The cDNA for human gamma-glutamyl carboxylase, which accomplishes the post-translational modification required for the activity of all of the vitamin K-dependent proteins, was cloned. The enzyme is a 758-residue integral membrane protein and appears to have three transmembrane domains near its amino terminus. The hydrophilic COOH-terminal half of the carboxylase has 19.3 percent identity with soybean seed lipoxygenase. Expression of the cloned cDNA resulted in an increase in carboxylase activity in microsomes of transfected cells compared to mock-transfected cells.
The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme ␥ -glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH 2 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 Ͼ 100% off warfarin and Ͻ 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 Ϫ 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 ␥ -carboxyglutamic acid protein. Measurement of CO 2 incorporation at a range of peptide concentrations yielded a V max of 343 cpm/min/reaction for the wild-type peptide, and V max values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The K m values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 M for wild-type, and 10.9 and 9.50 M, 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-10 → 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. ( J. Clin. Invest. 1996. 98:1619-1625.)
To identify potential mutations in the γ-glutamyl carboxylase gene, the sequence of all exons and intron/exon borders was determined in 4 patients from a consanguineous kindred with combined deficiency of all vitamin K-dependent procoagulants and anticoagulants and results were compared with normal genomic sequence. All 4 patients were homozygous for a point mutation in exon 9 that resulted in the conversion of an arginine codon (CTG) to leucine codon (CGG) at residue 394. Screening of this mutation based on introduction of Alu I site in amplified fragment from normal allele but not from the mutated allele showed that 13 asymptomatic members of the kindred were heterozygous for the mutation. The mutation was not found in 340 unrelated normal chromosomes. The segregation pattern of the mutation which is the first reported in the γ-glutamyl carboxylase gene fits perfectly with phenotype of the disorder and confirms the suggested autosomal recessive pattern of inheritance of combined deficiency of all vitamin K-dependent procoagulants and anticoagulants in this kindred. The mutated carboxylase protein expressed in Drosophila cells was stable but demonstrated threefold reduced activity compared with WT carboxylase, confirming that the L394R mutation results in a defective carboxylase.
Vitamin K-dependent carboxylase catalyzes the modification of specific glutamic acids to y-carboxyglutamic acid in several blood-coagulation proteins. This modification is required for the blood-clotting activity of these proteins and has thus been the subject of intense investigation. We have now identified the bovine vitamin K-dependent carboxylase and purified it to near homogeneity by an affinity procedure that uses the 59-amino acid peptide FIXQ/S (residues -18 to 41 of factor IX with mutations Arg --Gin at residue -4 and Arg -+ Ser at residue -1). The carboxylase as purified has a molecular weight of 94,000. It is also the major protein that can be cross-linked to iodinated FIXQ/S and is the only protein whose cross-linking is prevented by a synthetic factor IX propeptide. The degree of purification is about 7000-fold with reference to ammonium sulfate-fractionated microsomal protein from liver.A number of blood coagulation proteins require a posttranslational vitamin K-dependent modification for biological activity. Stenflo et al. (3) have reported that prothrombin, the prototype of these vitamin K-dependent proteins, contained the modified amino acid y-carboxyglutamic acid (Gla). Prothrombin from animals treated with the vitamin K antagonist warfarin lacked this Gla modification. It was inferred from these observations that the blood-clotting activity of the vitamin K-dependent proteins required y-carboxylation of specific glutamic acid residues. Shortly thereafter, Esmon et al. (4) demonstrated an enzyme activity, vitamin K-dependent carboxylase (hereafter called carboxylase), capable of making this Gla modification.After cDNA sequences were obtained for several of the vitamin K-dependent proteins, Pan and Price (5) compared the deduced amino acid sequences and suggested that the propeptide consensus sequence preceding the amino terminus of the vitamin K-dependent protein was a recognition site for the carboxylase. This suggestion was confirmed by Knobloch and Suttie (6), who demonstrated the importance ofthe propeptide in carboxylation by showing that the synthetic propeptide sequence of human factor X stimulated the activity of the carboxylase for a small substrate (Boc-Glu-GluLeu-OMe) in vitro. Jorgensen et al. (7) extended this observation by showing that factor IX with its propeptide deleted was not carboxylated.In spite of its importance, the carboxylase has not been previously purified. Purification of 400-fold was reported by Girardot (8 that an immobilized factor X antibody would bind the carboxylase, presumably through a factor X precursorcarboxylase complex, and that the bound carboxylase retained its activity for the synthetic peptide substrate FLEEL. Harbeck et al. (10) extended this method by eluting the carboxylase from a prothrombin antibody column with a synthetic propeptide achieving a 500-fold purification and a final specific activity of6.6 x 106 cpm per mg per hr. Hubbard et al. (11) reported the purification of the carboxylase to homogeneity using a synthetic propeptide ...
