The vitamin K-dependent (VKD) carboxylase is critical to hemostasis because it converts Glus to carboxylated Glus in VKD proteins to allow their binding to cell surfaces where hemostasis occurs. VKD proteins are carboxylated in the endoplasmic reticulum during their secretion, and a single carboxylase modifies all VKD proteins, many of which are coexpressed in tissue. In the previous grant period, we developed an approach to directly analyze intracellular carboxylation in mammalian cells, which showedthat the secretory process impacts carboxylation, that intracellular processing is not identical for all VKD proteins and that carboxylation is regulated by the availability of the reduced vitamin K cofactor required for Glu carboxylation. The studies also showed that the rate-limiting step in VKD turnover is different in cells than in an in vitro reaction, which may be due to post-translational modifications in the carboxylase. Other studies revealed that Leptospira, the bacterial pathogen that causes leptospirosis, contains an ortholog of the VKD carboxylase, which appears to have been acquired by horizontal gene transfer and to have been adapted for a role other than carboxylation. Our studies also implicated novel functional carboxylase residues, including the catalytic base that initiates carboxylation and residues whose substitution cause combined VKD coagulation factor deficiency. Our long-term goal is to understand the mechanism of carboxylation, including how it interfaces with the secretory machinery and how multiple VKD proteins are modified by one carboxylase to become fully-carboxylated and active. We propose: 1. To determine how the active site facilitates carboxylation. We will identify the catalytic base that initiates carboxylation and will determine how substitutions in carboxylase residues cause combined VKD factor deficiency. 2. To determine if turnovers of VKD proteins differ and are impacted by a second site of VKD protein-carboxylase interaction. We will determine whether the VKD proteins factor X and prothrombin are carboxylated with equal efficiencies, and whether having two sites of VKD protein-carboxylase interaction impacts efficiency. 3. To test our hypothesis that post-translational carboxylase modifications are important to VKD protein turnover. Sites of post-translational modification in the carboxylasewill be identified and mutated to determine if they impact VKD protein turnover. These studies will make important contributions to understanding carboxylation, which will be significant for developing superior anticoagulants and for producing VKD proteins for therapies in hemophilia and sepsis. Lav abstract. Vitamin K in the diet is used to activate a set of factors critical to blood clotting, and therefore it is important to understand how they become activated. The studies will impact the development of anticoagulants and the production of therapeutic proteins for treating hemophilia and septic shock.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Hemostasis and Thrombosis Study Section (HT)
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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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