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Article

The Vitamin K–Dependent Carboxylase¹

Department of Molecular Cardiology/NB50, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195

	ABSTRACT

TOP ABSTRACT INTRODUCTION VKD Protein/Carboxylase... Isolation and Characterization... How Are VKD Proteins... REFERENCES

 
The carboxylase is an integral membrane glycoprotein that uses vitamin K to modify clusters of glutamyl residues (glu’s) to -carboxylated glutamyl residues (gla’s) post-translationally in vitamin K–dependent (VKD) proteins as they pass through the endoplasmic reticulum. Carboxylation is required for VKD protein functions in hemostasis, bone metabolism, growth control and signal transduction. Carboxylation of multiple glu residues is accomplished via a processive mechanism, which occurs with at least some order and involves carboxylation of the carboxylase. The carboxylase has a high affinity binding site for VKD proteins, which in most cases is a VKD propeptide sequence; it also appears to have a low affinity site for those glu’s undergoing catalysis. The propeptide activates binding of the glu’s; together, the two contact points between the carboxylase and VKD protein increase the affinity of the carboxylase for vitamin K. Biochemical mapping to identify where these events occur in the carboxylase remains a challenge, despite the availability of recombinant protein. The affinity of the carboxylase for the propeptide of several VKD proteins that are coexpressed in liver varies over a 100-fold range. Treatment with anticoagulants such as warfarin that indirectly block carboxylation likely decreases the rate of VKD protein catalysis and increases the accumulation of VKD precursors, leading to a competitive state among these proteins, which results in the premature dissociation of undercarboxylated, inactive protein.

KEY WORDS: • vitamin K • vitamin K–dependent carboxylase

	INTRODUCTION

TOP ABSTRACT INTRODUCTION VKD Protein/Carboxylase... Isolation and Characterization... How Are VKD Proteins... REFERENCES

 
Theonly recognized function for vitamin K (vit K)² in higher organisms is as a cofactor for an enzymatic reaction that converts glutamyl residues (glu’s) to -carboxylated glutamyl residues (gla’s) in a class of proteins referred to as vitamin K–dependent (VKD) proteins. These proteins are modified by the VKD- or -carboxylase as they are secreted through the endoplasmic reticulum (ER). A model has been proposed in which a weak base (cys) generates a strong base (e.g., vit K alkoxide) that is sufficient for the stereospecific abstraction of a hydrogen ion from the -glutamyl position (1) . Subsequent addition of CO₂ to the carbanion intermediate produces the gla residue. Each cycle of glu to gla conversion results in the oxidation of vit K hydroquinone (KH₂) to vit K epoxide, and the carboxylase is also an epoxidase. Carboxylation, which is required for the biological activity of VKD proteins, occurs at multiple residues [3-12] within a region called the gla domain. Carboxylation results in Ca⁺⁺-binding and a conformational change in the gla domain, with consequent insertion of hydrophobic residues within this domain into phospholipid bilayers in which VKD proteins exert their biological effects (2) .

The VKD proteins presently comprise a family of ~12 proteins. The first such proteins identified were ones involved in hemostasis, including prothrombin (PT), factor IX (fIX), factor VII (fVII), factor X (fX), protein C (PC) and protein S (PS). Subsequent identification of other VKD proteins has shown that carboxylation is important to a broader range of biological functions, including bone morphogenesis [bone gla protein (BGP) and matrix gla protein (MGP)] and growth control [(gas)6] (3 ,4) . VKD proteins with potential functions in signal transduction [proline-rich gla protein (PRGP)-1 and PRGP-2] (5) and a VKD protein of unknown function (protein Z) have also been discovered. Finally, the carboxylase itself has been shown to be a VKD protein (6) . This observation was a surprise because the carboxylase does not share any homology with the other VKD proteins. It is very likely that other VKD proteins remain to be discovered. Tissue distribution of the carboxylase is widespread, thus accommodating such a possibility.

In vivo carboxylation of VKD proteins requires the continual recycling of vit K epoxide to KH₂. Epoxide reduction is accomplished by a reductase that has been proposed to exist as a multisubunit complex with the carboxylase (7) . This enzyme is inhibited by 4-OH coumarin analogs (e.g., warfarin), leading to the rapid depletion of the KH₂ supply. This extinction results in the accumulation of intracellular VKD precursors and, depending on the organism and the dose and duration of warfarin, to the secretion of undercarboxylated VKD proteins in vivo. Because of the requirement of several of the VKD proteins for normal hemostasis, these coumarins form the basis for anticoagulant therapy. Such therapy was developed initially in the absence of knowledge about VKD proteins required for biological processes other than hemostasis. Thus, long-term coumarin therapy has broader physiologic consequences than originally envisioned.

	VKD Protein/Carboxylase Interaction.

