constructed by inserting the PCR fragment (-730 to +52) of the E-selectin promoter into the SacI-HindIII site of pGL3 Basic vector (Promega, Madison, WI). Point mutations at the potential glycosylation sites (Asn26 and Asn114) of hMD-2 were created by PCR. One of either asparagines (N26Q or N114Q) or both (N26:114Q) was replaced with glutamine. The sequence of primers used for PCR were as follows: 5'-GGG TCT GCC AGT CAT CCG ATG CA-3' and 5'-AAT ACT GCT TCT GAG CTT CAG TAA ATA TGG-3' for N26Q and 5'-GAG ACT GTG CAG ACA ACA ATA TCA TTC TCC-3' and 5'-TCC CTT CAG AGC TCT GCA AAA AGA GT-3' for N114Q. All mutations were confirmed by automated DNA sequencing using a dye terminator cycle sequencing kit (Amersham Pharmacia Biotech, Piscataway, NJ) and an ABI Prism 310 genetic analyzer (PerkinElmer Applied Biosystems, Foster City, CA).
Glycosidase treatment of cellular extracts
After plating 293 cells in 6-cm dishes, the cells were transfected by the calcium phosphate precipitation method (18) with a hMD-2 expression plasmid (5 µg), and 24 h later, the cells were lysed with a lysis buffer (10 mM HEPES-KOH, 5 mM EDTA, 0.5% Nonidet P-40, and 10 mM KCl (pH 7.9)) containing a protease inhibitor mix (Boehringer Mannheim, Mannheim, Germany) on ice for 10 min. Following centrifugation at 1000 x g for 5 min, the supernatant obtained was used as the cellular extract and boiled for 10 min in 0.5% SDS and 1% 2-ME. After adding 1/10 vol of 0.5 M sodium phosphate (pH 7.5) and 10% Nonidet P-40, the boiled extract was incubated with PNGase F (final concentration of 10 U/ml) at 37°C for 1 h, and then subjected to SDS-PAGE. The hMD-2 molecule was detected by Western blot analysis. The signals were visualized with an ECL system (Amersham, Arlington Heights, IL).
Biotinylation of cell surface protein
After plating 293 cells in 6-cm dishes, they were transfected by the calcium phosphate precipitation method with hMD-2 expression plasmids (5 µg each), and 24 h later, the cells were washed with PBS twice and treated with 2 ml ice-cold 0.5 mg/ml sulfo-NHS-LC-LC-biotin, a membrane-impermeable biotinylation reagent (19, 20), in PBS at 4°C for 30 min. Then, after stopping the biotinylation reaction by incubating with 20 mM glycine in PBS at 4°C for 15 min, cellular extracts were prepared with 200 µl lysis buffer as described above. The cellular extracts were diluted to 500 µl with PBS containing 0.5% Nonidet P-40 and incubated with immobilized streptavidin agarose at 4°C for 1 h. After washing with PBS containing 0.5% Nonidet P-40 three times, the agarose was boiled in SDS-PAGE sample buffer. The supernatant obtained was subjected to SDS-PAGE and Western blot analysis, and hMD-2 was detected with a rabbit anti-EIAV antiserum as described above.
Immunoprecipitation of TLR4
After seeding 293 cells in 6-cm dishes, they were transfected by the calcium phosphate precipitation method with hMD-2 and TLR4 expression plasmids (5 µg each), and 24 h later, cellular extracts were prepared as described above. The cellular extracts were diluted to 500 µl with PBS containing 0.5% Nonidet P-40, and after adding an anti-human TLR4 Ab and protein A/G-Sepharose (Pierce), the diluted cellular extracts were incubated for 1 h at 4°C with rocking. After washing with PBS containing 0.5% Nonidet P-40, the Sepharose beads were boiled in SDS-PAGE sample buffer, and the supernatant was subjected to SDS-PAGE and Western blot analysis.
Purification of rhLBP
The coding region of hLBP minus its signal sequence was
amplified by RT-PCR from total RNA prepared from THP-1 and subcloned
into a yeast expression vector pGAPZ (Invitrogen, Carlsbad,
CA) with an N-terminal x6 histidine tag sequence. LBP was expressed in
a Pichia expression system according to the recommendation of the
manufacturer (Invitrogen) and was purified on a
Ni2+ column (Novagen, Madison, WI).
