LPS and Toll-like Receptor
	Lipopolysaccharide (LPS) is the principal outer membrane component of Gram-negative bacteria and a strong stimulator of the innate immunity of their host (1). LPS is an amphipathic molecule consisting of a hydrophilic polysaccharide portion in the core region and O-antigen, and a hydrophobic glycolipid anchor t ermed lipid A (Fig. 1). O-antigen is composed of a sequence of repeating units of oligosaccharide specific to bacterial species and/or strains, and defines the serologic classification of Gram-negative bacteria. The core region is an oligosaccharide containing characteristic sugar residues, Kdo and heptose, and its chemical variation is more limited than that of O-antigen. The core region is also considered to possess immunogenic properties. However, the details remain unknown. Lipid A is composed of a phosphorylated diglucosamine carrying acyl residues, and is recognized as a primary immunostimulatory center of LPS. Many kinds of lipid As and their analogues have been synthesized, and their structure-activity relationships thoroughly evaluated. The activity of lipid A is strictly dependent on the chemical structure, e.g., chain length and number, the positions of the acyl groups, as well as the number of phosphate groups. Furthermore, the activity depends on the particular mammalian species investigated. In the case of human cells, Escherichia coli-type lipid A consisting of glucosamine disaccharide, two phosphates and six acyl groups (Fig. 2A) showed the strongest immuno-stimulating activity, while its counterparts possessing different kinds or number of substituents display reduced activity. Remarkably, the biosynthetic precursor of the lipid A having tetraacyl groups (Fig. 2B) did not show the activity and inhibited the stimulartory activity of LPS or E. coli -type lipid A. However, both hexaacyl and tetraacyl compounds showed similar stimulating activities in the case of murine cells. These observations indicate that lipid A molecules are precisely recognized by a receptor or its complex on the host cells. CD14 was previously reported to be a LPS-binding receptor which is distributed on the cell surface and enhances the LPS signals. But since CD14 lacks a cytoplasmic domain, it may not be responsible directly for the signal transduction. Recently, Toll-like receptor (TLR) family, which is a type-I transmembrane protein expressed on immune cells such as macrophages and dendritic cells, has been found as a major signaling receptor of the innate immune system (2). TLRs recognize pathogen-specific molecules by their extracellular leucine-rich repeat (LRR) domain, activate the signaling cascade involving MyD88, IRAK, TRAF6, and NF-B via its cytoplasmic Toll/IL-1 receptor (TIR) domain, and lead to the synthesis of inflammatory mediators, like cytokines or nitric oxide. To date, more than ten members of the TLR family have been found. Among them, TLR4 is recognized as LPS receptors by the following facts. LPS-nonresponsive mouse was found to carry a point mutation in the TIL domain of the TLR4 gene. TLR4 deficient (TLR4-/-) cells were defective in their responses to LPS or lipid A. Furthermore, MD-2, a lipid binding protein associated with the LRR domain of TLR4, has been identified. MD-2-deficient (MD-2-/-) cells were shown to be LPS-unresponsive, indicating the essential role of MD-2 for the signal transduction. It was reported that MD-2 comes into physical contact with LPS and is responsible for the species-dependent activation by lipid A derivatives. These observations reveal that MD-2 acts as an adapter molecule of TLR4 and supports the recognition of LPS or lipid A. Ligand recognition using an adapter molecule is characteristic only of TLR4. However, direct evidence of binding between TLR4, MD-2 and LPS or lipid A has not yet been reported. Much remains to be elucidated concerning the molecular mechanisms of LPS and lipid A recognition.
Fig. 1 Schematic structure of LPS.
Fig. 2 Chemical structure of lipid A.
Fig. 3 Recognition and signaling pathway of LPS and lipid A.
	Masahito Hashimoto (Asahi University School of Dentistry)
	References (1) Ulmer, A.J., Rietschel, E.T., Zhringer, U., Hein, H. Lipopolysaccharide: structure, bioactivity, receptors, and signal transduction. Trends Glycosci. Glycotechnol., 14, 53-68, 2002; Alexander, C., Zhringer, U. Chemical structure of lipid A - the primary immunomodulatory center of bacterial lipopolysaccharides. ibid, 69-86; Takada, H., Kotani, S. Structural requirements of lipid A for endotoxicity and other biological activities. Crit. Rev. Microbiol., 16, 477-523, 1989. (2) Akira, S., Takeda, K., Kaisho, T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol., 2, 675-680, 2001. (3) Miyake, K. Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2. Int. Immunopharmacol., 3, 119-128, 2003.
Nov. 12, 2003