RESUMEN

γ-Carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively charged residue. Gla is generated by a single enzyme, the γ-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins.

Asunto(s)

Ligasas de Carbono-Carbono/análisis , Ácido Glutámico/metabolismo , Fragmentos de Péptidos/análisis , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Ligasas de Carbono-Carbono/genética , Ligasas de Carbono-Carbono/metabolismo , Línea Celular , Cromatografía Liquida , Cricetinae , Electroforesis en Gel de Poliacrilamida , Ácido Glutámico/análisis , Humanos , Espectrometría de Masas , Metilación , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Proteolisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tripsina/química

RESUMEN

Recent advances in the discovery of new functions for vitamin K-dependent (VKD) proteins and in defining vitamin K nutriture have led to a substantial revision in our understanding of vitamin K physiology. The only unequivocal function for vitamin K is as a cofactor for the carboxylation of VKD proteins which renders them active. While vitamin K was originally associated only with hepatic VKD proteins that participate in hemostasis, VKD proteins are now known to be present in virtually every tissue and to be important to bone mineralization, arterial calcification, apoptosis, phagocytosis, growth control, chemotaxis, and signal transduction. The development of improved methods for analyzing vitamin K has shed considerable insight into the relative importance of different vitamin K forms in the diet and their contribution to hepatic vs. non-hepatic tissue. New assays that measure the extent of carboxylation in VKD proteins have revealed that while the current recommended daily allowance for vitamin K is sufficient for maintaining functional hemostasis, the undercarboxylation of at least one non-hemostatic protein is frequently observed in the general population. The advances in defining VKD protein function and vitamin K nutriture are described, as is the potential impact of VKD proteins on atherosclerosis. Many of the VKD proteins contribute to atherogenesis. Recent studies suggest involvement in arterial calcification, which may be influenced by dietary levels of vitamin K and by anticoagulant drugs such as warfarin that antagonize vitamin K action.

Asunto(s)

Anticoagulantes/farmacología , Arteriosclerosis/tratamiento farmacológico , Arteriosclerosis/patología , Vitamina K/farmacología , Vitamina K/fisiología , Animales , Quimiotaxis , Hemostasis , Humanos , Hígado/metabolismo , Modelos Biológicos , Modelos Químicos , Fenómenos Fisiológicos de la Nutrición , Estructura Terciaria de Proteína , Factores de Riesgo , Transducción de Señal , Vitamina K/metabolismo

RESUMEN

The vitamin K-dependent (VKD) carboxylase converts clusters of Glu residues to gamma-carboxylated Glu residues (Glas) in VKD proteins, which is required for their activity. VKD precursors are targeted to the carboxylase by their carboxylase recognition site, which in most cases is a propeptide. We have identified a second tethering site for carboxylase and VKD proteins that is required for carboxylase activity, called the vitamin K-dependent protein site of interaction (VKS). Several VKD proteins specifically bound an immobilized peptide comprising amino acids 343-355 of the human carboxylase (CVYKRSRGKSGQK) but not a scrambled peptide containing the same residues in a different order. Association with the 343-355 peptide was independent of propeptide binding, because the VKD proteins lacked the propeptide and because the 343-355 peptide did not disrupt association of a propeptide factor IX-carboxylase complex. Analysis with peptides that overlapped amino acids 343-355 indicated that the 343-345 CVY residues were necessary but not sufficient for prothrombin binding. Ionic interactions were also suggested because peptide-VKD protein binding could be disrupted by changes in ionic strength or pH. Mutagenesis of Cys(343) to Ser and Tyr(345) to Phe resulted in 7-11-fold decreases in vitamin K epoxidation and peptide (EEL) substrate and carboxylase carboxylation, and kinetic analysis showed 5-6-fold increases in K(m) values for the Glu substrate. These results suggest that Cys(343) and Tyr(345) are near the catalytic center and affect the active site conformation required for correct positioning of the Glu substrate. The 343-355 VKS peptide had a higher affinity for carboxylated prothrombin (K(d) = 5 microm) than uncarboxylated prothrombin (K(d) = 60 microm), and the basic VKS region may also facilitate exiting of the Gla product from the catalytic center by ionic attraction. Tethering of VKD proteins to the carboxylase via the propeptide-binding site and the VKS region has important implications for the mechanism of VKD protein carboxylation, and a model is proposed for how the carboxylase VKS region may be required for efficient and processive VKD protein carboxylation.