NF-B reporter assay
After plating 293 cells in 6-well dishes (4 x 105/well), they were transfected by the calcium phosphate precipitation method with the expression plasmids indicated (0.02 µg), 0.2 µg of pELAM-L luciferase reporter plasmid, and 0.05 µg of pRL-TK (Renilla luciferase-thymidine kinase) (Promega) for normalization. After 24 h, cells were stimulated for 6 h in serum-free DMEM containing 100 ng/ml of human LBP, and the reporter gene activity was measured according to the recommendation of the manufacturer (Promega). Results were normalized by thymidine kinase reporter activity.
Tunicamycin treatment
After plating 293 cells (4 x 105/well) in 6-well dishes, they were transfected with hTLR4 and hMD-2 expression plasmid (2 ng) together with luciferase plasmid as described above. At 8 h after transfection, the medium was replaced with normal culture medium containing the indicated concentration of tunicamycin (WAKO Pure Chemical), and the cells were incubated at 37°C for 16 h. The cells were then stimulated with LPS for 6 h in DMEM containing the concentration of tunicamycin indicated and 10% FCS, and the relative luciferase activity was measured as described above. Results were normalized by thymidine kinase reporter activity.
LPS-binding assay
After plating 293 cells onto a coverslip precoated with rat tail collagen type I (BD Biosciences, San Jose, CA) and transfection with the plasmid indicated (5 µg), the cells were incubated with 100 ng/ml of Alexa fluor 594-conjugated LPS in DMEM containing 10% FCS at 37°C for 1 h. The cells were then washed with PBS three times and fixed with 3% paraformaldehyde in PBS (pH 7.6) for 20 min. After washing with PBS, the coverslip was mounted on a slide glass with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA) and viewed under a fluorescence microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An expression vector containing hMD-2 cDNA was transiently
transfected in human kidney 293 cells, and cellular extracts were
prepared from the cells (Fig. 1
). Western
blotting analysis of hMD-2 detected three forms of proteins with
different electrophoretic mobility (Fig. 1, left four
lanes). Analysis of the cellular extract prepared from the cells
transfected with the control vector gave no protein signals around that
molecular mass region, indicating that these signals originated from
hMD-2. The apparent molecular mass of these protein signals estimated
by a prestained molecular mass marker were found to be 
14, 18, and
23 kDa. To investigate the involvement of glycosylation in
the appearanceof the multiple forms, we treated cellular extracts
prepared from 293 cells transiently expressing hMD-2 with an
N-glycosidase, PNGase. The PNGase-treated cellular
extract was analyzed for hMD-2 protein by Western blotting (Fig. 1, left). Only the fastest mobility form of hMD-2 was detected
in the PNGase-treated extract, although three forms of hMD-2 were
detected in the cellular extract treated in parallel without PNGase.
Phosphatase treatment did not affect the mobility pattern (our
unpublished observation). These findings suggest that
glycosylation is responsible for the appearance of these forms. To
confirm this, we generated hMD-2 mutant plasmids lacking one or both
potential N-linked glycosylation sites, and these mutants
were expressed in 293 cells (Fig. 1, right five lanes). The
single mutation at Asn26 or
Asn114 (named N26Q or N114Q) resulted in the
disappearance of the slowest mobility form. Double mutation of both
Asn26 and Asn114 (named
N26:114Q) resulted in the disappearance of both the slowest and the
intermediate mobility. Taken together, these results indicate that the
N-linked glycosylation either at Asn26
or Asn114 is responsible for the appearance of
the intermediate mobility form, and that the slowest mobility form is
resulted from the glycosylation at both Asn26 and
Asn114.
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The effect of N-linked glycosylation of hMD-2 on
LPS-induced activation of NF-B via human TLR4 was investigated by an
NF-B-dependent luciferase reporter assay. Although 293 cells
expressing only TLR4 and CD14 hardly responded to LPS, coexpression of
wild-type hMD-2 resulted in strong induction of reporter activity in
response to LPS. Expression of the N26Q or N114Q mutant instead of
wild-type hMD-2 resulted in partial induction, and only slight
induction was observed in cells expressing the double mutant N26:114Q
(Fig. 2). To confirm that the lack of
glycosylation of hMD-2 was responsible for the impaired activity, we
next examined the effect of tunicamycin, which prevents the addition of
N-linked glycan to polypeptide (21). The 293
cells were transiently expressed with TLR4 and hMD-2 and treated with
varying concentrations of tunicamycin followed by LPS or TNF-. In
this experiment, we did not express CD14 because tunicamycin prevents
the glycosylation of CD14, which may release GPI-anchored CD14 from the
cell membrane and cause reduction of LPS response. Instead, we used
10% FCS as a source of soluble CD14 and LBP. Tunicamycin inhibited the
LPS-induced activation of NF-B in a concentration-dependent manner,
whereas the TNF--induced activation was unaffected by the
tunicamycin treatment (Fig. 3).
