血栓性血小板减少性紫癜发生的新机制：ADAMTS13 C甘露糖化修饰缺失

项目名称： 血栓性血小板减少性紫癜发生的新机制：ADAMTS13 C甘露糖化修饰缺失

项目编号： No.81500107

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 凌婧

作者单位： 苏州大学

项目金额： 18万元

中文摘要： 血栓性血小板减少性紫癜(TTP)是一种致死性疾病,其发生机制为ADAMTS13质和量的异常。ADAMT13是高度糖基化的蛋白，糖基化修饰是影响ADAMTS13结构和功能的重要因素，但C甘露糖化修饰对ADAMTS13结构和功能的影响目前尚不清楚。我们前期实验确证ADAMTS13的TSR1结构域中WXXW基序发生了C甘露糖化修饰，且在ADAMTS13的分泌及功能发挥中具有重要作用。我们将进一步通过ELISA、western blot、免疫荧光、晶体结构分析及模型小鼠的构建，探讨其对ADAMTS13与VWF相互作用的影响从而导致TTP发生的机制。本课题将通过研究TSR1结构域的C甘露糖化修饰对ADAMTS13分泌及功能的影响，阐明TSR1结构域C甘露糖化修饰缺失导致TTP发生的机制。本假说一经证实，丰富了ADAMTS13作用机制研究，并为TTP治疗提供新靶点和新策略。

中文关键词： 血栓性血小板减少性紫癜；血管性血友病因子裂解酶；C-甘露糖化；血管性血友病因子；结合能力

英文摘要： ADAMTS13 belongs to a family of proteases called ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs).ADAMTS13 cleaves the bond between the Tyr1605 and Met1606 residues in the central A2 domain of VWF to decrease the activity of VWF. A deficiency of ADAMTS13 or the presence of autoantibodies against ADAMTS13 causes congenital or acquired idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 is a highly glycosylated protein, the N-glycosylation and O-fucosylation of the protein have been reported, and they are thought to be important for ADAMTS13's secretion and proteolytic activity. But there are still other post-translational modifications should play an important role in the physiological functions of ADAMTS13. From the NetCGlyc 1.0 server, which predicts glycosylation sites, we found that the Trp387 and the Trp511 are potential sites for C-mannosylation, which is a novel type of protein glycosylation. However, the function of C-mannosylation is unclear. In the present study, we are going to demonstrate that ADAMTS13 is C-mannosylated, and the C-mannosylation is important for the secretion of ADAMTS13 and that it modulates the proteolytic cleavage of VWF by ADAMTS13. At present, we have generated wild-type and mutant (W390A, W511A) constructs of ADAMTS13 by PCR site-directed mutagenesis and expressed these constructs in cell lines, purificated the proteins, and further investigations should be carried out in vitro and in vivo. Due to the C-mannosylation of ADAMTS13 and its function have not been reported, so this study has obvious innovation.

英文关键词： TTP;ADAMTS13;C-mannosylation;VWF;Binding