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Structure-function analysis of Pattem-recognition Proteins

模式识别蛋白的结构功能分析

基本信息

批准号：
13143203
负责人：
KAWABATA Shunichiro
金额：
$ 66.3万
依托单位：
KYUSHU UNIVERCTIY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2005
项目状态：
已结题

项目摘要

In the horseshoe crab, LPS induces hemocyte exocytotic degranulation, resulting in the secretion of various defense molecules, such as coagulation factors, antimicrobial peptides, and lectins. Recent studies have demonstrated that the zymogen form of the serine protease factor C, a major granular component of hemocyte, also exists on the hemocyte surface and functions as a biosensor for LPS. The proteolytic activity of activated factor C initiates hemocyte exocytosis via a G protein mediated signal transduction pathway. Furthermore, it has become clear that an endogenous mechanism for the feedback amplification of the innate immune response exists and is dependent upon a granular component of the horseshoe crab hemocyte. We report that Factor C associates with LPS on the hemocyte surface and directly recognizes Gram-negative bacteria. Structure-function analyses reveal that the LPS-binding site is present in the amino-terminal cysteine-rich (Cys-rich) region of the molecule, and that it contains a tri-peptide sequence consisting of an aromatic residue flanked by two basic residues, which is conserved in other mammalian LPS-recognizing proteins. Moreover, we demonstrate that the Cys-rich region specifically binds to LPS on Gram-negative bacteria, and that mutations in the tri-peptide motif abrogate its association with both LPS and Gram-negative bacteria, underscoring the importance of the tri-peptide in LPS interaction. Although the innate immune response to LPS in the horseshoe crab is distinct from that of mammals, it appears to rely on structural features that are conserved among LPS-recognizing proteins from diverse species.

在鲎中，LPS诱导血细胞胞吐脱颗粒，导致各种防御分子的分泌，如凝血因子、抗菌肽和凝集素。最近的研究表明，酶原形式的丝氨酸蛋白酶因子C，血细胞的主要颗粒组分，也存在于血细胞表面，并作为LPS的生物传感器的功能。活化的C因子的蛋白水解活性通过G蛋白介导的信号转导途径启动血细胞胞吐。此外，已经清楚的是，存在先天免疫应答的反馈放大的内源性机制，并且依赖于鲎血细胞的颗粒组分。我们报告，C因子与血细胞表面的LPS相关，并直接识别革兰氏阴性菌。结构-功能分析表明，LPS结合位点存在于分子的氨基末端富含半胱氨酸（Cys-丰富）的区域，并且它包含由两侧为两个碱性残基的芳香残基组成的三肽序列，这在其他哺乳动物LPS识别蛋白中是保守的。此外，我们证明了富含Cys的区域特异性地结合到革兰氏阴性菌上的LPS，并且三肽基序中的突变废除了其与LPS和革兰氏阴性菌的关联，强调了三肽在LPS相互作用中的重要性。虽然鲎对LPS的先天免疫反应与哺乳动物不同，但它似乎依赖于来自不同物种的LPS识别蛋白之间保守的结构特征。