Tunicamycin was cytotoxic at concentrations above 100 ng/ml. These
results indicate the important role of N-linked
glycosylation of hMD-2 for LPS-induced activation of NF-B via
TLR4.
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Glycosylation is known to often play a crucial role in the folding
and trafficking of membrane glycoproteins (22, 23, 24, 25).
Therefore, to determine the influence of N-linked
glycosylation on cell surface expression of hMD-2, we expressed the
wild-type and mutant hMD-2 proteins into 293 cells and labeled cell
surface proteins with a membrane-impermeable biotin. The biotinylated
proteins were collected with streptavidin-agarose, electrophoresed by
SDS-PAGE, and finally, hMD-2 was detected by Western blotting. A
comparable amount of hMD-2 proteins was detected in the
streptavidin-precipitates obtained from cells expressing either
wild-type or mutant MD-2 proteins, whereas MD-2 was not detected
without biotinylation (Fig. 4). This
result demonstrates that hMD-2 is still expressed on the cell surface
without glycosylation at Asn26 and
Asn114.
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To determine whether N-linked glycosylation
participates in the association of hMD-2 with TLR4, immunoprecipitation
was conducted by using an anti-human TLR4 Ab. The wild-type or
mutant (N26Q, N114Q, and N26:114Q) MD-2 proteins were expressed in 293
cells with or without expression of TLR4 (Fig. 5, lower lane). TLR4 was
immunoprecipitated with its Ab, and coprecipitated hMD-2 was detected
by Western blotting. The hMD-2 molecule was not coprecipitated when
TLR4 alone or hMD-2 alone was expressed (Fig. 5, upper panel,
lanes 1 and 2). When both TLR4 and MD-2 were expressed,
three forms of hMD-2 proteins were coprecipitated. The hMD-2 mutants
that lack one or both N-glycosylation site(s) were also
coprecipitated with TLR4. These results indicate that hMD-2 that has
not been glycosylated at Asn26 and
Asn114 is still capable of associating with
TLR4.
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We next examined LPS binding by using Alexa fluor 594-labeled LPS
(Fig. 6), because lack of glycosylation
of the hMD-2 molecule may affect LPS-binding ability. The binding study
was conducted in the presence of 10% FCS to prevent nonspecific
binding of labeled LPS. No detectable bindings were observed in 293
cells transfected with either a vector control, TLR4, wild-type hMD-2,
N26:114Q, wild-type hMD-2 plus TLR4, or N26:114Q plus TLR4. However,
the bindings were clearly detectable in 293 cells expressing either
wild-type hMD-2 plus CD14 or wild-type hMD-2 plus TLR4 plus CD14. These
bindings were still detectable when N26:114Q was expressed instead of
wild-type hMD-2, indicating no significant role of glycosylation of
hMD-2 in the LPS binding. Taken together, these findings indicate that
N-linked glycosylations at Asn26 and
Asn114 in hMD-2 are crucial for TLR4-mediated
signal transduction of LPS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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14, 18, and 23 kDa,
respectively. Shimazu et al. (13) reported that hMD-2 is
detectable as multiple bands on Western blot analysis. The PROSIT scan
analysis revealed that there are two potential N-linked
glycosylation sites and several phosphorylation sites in the amino acid
sequence of hMD-2. N-glycosidase treatment, but not
phosphatase treatment, reduced the molecular mass of the two slower
mobility forms, resulting in the detection of only the fastest mobility
form. In addition, our mutational analysis at the potential
glycosylation sites clearly demonstrated that the glycosylation at
Asn26 and Asn114 caused the
molecular mass shift.