TOP ABSTRACT INTRODUCTION VKD Protein/Carboxylase... Isolation and Characterization... How Are VKD Proteins... REFERENCES

 
All VKD proteins have in common a homologous ~18 amino acid sequence; in most cases, this sequence is a propeptide juxtaposed immediately upstream of the gla domain, which targets the VKD protein to the carboxylase (8) . Three of the propeptide residues (phe at -16, ala at -10 and leu at -6) are highly conserved, and mutations at these positions decrease carboxylase affinity for the VKD proteins. However, other residues are clearly important to binding. In a recent study that included the analysis of six hemostatic VKD propeptides, all possessing the three invariant residues, a 100-fold range in carboxylase affinities was observed (9) .

In addition to its docking function, the propeptide is also an allosteric activator (10) . Thus, incubation of a free propeptide sequence with a peptide derived from the gla domain (Fig. 1A ) effects an increase in affinity for the gla domain peptide, but not for any of the cofactors (KH₂, O₂, CO₂). Covalent attachment of the two sequences causes a dramatic decrease in K_m for the gla domain, from the millimolar range into the low- or submicromolar range. Most likely, propeptide tethering increases the local concentration of the gla domain with respect to the carboxylase, and the K_m for carboxylation is governed by the propeptide affinity.

View larger version (18K): [in this window] [in a new window]   Figure 1. The carboxylase active site. Most vitamin K–dependent (VKD) proteins bind to the carboxylase through the propeptide (PRO), and there may also be a second point of contact for the glutamyl (glu; E) residues, as illustrated by the pocket. Covalent attachment of the propeptide and gla domain increases the affinity of KH2 binding, as contrasted in A vs. B. As shown in B and C, multiple carboxylations are accomplished via "tethered processivity," in which the propeptide remains bound throughout the reaction, while the gla domain undergoes intramolecular movement to reposition the glu’s for conversion to gla’s (). During the reaction, the carboxylase is also carboxylated.

Studies with BGP carboxylation support the concept of a glu-binding site. The carboxylase has high affinity for the BGP gla domain (Km = 0.2 µmol/L), at least 850-fold higher than its affinity for the fIX gla domain (14 ,15) . In contrast, the BGP propeptide exhibits poor binding, orders of magnitude less than that observed with other VKD propeptides. The high affinity for the BGP gla domain strongly suggests a binding site for the gla domain of VKD proteins in the carboxylase (Fig. 1) . All VKD proteins, then, appear to bind to the carboxylase through low affinity and high affinity sites, with the BGP propeptide and gla domain exhibiting reversed affinity to that of other VKD proteins.

	Isolation and Characterization of Recombinant (r)-Carboxylase.

TOP ABSTRACT INTRODUCTION VKD Protein/Carboxylase... Isolation and Characterization... How Are VKD Proteins... REFERENCES

 
Identification of the propeptide as a high affinity ligand for the carboxylase led to its use in purifying the enzyme and cDNA (16 17 18) . The human carboxylase cDNA predicts a unique 758 amino acid protein. An integral membrane protein is predicted; however, the topology of the carboxylase in the membrane is unknown, with different algorithms predicting three or five membrane-spanning sequences in the N-terminus. On the basis of the distribution of predicted glycosylation sites and the large discrepancy between observed (95 kDa) and predicted (78 kDa) molecular weight, the C-terminal half of the carboxylase must be localized in the ER lumen.

Despite the availability of r-carboxylase, the amount of structural information that has been acquired is limited. Given the predicted topologies, a secreted form is probably not obtainable; thus large quantities of protein are still difficult to generate. In addition, the requirement for isolating the carboxylase in a micelle complicates microsequence analysis. To date, mapping has been low in resolution and there have been no reports that biochemically identify functionally relevant amino acids. Binding of a suicide substrate was mapped to the first third of the carboxylase (19) . Propeptide binding has been mapped both to amino acids 50–225 by one group (20) and to amino acids 349–500 by another (21) . Clearly, a complete understanding of carboxylase structure and function requires the development of techniques that circumvent the problems with mapping residues.

	How Are VKD Proteins Multiply Carboxylated?

TOP ABSTRACT INTRODUCTION VKD Protein/Carboxylase... Isolation and Characterization... How Are VKD Proteins... REFERENCES

 
Secreted VKD proteins are normally fully carboxylated, indicating that some mechanism exists to ensure comprehensive carboxylation. Two very different mechanisms could account for full carboxylation. In the first instance, a quality control process could prevent the secretion of uncarboxylated forms. Evidence for cellular regulation exists for rat PT and human PC, in which undercarboxylated forms have been shown to be rapidly degraded (22 ,23) . Rat PT is secreted only in fully carboxylated form. In contrast, undercarboxylated human PT can be secreted, and this specificity is due to sequence differences in the kringle region (24) .

