ER-to-Golgi transport of coagulation factors V and VIII

描述（由申请方提供）：凝血因子V（FV）和因子VIII（FVIII）均为分泌型糖蛋白，在止血和血栓形成中具有关键作用。虽然许多分泌蛋白（称为货物）被认为需要转运受体的有效ER-高尔基体运输，只有有限数量的这样的受体已被描述，主要是在酵母。哺乳动物货物受体存在的证据出乎意料地来自对人类遗传性疾病FV和FVIII联合缺乏的研究，该研究确定LMAN 1和MCFD 2中的突变是该疾病的原因。FV和FVIII联合缺乏是一种罕见的出血性疾病，其特征是FV和FVIII均协调降低至正常值的5-30%。LMAN 1和MCFD 2在ER-高尔基体中间隔室中形成与FV和FVIII相互作用的Ca 2+依赖性蛋白复合物，表明LMAN 1-MCFD 2复合物是FV和FVIII从ER有效转运到高尔基体所需的货物受体。LMAN 1缺陷的小鼠模拟人类疾病，但也表现出菌株特异性围产期致死性，这不能通过降低的FV/FVIII水平来解释，表明LMAN 1的额外功能。使用LMAN 1和MCFD 2缺陷小鼠，拟议的研究将阐明LMAN 1-MCFD 2受体复合物的结构-功能关系，以及其对体内分泌FV和FVIII以及其他潜在货物蛋白的需求。拟议的研究不仅将回答有关LMAN 1-MCFD 2受体介导的FV和FVIII分泌机制的基本问题，而且还将为哺乳动物ER至高尔基体蛋白转运的一般机制提供基本的新见解。FVIII的遗传缺陷导致血友病A，其影响约1/5000的男性。另一方面，FV功能获得性突变（FV Leiden）和FVIII活性增加是静脉血栓形成的主要风险因素，在美国每年影响约1：1，000的个体。这一发现将对改善FVIII表达具有实际意义，并可能加快血友病A体细胞基因治疗的最终目标，以及限制血栓前状态下FV和FVIII产生的新方法。公共卫生相关性：该项目将研究一种转运受体，当缺乏时，会导致血液凝固因子V和VIII水平低的人类遗传疾病。目的是了解这种转运受体如何控制两种不同的凝血因子，并希望利用这些知识使血友病A和静脉血栓形成患者受益。

Description (provided by the applicant): Coagulation factors V (FV) and factor VIII (FVIII) are both secreted glycoproteins that share pivotal roles in both hemostasis and thrombosis. Although many secreted proteins (referred to as cargo) are believed to require transport receptors for efficient ER-to-Golgi transport, only a limited number of such receptors have been described, mostly in yeast. Evidence for the existence of mammalian cargo receptors came unexpectedly from studies of the human genetic disorder combined deficiency of FV and FVIII, which identified mutations in LMAN1 and MCFD2 as the cause of the disorder. Combined deficiency of FV and FVIII is a rare bleeding disorder characterized by coordinate reduction of both FV and FVIII to the range of 5-30% of normal. LMAN1 and MCFD2 form a Ca2+-dependent protein complex in the ER-Golgi intermediate compartment that interacts with FV and FVIII, suggesting that the LMAN1-MCFD2 complex is a cargo receptor required for efficient transport of FV and FVIII from the ER to the Golgi. Mice deficient in LMAN1 model the human disorder but also exhibit a strain-specific perinatal lethality that is not explained by the reduced FV/FVIII levels, indicating additional functions of LMAN1. Using LMAN1 and MCFD2 deficient mice, proposed research will elucidate structure-function relationship of the LMAN1-MCFD2 receptor complex, and its requirement for the secretion of FV and FVIII and other potential cargo proteins in vivo. Proposed studies will not only answer fundamental questions regarding the mechanism of LMAN1-MCFD2 receptor-mediated secretion of FV and FVIII, but will also provide fundamental new insights into general mechanism of mammalian ER-to-Golgi protein transport. Genetic deficiency of FVIII results in hemophilia A, which affects ~1 in 5000 males. On the other hand, a gain-of-function mutation in FV (FV Leiden) and increased FVIII activity are major risk factors for venous thrombosis, which affects ~1:1,000 individuals in the US per year. The findings will have practical importance for improving FVIII expression and may expedite the eventual goal of somatic cell gene therapy for hemophilia A, as well as new approaches to limiting FV and FVIII production in prothrombotic states. PUBLIC HEALTH RELEVANCE: The project will study a transport receptor that when absent, cause a human genetic disorder with low levels of blood coagulation factors V and VIII. The goal is to understand how this transport receptor controls two different blood coagulation factors and hopefully use the knowledge to benefit people with hemophilia A and venous thrombosis.