RESUMEN
Cytochrome P-450 (CYP) 2C19 is responsible for the metabolism of a number of therapeutic agents such as S-mephenytoin, omeprazole, proguanil, certain barbiturates, diazepam, propranolol, citalopram and imipramine. Genetic polymorphisms in this enzyme are responsible for the poor metabolizers (PM) of mephenytoin, which represent approximately 13-23% of Asians and 3-5% of Caucasians. Several polymorphisms contribute to this phenotype. We have isolated two new allelic variants that contribute to the PM phenotype in Caucasians. CYP2C19*7 contained a single T --> A nucleotide transversion in the invariant GT at the 5' donor splice site of intron 5. The second PM allele, CYP2C19*8, consisted of a T358C nucleotide transition in exon 3 that results in a Trp120Arg substitution. In a bacterial expression system, CYP2C198 protein exhibited a dramatic (approximately 90% and 70%) reduction in the metabolism of S-mephenytoin and tolbutamide, respectively, when compared with the wild-type CYP2C191B protein. Restriction fragment length polymerase chain reaction tests were developed to identify the new allelic variants.
Asunto(s)
Anticonvulsivantes/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Mefenitoína/metabolismo , Oxigenasas de Función Mixta/genética , Alelos , Citocromo P-450 CYP2C19 , Inhibidores Enzimáticos del Citocromo P-450 , Escherichia coli/genética , Escherichia coli/metabolismo , Exones , Francia , Genotipo , Humanos , Intrones , Pulmón/enzimología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Fenotipo , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Población BlancaRESUMEN
A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large interracial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. In the present study, we identified two new mutations in CYP2C19 in a single Swiss Caucasian PM outlier (JOB 1) whose apparent genotype (CYP2C19*1/CYP2C19*2) did not agree with his PM phenotype. These mutations consisted of a single base pair mutation (G395A) in exon 3 resulting in an Arg132-->Gln coding change and a (G276C) mutation in exon 2 resulting in a coding change Glu92-->Asp. However, the G276C mutation and the G395A mutation resided on separate alleles. Genotyping tests of a family study of JOB1 showed that the exon 2 change occurred on the CYP2C19*2 allele, which also contained the known splice mutation in exon 5 (this variant is termed CYP2C19*2B to distinguish it from the original splice variant now termed CYP2C19*2A). The exon 3 mutation resided on a separate allele (termed CYP2C19*6). In all other respects this allele was identical to one of two wild-type alleles, CYP2C19*1B. The incidence of CYP2C19*6 in a European Caucasian population phenotyped for mephenytoin metabolism was 0/344 (99% confidence limits of 0 to 0.9%). Seven of 46 Caucasian CYP2C19*2 alleles were CYP2C19*2B(15%) and 85% were CYP2C19*2A. The Arg132Gln mutation was produced by site-directed mutatgenesis and the recombinant protein expressed in a bacterial cDNA expression system. Recombinant CYP2C19 6 had negligible catalytic activity toward S-mephenytoin compared with CYP2C19 1B, which is consistent with the conclusion that CYP2C19*6 represents a PM allele. Thus, the new CYP2C19*6 allele contributes to the PM phenotype in Caucasians.
Asunto(s)
Alelos , Anticonvulsivantes/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Mefenitoína/metabolismo , Oxigenasas de Función Mixta/genética , Población Blanca/genética , Citocromo P-450 CYP2C19 , Humanos , Mutagénesis Sitio-DirigidaRESUMEN
Specificity of human CYP2C9 for two substrates, diclofenac and ibuprofen, was studied using chimeras and site-directed mutants of CYP2C9 and the highly related CYP2C19 expressed in Escherichia coli. Data were correlated with the presence of putative substrate recognition sites (SRS). A CYP2C19 chimera containing residues 228-340 (SRS 3 and 4) of 2C9 conferred both diclofenac hydroxylation and 2- and 3-hydroxylation of ibuprofen. The regiospecificity of this construct for metabolism of ibuprofen differed from that of CYP2C9 by favoring 2-hydroxylation over 3-hydroxylation. A CYP2C9 construct containing residues 228-340 of CYP2C19 lacked both diclofenac and ibuprofen hydroxylase activities. When residues 228-282 (containing SRS 3) of CYP2C9 were replaced by those of CYP2C19, the chimera retained appreciable activity for diclofenac and ibuprofen, and tolbutamide activity was inhibited by a specific CYP2C9 inhibitor, sulfaphenazole. This suggested that SRS 3 is not important in conferring specificity. CYP2C9 and CYP2C19 differ in five residues within the region 283-340 (within SRS 4). Mutations to analyze SRS 4 were made on a CYP2C19 chimera containing residues 228-282 of CYP2C9. A single I289N mutation conferred a dramatic increase in diclofenac hydroxylation and a small increase in ibuprofen 2-hydroxylation. A second mutation (N286S and I289N) increased diclofenac hydroxylation and conferred a dramatic increase in ibuprofen 2-hydroxylation. A V288E mutation did not increase activity toward either substrate and decreased activity toward the two substrates in combination with the I289N or the N286S, I289N mutants. Therefore residues 286 and 289 of CYP2C9 are important in conferring specificity for diclofenac and ibuprofen.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Diclofenaco/metabolismo , Ibuprofeno/metabolismo , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/metabolismo , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Escherichia coli/genética , Humanos , Hidroxilación , Oxigenasas de Función Mixta/genética , Mutagénesis Sitio-Dirigida , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Proteínas Recombinantes de Fusión/genética , Esteroide Hidroxilasas/antagonistas & inhibidores , Esteroide Hidroxilasas/genética , Especificidad por Sustrato/genética , Tolbutamida/metabolismoRESUMEN
