髓样分化蛋白-2在识别和转导内毒素信号中的作用

脂多糖(LPS)通过TLR4介导细胞炎症反应.研究表明,髓样分化蛋白-2(MD-2)通过与TLR4形成复合物参与LPS诱导的细胞信号过程.TLR4/MD-2复合物中的MD-2结合LPS后,引起TLR4低聚化,进而激发下游信号.MD-2合成后,大部分在内质网/高尔基体和TLR4结合,然后以TLR4/MD-2复合物的形式在细胞表面表达.这既能调节TLR4的胞内分布,又能辅助TLR4识别LPS.还有一部分MD-2释放到血浆中,形成可溶性的MD-2(sMD-2).sMD-2在CD14参与下,能结合血浆中的LPS,形成LPS-sMD-2复合物从而辅助只表达TLR4而不表达MD-2的细胞识别LPS,但过度表达的sMD-2又能抑制LPS信号.MD-2在TLR4介导的内毒素识别和信号转导过程中发挥了重要的调控作用.     

The lipopolysaccharide-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2

We analysed the lipopolysaccharide (LPS)-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2. When TLR4 and CD14 were transiently expressed in HEK293 cells, cell-surface expression of TLR4 was observed, although the expression level was lower than that in cells coexpressing MD-2. We found that membrane CD14-TLR4 complexes were formed in these cells in response to LPS stimulation even in the absence of MD-2 expression, although NF-kappaB-dependent reporter activity was not induced. A strong activation of NF-kappaB was observed when these cells were stimulated with LPS followed by soluble MD-2 in this order, even when excess LPS was removed after formation of the CD14-TLR4 complex by washing cells prior to sMD-2 addition. From these results, we propose an additional LPS-recognition mechanism. In cells expressing TLR4 and CD14 but lacking MD-2, LPS is first transferred to membrane CD14 with the aid of LPS binding protein, which leads to the formation of the TLR4-CD14 complex. Then, the binding of soluble MD-2 to this complex triggers the transmembrane signal transduction. Cells expressing TLR4 and CD14 but lacking MD-2, such as airway epithelial cells, may be activated in response to LPS by this mechanism.     

N-linked glycosylations at Asn(26) and Asn(114) of human MD-2 are required for toll-like receptor 4-mediated activation of NF-kappaB by lipopolysaccharide.

MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn(26) and Asn(114)) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn(26) and Asn(114). Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-kappaB 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 Asn(26) and Asn(114), and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.     

利用荧光共振能量转移技术研究活细胞TLR4与MD-2作用结构域

脂多糖(LPS)的识别和信号转导是宿主发生防御反应的关键,Toll样受体4(TLR4)与髓样分化蛋白-2(MD-2)形成复合物在LPS的识别及其信号转导中发挥了重要作用．研究TLR4与MD-2结合的功能结构域,对于深入了解LPS信号转导机制及其内毒素休克的防治具有重要意义．运用基于强度的三通道荧光共振能量转移技术(FRET)及基因突变和转染技术,研究了活细胞TLR4与MD-2作用的结构域．结果表明：N端Glu²⁴～Met⁴¹缺失使TLR4与MD-2结合能力明显下降;LPS刺激后TLR4聚合迅速增加,而缺失Glu²⁴～Met⁴¹的TLR4不能聚合．上述结果提示,TLR4的Glu²⁴～Met⁴¹不仅是结合MD-2的区域,并且还参与了LPS刺激后TLR4的聚合作用．     

Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2

The detection of Gram-negative LPS depends upon the proper function of the TLR4-MD-2 receptor complex in immune cells. TLR4 is the signal transduction component of the LPS receptor, whereas MD-2 is the endotoxin-binding unit. MD-2 appears to activate TLR4 when bound to TLR4 and ligated by LPS. Only the monomeric form of MD-2 was found to bind LPS and only monomeric MD-2 interacts with TLR4. Monomeric MD-2 binds TLR4 with an apparent Kd of 12 nM; this binding avidity was unaltered in the presence of endotoxin. E5564, an LPS antagonist, appears to inhibit cellular activation by competitively preventing the binding of LPS to MD-2. Depletion of endogenous soluble MD-2 from human serum, with an immobilized TLR4 fusion protein, abrogated TLR4-mediated LPS responses. By determining the concentration of added-back MD-2 that restored normal LPS responsiveness, the concentration of MD-2 was estimated to be approximately 50 nM. Similarly, purified TLR4-Fc fusion protein, when added to the supernatants of TLR4-expressing cells in culture, inhibited the interaction of MD-2 with TLR4, thus preventing LPS stimulation. The ability to inhibit the effects of LPS as a result of the binding of TLR4-Fc or E5564 to MD-2 highlights MD-2 as the logical target for drug therapies designed to pharmacologically intervene against endotoxin-induced disease.     

MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor.

The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn(26) and Asn(114), whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn(526) or Asn(575) failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor.     

CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern of Gram-negative bacterial infections, and includes smooth (S-LPS) and rough (R-LPS) chemotypes. Upon activation by LPS through CD14, TLR4/MD-2 heterodimers sequentially induce two waves of intracellular signaling for macrophage activation: the MyD88-dependent pathway from the plasma membrane and, following internalization, the TRIF-dependent pathway from endosomes. We sought to better define the role of scavenger receptors CD36 and CD204/SR-A as accessory LPS receptors that can contribute to pro-inflammatory and microbicidal activation of macrophages. We have found that CD36 differently regulates activation of mouse macrophages by S-LPS versus R-LPS. The ability of CD36 to substitute for CD14 in loading R-LPS, but not S-LPS onto TLR4/MD-2 allows CD14-independent macrophage responses to R-LPS. Conversely, S-LPS, but not R-LPS effectively stimulates CD14 binding to CD36, which favors S-LPS transfer from CD14 onto TLR4/MD-2 under conditions of low CD14 occupancy with S-LPS in serum-free medium. In contrast, in the presence of serum, CD36 reduces S-LPS binding to TLR4/MD-2 and the subsequent MyD88-dependent signaling, by mediating internalization of S-LPS/CD14 complexes. Additionally, CD36 positively regulates activation of TRIF-dependent signaling by both S-LPS and R-LPS, by promoting TLR4/MD-2 endocytosis. In contrast, we have found that SR-A does not function as a S-LPS receptor. Thus, by co-operating with CD14 in both R- and S-LPS loading onto TLR4/MD-2, CD36 can enhance the sensitivity of tissue-resident macrophages in detecting infections by Gram-negative bacteria. However, in later phases, following influx of serum to the infection site, the CD36-mediated negative regulation of MyD88-dependent branch of S-LPS-induced TLR4 signaling might constitute a mechanism to prevent an excessive inflammatory response, while preserving the adjuvant effect of S-LPS for adaptive immunity.     

Novel roles in human MD-2 of phenylalanines 121 and 126 and tyrosine 131 in activation of Toll-like receptor 4 by endotoxin

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E.CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E.CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E.CD14 +/- TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe(121) and Tyr(131) are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E.MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E.CD14 +/- TLR4. E.MD-2(F126A) binds TLR4 with high affinity (K(d) approximately 200 pm) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E.MD-2. These findings reveal roles of Phe(121) and Tyr(131) in TLR4-independent interactions of human MD-2 with E.CD14 and, together with Phe(126), in activation of TLR4 by bound E.MD-2. These findings strongly suggest that the structural properties of E.MD-2, not E alone, determine agonist or antagonist effects on TLR4.     

Kinetics of binding of LPS to recombinant CD14, TLR4, and MD-2 proteins

TLR4 together with CD14 and MD-2 forms a pattern recognition receptor that plays an initiating role in the innate immune response to Gram-negative bacteria. Here, we employed the surface plasmon resonance technique to investigate the kinetics of binding of LPS to recombinant CD14, MD-2 and TLR4 proteins produced in insect cells. The dissociation constants (KD) of LPS for immobilized CD14 and MD-2 were 8.7 microM, and 2.3 microM, respectively. The association rate constant (Kon) of LPS for MD-2 was 5.61 x 10(3) M-1S-1, and the dissociation rate constant (Koff) was 1.28 10 2 S 1, revealing slow association and fast dissociation with an affinity constant KD of 2.33 x 10-6 M at 25 degreesC. These affinities are consistent with the current view that CD14 conveys LPS to the TLR4/MD-2 complex.     

Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.     

