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

描述(申请人提供)：调节凝血因子V和凝血因子VIII的ER-高尔基转运体受体凝血因子V(FV)和凝血因子VIII(FVIII)都是分泌的糖蛋白，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均下降到正常的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。对跨膜成分(LMAN1)和可溶性辅因子(MCFD2)的要求表明了高等真核生物货物运输的更复杂机制，并可能为其他哺乳动物货物受体的组织提供了一种范例。我们假设FV和FVIII上的双重分类信号分别被LMAN1和MCFD2识别。这些研究将确定这些调节货物结合和释放的分选信号，调查内质网到高尔基体转运缺陷作为血友病A的新机制，并使用小鼠模型研究体内LMAN1-MCFD2介导的FV和FVIII的分泌。这些结果不仅将回答有关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 applicant): ER-to-Golgi transport receptor in regulating coagulation factors V and VIII Coagulation factor 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 the reduction of both FV and FVIII to 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. The requirement of both a transmembrane component (LMAN1) and a soluble cofactor (MCFD2) suggests a more sophisticated mechanism for cargo trafficking in higher eukaryotes, and could represent a paradigm for the organization of other mammalian cargo receptors. We hypothesize that dual sorting signals on FV and FVIII are separately recognized by LMAN1 and MCFD2. The proposed studies will identify these sorting signals that regulate cargo binding and release, investigate the ER-to-Golgi transport deficiency as a novel mechanism of hemophilia A and use mouse models to investigate LMAN1-MCFD2 mediated secretion of FV and FVIII in vivo. Results 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 insight 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.