Effect of propeptide mutations on post-translational processing of factor IX. Evidence that beta-hydroxylation and gamma-carboxylation are independent events

Post-translational processing of Factor IX includes glycosylation, cleavage of the signal peptide and propeptide, vitamin K-dependent carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acid, and beta-hydroxylation of aspartic acid at residue 64 to form beta-hydroxyaspartic acid. The human Factor IX cDNA coding sequence was modified in the propeptide region (residue -18 to -1) using oligonucleotide-directed site-specific mutagenesis, and the altered Factor IX cDNA was expressed in Chinese hamster ovary cells. The effects of the mutations on proteolytic processing, gamma-carboxylation, and beta-hydroxylation were assessed by direct structural analysis. After purification, the molecular weight of each of the recombinant Factor IX species and its NH2-terminal amino acid sequence were shown to be identical to those of plasma Factor IX. gamma-Carboxyglutamic acid and beta-hydroxyaspartic acid analyses revealed that recombinant wild-type Factor IX contained 9.2 gamma-carboxyglutamic acid and 0.3 beta-hydroxyaspartic acid residues/molecule compared with 11.4 gamma-carboxyglutamic acid and 0.39 beta-hydroxyaspartic acid residues in plasma Factor IX. When the 18-residue propeptide was deleted or when the cells were grown in the presence of sodium warfarin, secreted Factor IX contained no detectable gamma-carboxyglutamic acid but 0.36 and 0.40 residues of beta-hydroxyaspartic acid, respectively. Point mutations leading to substitution of alanine for phenylalanine at residue -16 or glutamic acid for alanine at residue -10 contained 0.2 and 1.7 gamma-carboxyglutamic acid residues, respectively, and 0.2 residues of beta-hydroxyaspartic acid. These data confirm that the propeptide mutations made do not interfere with proteolytic processing and that the Factor IX propeptide contains a recognition site that designates the adjacent glutamic acid-rich domain for gamma-carboxylation. In contrast, beta-hydroxylation of aspartic acid 64 is an independent process which does not require vitamin K and is mediated through a hydroxylation recognition site in the mature Factor IX, not in the propeptide.     

Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior

Deficiency in coagulation factor IX, a plasma glycoprotein constituent of the clotting cascade, results in hemophilia B, an inherited recessive X-linked bleeding disorder. Some affected individuals, referred to as antigen positive or CRM+, express an inactive factor IX gene product at normal levels and are expected to have natural mutations altering domains of the molecule that are critical for its correct function. The serine protease catalytic domain of activated factor IX, encoded by exons VII and VIII of the gene, is a possible target for such mutations. We designed a strategy allowing rapid analysis of this region through enzymatic amplification of genomic DNA, analysis of the amplification products by denaturing gradient gel electrophoresis, and direct sequencing of the fragments displaying an altered melting behavior. This procedure permitted us to characterize two previously undescribed mutations. Factor IX Angers is a G-to-A substitution generating an Arg in place of a Gly at amino acid 396 of the mature factor IX protein. Factor IX Bordeaux is an A-to-T substitution introducing a nonsense codon in place of the normal codon for Lys at position 411. Moreover, the already described factor IX Vancouver defect was found in three apparently independent families. These results provide further insight into the molecular heterogeneity of hemophilia B. In addition, we demonstrate the usefulness of this rapid screening procedure, which has broad applications in human genetics and can be used as an alternative to RFLP analysis in carrier detection or prenatal diagnosis studies.     

Replacement of isoleucine-397 by threonine in the clotting proteinase factor IXa (Los Angeles and Long Beach variants) affects macromolecular catalysis but not L-tosylarginine methyl ester hydrolysis. Lack of correlation between the ox brain prothrombin time and the mutation site in the variant proteins.

Previously, from the plasma of unrelated haemophilia-B patients, we isolated two non-functional Factor IX variants, namely Los Angeles (IXLA) and Long Beach (IXLB). Both variants could be cleaved to yield Factor IXa-like molecules, but were defective in catalysing the cleavage of Factor X (macromolecular substrate) and in binding to antithrombin III (macromolecular inhibitor). In the present study we have identified the mutation of IXLA by amplifying the exons (including flanking regions) as well as the 5' end of the gene by polymerase-chain-reaction (PCR) method and sequencing the amplified DNA by the dideoxy chain-termination method. Comparison of the normal IX and IXLA sequences revealed only one base substitution (T----C) in exon VIII of IXLA, with a predicted replacement of Ile-397 to Thr in the mature protein. This mutation is the same as found recently for IXLB. The observation that IXLB and IXLA have the same mutation is an unexpected finding, since, on the basis of their ox brain prothrombin time (PT, a test that measures the ability of the variant Factor IX molecules to inhibit the activation of Factor X by Factor VIIa-tissue factor complex), these variants have been classified into two different groups and were thought to be genetically different. Our observation thus suggests that the ox brain PT does not reflect the locus of mutation in the coding region of the variant molecules. However, our analysis suggests that the ox brain PT is related to Factor IX antigen concentration in the patient's plasma. Importantly, although the mutation in IXLA or IXLB protein is in the catalytic domain, purified IXaLA and IXaLB hydrolyse L-tosylarginine methyl ester at rates very similar to that of normal IXa. These data, in conjunction with our recent data on Factor IXBm Lake Elsinore (Ala-390----Val mutant), strengthen a conclusion that the peptide region containing residues 390-397 of normal Factor IXa plays an essential role in macromolecular substrate catalysis and inhibitor binding. However, the two mutations noted thus far in this region do not distort S1 binding site in the Factor IXa enzyme.     

