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. 1994 Mar;3(3):421-8.
doi: 10.1093/hmg/3.3.421.

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

C Hassett  1 , L Aicher, J S Sidhu, C J Omiecinski
Affiliations

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

C Hassett et al. Hum Mol Genet. 1994 Mar.

Erratum in

  • Hum Mol Genet 1994 Jul;3(7):1214

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.

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Figures

Figure 1
Figure 1
Alignment of human mEH amino acid sequences predicted from six DNA clones. The locations of intron/exon boundaries are indicated by vertical arrows above the amino acids (shown in single-letter code) nearest to the respective junction. Sequences 1, 2, and 3 are from references 6, 7, and 8, respectively. Sequence 4 is compiled from the translated exons determined from the human gene. Sequences 5 and 6 are identified in the text as clones HEH/p33 and HEH/p53, respectively. Residues which are invariant between sequences are not displayed. Nonconservative amino acid substitutions are designated in bold face and with an asterisk below the variant amino acid.
Figure 2
Figure 2
Exon 3 genotype analysis by dot blot hybridization. Thirteen human liver DNA samples were PCR amplified using primers flanking exon 3, denatured, and applied to a nylon membrane in duplicate. Nucleotides encoding amino acid 113 alleles were distinguished using oligomers with sequences diagnostic for either His113 (top) or Tyr113 (bottom). In this figure, five individuals are Tyr/His113 and eight are Tyr/Tyr113.
Figure 3
Figure 3
Exon 4 genotype determination by RFLP analysis. Lymphocyte DNA from 25 individuals was PCR amplified using primers flanking exon 4. DNA was digested with RsaI and size-separated on a 2% agarose gel. His 139 alleles are identified by two DNA bands (295 and 62 bp), whereas Arg139 alleles produce three bands (174, 121, and 62 bp) following digestion. Heterozygotes display a combination of all four DNA fragments.
Figure 4
Figure 4
(A) Northern blot analysis of mEH RNA obtained from in vitro expressed allelic variants. Five μg of total RNA was size-separated in an agarose gel, transferred to GeneScreen Plus, and probed with an oligomer specific for the detection of mEH RNA. Clones containing the variant amino acid(s) used in the transfection experiment are designated above each lane by the allelic amino acid(s). COS-1 mock represents RNA isolated from COS-1 cells which were subjected to transfection conditions without a plasmid. The locations of 18S and 28S rRNA are indicated. (B) Northern analysis of 18S rRNA obtained from allelic variants expressed in vitro. The membrane used to generate (A) was dehybridized, and re-probed with an oligomer complementary to 18S rRNA.
Figure 5
Figure 5
Western blot analysis of S9 protein. COS-1 cells were transfected with plasmids containing each of the mEH variant amino acids and 5 μg of S9 protein was electrophoretically separated, transferred to Immobilon-P, and reacted with antibody specific for mEH protein. The mEH standard is 100 ng of purified mEH protein. The size of the protein was estimated from molecular standards, run concurrently.
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