2007 Fiscal Year Final Research Report Summary
X-Ray Crystal Structure Analyses of Inante Immunological TLR-Related Receptor Proteins for Sepsis Treatments
| Project/Area Number |
17390010
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SATO Yoshinori The University of Tokyo, Graduate School of Pharmaceutical Sciences, Professor (30150014)
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| Co-Investigator(Kenkyū-buntansha) |
NOGUCHI Shuji The University of Tokyo, Graduate School of Engineering, Assistant Professor (60237823)
TOMA Sachiko The University of Tokyo, Graduate School of Pharmaceutical Sciences, Assistant Professor (40363535)
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| Project Period (FY) |
2005 – 2007
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| Keywords | Innate Immunity / Toll-Like-Receptor / MD-2 / Septis / Endotoxin / Lipopolysaccharide / X-Ray Crystal Structure / Structure Biology |
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| Research Abstract |
Innate immunity is the first line of defense against microbial infections. Among microbial components recognized by pathogen sensors, lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is a potent stimulant of immune responses. Excessive responses to the endotoxic LPS frequently result in severe sepsis, a rapidly progressing inflammatory disease with very high mortality.
MD-2 forms a stable complex with Toll-like receptor 4 (TLR4) on the cell surface. Lipid A, the primary immunostimulatory core of LPS, is diverse in several species, and these variations are discriminated by the TLR4/MD-2 complex as endotoxic or as anti-endotoxic. Its precursor lipid IVa, the tetra-acylated form of lipid A, acts as an antagonist in human cells but as an agonist in mouse cells.
Human MD-2, encoded as a 160 amino-acid glycoprotein with a 16 amino-acid secretion signal, was expressed in methyltropic yeast Pichia pastoris. Polysaccharide moieties of MD-2 were trimmed off with endoglycosidase treatment which leaves a single N-acetyl-glucosamine at each glycosylation site. Thus obtained MD-2 with residues 17-160 was successfully crystallized, but showed crystal twinning. Twinned crystals were transformed into single crystals through optimization of cryoprotectant. The structure of the native MD-2 crystal was determined at 2.0 A resolution. Cocrystals with the lipid IVa complex were obtained from a mixture of MD-2 and lipid IVa. The structure of the complex was refined at 2.2 A resolution.
MD-2 shows a deep hydrophobic cavity sandwiched by two (3-sheets, in which four acyl chains of the lipid IVa are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. In the native structure, unexpected electron-densities were observed in the cavity, and were attributed to bound lipidic molecules. These structures suggest that MD-2 plays a principal role in endotoxin recognition, and provide a basis for antiseptic drug development.
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