In the second instance, full carboxylation could be achieved if the carboxylase is processive, i.e., effecting all modifications as a consequence of a single binding event. Carboxylase processivity has been assessed by analyzing the products of an in vitro reaction between a fIX peptide and the carboxylase (25) . A small population of fully carboxylated peptide was detected among a large population of undercarboxylated intermediates, and a probability argument that only processive carboxylation would yield fully carboxylated fIX was used to conclude that the carboxylase is processive. The limited amount of fully carboxylated product is interesting, raising the following questions: Is a cofactor required? Do all VKD proteins demonstrate equal processivity?

Our approach to assess processivity was to develop a direct test in which the carboxylation of a fIX-carboxylase complex was challenged with a distinguishable fIX substrate. We found that carboxylation among individual fIX-carboxylase complexes was nonsynchronous and fully processive, resulting in the comprehensive carboxylation of fIX before the onset of carboxylation of the challenge substrate (Berkner et al., unpublished data). Thus, the VKD protein remains tethered to the carboxylase via the propeptide throughout the reaction, with gla domain intramolecular movement repositioning the glu’s for catalysis (Fig. 1) . Interestingly, the rate of fIX carboxylation was linear over most of the reaction, which indicates a driving force. The carboxylase must therefore position glu’s preferentially for catalysis and hence must distinguish a glu from a gla. Differentiation, e.g., by charge or size, could be accomplished by the proposed glu-binding site.

The concept of a carboxylase site that binds a glu more strongly than a gla is attractive in its ability to explain how the carboxylase modifies the gla domain comprehensively. Processive carboxylation means that the carboxylase does not release a VKD protein until it is fully modified; thus it is able to distinguish "fully" from "partially" modified forms. How this distinction is accomplished is unknown. Full carboxylation would result in the loss of glu binding (Fig. 1C ). This change could effect consequent release of the VKD protein, e.g., due to a conformational change that weakens propeptide binding or due to competition by glu-containing substrates.

Whether the gla domain itself contributes to efficient carboxylation is unknown. Most of the VKD proteins have a highly homologous gla domain, but conservation of structure could be due to a commonality required for biological function rather than carboxylation. Some mutations in the gla domain of r-PC result in the secretion of undercarboxylated protein (26) , suggesting that the gla domain is important to carboxylation. However, in another study, a r-PT truncation with the gla domain deleted and the propeptide fused to the serine protease domain was secreted as extensively carboxylated protein (27) . Interpretation of these and other studies of secreted VKD proteins is complicated because the ER quality control system may allow only a subset of the intracellular population to be secreted. In an in vitro study, carboxylation of a substrate with the propeptide attached to a random glu-containing sequence resulted in a large proportion of highly carboxylated (i.e., 4 of 5) glu’s (15) . Thus, the propeptide confers upon a substrate the ability to be multiply modified. However, whether the gla domain contributes to more comprehensive and/or efficient carboxylation remains to be determined.

An in vitro study with BGP indicates that there is some order to the carboxylation of multiple glu’s (14) . Analysis of the reaction intermediates of BGP, which has only three gla residues per molecule, indicated a preferential order of carboxylation, from the C- to the N-terminus. Given the uniqueness of BGP among the VKD proteins, it will be of interest to determine whether the pattern of carboxylation is the same with other VKD proteins.

Intracellular processing of VKD proteins.

How VKD proteins are bound by the carboxylase in the ER, then modified and released, is not well defined (Fig. 2 ). VKD protein-carboxylase association is likely mediated by chaperones, as observed with other multisubunit systems. After carboxylation and release, the VKD protein transits through the Golgi, where propeptide processing occurs. Processing is required for the Ca⁺⁺-dependent conformational change in the VKD gla domains that results in subsequent membrane binding (28) .

View larger version (20K): [in this window] [in a new window]   Figure 2. Carboxylation and secretion of vitamin K–dependent (VKD) proteins. Normally, VKD proteins undergo comprehensive carboxylation [k1–3 in this example for a VKD protein with 3 -carboxylated glutamyl (gla) residues], release (k4) and subsequent propeptide processing in the Golgi. Decarboxylation (i.e., k-1 to k-3) is unlikely to be physiologically relevant. Warfarin effects premature release (k5–7) of undercarboxylated forms. This effect could be due to a decrease in k1–3 as well as a build-up of VKD precursors that accelerate release. Most VKD proteins also undergo other post-translational modifications, including N-glycosylation, O-glycosylation, ß-aspartyl hydroxylation and proteolytic cleavage at internal sites within the mature protein.

