Synthesis of [3H]-Labeled Bioactive Lipid A Analogs and Their Use for Detection of Lipid A-Binding Proteins on Murine Macrophages

Fukase, Koichi; Kirikae, Teruo; Kirikae, Fumiko; Somorjai, Gábor A.; Oikawa, Masato; Suda, Yasuo; Kurosawa, Motohiro; Fukase, Yoshiyuki; Yoshizaki, Hiroaki; Kusumoto, Shoichi

doi:10.1246/bcsj.74.2189

Cited by 27 publications

(22 citation statements)

References 37 publications

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“…Both Lipid A and LPS did not contain TLR2 ligands, because both did not stimulate TLR4-deficient mice, which were able to respond to TLR2 ligands (data not shown). 3 H-labeled lipid A was described previously (25). Anti-Flag Ab and antiFlag agarose were purchased from Sigma-Aldrich.…”

Section: Reagentsmentioning

confidence: 99%

Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

Kobayashi

Saitoh

Tanimura

et al. 2006

The Journal of Immunology

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147

108

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LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe119 to Lys132 (Arg132 in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly59 was found to be a novel critical amino acid for LPS binding outside the region 119–132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe126 or Gly129 impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.

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Section: Reagentsmentioning

confidence: 99%

Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

Kobayashi

Saitoh

Tanimura

et al. 2006

The Journal of Immunology

Self Cite

147

108

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“…Either one or both of the phosphate groups can synthetically be substituted by other acidic groups without loosing the original biological activity (21,(34)(35)(36) substitution of the glycosyl phosphate groups improved the stability of the molecules so that their synthesis and purification are much easier than the corresponding compounds with the glycosyl phosphates. This merit was utilized in the synthesis of pure tritium-labeled bioactive lipid A derivatives (3a and 4a) with high specific radioactivity (37). The PE analogues (3 and 4) show indistinguishable activity with those of natural type biosynthetic precursor and mature E. coli lipid A (1 and 2), respectively: the former was antagonistic and the latter endotoxic (36).…”

Section: Synthesis Of Structural Analogues and Derivatives Of Lipid Amentioning

confidence: 99%

Synthesis and Functional Study of Bacterial Glycoconjugates Triggering the Innate Immune System of Higher Animals

Kusumoto

2010

TIGG

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Typical bacterial glycoconjugates, lipopolysaccharide (LPS) and peptidoglycan (PGN), which ubiquitously occur in a wide range of bacterial cells as important components of their cell envelope, have long been known to enhance the immunological responses of higher animals. Synthetic works and related biological investigations on LPS are reviewed which were performed to understand the molecular basis of this phenomena mainly by the author's and collaborating groups. Structural study followed by chemical synthesis of glycolipid part of LPS, named lipid A, proved it to exhibit all the biological activity described for LPS. Synthetic homogeneous lipid A and its derivatives were utilized for precise studies of their biological functions and interaction with receptor proteins. Similar research on PGN led to conclude its minimum active structure as muramyl dipeptide. Chemical synthesis gave unequivocal evidences for the concept that definite small molecular parts of LPS and PGN are recognized by its specific receptors and trigger our defense system which is now recognized as innate immunity.

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“…61) These labeled compounds were subjected to experiments aiming at direct identication of lipid A-binding proteins on animal cell but the attempt failed to obtain clear-cut informationn. 62) The labeled compounds were utilized in analyzing the interaction of lipid A and its binding receptor complex discovered after ve years as described later. …”

Section: )-42)mentioning

confidence: 99%

Erratum to “Key structures of bacterial peptidoglycan and lipopolysaccharide triggering the innate immune system of higher animals: Chemical synthesis and functional studies”

Kusumoto¹,

Fukase²,

Shiba³

2010

Proceedings of the Japan Academy. Ser. B: Physical and Biologic

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Chemistry-based investigation is reviewed which led to identication of the active entities responsible for the immunostimulating potencies of peptidoglycan and lipopolysaccharide. Though these glycoconjugates which ubiquitously occur in wide range of bacteria as the essential components of their cell envelopes have long been known to enhance the immunological responses of higher animals, neither the precise chemical structures required nor the mechanism of their action had been elucidated until early 1970s. Chemical synthesis of partial structures of peptidoglycan proved N-acetylmuramyl-L-alanyl-D-isoglutamine to be the minimum structure responsible for the activity and led to later identication of its receptor protein Nod2 present in animal cells. Another active partial structure of peptidoglycan, g-D-glutamylmeso-diaminopimelic acid, and its receptor Nod1 were also identied as well. With regard to lipopolysaccharide, its glycolipid part named lipid A was puried and the structure studied. Chemically synthesized lipid A according to the newly elucidated structure exhibited full activity described for lipopolysaccharide known as endotoxin. Synthetic homogeneous lipid A and its structural analogues and labeled derivatives enabled precise studies of their interaction with receptor proteins and the mechanism of their action. Chemical synthesis of homogeneous partial structures of peptidoglycan and lipopolysaccharide gave unequivocal evidences for the concept that denite small molecular parts of these complex macromolecular bacterial glycoconjugates are specically recognized by their respective receptors and trigger our defense system now widely recognized as innate immunity.

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Synthesis of [3H]-Labeled Bioactive Lipid A Analogs and Their Use for Detection of Lipid A-Binding Proteins on Murine Macrophages

Cited by 27 publications

References 37 publications

Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

Synthesis and Functional Study of Bacterial Glycoconjugates Triggering the Innate Immune System of Higher Animals

Erratum to “Key structures of bacterial peptidoglycan and lipopolysaccharide triggering the innate immune system of higher animals: Chemical synthesis and functional studies”

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