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

人甲酰肽受体(Fpr)介导了一系列重要的宿主防御功能。虽然目前已经对FPR的配体结合部位进行了研究，但其在细胞表面的动力学行为尚不清楚。近年来，来自GPCRs跨膜区的多肽被证明可以干扰受体二聚体的形成，从而导致受体功能的特异性调节。为了揭示FPRTM结构域的功能，合成了来源于人FPRTM结构域的hFPRTM多肽，并对其在人中性粒细胞上的生物学活性进行了评价。以Fmoc氨基酸为原料，在低取代度树脂上，采用固相法逐步合成TM多肽。采用水/异丙醇/甲酸体系制备反相高效液相色谱分离纯化TM多肽粗品。合成的hFPRTM多肽对人中性粒细胞产生超氧阴离子没有明显的拮抗或拮抗作用。然而，与未经处理的细胞相比，经hFPRTM4处理的人中性粒细胞产生4倍的超氧阴离子。HFPRTM4不能促进超氧阴离子的产生，提示hFPRTM4不是一种配体。圆二色谱和荧光光谱表明hFPRTM多肽插入到膜中，并与膜蛋白的TM结构域相互作用。HFPRTM4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某些膜蛋白。本研究表明，fpr的第四个TM结构域具有与启动效应相关的新功能。

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.