The binding of calcium to the activation products of bovine factor IX.

Binding isotherms of Ca2+ to the bovine Factor IX activation intermediates and products, i.e. Factor IXalpha, Factor IXa alpha, and Factor IXa beta have been examined. At pH 7.4, Factor IX alpha possesses at least two strong Ca2+ sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 3.7 mM. Bovine Factor IXa alpha also contains at least two Ca2+ binding sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 1.3 mM. Factor IXa beta, the ultimate activation product of Factor IX, in the intrinsic system, likewise contains at least two strong Ca2+ sites, of average KD 0.1 mM, as well as seven additional weaker sites, possessing an average KD of 1.0 mM. The Ca2+-binding properties of the above proteins are similar to those of their precursor molecule, Factor IX, which we have earlier shown to possess at least two strong Ca2+ sites, with an average KD of 0.1 mM, and 11 weaker sites, of average KD 1.3 mM (Amphlett, G.W., Byrne, R., and Castellino, F.J. (1978) J. Biol. Chem. 253, 6774-6779). Circular dichroism analysis of all of the above proteins was consistent with the molecules possessing a low alpha-helical content, and a high quantity of beta structure and random coil conformations.     

Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells

Factor IX has been expressed to high levels within a recombinant host cell and the biologically active fraction subsequently purified to homogeneity for characterization. The coding sequence for Factor IX was inserted into a mammalian cell expression vector and transfected into dihydrofolate reductase-deficient Chinese hamster ovary cells. The integrated DNA was amplified to a high copy number by selection for increasingly higher expression levels of the marker gene, dihydrofolate reductase, contained within a co-transfected plasmid. Cloned cell lines secreting over 100 micrograms/ml Factor IX antigen and up to 1.5 microgram/ml native Factor IX antigen have been obtained. Expression of biologically active Factor IX was dependent on the presence of vitamin K in the culture media. The gamma-carboxylated Factor IX was isolated from cell culture fluid by immunoaffinity chromatography using antibodies conformation-specific for the metal-stabilized conformer of Factor IX. This conformation is dependent upon metal ions and gamma-carboxyglutamic acid. Purified recombinant Factor IX migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an electrophoretic mobility equivalent to plasma-derived Factor IX. The purified recombinant Factor IX demonstrated Factor IX coagulant activity, measured in Factor IX-deficient plasma, of 35-75 units/mg. Amino acid analysis of the alkaline hydrolysate of recombinant Factor IX demonstrated an average of 6-7 mol of gamma-carboxyglutamic acid per mol of Factor IX. NH2-terminal sequence analysis of the first 17 residues revealed equivalent amino acid sequences for both purified recombinant and plasma-derived Factor IX. The results represent the first purification and characterization of a biologically active, gamma-carboxylated vitamin K-dependent protein expressed in a recombinant DNA system.     

Molecular analysis of hemophilia B in Poland: 12 novel mutations of the factor IX gene

We examined the molecular basis of factor IX deficiency in 53 unrelated Polish patients with hemophilia B. Heteroduplex analysis and direct sequencing of polymerase chain reaction (PCR) products were applied to identify the gene defect. Forty-three different point mutations were detected in the factor IX gene of 47 patients. There were 29 missense mutations, 9 nonsense mutations, 4 splice site mutations and 1 point mutation in the promoter region. Twelve mutations were novel. The results of this study emphasize a very high degree of heterogeneity of hemophilia B.     

Factor IX San Dimas. Substitution of glutamine for Arg-4 in the propeptide leads to incomplete gamma-carboxylation and altered phospholipid binding properties

DNA sequence analysis of the Factor IX gene from a hemophilia B patient (98% Factor IX antigen; less than 0.01 unit/ml clotting activity) has identified a point mutation in exon II. A guanine to adenine transition causes the substitution of a glutamine codon for an arginine codon at -4 in the propeptide of Factor IX. This variant, termed Factor IX San Dimas, circulates in the plasma as proFactor IX with a mutant 18-amino acid propeptide still attached. Like Factor IX Cambridge (Arg-1----Ser), Factor IX San Dimas is unable to express metal-induced epitopes recognized by conformation-specific polyclonal antibodies. Amino acid analysis of the alkaline hydrolysate indicates that purified Factor IX San Dimas contains a reduced number of gamma-carboxyglutamyl residues compared to Factor IX. However, this protein undergoes metal-induced quenching of the intrinsic fluorescence. In addition, Factor IX San Dimas is unable to interact with phospholipid vesicles. The absence of coagulant activity in Factor IX San Dimas can be attributed to impaired calcium-induced conformational changes and loss in the ability to bind phospholipid vesicles in the presence of calcium ions.     

