Functional study of the vitamin K cycle in mammalian cells

Abstract

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...

The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation

“…VKOR exists in mammals to bacteria, and our results indicate that mammalian VKORC1 acquired a new function (reducing KO to K) rather than losing an ancestral function (reducing K to KH 2 ), as proposed previously (17). That proposal was based on the observation that KH 2 production by purified VKORC1 was inefficient, i.e.…”

“…1), and VKORC1 can only provide two electrons for a single reaction. Although it is well established that VKORC1 is responsible for KO reduction to the vitamin K quinone (K) intermediate, it has recently been proposed that an enzyme other than VKORC1 performs the reduction of K to KH 2 (17,18). A non-VKORC1 vitamin K quinone reductase exists, as bleeding defects in patients undergoing warfarin therapy can be corrected by the administration of K. Treatment involves large amounts of vitamin K, and in vitro studies suggest a high K m for the quinone reductase (19), which has not yet been identified.…”

Recent trends in the metabolism and cell biology of vitamin K with special reference to vitamin K cycling and MK-4 biosynthesis

“…These experiments confi rmed that HEK293 cells (but not AV12 cells) possess signifi cant warfarin resistant "antidotal vitamin K reductase activity" that is able to maintain suffi cient KH 2 production to sustain ␥ -glutamyl carboxylation when the VKOR is completely inhibited. Another implication of these results is that AV12 cells contain a warfarin-sensitive enzyme other than VKOR that converts K to KH 2 ( 85 ).…”

“…In order to circumvent the problems of studying the vitamin K-epoxide cycle in isolated cell fractions, Stafford and colleagues recently developed a cell-based reporter assay system ( 85 ). The reporter protein in this assay was a chimeric VKD protein in which the Gla domain of protein C (PC) had been exchanged with the Gla domain of factor IX ( 85 ).…”

“…The reporter protein in this assay was a chimeric VKD protein in which the Gla domain of protein C (PC) had been exchanged with the Gla domain of factor IX ( 85 ). This replacement enabled the ready detection of the factor IXgla-PC fusion protein using a monoclonal antibody that was specifi c for the carboxylated Gla domain of factor IX ( 85 ). The chimeric reporter protein was then stably expressed in HEK293 cells and in AV12 cells.…”