Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
J Thromb Haemost. 2013 Aug;11(8):1556-64. doi: 10.1111/jth.12306.
Single nucleotide polymorphisms in the vitamin K epoxide reductase (VKOR) gene have been successfully used for warfarin dosage prediction. However, warfarin resistance studies of naturally occurring VKOR mutants do not correlate with their clinical phenotype. This discrepancy presumably arises because the in vitro VKOR activity assay is performed under artificial conditions using the non-physiological reductant dithiothreitol.
The aim of this study is to establish an in vivo VKOR activity assay in mammalian cells (HEK293) where VKOR functions in its native milieu without interference from endogenous enzymes.
Endogenous VKOR activity in HEK293 cells was knocked out by transcription activator-like effector nucleases (TALENs)-mediated genome editing.
Knockout of VKOR in HEK293 cells significantly decreased vitamin K-dependent carboxylation with vitamin K epoxide (KO) as substrate. However, the paralog of VKOR, VKORC1L1, also exhibits substantial ability to convert KO to vitamin K for carboxylation. Using both VKOR and VKORC1L1 knockout cells, we examined the enzymatic activity and warfarin resistance of 10 naturally occurring VKOR mutants that were reported previously to have no activity in an in vitro assay. All 10 mutants are fully active; five have increased warfarin resistance, with the order being W59R>L128R≈W59L>N77S≈S52L. Except for the L128R mutant, this order is consistent with the clinical anticoagulant dosages. The other five VKOR mutants do not change VKOR's warfarin sensitivity, suggesting that factors other than VKOR play important roles. In addition, we confirmed that the conserved loop cysteines in VKOR are not required for active site regeneration after each cycle of oxidation.
维生素 K 环氧化物还原酶（VKOR）基因中的单核苷酸多态性已成功用于华法林剂量预测。然而，天然 VKOR 突变体的华法林耐药性研究与它们的临床表型并不相关。这种差异可能是由于在体外 VKOR 活性测定中使用非生理还原剂二硫苏糖醇（DTT）在人为条件下进行。
本研究旨在建立哺乳动物细胞（HEK293）中的体内 VKOR 活性测定方法，其中 VKOR 在其天然环境中发挥作用，不受内源性酶的干扰。
通过转录激活因子样效应物核酸酶（TALENs）介导的基因组编辑敲除 HEK293 细胞中的内源性 VKOR 活性。
HEK293 细胞中 VKOR 的敲除显著降低了维生素 K 依赖的羧化作用，以维生素 K 环氧化物（KO）为底物。然而，VKOR 的同工酶 VKORC1L1 也具有将 KO 转化为维生素 K 进行羧化的显著能力。使用 VKOR 和 VKORC1L1 敲除细胞，我们研究了 10 种先前报道在体外测定中无活性的天然 VKOR 突变体的酶活性和华法林耐药性。这 10 种突变体均具有完全活性；其中 5 种具有增加的华法林耐药性，其顺序为 W59R>L128R≈W59L>N77S≈S52L。除了 L128R 突变体外，这个顺序与临床抗凝剂剂量一致。另外 5 种 VKOR 突变体不改变 VKOR 的华法林敏感性，表明除 VKOR 之外的其他因素也起着重要作用。此外，我们证实 VKOR 中的保守环半胱氨酸在每个氧化循环后不需要用于活性位点再生。