N-linked glycosylation is a cotranslational modification
found in most cell surface proteins, but the precise function of the
carbohydrate on these proteins is not well understood
(26). Until now, at least two categories of functions of
glycosylation were known. One consists of physicochemical functions,
which include modifications of solubility, electrical charge, and mass,
and control of protein folding and stabilization of protein
conformation (14), all of which are important for
trafficking and correct folding of membrane glycoproteins. The other
consists of biological functions. Recent reports have provided evidence
that N-linked glycosylation may be required for ligand
recognition or signaling (27, 28, 29, 30). However, what role
N-linked glycosylation plays in ligand binding and signaling
remains uncertain. N-linked glycosylation may stabilize a
conformation required for binding, or oligosaccharides themselves may
be an essential part of the binding site. To explore the function of
glycosylation of hMD-2, we generated hMD-2 mutants lacking one or both
potential N-linked glycosylation sites. The ability of hMD-2
to complement LPS-induced NF-
activation via TLR4 was
substantially impaired when the hMD-2 mutant lacking both
N-linked glycosylation sites was expressed instead of
wild-type hMD-2. The possibility that lack of glycosylation of hMD-2
impaired its membrane expression can be excluded, because a comparable
level of the membrane surface expression was observed between the
wild-type and mutant hMD-2 proteins (Fig. 4
). It is unlikely that the
mutations at Asn26 and/or
Asn114 of hMD-2 disrupted its secondary
structure, leading to loss of function of hMD-2, because hMD-2 carrying
these mutations still retained the ability to associate with TLR4 (Fig. 5). In addition, tunicamycin treatment, which prevents
N-linked glycosylation, greatly inhibited the LPS-induced
NF-B activation in cells expressing TLR4 and MD-2. Thus, these
results indicate that the glycosylation of hMD-2 plays a crucial role
in the LPS response via TLR4. TLR4 also possesses potential
glycosylation sites. Overexpression of TLR4 is known to increase
constitutive NF-B activity (31). We observed that
tunicamycin treatment did not inhibit the constitutive activation of
NF-B induced by overexpression of TLR4 (data not shown). This
indicates that the lack of glycosylation of TLR4 does not affect at
least one of the functions of TLR4, although this does not exclude the
possibility that the lack of glycosylation of TLR4 may affect
LPS-induced activation of TLR4. In contrast, TNF--induced activation
of NF-B was unaffected by tunicamycin treatment (Fig. 3), even
though the p60 TNFR, which is reported to be involved in the activation
of NF-B (32), possesses three potential
N-linked glycosylation sites (33). This may
reflect a selective role of glycosylation of MD-2 in the LPS
response.
Shimazu et al. (13) failed to detect cell surface
expression of hMD-2 when it was stably expressed in Ba/F3 cells without
expression of TLR4, suggesting that MD-2 is anchored in the membrane
via physical association with TLR4. However, in our study, we detected
cell surface expression of hMD-2 without TLR4 in 293 cells. It is
unlikely that the biotinylation reagent we used labeled intercellularly
located MD-2 because of the following reasons: 1) This reagent is well
characterized (19, 20) not to pass through cell membrane
due to its strong negative charge
(SO3-); 2) we performed the
biotinylation at 4°C to prevent endocytosis, and we stopped the
biotinylation reaction by adding glycine before lysing the cells; and
3) we were unable to detect the biotinylation of a cytosolic protein,
IB-, with this biotinylation reagent. Recently, da Silva Correia
et al. (34) also observed that MD-2 was clearly detectable
on the cell surface without TLR4 expression using FACS analysis when
MD-2 was transiently expressed in 293 cells. Therefore, the difference
between our result and the result of Shimazu et al. (13)
seems to be attributable to the difference in the level of MD-2
expression between stable and transient transfection. Higher expression
can be obtained with a transient transfection system, and that may have
led to the detection of MD-2 expressed on the cell surface without
anchoring to TLR4, although coexpression of TLR4 may increase cell
surface expression of MD-2.
It is unknown how the glycosylation of MD-2 participates in the LPS
signaling via TLR4. At least the mutations of potential glycosylation
sites of MD-2 did not affect the ability of MD-2 to associate with TLR4
or LPS binding to cells expressing TLR4, CD14, and MD-2 (Figs. 5 and 6). LPS binding was inhibited by a 100-fold excess of unlabeled LPS
(data not shown), and no binding was detected in 293 cells transfected
with vector alone (Fig. 6). These results indicate that the binding we
detected reflects the specific binding of LPS. Akashi et al.
(35) reported that a conformational change may occur in
mouse TLR4 as a result of the association of MD-2. The glycosylation of
hMD-2 may participate in stabilizing a certain conformation of TLR4
that allows TLR4 to transmit LPS signaling. This remains to be
clarified.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: LBP, LPS-binding protein; TLR, Toll-like receptor; EIAV, equine infectious anemia virus; ELAM, endothelial cell-leukocyte adhesion molecule; h, human; sulfo-NHS-LC-LC-biotin, sulfosuccinimidyl-6-(biotin-amido)-6-hexanamido hexanoate; RL-TK, Renilla luciferase-thymidine kinase.
