Impact of gamma-glutamyl carboxylase processivity on vitamin K-dependent protein modification and function in human health and disease

膳食中的维生素K被γ-谷氨酰羧基酶用来将谷氨酸簇转化为γ-谷氨酰羧基酶。 维生素K依赖(VKD)蛋白中的羧化谷氨酸(Gls)，几乎存在于人体所有组织中。第一 VKD蛋白被鉴定为凝血因子；然而，非止血VKD蛋白的鉴定 已经揭示了额外的作用，例如调节钙化。羧化通过以下途径激活VKD蛋白 产生其功能所需的钙结合模块和单一的伽马-谷氨酰羧基酶 修饰所有的VKD蛋白。自然产生的羧基酶突变会导致两种疾病：维生素K 凝血因子缺乏症1，与严重的出血缺陷和弹性假性黄瘤样有关 (PXE样)，伴有轻度出血但软组织过度钙化。这些羧基酶是如何 突变导致类似PXE的原因以前是未知的。我们研究了PXE中存在的两个羧基酶突变- 就像耐心一样。一种VKD凝血因子(因子IX)和一种抑制钙化的VKD蛋白(基质玻璃)的分析 蛋白质)表现出部分羧化，这是由于羧基酶的加工性缺陷。 加工性指的是羧基酶保持与VKD蛋白的结合，直到多个Glu残基 羧化。我们开发了一种新的检测方法来监测进行性羧化，并发现野生型 羧基酶保护VKD蛋白，即限制其他VKD蛋白进入活性部位，直到VKD 蛋白质被广泛的羧化。相反，类似PXE的突变体允许VKD的混杂访问 蛋白质底物进入活性部位，导致产生部分羧化的VKD蛋白。我们的 研究还发现，一个单一的野生型羧基酶同时结合两个VKD蛋白。作为组织 表达多个被认为具有极大不同亲和力的VKD蛋白，如何充分羧化所有VKD 蛋白质是如何实现的是一个悬而未决的问题。 我们的长期目标是了解部分VKD蛋白羧化如何影响人体生理。 中心问题是是否使用限制VKD蛋白的抗凝剂华法林进行治疗 羧化，产生部分羧化的蛋白质，以及华法林是否引起PXE样表型。 我们将结合蛋白质图谱和活性分析来解决这些问题，以确定 PXE样羧基酶的部分羧化如何影响VKD蛋白功能(目标1)，决定是否 VKD蛋白的羧化受到不同VKD蛋白存在的影响(目标2)，以及 检测华法林治疗和PXE样变异体对VKD蛋白羧化和 体内功能(目标3)。这些研究的结果将提供第一批将蛋白质含量与 不同表型结果的羧化。

Dietary vitamin K is used by the gamma-glutamyl carboxylase to convert clusters of Glus to gamma- carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins in virtually all tissues of the body. The first VKD proteins identified were coagulation factors; however, the identification of nonhemostatic VKD proteins has revealed additional roles, e.g. the regulation of calcification. Carboxylation activates VKD proteins by generating a calcium-binding module required for their function, and a single gamma-glutamyl carboxylase modifies all VKD proteins. Naturally occurring mutations in the carboxylase cause two diseases: vitamin K clotting factor deficiency 1 that is associated with severe bleeding defects, and pseudoxanthoma elasticum-like (PXE-like) that is associated with mild bleeding but excessive soft tissue calcification. How these carboxylase mutations cause PXE-like was previously unknown. We studied two carboxylase mutations present in a PXE- like patient. Analysis of a VKD clotting factor (factor IX) and a VKD protein that inhibits calcification (Matrix Gla Protein) revealed partial carboxylation due to a defect in carboxylase processivity. Processivity refers to the carboxylase remaining bound to a VKD protein until the multiple Glu residues are carboxylated. We developed a novel assay to monitor processive carboxylation, and found that the wild type carboxylase shields the VKD protein, i.e. limiting access of other VKD proteins into the active site until the VKD protein is extensively carboxylated. In contrast, the PXE-like mutants allowed promiscuous access of VKD protein substrates into the active site, resulting in the production of partially carboxylated VKD proteins. Our studies also revealed that a single wild type carboxylase binds two VKD proteins at the same time. As tissues express multiple VKD proteins thought to have widely different affinities, how full carboxylation of all VKD proteins is achieved is an open question. Our long-term goal is to understand how partial VKD protein carboxylation impacts human physiology. Central questions are whether treatment with the anticoagulant warfarin, which limits VKD protein carboxylation, generates partially carboxylated proteins, and whether warfarin evokes PXE-like phenotypes. We will approach these questions using a combination of protein mapping and activity assays to determine how partial carboxylation by PXE-like carboxylases impacts VKD protein function (Aim 1), determine whether the carboxylation of a VKD protein is impacted by the presence of a different VKD protein (Aim 2), and examine the consequence of warfarin therapy and a PXE-like mutant on VKD protein carboxylation and function in vivo (Aim 3). Results from these studies will provide the first insights that link the extent of protein carboxylation to different phenotypic outcomes.