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

凝血因子V和VIII的ER-高尔基体转运 凝血因子V（FV）和凝血因子VIII（FVIII）都是分泌的糖蛋白，它们在凝血过程中具有关键作用。 止血和血栓形成。FVIII的遗传缺陷导致血友病A，其影响约1/5000 男性。另一方面，FV的功能获得性突变（FV Leiden）和FVIII活性增加是主要原因。 静脉血栓形成的风险因素，每年影响美国约1：1，000人。尽管许多 分泌的蛋白质（称为货物）被认为需要转运受体来有效地进行内质网转运。 尽管已经描述了内质网（ER）到高尔基体的转运，但是只有有限数量的这样的受体被描述，大部分在 酵母哺乳动物货物受体存在的证据出乎意料地来自对哺乳动物的研究。 人类遗传性疾病合并FV和FVIII缺陷（F5 F8 D），其鉴定了LMAN 1突变 和MCFD 2作为疾病的原因。F5 F8 D是一种罕见的出血性疾病，其特征是 FV和FVIII均降至正常值的5-30%。LMAN 1和MCFD 2在ER-1中形成Ca 2+依赖性蛋白复合物。 与FV和FVIII相互作用的高尔基体中间室，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。的要求 跨膜成分（LMAN 1）和可溶性辅因子（MCFD 2）都表明， 在高等真核生物的货物运输机制，并可能代表了一个范例的组织 其他哺乳动物货物受体。在本研究中，我们将研究受体介导的内质网-内质网转化的机制， FV和FVIII的高尔基体转运。在目标1中，我们将鉴定FV/FVIII上的N-连接糖基化位点， 与LMAN 1，研究Ca 2+在调节货物的结合和释放，并增加体外FVIII 通过增强LMAN 1-MCFD 2分泌途径来生产。在aim 2中，我们将识别 FVIII被MCFD 2识别，研究ER到高尔基体转运缺陷作为一种新的机制， 血友病A，并鉴定控制FV/FVIII运输的其他组分/替代途径。在目标3中，我们 将测试LMAN 1-MCFD 2分泌途径作为血栓形成倾向的治疗靶点，并创建一个小鼠 模型来表征B结构域在FVIII生物合成和LMAN 1-MCFD 2介导的分泌中的作用。 vivo.结果不仅将回答关于LMAN 1-MCFD 2机制的基本问题， 受体介导的FV和FVIII分泌，但也将提供基本的新见解，一般 哺乳动物内质网-高尔基体蛋白转运的机制。研究结果将具有实际意义， 改善FVIII表达，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。

ER-to-Golgi transport of 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. 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. Although many secreted proteins (referred to as cargo) are believed to require transport receptors for efficient endoplasmic reticulum (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 (F5F8D), which identified mutations in LMAN1 and MCFD2 as the cause of the disorder. F5F8D 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. In this proposal, we will study the mechanism of receptor-mediated ER-to- Golgi transport of FV and FVIII. In aim 1, we will identify N-linked glycosylation sites on FV/FVIII that interact with LMAN1, investigate Ca2+ in regulating the binding and release of cargo, and increase in vitro FVIII production by enhancing the LMAN1-MCFD2 secretion pathway. In aim 2, we will identify sorting signals in FVIII that are recognized by MCFD2, investigate the ER-to-Golgi transport deficiency as a novel mechanism of hemophilia A, and identify other components/alternative pathways that control FV/FVIII trafficking. In aim 3, we will test the LMAN1-MCFD2 secretion pathway as a therapeutic target for thrombophilia and create a mouse model to characterize the role of B domain in FVIII biosynthesis and LMAN1-MCFD2 mediated secretion 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. 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.