Asunto(s)

Ligasas de Carbono-Carbono/química , Vitamina K/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Baculoviridae/metabolismo , Sitios de Unión , Western Blotting , Catálisis , Dominio Catalítico , Línea Celular , Cisteína/química , Cisteína/metabolismo , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Concentración de Iones de Hidrógeno , Insectos , Iones/metabolismo , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Fenilalanina/química , Unión Proteica , Precursores de Proteínas/metabolismo , Estructura Terciaria de Proteína , Protrombina/química , Protrombina/metabolismo , Conejos , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Serina/química , Tirosina/química , Tirosina/metabolismo

RESUMEN

The vitamin K-dependent (VKD) carboxylase binds VKD proteins via their propeptide and converts Glu's to gamma-carboxylated Glu's, or Gla's, in the Gla domain. Multiple carboxylation is required for activity, which could be achieved if the carboxylase is processive. In the only previous study to test for this capability, an indirect assay was used which suggested processivity; however, the efficiency was poor and raised questions regarding how full carboxylation is accomplished. To unequivocally determine if the carboxylase is processive and if it can account for comprehensive carboxylation in vivo, as well as to elucidate the enzyme mechanism, we developed a direct test for processivity. The in vitro carboxylation of a complex containing carboxylase and full-length factor IX (fIX) was challenged with an excess amount of a distinguishable fIX variant. Remarkably, carboxylation of fIX in the complex was completely unaffected by the challenge protein, and comprehensive carboxylation was achieved, showing conclusively that the carboxylase is processive and highly efficient. These studies also showed that carboxylation of individual fIX/carboxylase complexes was nonsynchronous and implicated a driving force for the reaction which requires the carboxylase to distinguish Glu's from Gla's. We found that the Gla domain is tightly associated with the carboxylase during carboxylation, blocking the access of a small peptide substrate (EEL). The studies describe the first analysis of preformed complexes, and the rate for full-length, native fIX in the complex was equivalent to that of the substrate EEL. Thus, intramolecular movement within the Gla domain to reposition new Glu's for catalysis is as rapid as diffusion-limited positioning of a small substrate, and the Gla domain is not sterically constrained by the rest of the fIX molecule during carboxylation. The rate of carboxylation of fIX in the preformed complex was 24-fold higher than for fIX modified by free carboxylase, which supports carboxylase processivity and which indicates that binding and/or release is the rate-limiting step in protein carboxylation. These data indicate a model of tethered processivity, in which the VKD proteins remain bound to the carboxylase throughout the reaction via their propeptide, while the Gla domain undergoes intramolecular movement to reposition new Glu's for catalysis to ultimately achieve comprehensive carboxylation.

Asunto(s)

Ligasas de Carbono-Carbono/química , Ligasas de Carbono-Carbono/metabolismo , Factor IX/química , Factor IX/metabolismo , Ácido Glutámico , Animales , Línea Celular , Clonación Molecular , Cricetinae , Humanos , Cinética , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Especificidad por Sustrato , Transfección

RESUMEN

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH(2)) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO(2), generating the gamma-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684-1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH(2)) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH(2) preincubation. Amino acid analysis of (14)C- N-ethyl maleimide-modified human carboxylase revealed 1.8-2.3 reactive residues and a specific activity of 7 x 10(8) cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0. 2% (Cys-99) or 1% (Cys-450), and increased the K(m)s for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH(2) oxygenation.

Asunto(s)

Ligasas de Carbono-Carbono/química , Ligasas de Carbono-Carbono/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Radioisótopos de Carbono , Cisteína , Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Etilmaleimida/farmacocinética , Humanos , Cinética , Fragmentos de Péptidos/química , Mapeo Peptídico , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Tripsina

RESUMEN

The carboxylase is an integral membrane glycoprotein that uses vitamin K to modify clusters of glutamyl residues (glu's) to gamma-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.

