Functional characterization of the chlorzoxazone 6-hydroxylation activity of human cytochrome P450 2E1 allelic variants in Han Chinese

• Published in: PeerJ (San Francisco, CA) CA), 2020-07, Vol.8, p.e9628, Article e9628
• Main Authors: Wang, Ting, Du, Huihui, Ma, Jingsong, Shen, Lu, Wei, Muyun, Zhao, Xianglong, Chen, Luan, Li, Mo, Li, Guorong, Xing, Qinghe, He, Lin, Qin, Shengying
• Format: Article
• Language: English
• Subjects: Alleles; Amino acid sequence; Amino acids; Carcinogens; Chlorzoxazone; Codons; CYP2E1 gene; Cytochrome; Cytochrome P-450; Cytochrome P450; Cytochrome P450 2E1 (CYP2E1); Enzymatic activity; Enzyme activity; Enzymes; Gene frequency; Gene polymorphism; Genes; Genetic aspects; Genetic diversity; Genetic polymorphisms; Genetics; Genomics; Hydroxylation; Investigations; Metabolism; Metabolites; Mutagenesis; Physiological aspects; Plasmids; Population; Proteins; Single nucleotide polymorphisms; Single-nucleotide polymorphism; Stop codon; Western blotting; Xenobiotics
• ISSN: 2167-8359; 2167-8359
• DOI: 10.7717/peerj.9628

Cytochrome P450 (P450) 2E1 is one of the primary enzymes responsible for the metabolism of xenobiotics, such as drugs and environmental carcinogens. The genetic polymorphisms of the gene in promoter and coding regions have been identified previously in the Han Chinese population from four different geographic areas of Mainland China. To investigate whether genetic variants identified in the coding region affect enzyme function, the enzymes of four single nucleotide polymorphism (SNP) variants in the coding region (novel c.1009C>T, causing p.Arg337X, where X represents the translational stop codon; c.227G>A, causing p.Arg76His; c.517G>A, yielding p.Gly173Ser; and c.1263C>T, presenting the highest allele frequency), two novel alleles (c.[227G>A;1263C>T] and c.[517G>A;1263C>T]), and the wild-type were heterologously expressed in COS-7 cells and functionally characterized in terms of expression level and chlorzoxazone 6-hydroxylation activity. The impact of the CYP2E1 variant sequence on enzyme activity was predicted with three programs: Polyphen 2, PROVEAN and SIFT. The prematurely terminated p.Arg337X variant enzyme was undetectable by western blotting and inactive toward chlorzoxazone 6-hydroxylation. The c.1263C>T and c.[517G>A;1263C>T] variant enzymes exhibited properties similar to those of the wild-type CYP2E1. The variants c.227G>A and c.[227G>A;1263C>T] displayed significantly reduced enzyme activity relative to that of the wild-type enzyme (decreased by 42.8% and 32.8%, respectively;  A transfectant was increased by 31% compared with the wild-type CYP2E1 enzyme (  T (p.Arg337X), c.227G>A (p.Arg76His), and c.517G>A (p.Gly173Ser), could influence the metabolism of CYP2E1 substrates, such as chlorzoxazone.