Human MD-2 confers on mouse Toll-like receptor 4 species-specific lipopolysaccharide recognition

Akashi, Shigeo; Nagai, Yoshinori; Ogata, Hirotaka; Oikawa, Masato; Fukase, Koichi; Kusumoto, Shoichi; Kawasaki, Kiyoshi; Nishijima, Masahiro; Hayashi, Shinichiro; Kimoto, Masao; Miyake, Kensuke

doi:10.1093/intimm/13.12.1595

Cited by 233 publications

(182 citation statements)

References 24 publications

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“…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.…”

Section: Identification Of Mouse Md-2 Residues Important Formentioning

confidence: 95%

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

Kawasaki

Nogawa

Nishijima

2003

The Journal of Immunology

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The expression of MD-2, which associates with Toll-like receptor (TLR) 4 on the cell surface, confers LPS and LPS-mimetic Taxol responsiveness on TLR4. Alanine-scanning mutagenesis was performed to identify the mouse MD-2 residues important for conferring LPS and Taxol responsiveness on mouse TLR4, and for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 Ab MTS510. Single alanine mutations were introduced into mouse MD-2 (residues 17–160), and the mutants were expressed in a human cell line expressing mouse TLR4. Mouse MD-2 mutants, in which a single alanine mutation was introduced at Cys37, Leu71, Leu78, Cys95, Tyr102, Cys105, Glu111, Val113, Ile117, Pro118, Phe119, Glu136, Ile138, Leu146, Cys148, or Thr152, showed dramatically reduced ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, and the mutants also showed reduced ability to confer LPS and Taxol responsiveness. In contrast, mouse MD-2 mutants, in which a single alanine mutation was introduced at Tyr34, Tyr36, Gly59, Val82, Ile85, Phe126, Pro127, Gly129, Ile153, Ile154, and His155 showed normal ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, but their ability to confer LPS and Taxol responsiveness was apparently reduced. These results suggest that the ability of MD-2 to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510 is essential for conferring LPS and Taxol responsiveness on TLR4, but not sufficient. In addition, the required residues at codon numbers 34, 85, 101, 122, and 153 for the ability of mouse MD-2 to confer LPS responsiveness are partly different from those for Taxol responsiveness.

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Section: Identification Of Mouse Md-2 Residues Important Formentioning

confidence: 95%

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

Kawasaki

Nogawa

Nishijima

2003

The Journal of Immunology

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“…On the other hand, an antagonistic effect of the tetraacylated biosynthetic precursor of lipid A, lipid IVa, on LPS-induced cell activation has been indicated [25]. Interestingly, the lipid A analog has been shown to be antagonistic toward human cells and agonistic with murine cells [26]; it was recently demonstrated that MD-2 is associated with this species-specific recognition of LPS [27]. To determine whether Tm-Gp has an antagonistic effect on TLR4/MD-2 and TLR2, we examined the effect of Tm-Gp on NF-‹ B activation in Ba/mTLR4/mMD-2 cells stimulated with various TLR4 ligands, including LPS, Escherichia colitype synthetic lipid A (compound 506), and Taxol, and PGN as a TLR2 ligand (Fig.…”

Section: Effect Of Tm-gp On Cell Activation By Tlr4 and Tlr2 Ligandsmentioning

confidence: 99%

Treponemal glycoconjugate inhibits Toll‐like receptor ligand‐induced cell activation by blocking LPS‐binding protein and CD14 functions