Asunto(s)

Ligasas de Carbono-Carbono/metabolismo , Vitamina K/fisiología , Animales , Sitios de Unión , Retículo Endoplásmico/metabolismo , Humanos , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

Vitamin K-dependent (VKD) proteins require modification by the VKD-gamma-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with 14CO2 and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction. Carboxylase carboxylation also was detected in baculovirus-(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.

Asunto(s)

Ligasas de Carbono-Carbono/metabolismo , Vitamina K/metabolismo , Línea Celular , Cromatografía Líquida de Alta Presión , Humanos , Cinética , Especificidad por Sustrato

Asunto(s)

Ligasas de Carbono-Carbono/aislamiento & purificación , Ligasas de Carbono-Carbono/metabolismo , Microsomas/enzimología , Animales , Ligasas de Carbono-Carbono/biosíntesis , Línea Celular , Células Cultivadas , Cromatografía de Afinidad/métodos , Cricetinae , Electroforesis en Gel de Poliacrilamida/métodos , Factor IX/biosíntesis , Humanos , Indicadores y Reactivos , Riñón , Cinética , Mamíferos , Peso Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera , Transfección/métodos

RESUMEN

A model system for the analysis of intracellular events governing the modification of individual vitamin K-dependent (VKD) proteins by the carboxylase has been developed using recombinant VKD protein-transfected cell lines. When untransfected 293 cells were analyzed by in vitro carboxylation followed by SDS-PAGE, endogenous VKD proteins were not detected. With 293 cells stably-transfected with recombinant native factor IX, most (> 95%) of the carboxylase was in complex with the factor IX, as assayed by adsorption of carboxylase activity to immobilized anti-factor IX antibody. In contrast, with 293 cells stably-transfected with recombinant factor IX deleted in the propeptide sequence (amino acids -18 to -4, delta pro factor IX), no association of factor IX with the carboxylase was observed. This observation was used to specifically isolate and identify the human carboxylase, and carboxylase-associated protein. When the carboxylase was purified from solubilized microsomes from either native factor IX, or delta pro factor IX, stably-transfected 293 cells, a single 98 kDa band was specifically obtained from native factor IX microsomes, but not from delta pro factor IX microsomes. This band was subsequently shown by Western and microsequencing analysis to comprise both the carboxylase and carboxylase-associated protein. This isolation, which represents the first isolation to near homogeneity of both the human carboxylase and the carboxylase from cell lines, will be valuable in isolating enzymatically active recombinant carboxylase, which has been refractile to other purification attempts. This system was also used to show that the human carboxylase in 293 cells is capable of binding and modifying two different liver-derived proteins. Protein C-producing 293 cells were generated from the same 293 progenitor cell line used to created the factor IX-expressing cells. With both factor IX- and protein C-transfected 293 cells, the secreted proteins were almost completely carboxylated, and in microsomes from each cell line the carboxylase was found in near quantitative complex with the two different VKD proteins. Thus the carboxylase modifies both VKD proteins. The approach described here for the analysis of the carboxylase from recombinant VKD protein-transfected cell lines should provide an important new system for studying protein carboxylation and VKD protein-carboxylase interaction.

Asunto(s)

Ligasas de Carbono-Carbono , Factor IX/metabolismo , Ligasas/aislamiento & purificación , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Factor IX/genética , Humanos , Ligasas/genética , Ligasas/metabolismo , Microsomas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Proteína C/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo

Asunto(s)

Factor IX/biosíntesis , Factor VII/biosíntesis , Proteína C/biosíntesis , Animales , Carcinoma Hepatocelular , Línea Celular , Cromatografía Líquida de Alta Presión/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Factor IX/genética , Factor IX/metabolismo , Factor VII/genética , Factor VII/aislamiento & purificación , Expresión Génica , Vectores Genéticos , Humanos , Indicadores y Reactivos , Neoplasias Hepáticas , Peso Molecular , Proteína C/genética , Proteína C/aislamiento & purificación , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección/métodos , Células Tumorales Cultivadas , Vitamina K/metabolismo

