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Structural analysis of lipope microbes responsible for binding to Toll-like receptors

负责与 Toll 样受体结合的脂质微生物的结构分析

基本信息

批准号：
13671891
负责人：
SHIBATA Ken-ichiro
金额：
$ 1.98万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671891/
关键词：
Lipoproteins lipopeptides Toll-like receptors 構造と生物活性

项目摘要

The synthetic lipopeptide FSL-1 [S-(2,3-bispalmitoyioxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam_2 CGDPKHPKSF] synthesized on the basis of the structure of the N-terminal lipopeptide moiety of a Mycoplasma salivarium lipoprotein activated human gingival fibroblasts to induce the cell surface expression of ICAM-1 and the production of IL-6, IL-8 and monocyte chemoattractant protein-1. FSL-1 also activated THP-1 cells, a monocytic cell line, to produce TNF-α. Both the diacylglyceryl cysteine (Pam_2C) and peptide (CGDPKHPKSF) portions of FSL-1 failed to activate THP-1 cells. A single amino acid substitution from Phe to Arg at the C terminus of FSL-1 and a fatty acid substitution from pralmitic acid to stearic acid drastically reduced the activity. The data obtained by experiments using Chinese hamster ovary cells transfected with CD14, CD14/Toll-like receptor 2 or CD14/Toll-like receptor 4 genes suggested that signaling by FSL-1 was mediated by Toll-like receptor 2. Mycoplasmal diacylated lipopeptides, FSL-1 and MALP-2 (Pam_2CGNNDESNISFKEK) and bacterial triacylated lipopeptides, S-(2 ,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-Ser-Asn-Asn-Ala (Pam_3 CSNNA) and Pam_3CSK_4 were examined for their abilities to activate THP-1 cells. FSL-1, MALP-2, Pam_3CSK_4 and Pam_3CSNNA had the activitiy to induce TNF-α Production by THP-1 cells, but the level of the activity decreased in this order.These results suggest that both the diacylglyceryl and peptide portions of FSL-1 are essential for the expression of biological activity, that the differences in fatty acid in the diacylglycerol portion and amino acid sequence of the peptide portion affect the biological activity, and that the electrostatic interaction plays an important role in the recognition of microbial lipopeptides by Toll-like receptor 2.

根据唾液支原体脂蛋白N端脂多肽的结构合成的脂肽FSL-1、[S-(2，3-bispalmitoyioxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe，、PAM_2、CGDPKHSF可激活人牙龈成纤维细胞，诱导细胞表面ICAM-1的表达，并产生IL-6、IL-8和单核细胞趋化蛋白-1。FSL-1还能激活单核细胞系THP-1细胞产生肿瘤坏死因子-α。FSL-1的二甘油基半胱氨酸(PAM_2C)和多肽(CGDPKHPKSF)部分均不能激活THP-1细胞。FSL-1 C端从Phe到Arg的单一氨基酸替换以及从Pralmitic酸到硬脂酸的脂肪酸替换显著降低了该酶的活性。用转染CD14、CD14/Toll样受体2或CD14/Toll样受体4基因的中国仓鼠卵巢细胞进行的实验结果表明，Fsl-1的信号转导是由Toll样受体2介导的。检测了支原体双酰化脂肽Fs1和MALP-2(Pam_2CGNNDESNISFKEK)和细菌三酰化脂肽S-(2，3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-Ser-Asn-Asn-Ala，Pam_3CSNNA)和Pam_3CSK_4对THP-1细胞的激活作用。FsL-1、MALP-2、Pam_3CSK_4和Pam_3CSNNA具有诱导THP-1细胞产生肿瘤坏死因子-α的活性，但活性依次降低。这些结果表明，FsL-1的二酰甘油和多肽部分都是生物活性表达所必需的，二甘油部分的脂肪酸和多肽部分的氨基酸序列的差异影响了生物活性，静电相互作用在Toll样受体2识别微生物脂肽的过程中起着重要作用。