Human microsomal epoxide hydrolase: genetic poloymorphism and functional expression in vitro of amino acid variants

Abstract

Human microsomal epoxide hydrolase (mEH) is a biotransformation enzyme that metabolizes reactive epoxide intermediates to more water-soluble trans-dihydrodiol derivatives. We compared protein-coding sequences from six full-length human mEH DNA clones and assessed potential amino acid variation at seven positions. The prevalence of these variants was assessed in at least 37 unrelated individuals using polymerase chain reaction experiments. Only Tyr/His 113 (exon 3) and His/Arg 139 (exon 4) variants were observed. The genotype frequencies determined for residue 113 alleles indicate that this locus may not be in Hardy-Weinberg equilibrium, whereas frequencies observed for residue 139 alleles were similar to expected values. Nucleotide sequences coding for the variant amino acids were constructed in an mEH cDNA using site-directed mutagenesis, and each was expressed in vitro by transient transfection of COS-1 cells. Epoxide hydrolase mRNA level, catalytic activity, and immunoreactive protein were evaluated for each construct. The results of these analyses demonstrated relatively uniform levels of mEH RNA expression between the constructs. mEH enzymatic activity and immunoreactive protein were strongly correlated, indicating that mEH specific activity was similar for each variant. However, marked differences were noted in the relative amounts of immunoreactive protein and enzymatic activity resulting from the amino acid substitutions. These data suggest that common human mEH amino acid polymorphisms may alter enzymatic function, possibly by modifying protein stability.

Genetic polymorphism and gene expression of microsomal epoxide hydrolase in non-small cell lung cancer

“…In a mutagenesis study, Hasset et al (8) suggested that the protection effect could be due to the 'allelic selection' of gene expression. By showing that high activity mEH with Tyr113 exon 3 /Arg139 exon 4 was barely detected in NSCLC patients, our results indicated that selection pressure could be from air pollution, in particular, cigarette smoking or heavy oil fumes from the traditional Chinese cooking method (such as frying fish) (26).…”

“…When 295-and 62-bp fragments appeared, the patient had homozygous allele of His139. When 174-, 121-and 62-bp bands appeared, the patient was identified as homozygous Arg139 allele, and when all four DNA fragments appeared, the patient was heterozygous with His139/Arg139 (1,(8)(9)(10)(11)(12)(13)(14)(15).…”

“…Two polymorphic sites have been reported within the coding region of EPHX1. One is located in exon 3 at amino acid residue 113, in which tyrosine (Tyr113, TAC) or histidine (His113, CAC) is expressed, and the other is located in exon 4 at amino acid residue 139, in which histidine (His139) or arginine (Arg139) is expressed (1,8,9). The enzyme with Tyr113 (fast type) has 50% higher activity than that with His113 (slow type), whereas mEH with His139 (slow type) has only 25% activity of that with Arg139 (fast type) (8).…”

“…One is located in exon 3 at amino acid residue 113, in which tyrosine (Tyr113, TAC) or histidine (His113, CAC) is expressed, and the other is located in exon 4 at amino acid residue 139, in which histidine (His139) or arginine (Arg139) is expressed (1,8,9). The enzyme with Tyr113 (fast type) has 50% higher activity than that with His113 (slow type), whereas mEH with His139 (slow type) has only 25% activity of that with Arg139 (fast type) (8). Previous studies indicate that the mEH enzyme with Tyr113 (fast type) is closely associated with the increased risk of lung, breast, laryngeal and colon cancers (10)(11)(12)(13)(14)(15).…”

Genetic variation of genes for xenobiotic-metabolizing enzymes and risk of bronchial asthma: the importance of gene–gene and gene–environment interactions for disease susceptibility

“…All DNA samples were genotyped for 25 SNPs of 18 XME genes by PCR-restriction fragment length polymorphism (RFLP) assays according to the array protocols available from the publications. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] Supplementary Table 3 shows SNPs that have been analyzed in the study. PCR was performed in a final volume equal to 25 ml of reaction mixture containing 1.5 U of thermostable Taq DNA polymerase (Lytech, Moscow, Russia), B1 mg DNA, 0.25mM of each primer, 250 mM of dNTPs, 1.5-3.5 mM of MgCl 2 and 1Â PCR buffer (67 mM Tris-HCl pH 8.8, 16.6 mM (NH 4 ) 2 SO 4 , 0.01% Tween-20).…”