The inhibitory effect of calumenin on the vitamin K-dependent gamma-carboxylation system. Characterization of the system in normal and warfarin-resistant rats.
The inhibitory effect of calumenin on the vitamin K-dependent gamma-carboxylation system. Characterization of the system in normal and warfarin-resistant rats.
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DOI：
10.1074/jbc.m401645200
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发表时间：
2004-06
期刊：
影响因子：
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通讯作者：
N. Wajih;D. Sane;S. Hutson;R. Wallin
中科院分区：
文献类型：
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作者：
N. Wajih;D. Sane;S. Hutson;R. Wallin
维生素K依赖的伽马-羧化系统负责维生素K依赖的蛋白质的翻译后修饰，将它们转化为含有GLA的蛋白质。该系统由位于内质网膜上的完整膜蛋白组成，包括γ-羧基酶和华法林敏感酶维生素K(1)2，3-环氧化物还原酶(VKOR)，后者为γ-羧基酶提供还原的维生素K(1)辅因子。在这项工作中，我们设计了一个体外伽玛-羧化系统，用来了解VKOR和伽马-羧基酶是如何作为一个系统一起工作的，并确定可以调节该系统活性的因素。结果表明，内质网伴侣蛋白calumenin与γ-羧基酶有关，并抑制其活性。用siRNA沉默calumenin基因导致伽玛-羧基酶活性增加5倍。这一结果首次发现了一种可以调节伽马-羧化系统活性的蛋白质。依赖维生素K的蛋白质的前肽可以刺激伽玛-羧基酶的活性。在这里，我们证明了因子X和凝血酶原多肽不会增加VKOR在VKOR中减少的维生素K(1)辅因子的产生，其中VKOR是伽玛-羧化的限速步骤。这些发现使Calumenin在调节维生素K依赖的蛋白质的伽马-羧化方面处于中心位置。在华法林耐药大鼠制备的体外伽玛羧化系统中，VKOR和伽马-羧基酶之间减少的维生素K(1)辅因子转移被证明是显著受损的。此外，VKOR酶复合体的18 kDa亚基1的序列在两个品系中是相同的。这一发现支持这样一种观点，即存在不同形式的华法林基因抗性。
The vitamin K-dependent gamma-carboxylation system is responsible for post-translational modification of vitamin K-dependent proteins, converting them to Gla-containing proteins. The system consists of integral membrane proteins located in the endoplasmic reticulum membrane and includes the gamma-carboxylase and the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase (VKOR), which provides gamma-carboxylase with reduced vitamin K(1) cofactor. In this work, an in vitro gamma-carboxylation system was designed and used to understand how VKOR and gamma-carboxylase work together as a system and to identify factors that can regulate the activity of the system. Results are presented that demonstrate that the endoplasmic reticulum chaperone protein calumenin is associated with gamma-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in gamma-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the gamma-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate gamma-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K(1) cofactor production by VKOR in the system where VKOR is the rate-limiting step for gamma-carboxylation. These findings put calumenin in a central position concerning regulation of gamma-carboxylation of vitamin K-dependent proteins. Reduced vitamin K(1) cofactor transfer between VKOR and gamma-carboxylase is shown to be significantly impaired in the in vitro gamma-carboxylation system prepared from warfarin-resistant rats. Furthermore, the sequence of the 18-kDa subunit 1 of the VKOR enzyme complex was found to be identical in the two rat strains. This finding supports the notion that different forms of genetic warfarin resistance exist.