A Novel Immunomodulatory Protein from Poria cocos Induces Toll-like Receptor 4-Dependent Activation within Mouse Peritoneal Macrophages
A Novel Immunomodulatory Protein from Poria cocos Induces Toll-like Receptor 4-Dependent Activation within Mouse Peritoneal Macrophages
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DOI：
10.1021/jf9011399
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发表时间：
2009-07-22
影响因子：
6.1
通讯作者：
Sheu, Fuu
中科院分区：
文献类型：
--
作者：
Chang, Hui-Hsin;Yeh, Chen-Hao;Sheu, Fuu
茯苓是一种重要的东方药用真菌，具有多种功能，但其生物活性物质及其作用机理尚未完全阐明。从Cocos(Schw.)的干燥菌核中分离纯化出一种新的免疫调节蛋白--P.cos免疫调节蛋白。Wolf采用DE-52纤维素层析和凝胶过滤层析。层析和电泳结果表明，天然PCP为二硫键L标记的异二聚体糖蛋白，由14.3kDa和21.3kDa亚基组成，N-糖基化和O-糖基化。PCP可通过诱导肿瘤坏死因子-α和白介素1-β以及调节核因子-kappaB相关基因的表达，在体外刺激RAW-264.7巨噬细胞。在原代小鼠巨噬细胞中，PCP直接激活腹腔巨噬细胞，诱导Toll样受体4(TLR4)介导的髓系分化因子88(MyD88)依赖的信号转导。本研究证实了PCP与TLR4细胞表面的相互作用以及PCP对TLR4酪氨酸磷酸化的能力。用TLR4基因缺陷的C57BL/10ScN小鼠的腹膜巨噬细胞获得的结果表明，PCP诱导的激活和PCP细胞表面的结合明显减弱。此外，酶去糖基化降低了PCP介导的反应，表明PCP的糖基化部分是通过TLR4在腹膜巨噬细胞中传递PCP信号的关键因素。这些发现表明PCP是一种新的潜在的免疫刺激因子，TLR4在小鼠巨噬细胞的PCP信号转导中起主要作用。
Poria cocos is an important Oriental medical fungus with multiple functionalities, yet its bioactive substances and the mechanisms involved have not been fully characterized. A novel immunomodulatory protein (P. cocos immunomodulatory protein; PCP) was purified from the dried sclerotium of P. cocos (Schw.) Wolf using DE-52 cellulose and gel filtration chromatography. Chromatography and electrophoresis results indicated that the native PCP (35.6 kDa) is a disulfide-l inked heterodimeric glycoprotein consisting of 14.3 and 21.3 kDa subunits with N- and O-glycosylation. PCP was capable of stimulating RAW 264.7 macrophages in vitro through the induction of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) as well as the regulation of nuclear factor-kappa B (NF-kappa B)-related gene expression. In primary mouse macrophages, PCP directly activated peritoneal cavity macrophages to induce Toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling. This study demonstrated the cell surface interactions of PCP with TLR4 and the capacity of PCP for TLR4 tyrosine phosphorylation. Results obtained with peritoneal macrophages from TLR4-deficient C57BL/10ScN mice revealed that PCP-induced activation and PCP cell surface binding were significantly attenuated. Moreover, enzymatic deglycosylation decreased PCP-mediated responses, indicating that the glycosylated portion of PCP was a key factor in PCP signaling through TLR4 in peritoneal macrophages. These findings suggest that PCP is a new potential immune stimulator within P. cocos and that TLR4 is primarily responsible for PCP signaling in murine macrophages.