C型凝集素LSECtin作为丙型肝炎病毒受体的系统分析

【作者】 李怡；

【导师】 贺福初；

【作者基本信息】 中国人民解放军军事医学科学院 ， 细胞生物学， 2006， 博士

【摘要】 丙型肝炎病毒(HCV)是导致肝脏炎症的主要病毒之一。目前全世界有1.7亿人感染HCV，大部分患者发展成慢型肝炎，有些进一步发展为肝硬化和肝细胞性肝癌。HCV引起肝病的机制还不是很清楚。研究者利用病毒样颗粒、可溶性HCV膜糖蛋白E2和HCV假型病毒颗粒发现了CD81、SR-Bl等潜在的HCV受体，然而这些受体在人体内广泛表达，不能解释HCV感染的组织特异性。最近的研究发现一些肝脏特异表达的分子虽然不是感染所必需的，但是能够有效地以反式感染方式提高HCV感染效率如DC-SIGNR。LSECtin是我室克隆鉴定的一种新的Ⅱ型C型凝集素，特异性地表达在肝窦和淋巴结窦内皮细胞表面。LSECtin能够结合甘露糖、岩藻糖和N-乙酰葡糖胺，与同一家族的HCV受体DC-SIGNR具有相似的基因和蛋白结构，共定位于肝窦内皮细胞。这提示LSECtin可能结合HCV，并且与DC-SIGNR相互作用参与结合HCV，参与感染过程。我们用HCV患者血清、可溶性糖蛋白E2和HCV假病毒颗粒检测HCV与LSECtin的结合，并且采用免疫共沉淀技术和细胞粘附实验检测LSECtin与DC-SIGNR的相互作用。结果表明LSECtin能够结合血清中的病毒颗粒，这种结合可能是通过与HCV表面的糖蛋白相互作用实现的，LSECtin结合HCV假病毒颗粒的能力低于DC-SIGNR。结合抑制实验显示其结合受到钙离子拮抗剂EGTA抑制，但是不能被甘露聚糖抑制，提示LSECtin是通过其C型凝集素样结构域(CTLD)识别E2糖蛋白上非甘露糖的特异性糖基。同时还发现LSECtin和DC-SIGNR存在相互作用，LSECtin颈区的coiled-coil域为相互作用所必需。LSECtin和DC-SIGNR共表达的细胞结合HCV能力较单独表达DC-SIGNR的细胞降低，提示二者的相互作用可能是改变细胞结合能力的原因。本研究鉴定了一个新的HCV受体——LSECtin，它在体内肝窦内皮细胞上与DC-SIGNR共同参与与HCV的结合，这提示HCV反式感染可能是一个更为复杂的过程，并且为研究抑制HCV入胞药物提供了新的药物靶点。更多还原

【Abstract】 Hepatitis C virus (HCV) is a major cause of liver disease. However the detail mechanism of hepatocyte infection remains unknown. A novel C-type lectin LSECtin is expressed on the liver and lymph node sinusoidal endothelial cell and has a similar structure to DC-SIGNR, a binding receptor for HCV. Previous report suggest that LSECtin may bind HCV by interacting with glycoprotein E2 and function as a capture receptor in a manner similar to DC-SIGNR . We used three methods including the soluble E2 glycoprotein binding assay, the HCV pseudotype particles binding assay and the HCV virions binding assay to evidence the hypothesis that HCV binds to LSECtin. Moreover, similar protein structure and expression profile of LSECtin and DC-SIGNR urged us to investigate whether there is an interaction between LSECtin and DC-SIGNR and how the interaction affects the adhesion of HCV. The interaction between LSECtin and DC-SIGNR was studied by co-immunoprecipitation and cell adhesion assays. Here we demonstrated that the E2 glycoprotein and HCV pseudotype particles bind the LSECtin expressed cells, which was blocked by EGTA but not mannan. Furthermore HCV virions present in the sera of infected patients confirmed the interaction. We also showed that LSECtin interacts with DC-SIGNR, in which the neck region of LSECtin is necessary for the interaction. The interaction may induce the decrease of HCV affinity binding to the DC-SIGNR and LSECtin co-expressed cells. This study indicates LSECtin is a novel to the binding receptors on the liver node sinusoidal endothelial cells and that there is the more complicated mechanism of HCV infection.更多还原