Regions of the mouse CD14 molecule required for toll-like receptor 2- and 4-mediated activation of NF-kappa B

Regions of mouse CD14 required for Toll-like receptor 2 (TLR2)- and TLR4-mediated activation of NF-kappaB were studied in transiently transfected 293 cells. Wild-type CD14 enhanced lipopolysaccharide (LPS)-induced NF-kappaB-dependent reporter activity in cells expressing TLR4/MD-2, and deletion of amino acid regions 35-44, 144-153, 235-243, and 270-275 impaired the TLR4-mediated activation. Unlike human CD14, mouse CD14 truncated at amino acid 151 lost the activity. Deletion of amino acids 35-44 or 235-243 also abrogated TLR2-mediated activation of NF-kappaB, whereas mutants lacking 144-153 and 270-275 retained the activity. Deletion and alanine substitution experiments revealed that amino acids 151-153 and 273-275 were required for the TLR4-mediated activation. Both deletion mutants lacking amino acids 35-44 and 235-243 and alanine substitution mutants in regions 151-153 and 273-275 were expressed on the cell surface and retained the ability to associate with TLR4. A cross-linking study with photoreactive LPS showed that the labeling intensities to CD14 mutants/TLR4/MD-2 were paralleled by the ability of CD14 mutants to increase TLR4-mediated activation. These results indicate that different regions of mouse CD14 are required for TLR4- and TLR2-mediated activation of NF-kappaB and suggest that amino acids 35-44, 151-153, 235-243, and 273-275 of mouse CD14 play an important role in LPS binding and its transfer to TLR4/MD-2.     

Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2

The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation of Drosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated cross-linking group ((125)I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex.     

Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness

Cellular responses to LPS are mediated by a cell surface receptor complex consisting of Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4. Site-directed mutagenesis was used to identify the regions of human MD-2 involved in its ability to bind TLR4 and confer LPS responsiveness. A separate region of MD-2 was found to mediate each function. MD-2 binding to TLR4 was dependent on Cys(95) and Cys(105), which might form an intramolecular disulfide bond. Hydrophilic and charged residues surrounding this area, such as R90, K91, D100, and Y102, also contributed to the formation of the TLR4-MD-2 complex. A different region of MD-2 was found to be responsible for conferring LPS responsiveness. This region is not involved in TLR4 binding and is rich in basic and aromatic residues, several of which cooperate for LPS responsiveness and might represent a LPS binding site. Disruption of the endogenous MD-2-TLR4 complex by expression of mutant MD-2 inhibited LPS responses in primary human endothelial cells. Thus, our data indicate that MD-2 interaction with TLR4 is necessary but not sufficient for cellular response to LPS. Either of the two functional domains of MD-2 can be disrupted to impair LPS responses and therefore represent attractive targets for therapeutic interventions.     

Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling

TLRs have been implicated in recognition of pathogen-associated molecular patterns. TLR4 is a signaling receptor for LPS, but requires MD-2 to respond efficiently to LPS. The purposes of this study were to examine the interactions of the extracellular TLR4 domain with MD-2 and LPS. We generated soluble forms of rTLR4 (sTLR4) and TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains. sTLR4 consisted of Glu(24)-Lys(631). MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14. BIAcore analysis demonstrated the direct binding of sTLR4 to MD-2 with a dissociation constant of K(D) = 6.29 x 10(-8) M. LPS-conjugated beads precipitated MD-2, but not sTLR4. However, LPS beads coprecipitated sTLR4 and MD-2 when both proteins were coincubated. The addition of sTLR4 to the medium containing the MD-2 protein significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu(24)-Lys(631) enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.     

Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp(299)Gly and Thr(399)Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-kappaB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-kappaB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-kappaB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.     