The metabolism of the anticonvulsant drug mephenytoin exhibits a genetic polymorphism in humans. This polymorphism exhibits marked racial heterogeneity, with the poor metabolizer PM phenotype representing 13-23% of oriental populations, but only 2-5% of Caucasian populations. Two defective CYP2C19 alleles (CYP2C19*2 and CYP2C19*3) have been described, which account for more than 99% of Oriental poor metabolizer alleles but only approximately 87% of Caucasian poor metabolizer alleles. Therefore, additional defects presumably contribute to the poor metabolizer in Caucasians. Recent studies have found a third mutation CYP2C19*4, which accounts for approximately 3% of Caucasian poor metabolizer alleles. A fourth rare mutation (CYP2C19*5A) (C99,A991,Ile331;C1297T,Arg433-->Trp) resulting in an Arg433 to Trp substitution in the heme-binding region has been reported in a single Chinese poor metaboliser outlier belonging to the Bai ethnic group. The present study identifies a second variant allele CYP2C19*5B (C99-->T; A991-->G, Ile331-->Val; C1297-T, Arg433-->Trp in one of 37 Caucasian poor metabolizers. The frequency of the CYP2C19*5 alleles is low in Chinese (approximately 0.25% in the Bai ethnic group) and Caucasians (< 0.9%). However, these alleles contribute to the poor metabolizer phenotype in both ethnic groups and increases the sensitivity of the genetic tests for identifying defective alleles to approximately 100% in Chinese poor metabolizers and 92% in Caucasian poor metabolizers genotyped in our laboratory. The Arg433 to Trp mutation in the heme-binding region essentially abolishes activity of recombinant CYP2C19*5A toward S-mephenytoin and tolbutamide, which is consistent with the conclusion that CYP2C19*5 represents poor metabolizer alleles.
Asunto(s)
Alelos , Anticonvulsivantes/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Mefenitoína/metabolismo , Oxigenasas de Función Mixta/genética , Población Blanca/genética , Sustitución de Aminoácidos , Secuencia de Bases , Citocromo P-450 CYP2C19 , Sistema Enzimático del Citocromo P-450/química , Cartilla de ADN , Humanos , Oxigenasas de Función Mixta/química , FenotipoRESUMEN
Human P450 2C19 is selective for 4'-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the structurally homologous P450 2C9 has low activity toward these substrates. To identify the critical amino acids that determine the specificity of human amino acids that determine the specificity of human P450 2C19, we constructed chimeras of p450 2C9 replacing various proposed substrate binding sites (SRS) with those of P450 2C19 and then replaced individual residues of P450 2C19 and then replaced individual residues of P450 2C9 by site-directed mutagenesis. The 339 NH2-terminal amino acid residues (SRS-1-SRS-4) and amino acids 160-383 (SRS-2-SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase activity to P450 2C9. In contract, the COOH terminus of P450 2C19 (residues 340-490 including SRS-5 and SRS-6), residues 228-339 (SRS-3 and SRS-4) and residues 292-383 (part of SRS-4 and SRS-5) conferred only modest increases in activity. A single mutation Ile99 --> His increased omeprazole 5-hydroxylase to approximately 51% of that of P450 2C19. A chimera spanning residues 160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylase activity which was dramatically enhanced by the mutation Ile99 --> His. A combination of two mutations, Ile99 --> His and Ser200 --> Pro, converted P450 2C9 to an enzyme with a turnover number of omeprazole 5-hyrdroxylation, which resembled that of P450 /c19. Mutation of Pro221 --> Thr enhanced this activity. Residue 99 is within SRS-1, but amino acids 220 and 221 are in the F-G loop and outside any known SRS. Mutation of these three amino acids did not confer significant S-mephenytoin 4'-hydroxylase activity to P450 2C9, although chimeras containing SRS-1-SRS-4 and SRS-2-SRS-5 of P450 2C19 exhibited activity toward this substrate. Our results thus indicate that amino acids 99, 220, and 221 are key residues that determine the specificity of P450 2C19 for omeprazole.