Characterization of Vitamin K Epoxide Reductase

描述(申请人提供)：本项目的长期目标是了解维生素K环氧化物还原酶(VKOR)的酶性质及其在维生素K依赖的羧化中的作用。羧化是蛋白质翻译后的重要修饰，在多种生理功能中起重要作用，包括凝血和骨代谢。最近，该实验室的研究人员发现了对华法林敏感的VKOR基因。VKOR是华法林的靶标，华法林是治疗血栓栓塞症的最广泛处方的抗凝剂。虽然据估计，华法林每次诱发出血可以预防20次中风，但由于担心出血，华法林没有得到充分利用。由这笔资金资助的研究有望导致治疗方法，缓解华法林治疗中涉及的一些问题。具体地说，我们建议在这笔赠款的有效期内完成以下工作：(1)表达VKOR基因的异构体，以确定哪些具有活性；(2)使用我们插入的氨基酸标签和该标签的抗体来纯化VKOR；(3)研究不同的细胞表达系统，以确定哪种重组VKOR最适合生产重组VKOR；(4)通过质谱仪研究半胱氨酸在VKOR中的地位、糖基化和其他潜在的翻译后修饰，以及华法林结合位点；以及(5)研究维生素K环氧化物还原酶(VKOR)基因多态性对华法林反应的影响。对VKOR的研究将有助于更好地理解华法林抑制的机制。它还可能导致更准确的华法林剂量和新的抗凝血剂的设计。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the enzymatic properties of the vitamin K epoxide reductase (VKOR) and its role in vitamin K-dependent carboxylation. Carboxylation is an essential post-translational modification for proteins important in several physiologic functions, including blood coagulation and bone metabolism. Recently, researchers in this laboratory identified the warfarin-sensitive VKOR gene. VKOR is the target of warfarin, the most widely prescribed anti-coagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-utilized because of fear of bleeding. Research funded by this grant hopefully will lead to therapies that will alleviate some of the problems involved in warfarin treatment. Specifically, we propose to accomplish the following during the tenure of this grant: (1) express the isoforms of the VKOR gene to determine which have activity; (2) purify the VKOR using the amino acid tag we have inserted and the antibody to this tag; (3) investigate different cell expression systems to determine which is best for production of recombinant VKOR; (4) investigate the status of cysteines in VKOR, the glycosylation sites and other potential post-translational modifications, and the warfarin binding site by mass spectrometry; and (5) investigate the effect of vitamin K epoxide reductase (VKOR) gene polymorphisms on response to warfarin. This study of VKOR should lead to a better understanding of the mechanism of warfarin inhibition. It may also result in more accurate dosing of warfarin and in the design of new anti-coagulants.