Mutational analysis of membrane and soluble forms of human MD-2

Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria. MD-2 is a 20-25-kDa extracellular glycoprotein that binds to Tolllike receptor 4 (TLR4) and LPS and is a critical part of the LPS receptor. Here we have shown that the level of MD-2 expression regulates TLR4 activation by LPS. Using site-directed mutagenesis, we have found that glycosylation has no effect on MD-2 function as a membrane receptor for LPS. We used alanine-scanning mutagenesis to identify regions of human MD-2 that are important for TLR4 and LPS binding. We found that mutation in the N-terminal 46 amino acids of MD-2 did not substantially diminish LPS activation of Chinese hamster ovary (CHO) cells co-transfected with TLR4 and mutant MD-2. The residues 46-50 were important for LPS activation but not LPS binding. The residues 79-83, 121-124, and 125-129 are identified as important in LPS activation but not surface expression of membrane MD-2. The function of soluble MD-2 is somewhat more sensitive to mutation than membrane MD-2. Our results suggest that the 46-50 and 127-131 regions of soluble MD-2 bind to TLR4. The region 79-120 is not involved in LPS binding but affects monomerization of soluble MD-2 as well as TLR4 binding. We define the LPS binding region of monomeric soluble MD-2 as a cluster of basic residues 125-131. Studies on both membrane and soluble MD-2 suggest that domains of MD-2 for TLR4 and LPS binding are separate as well as overlapping. By mapping these regions on a three-dimensional model, we show the likely binding regions of MD-2 to TLR4 and LPS.     

MD-2 binds to bacterial lipopolysaccharide

The exact roles and abilities of the individual components of the lipopolysaccharide (LPS) receptor complex of proteins remain unclear. MD-2 is a molecule found in association with toll-like receptor 4. We produced recombinant human MD-2 to explore its LPS binding ability and role in the LPS receptor complex. MD-2 binds to highly purified rough LPS derived from Salmonella minnesota and Escherichia coli in five different assays; one assay yielded an apparent KD of 65 nm. MD-2 binding to LPS did not require LPS-binding proteins LBP and CD14; in fact LBP competed with MD-2 for LPS. MD-2 enhanced the biological activity of LPS in toll-like receptor 4-transfected Chinese hamster ovary cells but inhibited LPS activation of U373 astrocytoma cells and of monocytes in human whole blood. These data indicate that MD-2 is a genuine LPS-binding protein and strongly suggest that MD-2 could play a role in regulation of cellular activation by LPS depending on its local availability.     

MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.     

Regulatory roles for CD14 and phosphatidylinositol in the signaling via toll-like receptor 4-MD-2

The complex consisting of Toll-like receptor 4 (TLR4) and associated MD-2 signals the presence of lipopolysaccharide (LPS) when it is expressed in cell lines. We here show that normal human mononuclear cells express TLR4 and signal LPS via TLR4. CD14 is a molecule that binds to LPS and facilitates its signaling. Little is known, however, about the relationship of CD14 with TLR4-MD-2. We show that CD14 helps TLR4-MD-2 to sense and signal the presence of LPS. CD14 has also been implicated in recognition of apoptotic cells, which leads to phagocytosis without activation. Membrane phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PtdIns) are thought to serve as the ligands for CD14 in apoptotic cells. We find that PtdIns acts as an LPS antagonist in the signaling via TLR4-MD-2. TLR4-MD-2 seems to discriminate LPS from phospholipids. The signaling via TLR4-MD-2 is thus regulated by CD14 and phospholipid such as PtdIns.     

Functional activity of MD-2 polymorphic variant is significantly different in soluble and TLR4-bound forms: decreased endotoxin binding by G56R MD-2 and its rescue by TLR4 ectodomain

MD-2 is an essential component of endotoxin (LPS) sensing, binding LPS independently and when bound to the ectodomain of the membrane receptor TLR4. Natural variation of proteins involved in the LPS-recognition cascade such as the LPS-binding protein, CD14, and TLR4, as well as proteins involved in intracellular signaling downstream of LPS binding, affect the cellular response to endotoxin and host defense against bacterial infections. We now describe the functional properties of two nonsynonymous coding polymorphisms of MD-2, G56R and P157S, documented in HapMap. As predicted from the MD-2 structure, the P157S mutation had little or no effect on MD-2 function. In contrast, the G56R mutation, located close to the LPS-binding pocket, significantly decreased cellular responsiveness to LPS. Soluble G56R MD-2 showed markedly reduced LPS binding that was to a large degree rescued by TLR4 coexpression or presence of TLR4 ectodomain. Thus, cells that express TLR4 without MD-2 and whose response to LPS depends on ectopically produced MD-2 were most affected by expression of the G56R variant of MD-2. Coexpression of wild-type and G56R MD-2 yielded an intermediate phenotype with responses to LPS diminished to a greater extent than that resulting from expression of the D299G TLR4 polymorphic variant.