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

说明(申请人提供)：凝血因子V(FV)和因子(FVIII)都是分泌型糖蛋白，在止血和血栓形成中发挥关键作用。尽管许多分泌蛋白(称为Cargo)被认为需要转运受体来有效地从内质网到高尔基体的运输，但目前只有有限数量的这种受体被描述，主要是在酵母中。哺乳动物货运受体存在的证据出人意料地来自对人类遗传性疾病Fv和FVIII缺陷的研究，研究发现LMAN1和MCFD2突变是导致这种疾病的原因。FV和FVIII的联合缺陷是一种罕见的出血性疾病，其特征是FV和FVIII的协调降低到正常范围的5-30%。LMAN1和MCFD2在内质网-高尔基体中间区形成一个钙依赖的蛋白质复合体，与Fv和FVIII相互作用，提示LMAN1-MCFD2复合体是Fv和FVIII从内质网高效运输到高尔基体所必需的货物受体。缺乏LMAN1的小鼠不仅表现出人类疾病的模型，而且还表现出菌株特异性的围产期致死性，这不能用Fv/FVIII水平的降低来解释，这表明LMAN1具有额外的功能。利用LMAN1和MCFD2缺陷小鼠，拟议的研究将阐明LMAN1-MCFD2受体复合体的结构和功能关系，以及它在体内对FV和FVIII和其他潜在货物蛋白分泌的需求。所提出的研究不仅将回答有关LMAN1-MCFD2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到高尔基体蛋白运输的一般机制提供基本的新见解。FVIII的遗传缺陷会导致血友病A，每5000名男性中就有1人受到影响。另一方面，Fv(Fv Leiden)功能获得突变和FVIII活性增加是静脉血栓形成的主要风险因素，在美国每年约有1：1000人受到影响。这些发现将对改善FVIII的表达具有实际意义，并可能加速血友病A的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。

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.