2000 Fiscal Year Final Research Report Summary
Activation Mechanisms of Defense Systems by Substances on the Surface of Pathogens
| Project/Area Number |
11680609
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
MUTA Tatsushi Graduate School of Medical Sciences, KYUSHU UNIVERSITY, Ass.Prof, 大学院・医学研究院, 助教授 (60222337)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Defense system / Innate immunity / Gram-negative bacteria / Lipopolysaccharide / Macrophage / Receptor / Inflammation / Transcription factor |
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| Research Abstract |
Although genetic studies have revealed a critical role for the toll-like receptor (TLR) 4 in the biological response to lipopolysaccharide (LPS), the activities of ectopically expressed TLR4 and TLR2 are controversial. We found that under appropriate transfection conditions, both TLR2 and TLR4 mediate LPS-induced NF-κB activation in human embryonic kidney 293 cells. The reconstitution systems we established in this study allowed direct biochemical characterization and comparison of activation of each receptor. Mutant TLRs harboring a point mutation in the cytoplasmic domain was inactive in transducing the signal upon stimulation, and acted as dominant-negative mutants specifically inhibiting the LPS-mediated activation by corresponding type of the receptor but not the othertype, indicating that each TLR independently transduces the LPS signal. TLR4 was 10-100-fold more sensitive to a commercial LPS preparation than TLR2. In contrast to the response to the commercial LPS preparations, TLR2 was unresponsive to re-purified LPS or synthetic lipid A.Nevertheless, the responses of both TLRs were strongly dependent on serum and CD14 and LPS-binding protein were essential for the activation. Furthermore, a lipid A-neutralizing reagent, polymyxin B, blocked the ability of LPS to stimulate TLR2 as well as TLR4. Therefore, despite the distinct and independent activation, critical involvement of an interaction between CD14 and lipid A was strongly suggested not only for the activation of TLR4 but also for that of TLR2.
In parallel with the above study, we screened molecules that was up-regulated upon LPS-stimulation and found a new molecule that would negatively regulate inflammation reactions.
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[Publications] Sano, H., Sohma, H., Muta, T., Nomura, S., Voelker, D.R., and Kuroki, Y.: "Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14."J.Immunol.. 163 (1). 387-395 (1999)
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[Publications] Gokudan, S., Muta, T., Tsuda, R., Koori, K., Kawahara, T., Seki, N., Mizunoe, Y., Wai, S.N., Iwanaga, S., and Kawabata, S.: "Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen."Proc.Natl.Acad.Sci.USA. 96 (18). 10086-10091 (1999)
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[Publications] Kawasaki, H., Nose, T., Muta, T., Iwanaga, S., Shimohigashi, Y., and Kawabata, S.: "Head-to-tail polymerization of coagulin, a clottable protein of the horseshoe crab."J.Biol.Chem.. 275 (45). 35297-35301 (2000)
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[Publications] Uchimura, K., Nakamuta, M., Enjoji, M., Irie, T., Sugimoto, R., Muta, T., Iwamoto, H., and Nawata, H.: "Activation of retinoic X recetor and peroxisome proliferator-activated receptor-γ inhibits nitric oxide and tumor necrosis factor-α production in rat Kupffer cells."Hepatology. 33 (1). 91-99 (2001)
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