The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the processive carboxylation of specific glutamates in a number of proteins related to blood coagulation and bone. To address the independent importance of the propeptide, gamma-carboxyglutamic acid (Gla) domain and elements beyond the Gla domain of factor IX in vitamin K-dependent carboxylation, we have examined the kinetics of carboxylation of peptides containing (1) propeptide and Gla domain, (2) the Gla domain alone, (3) uncarboxylated bone Gla protein, (4) propeptide followed by the entire uncarboxylated factor IX molecule, and (5) the factor IX propeptide followed by a non-Gla domain sequence. Our studies indicate that peptides with a covalently linked propeptide have Km values similar to the physiological substrate of the carboxylase. In contrast, the Gla domain of factor IX has a >/=230-fold higher Km for the carboxylase than the corresponding peptide with a covalently linked propeptide. This contrasts with bone Gla protein, another vitamin K-dependent protein, which appears not to require a covalently linked propeptide for high-affinity binding to the carboxylase. Analysis of the carboxylation products of a propeptide/non-Gla domain substrate indicate that it is carboxylated multiple times in a processive manner. These studies show that the perceived binding affinity of the carboxylase substrate and processivity is conferred by the propeptide without requiring the conserved Gla domain sequences and that factor IX and bone Gla protein may have distinct mechanisms of interacting with the carboxylase.
␥-Glutamyl carboxylase is an integral membrane protein required for the posttranslational modification of vitamin K-dependent proteins. The main recognition between the enzyme and its substrates is through an 18-amino acid propeptide. It has been reported that this binding site resides in the amino-terminal third of the ␥-glutamyl carboxylase molecule (Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie, B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry 34, 481-489). In contrast, we found the binding site in the carboxyl half of the ␥-glutamyl carboxylase. We show that the carboxylase may be cleaved by trypsin into an amino-terminal 30-kDa and a carboxyl-terminal 60-kDa fragment joined by a disulfide bond(s), and the propeptide binds to the 60-kDa fragment. The sequence of the amino terminus of the 60-kDa fragment reveals that the primary trypsin-sensitive sites are at residues 349 and 351. Furthermore, the tryptic fragment that cross-links to the propeptide also reacts with an antibody specific to the carboxyl portion of the ␥-glutamyl carboxylase. In addition, cyanogen bromide cleavage of bovine ␥-glutamyl carboxylase cross-linked to the peptide comprising residues TVFLDHENANKILNRPKRY of human factor IX yields a cross-linked fragment of 16 kDa from the carboxyl half of the molecule, the amino-terminal sequence of which begins at residue 438. Thus, the propeptide binding site lies carboxyl-terminal to residue 438 and is predicted to be in the lumen of the endoplasmic reticulum.␥-Glutamyl carboxylation, accomplished by the enzyme ␥-glutamyl carboxylase, is a posttranslational modification essential for the biological activities of a number of vitamin K-dependent proteins. The importance of ␥-glutamyl carboxylation is demonstrated by the various functions of the vitamin K-dependent proteins. The best characterized functions are related to coagulation and are exemplified by proteins such as prothrombin, factor VII, factor IX, factor X, protein C, and protein S. In addition, there are two bone proteins, osteocalcin and matrix Gla protein (1), and the newly discovered growth arrest-specific protein Gas 6 (2).The ␥-glutamyl carboxylation reaction occurs in the endoplasmic reticulum (3-5), where the enzyme ␥-glutamyl carboxylase uses the small substrates carbon dioxide, oxygen, and vitamin K hydroquinone to convert specific glutamic acid residues of the vitamin K-dependent protein into the ␥-carboxyl glutamic acid Gla. During the process of ␥-glutamyl carboxylation, the vitamin K hydroquinone is converted to vitamin K epoxide, which must be recycled to vitamin K hydroquinone by the enzyme epoxide reductase for the reaction to continue.The enzymatic activity of the ␥-glutamyl carboxylase was first discovered in the postmitochondrial supernatant of hepatocytes (6). Numerous early studies used microsomes as the crude enzyme sources and the pentapeptide FLEEL as the in vitro substrate to establish a basic understanding of ␥-glutamyl carboxylation (7). By comparing the cDNA-deduced amino acid sequences of di...
The human gene for γ-glutamyl carboxylase is 13 kb in length and contains 15 exons. Transcription starts at a cytosine 217 base pair upstream of the first codon. There are two major transcripts in all tissues examined. They are distinguished by the presence of an Alu sequence in the 3′ nontranslated end of the longer species. Relative mRNA levels for 12 bovine tissues are presented.
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