PDF(Pubmed)
Abstract：
γ-Glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M and S300F) whose combined heterozygosity in a patient causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation. Complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of the VKD protein in the complex. The VKD protein in the complex with wild-type carboxylase was carboxylated before challenge protein carboxylation occurred and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time and did not completely carboxylate FIX in the complex. S300F carboxylation was poor with both FIX and MGP. Additional studies analyzed FIX- and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The total amount of carboxylated peptide generated by the V255M mutant was higher than that of wild-type GGCX; however, the individual peptides were partially carboxylated. Analysis of the V255M mutant in FIX HEK293 cells lacking endogenous GGCX revealed poor FIX clotting activity. This study shows that disrupted processivity causes disease and explains the defect in the patient. Kinetic analyses also suggest that disrupted processivity may occur in wild-type carboxylase under some conditions (eg, warfarin therapy or vitamin K deficiency).
摘要：
γ-谷氨酰羧化酶（GGCX）在维生素K依赖性（VKD）蛋白中产生多种羧化Glus（Glas），这是其功能所必需的。GGCX是强制性的，在多个Glu羧化过程中保持与VKD蛋白的结合，这项研究揭示了持续合成能力对VKD蛋白功能的重要性。GGCX突变体(V255M和S300F),其在患者中的组合杂合性导致缺陷性凝血和钙化,使用模拟体内羧化的新测定法进行了研究。变体羧化酶和对止血(因子IX[FIX])或钙化(基质Gla蛋白[MGP])重要的VKD蛋白之间的复合物在可能潜在地干扰复合物中VKD蛋白的羧化的挑战VKD蛋白的存在下反应。在攻击蛋白羧化发生之前,与野生型羧化酶的复合物中的VKD蛋白被羧化并变成完全羧化。相比之下,V255M突变体同时羧化了两种形式，并且在复合物中没有完全羧化FIX。对于FIX和MGP两者,S300F羧化作用较差。另外的研究分析了含有与介导羧化酶结合的序列连接的Gla结构域的FIX和MGP衍生的肽。V255M突变体产生的羧化肽的总量高于野生型GGCX；然而，单个肽部分羧化。缺乏内源性GGCX的FIXHEK293细胞中V255M突变体的分析揭示了差的FIX凝血活性。这项研究表明，连续性中断会导致疾病，并解释了患者的缺陷。动力学分析还表明，在某些条件下，野生型羧化酶的持续合成能力可能会受到破坏（例如，华法林治疗或维生素K缺乏症)。
