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).

中文翻译：

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损害止血的 GGCX 突变体揭示了持续合成和完全羧化对 VKD 蛋白功能的重要性。

γ-谷氨酰羧化酶 (GGCX) 在维生素 K 依赖性 (VKD) 蛋白质中产生其功能所需的多个羧化 Glus (Glas)。GGCX 具有持续性，在多次 Glu 羧化过程中始终与 VKD 蛋白结合，这项研究揭示了持续性对 VKD 蛋白功能的重要性。使用模拟体内羧化的新测定法研究了 GGCX 突变体（V255M 和 S300F），其在患者中的组合杂合性导致凝血和钙化缺陷。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存在挑战 VKD 蛋白的情况下发生反应，该蛋白可能会干扰复合物中 VKD 蛋白的羧化。与野生型羧化酶形成的复合物中的VKD蛋白在攻击蛋白羧化发生之前被羧化并且变得完全羧化。相反，V255M突变体同时羧化两种形式并且没有完全羧化复合物中的FIX。FIX 和 MGP 的 S300F 羧化都很差。其他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 衍生肽。V255M突变体产生的羧化肽总量高于野生型GGCX；然而，单个肽被部分羧化。对缺乏内源性 GGCX 的 FIX HEK293 细胞中 V255M 突变体的分析显示 FIX 凝血活性较差。这项研究表明，破坏的持续性会导致疾病，并解释了患者的缺陷。动力学分析还表明，在某些条件下（例如华法林治疗或维生素 K 缺乏），野生型羧化酶可能会发生持续合成能力破坏。