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/química , Oxigenasas de Función Mixta/química , Secuencia de Bases , Citocromo P-450 CYP2C19 , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The cytochrome P4502C subfamily comprises a group of constitutive microsomal hemoproteins which are expressed primarily in liver. In humans, this subfamily is responsible for metabolism of a variety of therapeutic drugs such as warfarin, mephenytoin, omeprazole, and antiinflammatory drugs. In the present study, we analyzed the promoter activity of the 5'-flanking region of two human CYP2C genes, CYP2C9 and CYP2C18. The ability of the 2.2-kb 5'-flanking region of the CYP2C9 gene to direct expression of a luciferase reporter gene in HepG2 cells was 25 times greater than that of the 1.3-kb 5'-flanking region of CYP2C18. Deletional analysis of CYP2C9 indicated that the minimal promoter was located between the translation start site and nucleotide -155, and an HPF-1 domain consensus sequence was identified in this region. Gel shift analysis demonstrated that nuclear proteins from HepG2 cells had a high binding affinity for a 20-bp oligonucleotide containing the HPF-1 site of CYP2C9. Antiserum to rat HNF-4 supershifted this DNA--protein complex, and an oligonucleotide derived from an HNF-4 motif present in the human apolipoprotein CIII promoter competed for the supershifted complex. Cotransfection with an HNF-4 expression plasmid increased transcriptional activity of the CYP2C9 minimal promoter (approximately 2-fold) in HepG2 cells and elevated activity more substantially in nonhepatic NIH3T3 cells (26-fold) and Cos 1 cells (9-fold).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Fosfoproteínas , Regiones Promotoras Genéticas , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/genética , Transcripción Genética , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Carcinoma Hepatocelular , Citocromo P-450 CYP2C9 , ADN/química , ADN/metabolismo , Eliminación de Gen , Haplorrinos , Factor Nuclear 4 del Hepatocito , Humanos , Neoplasias Hepáticas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacología , Activación Transcripcional , Transfección , Células Tumorales CultivadasRESUMEN
N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-alkylpurines and other purine lesions induced in DNA by simple alkylating carcinogens. A mouse MPG cDNA clone was isolated from a lambda recombinant phage library of BALB/c mouse lung cell and characterized. Using the mouse MPG cDNA as a probe, the complete mouse MPG gene was isolated in two overlapping lambda recombinant genomic clones. The 6-kb gene has four exons containing 1,002 bp of coding sequence. The transcription start site was identified in the genomic sequence by primer extension of MPG mRNA from a mouse lung fibroblast cell line. The location of this transcription start site was confirmed by in vitro transcription with the promoter-containing plasmid template. Promoter function of the sequence 5' upstream of the transcription initiation site was shown by transient expression of the firefly luciferase reporter gene under the control of this sequence in transfected human and mouse cells. The mouse MPG promoter contains no TATA box, but has a CAAT element and is G.C-rich with putative AP2 elements and SP1-complementary sequences.
Asunto(s)
ADN Glicosilasas , Reparación del ADN/genética , N-Glicosil Hidrolasas/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Exones/genética , Biblioteca de Genes , Pulmón , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/química , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Mapeo Restrictivo , Análisis de Secuencia de ADN , Transcripción Genética/genéticaRESUMEN
A 871-base pair cDNA encoding the human N-methylpurine-DNA glycosylase (MPG) was cloned from a HeLa S3 cDNA expression library in a pUC vector by phenotypic screening of MPG-negative (tag- alkA-) Escherichia coli cells exposed to methylmethane sulfonate. The active MPG is expressed as a 31-kDa fusion protein. The human cDNA-encoded MPG releases 3-methyladenine, 7-methylguanine, and 3-methylguanine from DNA and thus has a substrate range similar to that of the indigenous enzyme and the E. coli AlkA protein. The cDNA hybridizes with distinct restriction fragments of mammalian DNAs but not with E. coli or yeast DNA. A search in the GenBank data bank failed to show any other cloned DNA with a similar sequence. Although the human protein has 62% sequence homology with the corresponding rat enzyme, only a few amino acid residues are conserved between the human protein and the E. coli and yeast MPGs. However, a conserved glutamine residue in all MPGs that release 3-alkyladenine and an arginine residue in eukaryotic MPGs and E. coli AlkA that act additionally on N-alkylguanines suggest that these residues are involved in recognition of adenine and guanine adducts in DNA, respectively. Although the 1.1-kilobase mRNAs of MPG from human and rodents are similar in size, liver and cultured cells of rat have much lower levels of MPG mRNA than do human and mouse cells. A hamster cell line variant isolated as being resistant to methylmethane sulfonate does not have a higher level of MPG mRNA than the parent cell line.
Asunto(s)
ADN Glicosilasas , Reparación del ADN , ADN/genética , N-Glicosil Hidrolasas/genética , Adenina/análogos & derivados , Adenina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Toxin A of Clostridium difficile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. difficile chromosomal DNA was developed which allowed for the construction of a genomic library in lambda gt11. Approx. 35,000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, lambda cd19. Verification of the immunological identity of the isolated toxin A gene fragment in lambda cd19 was determined by affinity purifying toxin A antibodies specific for lambda cd19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in lambda cd19 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. difficile. The peptide coded for by the toxin A gene fragment in lambda cd19 was not cytotoxic for 3T3 mammalian tissue culture cells.