Vitamin K-Dependent Protein Activation: Normal Gamma-Glutamyl Carboxylation and Disruption in Disease

Abstract

Vitamin K-dependent (VKD) proteins undergo an unusual post-translational modification, which is the conversion of specific Glu residues to carboxylated Glu (Gla). Gla generation is required for the activation of VKD proteins, and occurs in the endoplasmic reticulum during their secretion to either the cell surface or from the cell. The gamma-glutamyl carboxylase produces Gla using reduced vitamin K, which becomes oxygenated to vitamin K epoxide. Reduced vitamin K is then regenerated by a vitamin K oxidoreductase (VKORC1), and this interconversion of oxygenated and reduced vitamin K is referred to as the vitamin K cycle. Many of the VKD proteins support hemostasis, which is suppressed during therapy with warfarin that inhibits VKORC1 activity. VKD proteins also impact a broad range of physiologies beyond hemostasis, which includes regulation of calcification, apoptosis, complement, growth control, signal transduction and angiogenesis. The review covers the roles of VKD proteins, how they become activated, and how disruption of carboxylation can lead to disease. VKD proteins contain clusters of Gla residues that form a calcium-binding module important for activity, and carboxylase processivity allows the generation of multiple Glas. The review discusses how impaired carboxylase processivity results in the pseudoxanthoma elasticum-like disease.

Keywords: VKCFD; gamma-glutamyl carboxylase (GGCX); processivity; pseudoxanthoma elasticum-like (PXE-like); vitamin K; vitamin K oxidoreductase (VKORC1); vitamin K-dependent proteins; warfarin.

Figure 4

VKORC1 reduces vitamin K epoxide, and is inhibited by warfarin. (A) VKORC1 reduces vitamin K through an electron (e⁻) relay pathway. Redox protein generates thiols in VKORC1 located in the endoplasmic reticulum, which in turn generate membrane-embedded thiols that reduce vitamin K. (B) VKORC1 reduction of vitamin K epoxide (KO) to the vitamin K hydroquinone (KH₂) carboxylase cofactor occurs in two reactions that involve a vitamin K quinone (K) intermediate. VKORC1 exists as a dimer [75], which can explain how VKORC1 performs both reactions. (C) Sequestration of the K intermediate may increase the efficiency of VKD protein carboxylation [75]. One monomer converts KO to the K intermediate, which is then repositioned for further reduction to KH₂ by the second monomer. Warfarin uncouples the two VKORC1 reactions [76], which alters full reduction by inhibiting the K to KH₂ reaction much more strongly that the KO to K reaction. Full reduction during warfarin therapy may therefore result from a second reductase cooperating with VKORC1 to generate the KH₂ carboxylase cofactor.

Figure 1

Vitamin K-dependent proteins impact multiple physiologies. (A). Many of the vitamin K-dependent proteins are essential for blood clotting, either supporting (procoagulant) or attenuating (anticoagulant) hemostasis. (B). Msost of the coagulation factors, as well as Gas6, signal through various receptors on the cell surface. Some examples of receptors are tissue factor, platelet activated receptors (PARS) and receptor tyrosine kinases (RTKs), which mediate multiple physiologies. The scheme shows zymogen nomenclatures for simplicity; however, the function of VKD coagulation factors requires activation (e.g., of prothrombin to thrombin). (C,D). VKD proteins that have roles beyond hemostasis have also been identified. Some regulate calcification (C), while others have functions that remain to be determined (D). TMG stands for Transmembrane Gla Protein, a family of proline-rich proteins that are also referred to as PRGP.

Figure 2

Carboxylation of multiple glutamyl residues generates a calcium-binding module required for vitamin K-dependent protein activities. (A) Vitamin K-dependent (VKD) protein carboxylation occurs in the endoplasmic reticulum, where vitamin K (vit K) cycles between reduced vitamin K generated by a vitamin K epoxide reductase (VKORC1) and oxidized vitamin K produced by the carboxylase. VKORC1 becomes inactivated during vitamin K reduction, and activity is regenerated through electron (e⁻) flow from a redox protein. VKD proteins are targeted for carboxylation because they contain an exosite binding domain (EBD) that mediates binding to the carboxylase. Multiple Glu residues are carboxylated (arrowheads) to Glas (red Ys). Following carboxylation and release from the carboxylase, the VKD proteins exit the endoplasmic reticulum (ER) and traffic to the Golgi where additional post-translational modifications occur. Further secretion localizes VKD proteins in blood or extracellular matrix or the cell surface. (B) The carboxylase uses the epoxidation of vitamin K hydroquinone (KH₂) to vitamin K epoxide (KO) to replace a hydrogen in glutamic acid (Glu) residues by CO₂, forming carboxylated Glu (Gla). KO is then recycled by VKORC1, first to a quinone intermediate (K) and then to vitamin K hydroquinone (KH₂). (C) Multiple Gla residues (red Ys) coordinate several calcium ions to transform the Gla domain into a highly organized structure.

Figure 3

Quality control impacts the secretion of vitamin K-dependent proteins. (A) Newly-synthesized proteins undergo maturation that is mediated by chaperones and involves protein folding and, in some cases, disulfide bond formation and the assembly of complexes. Quality control mechanisms distinguish mature versus immature proteins, which are processed differently. Mature proteins exit the endoplasmic reticulum and traffic to the Golgi and beyond, while immature proteins are targeted for degradation through the ERAD pathway. (B) Vitamin K-dependent (VKD) protein maturation also includes Glu carboxylation to Gla that occurs in the endoplasmic reticulum. Cellular studies have shown that the fate of carboxylated and uncarboxylated proteins varies among individual VKD proteins. Specifically, both factor IX forms are secreted from cells, while only the carboxylated form is secreted in the case of protein C and protein Z.

Figure 5

Carboxylase processivity regulates vitamin K-dependent carboxylation. (A) To study carboxylase processivity, a complex between the carboxylase and a vitamin K-dependent (VKD) protein is incubated in the presence or absence of a distinguishable VKD protein, and carboxylation of both VKD forms is monitored [16]. The VKD protein in the study was full length factor IX (fIX) attached to the propeptide that mediates complex formation. The challenge protein was the propeptide-containing fIX light chain that has the entire Gla domain. (B) Carboxylation of fIX in the complex is the same in the presence or absence of the challenge protein. (C) Carboxylation of the challenge protein occurs after carboxylation of fIX in the complex. (D) Wild type carboxylase blocks the access of challenge protein during the carboxylation of VKD protein in the complex [16]. (E) Carboxylase processivity depends upon the relative rates of catalysis versus release. The time to fully carboxylate the Gla domain is 5–6 fold faster than release with wild type carboxylase [15]. A mutant carboxylase with impaired processivity shows similar rates of catalysis and release and generates partially carboxylated VKD protein [15]. Panels B and C are adapted with permission from Stenina et al. [16], copyright 2001 American Chemical Society.