Comparison of factor IX methylation on human active and inactive X chromosomes: implications for X inactivation and transcription of tissue-specific genes.

Maintenance of dosage compensation for housekeeping genes on the human X chromosome is mediated through differential methylation of clustered CpG nucleotides associated with these genes. To determine if methylation has a role in maintaining inactivity of X-linked genes which show tissue-specific expression, we examined the locus for blood clotting Factor IX. The analysis encompassed 91% of the HpaII and HhaI sites in the 41-kb region that includes the presumed promoter region, 5 kb of 5'- and 4 kb of 3'-flanking sequences. Although there are sex differences in methylation of the locus in leukocytes, the methylation pattern in liver, where the gene is expressed, is essentially the same for loci on the active and inactive X chromosome. The lack of differences in methylation of active and inactive genes makes it unlikely that methylation within the locus has a role in expression of the Factor IX gene. These findings, along with the absence of clustered CpG dinucleotides within the Factor IX locus, suggest that functional differences in DNA methylation related to X chromosome dosage compensation may be limited to CpG clusters. In any event, dosage compensation seems to be maintained regionally, rather than locus by locus.     

Crystal structure of the calcium-stabilized human factor IX Gla domain bound to a conformation-specific anti-factor IX antibody

The binding of Factor IX to membranes during blood coagulation is mediated by the N-terminal gamma-carboxyglutamic acid-rich (Gla) domain, a membrane-anchoring domain found on vitamin K-dependent blood coagulation and regulatory proteins. Conformation-specific anti-Factor IX antibodies are directed at the calcium-stabilized Gla domain and interfere with Factor IX-membrane interaction. One such antibody, 10C12, recognizes the calcium-stabilized form of the Gla domain of Factor IX. We prepared the fully carboxylated Gla domain of Factor IX by solid phase peptide synthesis and crystallized Factor IX-(1-47) in complex with Fab fragments of the 10C12 antibody. The overall structure of the Gla domain in the Factor IX-(1-47)-antibody complex at 2.2 A is similar to the structure of the Factor IX Gla domain in the presence of calcium ions as determined by NMR spectroscopy (Freedman, S. J., Furie, B. C., Furie, B., and Baleja, J. D. (1995) Biochemistry 34, 12126-12137) and by x-ray crystallography (Shikamoto, Y., Morita, T., Fujimoto, Z., and Mizuno, H. (2003) J. Biol. Chem. 278, 24090-24094). The complex structure shows that the complementarity determining region loops of the 10C12 antibody form a hydrophobic pocket to accommodate the hydrophobic patch of the Gla domain consisting of Leu-6, Phe-9, and Val-10. Polar interactions also play an important role in the antibody-antigen recognition. Furthermore, the calcium coordination network of the Factor IX Gla domain is different than in Gla domain structures of other vitamin K-dependent proteins. We conclude that this antibody is directed at the membrane binding site in the omega loop of Factor IX and blocks Factor IX function by inhibiting its interaction with membranes.     

Binding of the Ets factor GA-binding protein to an upstream site in the factor IX promoter is a critical event in transactivation.

Factor IX is an essential vitamin K-dependent serine protease that participates in the intrinsic pathway of coagulation. The protein is expressed exclusively in the liver. The rare Leyden form of hemophilia B (inherited factor IX deficiency) results from point mutations in three proximal promoter elements that decrease factor IX expression. Recovery of expression occurs following puberty, with factor IX protein levels rising into the normal range. We have previously implicated the PAR domain D-site-binding protein (DBP) as well as an upstream element, site 5, as playing important roles in the phenotypic recovery of hemophilia B Leyden. Here we demonstrate that site 5 binds both the CCAAT/enhancer-binding protein (C/EBPalpha) and the ubiquitous Ets factor GA-binding protein (GABPalpha/beta). Transactivation of the factor IX promoter by the PAR proteins DBP and hepatic leukemia factor (HLF) is dependent on the binding of GABPalpha/beta to site 5, and coexpression of these two factors is required for optimal activation of this promoter. The binding of C/EBPalpha to site 5 also augments the activity of GABPalpha/beta. Analysis of the developmental regulation of site 5-binding proteins in rat liver has shown that C/EBPalpha and the GABPbeta subunit increase markedly in the 2 weeks after birth. These observations establish a functional association between the Ets factor GABPalpha/beta and C/EBPalpha and indicate that the two PAR proteins, DBP and HLF, may play complementary roles in factor IX activation. Given the developmental changes exhibited by these proteins, it is likely that they play a role in regulation of the normal factor IX promoter as well as promoters carrying hemophilia B Leyden mutations.