Identification and functional characterization of human soluble epoxide hydrolase genetic polymorphisms.
Human soluble epoxide hydrolase (sEH), an enzyme directing the functional disposition of a variety of endogenous and xenobiotic-derived chemical epoxides, was characterized at the genomic level for interindividual variation capable of impacting function. RNA was isolated from 25 human liver samples and used to generate full-length copies of soluble epoxide hydrolase cDNA. The resulting cDNAs were polymerase chain reaction amplified, sequenced, and eight variant loci were identified. The coding region contained five silent single nucleotide polymorphisms (SNPs) and two variant loci resulting in altered protein sequence. An amino acid substitution was identified at residue 287 in exon 8, where the more common arginine was replaced by glutamine. A second variant locus was identified in exon 13 where an arginine residue was inserted following serine 402 resulting in the sequence, arginine 403-404, instead of the more common, arginine 403. This amino acid insertion was confirmed by analyzing genomic DNA from individuals harboring the polymorphic allele. Slot blot hybridization analyses of the liver samples indicated that sEH mRNA steady-state expression varied approximately 10-fold. Transient transfection experiments with CHO and COS-7 cells were used to demonstrate that the two new alleles possess catalytic activity using trans-stilbene oxide as a model substrate. Although the activity of the glutamine 287 variant was similar to the sEH wild type allele, proteins containing the arginine insertion exhibited strikingly lower activity. Allelic forms of human sEH, with markedly different enzymatic profiles, may have important physiological implications with respect to the disposition of epoxides formed from the oxidation of fatty acids, such as arachidonic acid-derived intermediates, as well in the regulation of toxicity due to xenobiotic epoxide exposures.
Sandberg
M
M
Department of Plant Biology, Uppsala Genetic Center, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden.
Hassett
C
C
Adman
E T
ET
Meijer
J
J
Omiecinski
C J
CJ
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D004851:Epoxide Hydrolases / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000032:analysis
D012333:RNA, Messenger; D004851:Epoxide Hydrolases