2003

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Spirochetes are well-known as causative agents of various chronic infectious diseases. Glycolipids of small-sized Treponema spirochetes have been shown to exhibit immunostimulating activities. In this study, we found that a glycoconjugate preparation obtained from Treponema medium (Tm-Gp), an intermediate-sized oral Treponema seen in subgingival plaque from patients with chronic periodontal diseases, diminished the LPS-induced activation of a human monocytic cell line, while TNF- § -induced activation was unaffected. NF-‹ B activation in Ba/F3 cells expressing murine Toll-like receptor (TLR) 4 and MD-2 was inhibited by Tm-Gp in a CD14/LPS-binding protein (LBP)-dependent manner when stimulated with LPS or its active center lipid A but not when stimulated with Taxol. Tm-Gp blocked the binding of LPS to immobilized CD14 and LBP and inhibited nitric oxide (NO) production by a murine M ¤ cell line following stimulation with peptidoglycan and LPS in the presence of FBS, while NO production in response poly (I:C) RNA or CpG DNA remained unaffected. The inhibitory effects of Tm-Gp seem to be attributable to the lipophilic portion of the glycoconjugate. These results indicate that T. medium contains a glycoconjugate possessing an inhibitory effect on TLR-mediated cell activation through interaction with LBP and CD14.

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“…Lipid A is an agonist in most species whereas lipid IVa, while being an agonist in the mouse, is an antagonist for human cells (21,24). This suggests that lipid IVa is able to bind to human TLR4/MD-2 but is 3 Address correspondence and reprint requests to Dr. Clare Bryant, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 OES, U.K. E-mail address: ceb27@cam.ac.uk 4 Abbreviations used in this paper: LRR, leucine-rich repeat; EH, chimera in which a human region is engineered into an equine framework; HE, chimera in which an equine region is engineered into a human framework.…”

mentioning

confidence: 99%

“…There are marked differences among mammalian species in the activities of different types of lipid A that behave as agonists or antagonists at the MD-2/TLR4 complex (21)(22)(23). Lipid A is an agonist in most species whereas lipid IVa, while being an agonist in the mouse, is an antagonist for human cells (21,24).…”

mentioning

confidence: 99%

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Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Walsh

Gangloff

Monie

et al. 2008

The Journal of Immunology

115

137

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LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure ( L ipopolysaccharide molecules are complex glycolipids that form the outer layer of the outer membrane of Gramnegative bacteria (1). The lipid A domain of LPS is responsible for cellular activation and consists of a disaccharide to which various substituents, including acyl chains of variable length and number, are attached (2). Escherichia coli lipid A is usually hexa-acylated whereas a tetra-acylated lipid A, lipid IVa, is also produced by E. coli as an intermediate in the lipid A biosynthetic pathway (2). Lipid IVa was originally identified as an inhibitor at the human LPS receptor and was considered a candidate to be developed for clinical use as an endotoxin antagonist. Lipid A signals to host cells through a transmembrane complex consisting of the lipid-binding protein MD-2 and the type 1 receptor TLR4 (1, 3-5). MD-2 is probably the key player in lipid A recognition whereas TLR4, unlike other TLRs, is thought not to participate directly in lipid A binding (5).LPS and lipid A are believed to be recognized by MD-2 following transfer from CD14, which does not participate in the signaling complex (6). Contrary to expectations, ligand binding does not significantly alter the overall structure of MD-2 (7, 8), but the ligands used in the crystallographic studies (lipid IVa and eritoran) are antagonists to human MD-2/TLR4, so it remains unclear what happens to MD-2 and TLR4 upon agonist binding and activation. Active ligands such a lipid A (9) presumably induce structural rearrangements that trigger dimerization of TLR4 and initiate signal transduction (7, 10 -13). Mutagenesis studies identified amino acids 79 -83, 121-124, 125-129 (12), K128, and K132 of human MD-2 as being important in lipid A binding (13), but in the crystal structure of the inactive MD-2-lipid IVa complex, only residues I46, L78, I80, and F121-I124 contact the ligand. The other residues...

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Human MD-2 confers on mouse Toll-like receptor 4 species-specific lipopolysaccharide recognition

Cited by 233 publications

References 24 publications

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

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

Treponemal glycoconjugate inhibits Toll‐like receptor ligand‐induced cell activation by blocking LPS‐binding protein and CD14 functions

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

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