Carboxylation of multiple VKD proteins occurs in at least two tissues. Liver synthesizes several VKD proteins (fVII, fIX, fX, PT, PC, PZ, PS and MGP) and osteoblasts express BGP and MGP during healing (31) . Tissue distribution of the carboxylase and of several VKD proteins (gas6, MGP, PRGP-1, PRGP-2) has been shown to be broad (4 ,5 ,32 ,33) . In addition, recent studies have demonstrated other synthetic sites for the VKD clotting factors (e.g., muscle for PT) (34) . Consequently, there may be other tissues that coexpress different VKD proteins.

Carboxylation of multiple VKD proteins in the same tissue suggests the need for regulation to ensure full carboxylation of individual proteins. Given the wide range of VKD propeptide affinities, binding and release of fully carboxylated protein vs. premature release of undercarboxylated protein could differ considerably among the VKD proteins. In vivo studies support the concept that the carboxylase does not process all VKD proteins equally, i.e., analysis of warfarin-treated rat cells expressing PT either alone or with fX suggested that fX outcompetes PT for the carboxylase (35) . This interpretation was subsequently supported by in vitro experiments showing a 100-fold difference between fX and PT propeptide affinities (9) .

Recent characterization of a hemophiliac highlights the importance of propeptide affinity in the hemostatic process (36) . The patient normally exhibited wild-type clotting, but responded to anticoagulation with a selective decrease in fIX activity. An ala to gly mutation at the highly conserved -10 position in the propeptide was identified; a peptide bearing this mutation exhibited a 30-fold decrease in propeptide affinity, but only a twofold decrease in KH₂ binding. To explain the puzzling observation that fIX showed a decrease in activity only during oral anticoagulation, the authors proposed that when KH₂ is limiting, the carboxylation rate slows down and thus the ratio of dissociation rate to catalytic rate (k_5–7 vs. k_1–3 in Fig. 2 ) increases. Thus, fIX would selectively respond to KH₂ depletion due to its lowered affinity.

An additional possibility is that the decreased availability of KH₂ also changes the normal balance between the VKD precursor pool and carboxylase, causing the premature dissociation (k_5–7, Fig. 2 ) of substrates by other VKD proteins with stronger affinities. Interestingly, PT carboxylation is also disrupted only upon anticoagulation, and the PT propeptide has an affinity that is much lower than that of other hepatic VKD propeptides and not very different from that of the fIX mutant (the PT propeptide affinity is 7-fold less than the fIX propeptide (9) , and the fIX mutant had a 30-fold lower affinity than native fIX (36) ). Under normal conditions, the levels of VKD proteins and carboxylase may be regulated to ensure complete carboxylation. Consequently, efficient carboxylation of VKD proteins with large differences in affinities occurs because a competitive state does not exist. When KH₂ is limiting, decreased VKD protein turnover traps the carboxylase in the complex, and this block results in the accumulation of VKD precursors. Competition between VKD precursors with varying carboxylase affinities can then lead to the displacement of lower affinity substrates such as PT or the fIX mutant.

A disruption in the normal equilibrium between VKD precursors and carboxylase may account for the poor carboxylation that often confounds mammalian expression of r-VKD proteins. Cell lines expressing increased levels of r-VKD proteins become saturated for carboxylation. Cognate cell lines expressing low or high amounts of r-VKD protein have the same amount of carboxylase and vit K; however, there is an increase in the ratio of VKD precursors to carboxylase, which likely compromises the normal secretory process as a result of competition that effects premature VKD protein dissociation. In addition, if k_cat in mammalian cells is slow, e.g., due to lack of a vit K reductase that efficiently recycles vit K epoxide, then VKD precursor build-up will compound the problem.

The fact that full carboxylation occurs under normal physiologic conditions is remarkable, and determining what mechanisms effect this process is central to understanding VKD protein carboxylation. For example, is the catalytic rate the same for all VKD proteins or do lower affinity proteins such as PT have a higher k_cat? Determining whether there is a control mechanism that regulates the amount of VKD protein available to the carboxylase, and whether other intracellular proteins participate in the process, will also be important in defining the overall process of carboxylation.

	FOOTNOTES

 
¹ Manuscript received 10 April 2000.

² Abbreviations used: BGP, bone gla protein; ER, endoplasmic reticulum; fIX, factor IX; fVII, factor VII; fX, factor X; gas, growth arrest specific; gla, -carboxylated glutamyl residues; glu, glutamyl residues; KH₂, vitamin K hydroquinone; MGP, matrix gla protein; PC, protein C; PRGP, proline-rich gla protein; PS, protein S; PT, prothrombin; PZ, protein Z; r-, recombinant-; vit K, vitamin K; VKD, vitamin K dependent.

	REFERENCES

TOP ABSTRACT INTRODUCTION VKD Protein/Carboxylase... Isolation and Characterization... How Are VKD Proteins... REFERENCES

 

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