Received for publication February 23, 2001. Accepted for publication July 10, 2001.
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subunit impair intracellular transport. Biochemistry 32:7818.[Medline]
subunit is essential for ligand binding and signal transduction. J. Biol. Chem. 270:24580. subunit are essential for transmembrane signaling. J. Biol. Chem. 267:17415.B by TNF muteins specific for the p60 and p80 TNF receptors. J. Immunol. 157:2410.[Abstract]
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T. L. Gioannini, A. Teghanemt, D. Zhang, N. P. Coussens, W. Dockstader, S. Ramaswamy, and J. P. Weiss Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations PNAS, March 23, 2004; 101(12): 4186 - 4191. [Abstract] [Full Text] [PDF]
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M. Muroi, T. Ohnishi, S. Azumi-Mayuzumi, and K.-i. Tanamoto Lipopolysaccharide-Mimetic Activities of a Toll-Like Receptor 2-Stimulatory Substance(s) in Enterobacterial Lipopolysaccharide Preparations Infect. Immun., June 1, 2003; 71(6): 3221 - 3226. [Abstract] [Full Text] [PDF]
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 T. Ohnishi, M. Muroi, and K.-i. Tanamoto MD-2 Is Necessary for the Toll-Like Receptor 4 Protein To Undergo Glycosylation Essential for Its Translocation to the Cell Surface Clin. Vaccine Immunol., May 1, 2003; 10(3): 405 - 410. [Abstract] [Full Text] [PDF]
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G. E. D. Mullen, M. N. Kennedy, A. Visintin, A. Mazzoni, C. A. Leifer, D. R. Davies, and D. M. Segal The role of disulfide bonds in the assembly and function of MD-2 PNAS, April 1, 2003; 100(7): 3919 - 3924. [Abstract] [Full Text] [PDF]
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K. Kawasaki, H. Nogawa, and M. Nishijima Identification of Mouse MD-2 Residues Important for Forming the Cell Surface TLR4-MD-2 Complex Recognized by Anti-TLR4-MD-2 Antibodies, and for Conferring LPS and Taxol Responsiveness on Mouse TLR4 by Alanine-Scanning Mutagenesis J. Immunol., January 1, 2003; 170(1): 413 - 420. [Abstract] [Full Text] [PDF]
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K. Miyake, Y. Nagai, S. Akashi, M. Nagafuku, M. Ogata, and A. Kosugi Essential role of MD-2 in B-cell responses to lipopolysaccharide and Toll-like receptor 4 distribution Innate Immunity, December 1, 2002; 8(6): 449 - 452. [Abstract] [PDF]
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M. Muroi and K.-i. Tanamoto The Polysaccharide Portion Plays an Indispensable Role in Salmonella Lipopolysaccharide-Induced Activation of NF-{kappa}B through Human Toll-Like Receptor 4 Infect. Immun., November 1, 2002; 70(11): 6043 - 6047. [Abstract] [Full Text] [PDF]
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A. E. Medvedev, A. Lentschat, L. M. Wahl, D. T. Golenbock, and S. N. Vogel Dysregulation of LPS-Induced Toll-Like Receptor 4-MyD88 Complex Formation and IL-1 Receptor-Associated Kinase 1 Activation in Endotoxin-Tolerant Cells J. Immunol., November 1, 2002; 169(9): 5209 - 5216. [Abstract] [Full Text] [PDF]
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M. Muroi, T. Ohnishi, and K.-i. Tanamoto Regions of the Mouse CD14 Molecule Required for Toll-like Receptor 2- and 4-mediated Activation of NF-kappa B J. Biol. Chem., October 25, 2002; 277(44): 42372 - 42379. [Abstract] [Full Text] [PDF]
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), wild-type hMD-2 (
), N26Q (•), N114Q (
), or N26:114Q
(
) plasmids, together with pELAM-L and pRL-TK. After 24 h,
cells were stimulated with LPS in serum-free DMEM containing human LBP
(100 ng/ml) for 6 h, and luciferase activity was measured.
Relative luciferase activity was normalized by thymidine kinase
promoter activity. Data are presented as the means ± SE from at
least three independent experiments.
