We describe a cell-based assay for studying vitamin K-cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO). However, neither cell line carboxylated the reporter when fed KO in the presence of warfarin. In the presence of warfarin, vitamin K rescued carboxylation in HEK293 cells but not in AV12 cells. Dicoumarol, an NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) inhibitor, behaved similarly to warfarin in both cell lines. Warfarin-resistant vitamin K epoxide reductase (VKOR-Y139F) supported carboxylation in HEK293 cells when fed KO in the presence of warfarin, but it did not in AV12 cells. These results suggest the following: (1) our cell system is a good model for studying the vitamin K cycle, (2) the warfarin-resistant enzyme reducing vitamin K to hydroquinone (KH 2 ) is probably not NQO1, (3) there appears to be a warfarin-sensitive enzyme other than VKOR that reduces vitamin K to KH 2 , and (4) the primary function of VKOR is the reduction of KO to vitamin K. (Blood. 2011;117(10):2967-2974) IntroductionVitamin K hydroquinone (KH 2 ) is a cofactor for ␥-glutamyl carboxylase (GGCX), which catalyzes the posttranslational carboxylation of specific glutamic acid residues to ␥-carboxyglutamic acid (gla) in a variety of vitamin K-dependent proteins. 1 ␥-Glutamyl carboxylation is essential for the biologic functions of vitamin K-dependent proteins involved in blood coagulation, bone metabolism, signal transduction, and cell proliferation. Concomitant with ␥-glutamyl carboxylation, KH 2 is oxidized to vitamin K 2,3-epoxide (KO). KO must then be converted back to vitamin K (the quinone form) and then to KH 2 by separate 2 electron reductions to support the carboxylation reaction. The cyclic production of KO and the conversion back to KH 2 constitutes the vitamin K cycle (Figure 1). The only enzymes unequivocally identified as part of the cycle are GGCX and vitamin K epoxide reductase (VKOR). 2 Sherman et al first proposed that the reduction of KO to vitamin K is carried out by a sulfhydryl-dependent epoxide reductase that is sensitive to warfarin inhibition. 3 This enzyme is probably VKOR, the only enzyme thus far shown to reduce KO to vitamin K. On the other hand, some reports suggest that the reduction of vitamin K to KH 2 can be accomplished by at least 2 microsomal enzymes called vitamin K reductases. 4 However, other studies 5,6 suggest that one enzyme serves as both the epoxide reductase and the vitamin K reductase, catalyzing both the reduction of KO to vitamin K and that of vitamin K to KH 2 .Wallin proposed that there are 2 enzymes that reduce vitamin K in support of vitamin K-dependent carboxylation. 7 One enzyme is inhibited by anticoagulant drugs such as warfarin, 8 while the other is an NADH-dependent reductase that is resistant to inhibition by warfarin. 4,[9][10][11] Consistent with the latte...
Summary Background 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. Objectives 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. Methods Endogenous VKOR activity in HEK293 cells was knocked out by transcription activator-like effector nucleases (TALENs)-mediated genome editing. Results and Conclusions 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 ten naturally occurring VKOR mutants that were reported previously to have no activity in an in vitro assay. All ten 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.
Vitamin K epoxide (or oxido) reductase (VKOR) is the target of warfarin and provides vitamin K hydroquinone for the carboxylation of select glutamic acid residues of the vitamin K-dependent proteins which are important for coagulation, signaling, and bone metabolism. It has been known for at least 20 years that cysteines are required for VKOR function. To investigate their importance, we mutated each of the seven cysteines in VKOR. In addition, we made VKOR with both C43 and C51 mutated to alanine (C43A/C51A), as well as a VKOR with residues C43-C51 deleted. Each mutated enzyme was purified and characterized. We report here that C132 and C135 of the CXXC motif are essential for both the conversion of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. Surprisingly, conserved cysteines, 43 and 51, appear not to be important for either reaction. For the in vitro reaction driven by dithiothreitol, the 43-51 deletion mutation retained 85% and C43A/C51A 112% of the wild-type activity. The facile purification of the nine different mutations reported here illustrates the ease and reproducibility of VKOR purification by the method reported in our recent publication [Chu, P.-H., Huang, T.-Y., Williams, J., and Stafford, D. W. (2006) Proc. Natl. Acad. Sci. U S A. 103, 19308-19313].
Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10 -29, 101-123, and 127-149, and membrane-integration of residues 75-97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM.The K vitamins, phylloquinone (K1), menaquinones (K2), and menadione (K3), are a family of structurally similar, fatsoluble, 2-methyl-1,4-naphthoquinones. The main function of vitamin K is to act as a co-factor for the ␥-glutamyl carboxylase that catalyzes the post-translational carboxylation of specific glutamic acid to ␥-carboxyglutamic acid (Gla) 1 of variety of vitamin K-dependent proteins (1). Members of the vitamin K-dependent protein family include coagulation factors (factor II, VII, IX, X) as well as several other proteins that function in bone metabolism (2) and signal transduction (3). Concomitant with ␥-glutamyl carboxylation, the reduced form of vitamin K (vitamin K hydroquinone) is converted to vitamin K 2,3-epoxide, which must be converted back to vitamin K hydroquinone for the reaction to continue because of limited vitamin K amounts in vivo (4). This cyclic conversion of vitamin K establishes a redox cycle known as the vitamin K cycle (5).VKOR is responsible for the conversion of vitamin K 2,3-epoxide into vitamin K and is highly sensitive to inhibition by coumarin drugs, such as R,S-warfarin (4-hydroxy-3-(3-oxo-1-phenylbutyl-coumarin)), the most commonly prescribed oral anticoagulant. Warfarin inhibition of VKOR reduces the availability of reduced vitamin K, which reduces the rate of carboxylation and leads to partially carboxylated, inactive vitamin K-dependent proteins. Since its discovery in 1970 (6), numerous futile attempts to purify the enzyme were reported (7-11). Attempts to understand the mechanism underlying warfarinsensitive vitamin K epoxide reduction have been somewhat more successful (8,(12)(13)(14)(15)....
Vitamin K epoxide reductase (VKOR) is essential for the production of reduced vitamin K that is required for modification of vitamin K-dependent proteins. Three- and four-transmembrane domain (TMD) topology models have been proposed for VKOR. They are based on in vitro glycosylation mapping of the human enzyme and the crystal structure of a bacterial (Synechococcus) homologue, respectively. These two models place the functionally disputed conserved loop cysteines, Cys-43 and Cys-51, on different sides of the endoplasmic reticulum (ER) membrane. In this study, we fused green fluorescent protein to the N or C terminus of human VKOR, expressed these fusions in HEK293 cells, and examined their topologies by fluorescence protease protection assays. Our results show that the N terminus of VKOR resides in the ER lumen, whereas its C terminus is in the cytoplasm. Selective modification of cysteines by polyethylene glycol maleimide confirms the cytoplasmic location of the conserved loop cysteines. Both results support a three-TMD model of VKOR. Interestingly, human VKOR can be changed to a four-TMD molecule by mutating the charged residues flanking the first TMD. Cell-based activity assays show that this four-TMD molecule is fully active. Furthermore, the conserved loop cysteines, which are essential for intramolecular electron transfer in the bacterial VKOR homologue, are not required for human VKOR whether they are located in the cytoplasm (three-TMD molecule) or the ER lumen (four-TMD molecule). Our results confirm that human VKOR is a three-TMD protein. Moreover, the conserved loop cysteines apparently play different roles in human VKOR and in its bacterial homologues.
Summary Vitamin K-dependent proteins require carboxylation of certain glutamates for their biological functions. The enzymes involved in the vitamin K-dependent carboxylation include: gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR) and an as-yet-unidentified vitamin K reductase (VKR). Due to the hydrophobicity of vitamin K, these enzymes are likely to be integral membrane proteins that reside in the endoplasmic reticulum. Therefore, structure-function studies on these enzymes have been challenging, and some of the results are notably controversial. Patients with naturally occurring mutations in these enzymes, who mainly exhibit bleeding disorders or are resistant to oral anticoagulant treatment, provide valuable information for the functional study of the vitamin K cycle enzymes. In this review, we discuss: (i) the discovery of the enzymatic activities and gene identifications of the vitamin K cycle enzymes; (ii) the identification of their functionally important regions and their active site residues; (iii) the membrane topology studies of GGCX and VKOR; and (iv) the controversial issues regarding the structure and function studies of these enzymes, particularly, the membrane topology, the role of the conserved cysteines and the mechanism of active site regeneration of VKOR. We also discuss the possibility that a paralogous protein of VKOR, VKOR-like 1 (VKORL1), is involved in the vitamin K cycle, and the importance of and possible approaches for identifying the unknown VKR. Overall, we describe the accomplishments and the remaining questions in regard to the structure and function studies of the enzymes in the vitamin K cycle.