RESUMEN

The microsomal gamma-carboxylase catalyzes modification of a limited set of glutamyl residues to gamma-carboxyglutamyl residues in a vitamin K-dependent reaction that also utilizes O2 and CO2. We report the purification to apparent homogeneity of the bovine liver microsomal carboxylase. Affinity chromatography exploiting the association of the carboxylase with prothrombin precursor and carboxylase binding to the propeptide sequence were combined with ion-exchange chromatography and fractionation using immobilized lectins. A 3.5 x 10(5)-fold purification was obtained, which is the highest purification, by a factor of 35, yet reported for this enzyme. A single 98-kDa protein is obtained from this isolation. Carboxylase activity is associated with this protein by two different criteria. Antibodies prepared against the carboxylase detected the 98-kDa protein when used in Western analysis. In addition, the single 98-kDa protein was shown to comigrate with activity when electrophoresed in a nondenaturing gel system. The availability of purified preparations of carboxylase will facilitate an increased understanding of the complex biochemical reaction carried out by this protein.

Asunto(s)

Ligasas de Carbono-Carbono , Ligasas/aislamiento & purificación , Microsomas Hepáticos/enzimología , Animales , Western Blotting , Bovinos , Ligasas/química , Ligasas/inmunología , Peso Molecular

Asunto(s)

Adenoviridae/genética , Expresión Génica , Vectores Genéticos , Línea Celular , Recombinación Genética , Transcripción Genética , Transformación Genética , Proteínas Virales/biosíntesis , Vacunas Virales/biosíntesis

RESUMEN

Previous studies demonstrated proteolytic activation of human blood coagulation factor VII by an unidentified protease following complex formation with tissue factor expressed on the surface of a human bladder carcinoma cell line (J82). In the present study, an active-site mutant human factor VII cDNA (Ser344----Ala) has been constructed, subcloned, and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity in a single step from serum-free culture supernatants by immunoaffinity column chromatography. Mutant factor VII was fully carboxylated, possessed no apparent clotting activity, and was indistinguishable from plasma factor VII by SDS-PAGE. Cell binding studies indicated that mutant factor VII bound to J82 tissue factor with essentially the same affinity as plasma factor VII and was cleaved by factor Xa at the same rate as plasma factor VII. In contrast to radiolabeled single-chain plasma factor VII that was progressively converted to two-chain factor VIIa on J82 monolayers, mutant factor VII was not cleaved following complex formation with J82 tissue factor. Incubation of radiolabeled mutant factor VII with J82 cells in the presence of recombinant factor VIIa resulted in the time-dependent and tissue factor dependent conversion of single-chain mutant factor VII to two-chain mutant factor VIIa. Plasma levels of antithrombin III had no discernible effect on the factor VIIa catalyzed activation of factor VII on J82 cell-surface tissue factor but completely blocked this reaction catalyzed by factor Xa. These results are consistent with an autocatalytic mechanism of factor VII activation following complex formation with cell-surface tissue factor, which may play an important role in the initiation of extrinsic coagulation in normal hemostasis.

Asunto(s)

Coagulación Sanguínea/fisiología , Factor VII/metabolismo , Secuencia de Aminoácidos , Animales , Antitrombina III/farmacología , Línea Celular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Factor VII/genética , Factor VII/aislamiento & purificación , Factor VIIa/metabolismo , Factor Xa/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Transfección , Neoplasias de la Vejiga Urinaria

RESUMEN

Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg152-Ile153. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, we have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg152----Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. Purified mutant factor VII was indistinguishable from plasma-derived or recombinant wild-type factor VII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated as a single band with an apparent molecular weight of 50,000. The average specific activity of several mutant factor VII preparations was 0.00025 unit/micrograms, or 0.01% of that observed for recombinant wild-type factor VII preparations. The clotting activity of mutant factor VII was, however, completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Arginina/genética , Compuestos de Dansilo , Factor VII/genética , Glutamatos/genética , Clorometilcetonas de Aminoácidos , Secuencia de Bases , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Factor VII/biosíntesis , Ácido Glutámico , Humanos , Datos de Secuencia Molecular , Mutación , Péptido Hidrolasas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética

RESUMEN

While a role has been ascribed to the gamma-carboxyglutamate (Gla) residues in vitamin K-dependent coagulation proteins and the enzyme catalyzing this posttranslational modification has been identified and partially characterized, both the functional significance of a second posttranslationally synthesized amino acid found in these proteins, beta-hydroxyaspartate (Hya), and the aspartyl beta-hydroxylating enzyme remain to be determined. We now report that inhibitors of 2-ketoglutarate-dependent dioxygenases, such as dipyridyl, o-phenanthroline, and pyridine 2,4-dicarboxylate, block hydroxylation of Asp64 in recombinant factor IX molecules produced in three different mammalian expression systems. This hydroxylation was not inhibited by the specific copper chelators 2,9-dimethylphenanthroline or D-penicillamine. The Gla levels in these proteins were unaffected by these compounds and demonstrate that carboxylation proceeds independently of hydroxylation. Using these Hya-deficient recombinant factor IX molecules we demonstrate that this residue does not play a significant role in factor IX binding to endothelial cells under equilibrium conditions. From additional binding studies we have concluded that the Gla domain of factor IX is a major cell binding domain of factor IX. Furthermore, in contrast to studies demonstrating a marked loss of one-stage clotting activity in recombinant factors IX following site-directed mutations of Asp64 to neutral or basic residues (Rees, D. J. G., Jones, I. M., Handford, P. A., Walter, S. J., Esnouf, M. P., Smith, K. J., and Brownlee, G. J. (1988) EMBO J. 7, 2053-2061), we have not found a decrease of one-stage clotting activity with Hya-deficient factor IX. Hya-deficient proteins produced in this manner may prove to be more appropriate to elucidate the function of Hya than those produced by site-directed mutagenesis.

Asunto(s)

2,2'-Dipiridil/farmacología , Factor IX/metabolismo , Hidrolasas/antagonistas & inhibidores , Fenantrolinas/farmacología , Piridinas/farmacología , Animales , Ácido Aspártico , Línea Celular , Cricetinae , Endotelio Vascular/metabolismo , Hidroxilación , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad

Asunto(s)

Adenoviridae , Regulación de la Expresión Génica , Vectores Genéticos , Transfección , Animales , ADN Recombinante , Humanos

RESUMEN

Protein C is one of a family of vitamin K dependent proteins, including blood coagulation factors and bone proteins, that contains gamma-carboxyglutamic acid. Sequence analysis of the cDNAs for these proteins has revealed the presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from the growing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. In the present study, deletion mutants have been constructed in the propeptide region of the cDNA for human protein C, and the cDNAs were then expressed in mammalian cell culture. These deletions included the removal of 4, 9, 12, 15, 16, or 17 amino acids comprising the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These experiments have shown that protein C is readily synthesized in mammalian cell cultures, processed, and secreted as a two-chain molecule with biological activity. Furthermore, the pre portion or signal sequence in human protein C is 18 amino acids in length, and the pro portion of the leader sequence is 24 amino acids in length. Also, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) is important for gamma-carboxylation of protein C, while the present data and those of others indicate that the carboxyl-terminal portion of the propeptide (residues -1 through -4) is important for the removal of the pro leader sequence by proteolytic processing.

Asunto(s)

Proteína C/genética , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Animales , Factores de Coagulación Sanguínea/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutación , Plásmidos , Proteína C/aislamiento & purificación , Proteína C/metabolismo , Precursores de Proteínas/metabolismo , Ratas , Proteínas Recombinantes/aislamiento & purificación , Transfección

RESUMEN

The ability of eukaryotic ribosomes to reinitiate translation at downstream AUG codons on polycistronic mRNAs was used to select transfected CHO clones that secreted a precursor to the human pancreatic polypeptide (PP). In the in vitro constructed transcription unit, a viral promoter directed the synthesis of a dicistronic mRNA. The PP cDNA was placed in the 5'-part of this transcript, while a DHFR cDNA was placed 3' to the PP. This dicistronic expression unit was transfected into CHO cells, and methotrexate-resistant colonies were selected. RNA-blots verified that the PP precursor and DHFR were expressed from the same dicistronic mRNA. The CHO cells synthesized the hormone precursor and secreted it through the constitutive secretory pathway.

