Structure and Function of ADAMTS13 Metalloprotease

ADAMTS13 金属蛋白酶的结构和功能

• 批准号: 6857422
• 负责人: X. Long Zheng
• 金额: $ 33.2万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6857422
• 关键词: MDCK cell; antigen antibody reaction; autoantibody; cellular polarity; clinical research; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; human subject; intracellular transport; metalloendopeptidases; monoclonal antibody; protein engineering; protein localization; protein purification; protein transport; proteolysis; radiotracer; surface plasmon resonance; thrombocytopenic purpura; thrombosis; thrombospondins; tissue /cell culture; von Willebrand factor

DESCRIPTION (provided by applicant): The catastrophic complications due to deficiency in plasma metalloprotease (ADAMTS13) highlight the biological importance of this protease in regulation of hemostasis and prevention of thrombosis in microvascular circulation that occurs in the thrombotic thrombocytopenic purpura (TTP) syndrome. Identification of the gene encoding ADAMTS13 metalloprotease and determination of the structure elements in the encoded protein have opened a new era in understanding of the roles of a zinc metalloprotease in regulation of hemostasis and thrombosis, and provided the essential tools to address the fundamental (patho)biological questions concerning ADAMTS13 including its biosynthesis, activation, substrate recognition, intracellular sorting and autoantibody interaction. The goals of the proposed research are: Specific Aim 1. To determine ADAMTS13 substrate recognition by 1) analyzing the proteolytic activity of wild type ADAMTS13 and various mutants of ADAMTS13 toward von Willebrand factor and its analog, VWF73; 2) determining the binding kinetics between VWF or VWF73 and wild type or mutant ADAMTS13 by radioligand binding and surface plasmon resonance assays. Specific Aim 2. To determine intracellular sorting of ADAMTS13 by 1) determining the polarity of full-length and mutant ADAMTS13 secretion in MDCK cells; 2) determining the sorting signal and mechanisms by which ADAMTS13 is sorted; 3) determining the effect of point mutations or truncations of ADAMTS13 gene found in patients with congenital TTP on the polarity of ADAMTS13 secretion. Specific Aim 3. To determine the domains (or sites) of ADATMTS13 to which anti-ADAMTS13 autoantibodies bind and the kinetic parameters (affinity and specificity) of interaction between ADAMTS13 metalloprotease and anti-ADAMTS13 autoantibodies, and to correlate the clinical course and outcome to the distinct type of anti-ADAMTS autoantibodies identified in patients with TTP.

描述（由申请人提供）：由于血浆金属蛋白酶缺乏（ADAMTS13）引起的灾难性并发症突出了这种蛋白酶在调节止血的调节和预防微血管循环中血栓形成血栓形成血栓性血栓细胞性紫罗兰（TTTP）综合体的生物学重要性。编码ADAMTS13的基因的识别金属蛋白酶和确定编码蛋白质中的结构元素的确定为理解锌金属蛋白酶在调节止血和剧动性调节中的作用方面开辟了一个新时代分类和自身抗体相互作用。拟议的研究的目标是：特定目的1。通过1）分析野生型ADAMTS13的蛋白水解活性和ADAMTS13对von Willebrand因子及其模拟因子VWF73的蛋白水解活性； 2）通过放射线结合和表面等离子体共振测定法确定VWF或VWF73与野生型或突变体Adamts13之间的结合动力学。具体目的2。确定ADAMTS13的细胞内分选1）确定MDCK细胞中全长和突变体Adamts13分泌的极性； 2）确定ADAMTS13分类的排序信号和机制； 3）确定先天性TTP患者ADAMTS13基因的点突变或截断对ADAMTS13分泌的极性的影响。具体目的3。确定抗ADAMTS13自身抗体结合的ADATMTS13的域（或位点），以及ADAMTSS13金属蛋白酶和抗ADAMTSS13自动抗体的ADAMTSS13 ADAMTSS13与临床类型相关的ADAMTS13金属蛋白酶之间的相互作用的动力学参数（亲和力和特异性），以实现临床类型，以与临床类型相关联。与TTP。