Key Points• CRISPR-Cas9-mediated GGCX knockout cell-based assay clarifies the correlation between GGCX genotypes and their clinical phenotypes.• A GGCX mutation decreases clotting factor carboxylation and abolishes MGP carboxylation, causing 2 distinct clinical phenotypes.Vitamin K-dependent coagulation factors deficiency is a bleeding disorder mainly associated with mutations in g-glutamyl carboxylase (GGCX) that often has fatal outcomes. Some patients with nonbleeding syndromes linked to GGCX mutations, however, show no coagulation abnormalities. The correlation between GGCX genotypes and their clinical phenotypes has been previously unknown. Here we report the identification and characterization of novel GGCX mutations in a patient with both severe cerebral bleeding disorder and comorbid Keutel syndrome, a nonbleeding malady caused by functional defects of matrix g-carboxyglutamate protein (MGP). To characterize GGCX mutants in a cellular milieu, we established a cell-based assay by stably expressing 2 reporter proteins (a chimeric coagulation factor and MGP) in HEK293 cells. The endogenous GGCX gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. Our results show that, compared with wild-type GGCX, the patient's GGCX D153G mutant significantly decreased coagulation factor carboxylation and abolished MGP carboxylation at the physiological concentration of vitamin K. Higher vitamin K concentrations can restore up to 60% of coagulation factor carboxylation but do not ameliorate MGP carboxylation. These results are consistent with the clinical results obtained from the patient treated with vitamin K, suggesting that the D153G alteration in GGCX is the causative mutation for both the bleeding and nonbleeding disorders in our patient. These findings provide the first evidence of a GGCX mutation resulting in 2 distinct clinical phenotypes; the established cell-based assay provides a powerful tool for studying the clinical consequences of naturally occurring GGCX mutations in vivo. (Blood. 2016;127(15):1847-1855 Introduction Vitamin K-dependent carboxylation is a posttranslational modification that converts specific glutamate (Glu) residues to g-carboxyglutamate (Gla) residues in vitamin K-dependent proteins. It is required for the functioning of numerous vitamin K-dependent proteins involved in a broad range of biological functions, including blood coagulation. Carboxylation is catalyzed by the enzyme g-glutamyl carboxylase (GGCX), which uses a reduced form of vitamin K (KH 2 ) as a cofactor. Concomitant with each glutamate modification, KH 2 is oxidized to vitamin K epoxide (KO). Because humans cannot synthesize vitamin K, KO must be converted back to KH 2 by the enzymes KO reductase and the as-yet unknown vitamin K reductase in a pathway known as the vitamin K cycle. 1Defects of vitamin K-dependent carboxylation have long been known to cause bleeding disorders, referred to as combined vitamin Kdependent coagulation factors deficiency (VKCFD). Patients with VKCFD have decreased levels of mul...
Vitamin K-dependent ␥-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to ␥-carboxyglutamate. Carboxylase has ten cysteine residues, but their form The vitamin K-dependent carboxylase is an integral membrane glycoprotein that catalyzes the post-translational modification of specific glutamic acid residues to ␥-carboxyglutamic acid (Gla) 1 (1, 2). The carboxylation reaction occurs in the lumen of the ER (3, 4) and uses the substrates carbon dioxide, oxygen, and vitamin K hydroquinone. During the process of carboxylation, the ␥-proton of the glutamic acid is abstracted, followed by the addition of carbon dioxide (5). Simultaneous with carboxylation, the vitamin K hydroquinone is converted to vitamin K epoxide, which is converted back to vitamin K by the enzyme epoxide reductase. The formation of vitamin K epoxide has sometimes been called an epoxidation reaction. Gla modification is critical for the function of more than a dozen proteins involved in blood coagulation and calcium homeostasis (6, 7). The importance of vitamin K-dependent proteins may be even greater than previously thought, as evidenced by the discovery of growth-arrest protein gas-6 (8), and the very recent identification of four putative vitamin K-dependent membrane Gla proteins PRGP1, PRGP2, TMG3, and TMG4 (9, 10). There are ten cysteine residues in the human carboxylase molecule. Our work on the topology of the carboxylase predicts that of these ten cysteines, two are located in the cytoplasm, three are buried in the ER membrane, and five are found in the lumen of the ER (11). Sulfhydryl groups and disulfide bonds are important for both the structure and function of proteins (12-15). For example, the natural abundance of cysteine is 1.2%, but these residues constitute 5.6% of enzyme catalytic sites (16). Therefore, identification of free cysteine residues or those involved in disulfide bond formation can give valuable information about the structure and function of proteins.Several studies have implicated cysteine in the function of carboxylase. Chemical modification of carboxylase by sulfhydryl-reactive reagents suggests that cysteine residues are important for the carboxylation reaction (17-21). Based on a non-enzymatic chemical model, Paul Dowd et al. (22) developed a "base strength amplification mechanism" for carboxylation. They proposed that two free cysteines are involved in the active site of carboxylase. Recently, Pudota et al. (21) analyzed the catalytically important cysteine residues of the carboxylase by modifying free cysteines with the radiolabeled sulfhydryl-reactive reagent 14 C-NEM. These authors reported that cysteine residues 99 and 450 are the active site residues of carboxylase (21). In contrast to the multiple studies on the importance of free cysteines in carboxylase, the only information about disulfide bridges in the structure of the carboxylase is the study by * This work was supported by National Institutes ...
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