Darrel W. Stafford

Identification of the gene for vitamin K epoxide reductase

Vitamin K epoxide reductase (VKOR) is the target of warfarin, the most widely prescribed anticoagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-used because of the difficulty of controlling dosage and the fear of inducing bleeding. Although identified in 1974 (ref. 2), the enzyme has yet to be purified or its gene identified. A positional cloning approach has become possible after the mapping of warfarin resistance to rat chromosome 1 (ref. 3) and of vitamin K-dependent protein deficiencies to the syntenic region of human chromosome 16 (ref. 4). Localization of VKOR to 190 genes within human chromosome 16p12-q21 narrowed the search to 13 genes encoding candidate transmembrane proteins, and we used short interfering RNA (siRNA) pools against individual genes to test their ability to inhibit VKOR activity in human cells. Here, we report the identification of the gene for VKOR based on specific inhibition of VKOR activity by a single siRNA pool. We confirmed that MGC11276 messenger RNA encodes VKOR through its expression in insect cells and sensitivity to warfarin. The expressed enzyme is 163 amino acids long, with at least one transmembrane domain. Identification of the VKOR gene extends our understanding of blood clotting, and should facilitate development of new anticoagulant drugs.

Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

The vitamin K cycle.

Summary.  Post‐translational modification of glutamate to gamma carboxyl glutamate is required for the activity of vitamin K‐dependent proteins. Carboxylation is accomplished by the enzyme gamma glutamyl carboxylase (GGCX) which requires the propeptide‐containing substrate and three co‐substrates: reduced vitamin K, CO2, and O2. Most propeptides bind tightly to GGCX and all of the Glu residues that will be modified are modified during one binding event. Complete carboxylation is thus dependent upon the rate of carboxylation and the dissociation rate constant of the substrate from the GGCX enzyme. If the propeptide is released before carboxylation is complete, partially carboxylated vitamin K‐dependent proteins are produced. The rate of carboxylation is mainly controlled by the level of reduced vitamin K available for the reactions while the dissociation rate constant is dependent upon both the propeptide and the Gla domain of the substrate. In addition, there are allosteric effects that increase the rate of dissociation of the fully carboxylated substrates. Carboxylation requires the abstraction of a proton from the 4‐carbon of glutamate by reduced vitamin K and results in the conversion of vitamin K to vitamin K epoxide. The vitamin K epoxide must be recycled to vitamin K before it can be reused, a reaction catalyzed by the enzyme vitamin K epoxide reductase (VKOR). The gene for VKOR has recently been identified but the enzyme itself has not been purified to homogeneity. It appears, however, that most of the variability observed in patients response to warfarin may be attributed to variability in the VKOR gene.

Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/cotranslocation

Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10–29, 101–123, and 127–149, and membrane-integration of residues 75–97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM.

A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism.

The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme gamma-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of > 100% off warfarin and < 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine-->adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue -10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution based on the sequence in bone gamma-carboxyglutamic acid protein. Measurement of C02 incorporation at a range of peptide concentrations yielded a Vmax of 343 cpm/min/reaction for the wild-type peptide, and Vmax values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The Km values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 microM for wild-type, and 10.9 and 9.50 microM, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-l0-->Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins.

Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.

The study of membrane protein structure and enzymology has traditionally been hampered by the inherent insolubility of membrane proteins in aqueous environments and experimental challenges in emulating an in vivo lipid environment. Phospholipid bilayer nanodiscs have recently been shown to be of great use for the study of membrane proteins since they offer a controllable, stable, and monodisperse model membrane with a nativelike lipid bilayer. Here we report the integration of nanodiscs with hydrogen exchange (HX) mass spectrometry (MS) experiments, thereby allowing for analysis of the native conformation of membrane proteins. gamma-Glutamyl carboxylase (GGCX), an approximately 94 kDa transmembrane protein, was inserted into nanodiscs and labeled with deuterium oxide under native conditions. Analytical parameters including sample-handling and chromatographic separation were optimized to measure the incorporation of deuterium into GGCX. Coupling nanodisc technology with HX MS offers an effective approach for investigating the conformation and dynamics of membrane proteins in their native environment and is therefore capable of providing much needed insight into the function of membrane proteins.

Changing Residue 338 in Human Factor IX from Arginine to Alanine Causes an Increase in Catalytic Activity

This study was designed to identify functionally important factor IX (FIX) residues. Using recombinant techniques and cell culture, we produced a mutant FIX with arginine at 338 changed to alanine (R338A-FIX). This molecule had approximately 3 times greater clotting activity than that of wild type FIX (wt-FIX) in the activated partial thromboplastin assay. R338A-FIX reacted normally with a panel of three FIX specific monoclonal antibodies and migrated on sodium dodecyl sulfate-polyacrylamide gels indistinguishably from wt-FIX. Using functional assays, we determined that R338A-FIXa’sK d for factor VIIIa (FVIIIa) was similar to that of wt-FIXa. Our kinetic analysis, using factor X as substrate, indicated that the mutation’s major effects were a 3-fold increase ink cat and a 2-fold decrease inK m both manifested only in the presence of FVIIIa. R338A-FIXa’s increased catalytic efficiency did not result from ablation of a thrombin sensitive site, reported to occur at arginine 338, since in our assays the thrombin inhibitor, hirudin, had no effect on activity of either wt-FIXa or R338A-FIXa. R338A-FIXa and wt-FIXa had equal activity, with or without FVIIIa, toward the synthetic substrate, methylsulfonyl-d-cyclohexylglycyl-arginine-p-nitroanilide. Interestingly, R338A-FIXa had reduced affinity for heparin. Therefore, we propose that R338A-FIXa’s increased activity is not due to an allosteric effect on the active site, but that the Arg-338 residue is part of an exosite that binds both factor X and the mucopolysaccharide, heparin.

The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase.

The vitamin K-dependent γ-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (K i ) toward a factor IX propeptide/γ-carboxyglutamic acid domain substrate. TheK i values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2–35 nm, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are ∼100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin K-dependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.

Purified vitamin K epoxide reductase alone is sufficient for conversion of vitamin K epoxide to vitamin K and vitamin K to vitamin KH2

More than 21 million prescriptions for warfarin are written yearly in the U.S. Despite its importance, warfarins target, vitamin K epoxide reductase (VKOR), has resisted purification since its identification in 1972. Here, we report its purification and reconstitution. HPC4, a calcium-specific antibody that recognizes a 12-aa tag, was used to purify and identify VKOR. Partial reconstitution is achieved on the column by washing with 0.4% dioleoylphosphatidylcholine/0.4% deoxycholate. Activity is completely recovered by dialysis against a buffer containing a reducing agent but lacking dioleoylphosphatidylcholine/deoxycholate. Removal of detergent from the eluted proteins apparently facilitates liposome formation. Purified recombinant VKOR with tag is ≈21 kDa, as expected; fully active; and >93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported that VKOR is a multisubunit enzyme. Our results, however, suggest that a single peptide can accomplish both the conversion of vitamin K epoxide to vitamin K and vitamin K to reduced vitamin K. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.

Concentration of DNA solutions by extraction with 2-butanol

Abstract A simple, efficient way to concentrate dilute solutions of DNA is given. This procedure is rapid, the DNA is quantitatively recovered, and there is no change in the single- or double-stranded molecular weight of the DNA.