The gamma-glutamyl carboxylase generates carboxylated Glus (Glas) in the Gla domain of vitamin K- dependent (VKD) proteins to render them active in a diverse range of functions that includes hemostasis, calcification, apoptosis and growth control. A single carboxylase modifies all VKD proteins, and this broad physiological impact makes it critical to understand the mechanisms of catalysis and regulation. Naturally occurring carboxylase mutations cause vitamin K-dependent clotting factor deficiency (VKCFD), which is associated with severe bleeding. Mutations have recently also been shown to cause a second disease, pseudoxanthoma elasticum (PXE), where bleeding is mild but calcification is defective. Why carboxylase mutations cause this second disease was unknown, and our studies on a carboxylase mutation that causes PXE indicate a defect in fully modifying the Gla domain of VKD proteins. The observation that carboxylase mutations cause PXE highlights the importance of understanding how carboxylation fully impacts human health. The role of carboxylation in hemostasis is well established;however, the carboxylase is expressed in virtually every tissue and nonhemostatic functions are very poorly understood. These functions are likely to be affected in the many patients treated with Warfarin, which inhibits carboxylation. Our goals in the next funding period are to understand the mechanism of VKD protein carboxylation, determine how carboxylase mutations cause two different diseases, and begin defining the role of carboxylation in different tissues.
In Aim 1, we will test a novel hypothesis or how the carboxylase initiates catalysis, and will also determine how two naturally occurring carboxylase mutations cause VKCFD.
Aim 2 will determine how undercarboxylation impacts PXE using a novel method we developed to analyze VKD proteins by mass spectrometry.
Aim 2 will also use an innovative mouse model to determine whether the carboxylase plays a role in calcification in soft tissue. The carboxylase contains Glas, of unknown function, and we have now identified the Gla domain.
In Aim 3, we will use a mutagenesis approach to test the hypothesis that the Gla domain regulates VKD protein carboxylation, and will use FRET to determine if the Gla domain facilitates phospholipid interaction. VKD proteins are carboxylated as part of a secretory process that impacts carboxylation, and Aim 4 will test the role of cellular components in mediating VKD protein trafficking. These studies will provide new fundamental information important for the development of mechanism-based inhibitors that lead to superior anticoagulants, and will also be significant for the production of therapeutic VKD proteins for applications in hemophilia, sepsis and tissue glues.

Public Health Relevance

Vitamin K is required for normal blood clotting, and recent studies have revealed new roles related to skin and cardiovascular disease. The studies proposed here will investigate how the body utilizes vitamin K, how defective utilization results i disease, and how these diseases inform us about the role of vitamin K in normal human physiology. The results of this work will lead to the development of superior drugs over ones like Warfarin, which controls bleeding but is compromised by serious side effects. The results will be important for improving therapeutic strategies to treat hemophilia and sepsis, and will also impact the recommended daily allowance for vitamin K in all individuals.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Link, Rebecca P
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Cleveland Clinic Lerner
Other Basic Sciences
Schools of Medicine
United States
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Hallgren, K W; Zhang, D; Kinter, M et al. (2013) Methylation of ?-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the ?-glutamyl carboxylase. J Proteome Res 12:2365-74
Rishavy, Mark A; Hallgren, Kevin W; Wilson, Lee A et al. (2013) The vitamin K oxidoreductase is a multimer that efficiently reduces vitamin K epoxide to hydroquinone to allow vitamin K-dependent protein carboxylation. J Biol Chem 288:31556-66
Rishavy, Mark A; Berkner, Kathleen L (2012) Vitamin K oxygenation, glutamate carboxylation, and processivity: defining the three critical facets of catalysis by the vitamin K-dependent carboxylase. Adv Nutr 3:135-48
Rishavy, Mark A; Hallgren, Kevin W; Berkner, Kathleen L (2011) The vitamin K-dependent carboxylase generates ?-carboxylated glutamates by using CO2 to facilitate glutamate deprotonation in a concerted mechanism that drives catalysis. J Biol Chem 286:44821-32
Rishavy, Mark A; Usubalieva, Aisulu; Hallgren, Kevin W et al. (2011) Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin K-dependent protein carboxylation. J Biol Chem 286:7267-78
Li, Qiaoli; Grange, Dorothy K; Armstrong, Nicole L et al. (2009) Mutations in the GGCX and ABCC6 genes in a family with pseudoxanthoma elasticum-like phenotypes. J Invest Dermatol 129:553-63
Rishavy, Mark A; Berkner, Kathleen L (2008) Insight into the coupling mechanism of the vitamin K-dependent carboxylase: mutation of histidine 160 disrupts glutamic acid carbanion formation and efficient coupling of vitamin K epoxidation to glutamic acid carboxylation. Biochemistry 47:9836-46
Rishavy, Mark A; Hallgren, Kevin W; Yakubenko, Anna V et al. (2006) Bronsted analysis reveals Lys218 as the carboxylase active site base that deprotonates vitamin K hydroquinone to initiate vitamin K-dependent protein carboxylation. Biochemistry 45:13239-48
Darghouth, Dhouha; Hallgren, Kevin W; Shtofman, Rebecca L et al. (2006) Compound heterozygosity of novel missense mutations in the gamma-glutamyl-carboxylase gene causes hereditary combined vitamin K-dependent coagulation factor deficiency. Blood 108:1925-31
Hallgren, Kevin W; Qian, Wen; Yakubenko, Anna V et al. (2006) r-VKORC1 expression in factor IX BHK cells increases the extent of factor IX carboxylation but is limited by saturation of another carboxylation component or by a shift in the rate-limiting step. Biochemistry 45:5587-98

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