Asunto(s)

Genes , Polipéptido Pancreático , Precursores de Proteínas/biosíntesis , ARN Mensajero/metabolismo , Ribosomas/fisiología , Animales , Autorradiografía , Línea Celular , Clonación Molecular , ADN , Humanos , Plásmidos , Biosíntesis de Proteínas , Precursores de Proteínas/genética , Tetrahidrofolato Deshidrogenasa/genética , Transcripción Genética , Transformación Genética

RESUMEN

The adenovirus Ad5(pymT) has been used to express middle T antigen at very high levels in 293 cells. The middle T antigen produced was localized to membranes and was modified in the same way as that expressed in polyoma virus-infected mouse cells. It was phosphorylated in vivo on serine residues and in vitro on tyrosine residues. The in vivo phosphorylations occurred between residues 223 and 275. The middle T antigen encoded by A d5(pymT) was phosphorylated in vitro in a complex with human pp60c-src. Interestingly, the extreme overexpression of middle T antigen did not cause a parallel increase in the amount of complex; most of the pp60c-src remained unassociated. Immunoaffinity purification resulted in approximately 100 micrograms of middle T antigen from a 100-mm tissue culture dish. Several cell proteins copurified with the Ad5(pymT)-derived middle T antigen. Two of these, the 74- and 63-kilodalton species, are of particular interest because they were also purified from mouse tumors expressing middle T antigen.

Asunto(s)

Adenovirus Humanos/genética , Antígenos Virales de Tumores/genética , Proteínas Oncogénicas Virales/genética , Poliomavirus/genética , Proteínas Quinasas/genética , Transcripción Genética , Aminoácidos/análisis , Animales , Antígenos Transformadores de Poliomavirus , Antígenos Virales de Tumores/aislamiento & purificación , Línea Celular , Proteínas Oncogénicas Virales/aislamiento & purificación , Fosforilación , Poliomavirus/enzimología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas pp60(c-src)

RESUMEN

A modular gene with a cDNA encoding the polyomavirus middle T antigen positioned behind the adenovirus type 2 major late promoter and tripartite leader was substituted for the E1a region in an adenovirus vector. Permissive human cells infected with this recombinant produce middle T protein at levels as high as those of the most abundant late adenoviral proteins, e.g., hexon or fiber. This level represents at least a 40-fold increase over that observed in a polyomavirus lytic infection of murine cells. Partial proteolytic mapping showed that this protein has the same primary structure as middle T protein produced in polyomavirus-infected murine cells. The adenovirus recombinant-generated middle T protein exhibited in vitro kinase activity, although at an approximately 10-fold-lower specific activity than that of middle T protein from polyomavirus-infected murine cells. Comparison of the expression levels of this middle T antigen-containing adenovirus vector with a similar construction encoding dihydrofolate reductase suggested that the translation efficiency of the inserted gene was dependent upon the proximity of its initiation codon to the tripartite leader. We tested this possibility by comparing three dihydrofolate reductase recombinants among which the spacing between the initiation codon and tripartite leader varied from 188 to 36 nucleotides. The efficiency of expression of dihydrofolate reductase protein dramatically increased as this spacing was reduced.

Asunto(s)

Adenovirus Humanos/genética , Antígenos Virales de Tumores/genética , Proteínas Oncogénicas Virales/genética , Poliomavirus/genética , Proteínas Quinasas/genética , Tetrahidrofolato Deshidrogenasa/genética , Transcripción Genética , Adenovirus Humanos/enzimología , Animales , Antígenos Transformadores de Poliomavirus , Línea Celular , ADN/metabolismo , Genes , Genes Virales , Vectores Genéticos , Plásmidos , Poliomavirus/enzimología