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Functional Structure Analysis of Ion-channel Oligomer and Production of regulating Peptides.

离子通道寡聚物的功能结构分析和调节肽的生产。

基本信息

批准号：
16550144
负责人：
KODAMA Hiroaki
金额：
$ 2.37万
依托单位：
Saga University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16550144/
关键词：
transmembrane peptide channel peptide receptor protein GPCR neutrophil superoxide production priming Intracelluler alcium ion イオンチャンネルペプチド合成 Aib 分子架橋二量体ペプチド

项目摘要

Human formyl peptide receptor (FPR) mediates a number of important host defense functions. Although studies have been made on ligand binding site of FPR, FPR dynamic behaviors on cell surface is unknown. Recently, peptides derived from transmembrane (TM) domain of GPCRs were shown to disrupt dimer formation of receptors and results in specific regulation of receptor function. To reveal the function of FPR TM domains, hFPRTM peptides derived from human FPR were synthesized, and their biological activities were evaluated on human neutrophils. TM peptides were synthesized through a stepwise solid phase method using Fmoc amino acid on low substituted resin. The purifications of crude TM peptides were carried out by preparative RP-HPLC using water/2-propanol/formic acid system. The homogeneities and structures of synthetic peptides were verified by analytical RP-HPLC and MALDI-TOF MS. Synthetic hFPRTM peptides did not exhibit agonistic or antagonistic activities on superoxide anion production in human neutrophils. However, human neutrophils treated with hFPRTM4 produced 4-folds superoxide anion compared with untreated cells. Short peptides fragments from fourth TM region of human FPR did not enhance superoxide anion production, which suggest that hFPRTM4 did not behave as a ligand. CD and fluorescence spectra suggested that hFPRTM peptides inserted into the membrane and interacted with TM domain of membrane proteins. Addition of hFPRTM4 increased intracellular calcium concentration, which meant the peptide activate some membrane protein on cell surface. Present study suggests that the fourth TM domain of FPR has novel function related to priming effect.

人甲酰肽受体（FPR）介导许多重要的宿主防御功能。尽管人们对FPR的配体结合位点进行了研究，但FPR在细胞表面的动力学行为尚不清楚。最近，来自GPCR的跨膜（TM）结构域的肽被证明可以破坏受体的二聚体形成，并导致受体功能的特异性调节。为了揭示FPR TM结构域的功能，合成了人FPR衍生的hFPR TM肽，并在人中性粒细胞上评价了其生物学活性。以Fmoc氨基酸为原料，在低取代度树脂上通过分步固相法合成TM肽。通过制备型RP-HPLC使用水/2-丙醇/甲酸系统进行粗TM肽的纯化。通过分析型RP-HPLC和MALDI-TOF MS验证了合成肽的均一性和结构。合成hFPRTM肽对人中性粒细胞中超氧阴离子产生没有表现出激动或拮抗活性。然而，与未处理的细胞相比，用hFPRTM 4处理的人中性粒细胞产生4倍的超氧阴离子。人FPRTM 4的第四个TM区的短肽片段没有增强超氧阴离子的产生，这表明hFPRTM 4没有作为配体的行为。CD和荧光光谱表明hFPRTM肽插入到膜上，并与膜蛋白的TM结构域相互作用。hFPRTM 4的加入增加了细胞内钙离子浓度，这意味着该肽激活了细胞表面的一些膜蛋白。本研究提示FPR第四TM结构域具有与启动效应相关的新功能。