X-Ray Crystal Structure Analyses of Inante Immunological TLR-Related Receptor Proteins for Sepsis Treatments

用于败血症治疗的 Inante 免疫学 TLR 相关受体蛋白的 X 射线晶体结构分析

• 批准号: 17390010
• 负责人: SATO Yoshinori
• 金额: $ 10.2万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390010/
• 关键词: Innate Immunity; Toll-Like-Receptor; MD-2; Septis; Endotoxin; Lipopolysaccharide; X-Ray Crystal Structure; Structure Biology; Toll-Like-Recetor; X線結晶構造析; Toll-Like-Receptor

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.

先天免疫是抵御微生物感染的第一道防线。在病原体传感器识别的微生物成分中，革兰氏阴性菌外膜中的脂多糖（LPS）是一种有效的免疫反应刺激物。对内毒素LPS的过度反应经常导致严重的败血症，这是一种快速发展的炎症性疾病，死亡率很高。MD-2在细胞表面与toll样受体4 （TLR4）形成稳定的复合物。脂质A是脂多糖的初级免疫刺激核心，在不同物种中存在差异，这些变化被TLR4/MD-2复合物区分为内毒或抗内毒。它的前体脂质IVa，脂质A的四酰化形式，在人类细胞中作为拮抗剂，但在小鼠细胞中作为激动剂。人MD-2编码为160个氨基酸的糖蛋白，具有16个氨基酸的分泌信号，在嗜甲基酵母毕赤酵母中表达。MD-2的多糖部分被内糖苷酶处理，在每个糖基化位点留下一个单一的n -乙酰氨基葡萄糖。由此得到残基为17-160的MD-2成功结晶，但出现了晶体孪晶。通过对冷冻保护剂的优化，将双晶转化为单晶。在2.0 A分辨率下测定了天然MD-2晶体的结构。从MD-2和脂质IVa的混合物中获得了具有脂质IVa复合物的共晶。该配合物的结构以2.2 A的分辨率进行了细化。MD-2显示一个由两层（3-sheet）夹在中间的深疏水腔，其中脂质IVa的四个酰基链被完全限制。磷酸化的氨基葡萄糖部分位于腔的入口。在天然结构中，在腔中观察到意想不到的电子密度，并归因于结合的脂质分子。这些结构表明，MD-2在内毒素识别中起主要作用，并为抗菌药物的开发提供了基础。

项目成果

Purification, characterization and crystallization of human β-galactosidase

人β-半乳糖苷酶的纯化、表征和结晶

• 发表时间: 2005
• 作者: Kimihito Usui;Toshinari Ochi;Ryuta Mizutani;Yoshinori Satow
• 通讯作者: Yoshinori Satow

Purification of human β2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris

• DOI: 10.1093/jb/mvj211
• 发表时间: 2006-12-01
• 期刊: JOURNAL OF BIOCHEMISTRY
• 影响因子: 2.7
• 作者: Noguchi, Shuji;Satow, Yoshinori
• 通讯作者: Satow, Yoshinori

ヒトHsp40 C末端側ドメインの精製および結晶化

人 Hsp40 C 末端结构域的纯化和结晶

• 发表时间: 2007
• 作者: 鈴木浩典;野口修治;佐藤能雅
• 通讯作者: 佐藤能雅

敗血症エンドトキシンに結合するヒトMD-2蛋白質の結晶構造

人MD-2蛋白与脓毒症内毒素结合的晶体结构

• 发表时间: 2007
• 作者: 大戸 梅治;佐藤 能雅
• 通讯作者: 佐藤 能雅

Structures of Human MD-2 Coreceptor and Its Complex with Antiendotoxic Lipid IVa Preventing Endotoxin Shock

人MD-2辅助受体及其与抗内毒素脂质IVa复合物预防内毒素休克的结构

• 发表时间: 2008
• 期刊: SPring-8 Research Frontiers 2007 (印刷中)
• 作者: Umeharu Ohto;Yoshinori Satow;Ikemoto Shigehiroら;Yoshinori Satow and Umeharu Ohto
• 通讯作者: Yoshinori Satow and Umeharu Ohto