title: Functional characterization of five novel CYP2C8 variants, G171S, R186X, R186G, K247R, and K383N, found in a Japanese population.
abstract: Cytochrome P450 2C8 is one of the primary enzymes responsible for the metabolism of a wide range of drugs such as paclitaxel, cerivastatin, and amiodarone. We have sequenced the CYP2C8 gene from 201 Japanese subjects and found five novel nonsynonymous single nucleotide polymorphisms (SNPs): 511G>A (G171S), 556C>T (R186X; X represents the translational stop codon), 556C>G (R186G), 740A>G (K247R), and 1149G>T (K383N), with the allele frequency of 0.0025. The CYP2C8 variants were heterologously expressed in COS-1 cells and functionally characterized in terms of expression level, paclitaxel 6alpha-hydroxylase activity, and intracellular localization. The prematurely terminated R186X variant was undetectable by Western blotting and inactive toward paclitaxel 6alpha-hydroxylation. The G171S, K247R, and K383N variants exhibited properties similar to those of the wild-type CYP2C8. Paclitaxel 6alpha-hydroxylase activity of the R186G transfectant was only 10 to 20% that of wild-type CYP2C8. Furthermore, the R186G variant displayed a lower level of protein expression in comparison to the wild type, which was restored by the addition of a proteasome inhibitor (MG-132; Z-Leu-Leu-Leu-aldehyde). The reduced CO-difference spectral analysis using recombinant proteins from an insect cell/baculovirus system revealed that the R186G variant has a minor peak at 420 nm in addition to the characteristic Soret peak at 450 nm, suggesting the existence of improperly folded protein. These results indicate that the novel CYP2C8 SNPs, 556C>T (R186X) and 556C>G (R186G), could influence the metabolism of CYP2C8 substrates such as paclitaxel and cerivastatin.
authors.affiliation: Project Team for Pharmacogenetics, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hichiya
authors.forename: Toshiko
authors.initials: T
authors.lastname: Tanaka-Kagawa
authors.forename: Akiko
authors.initials: A
authors.lastname: Soyama
authors.forename: Hideto
authors.initials: H
authors.lastname: Jinno
authors.forename: Satoru
authors.initials: S
authors.lastname: Koyano
authors.forename: Noriko
authors.initials: N
authors.lastname: Katori
authors.forename: Erika
authors.initials: E
authors.lastname: Matsushima
authors.forename: Shigehisa
authors.initials: S
authors.lastname: Uchiyama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tokunaga
authors.forename: Hideo
authors.initials: H
authors.lastname: Kimura
authors.forename: Narihiro
authors.initials: N
authors.lastname: Minami
authors.forename: Masaaki
authors.initials: M
authors.lastname: Katoh
authors.forename: Kenji
authors.initials: K
authors.lastname: Sugai
authors.forename: Yu-ichi
authors.initials: Y
authors.lastname: Goto
authors.forename: Tomohide
authors.initials: T
authors.lastname: Tamura
authors.forename: Noboru
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Ohe
authors.forename: Hideo
authors.initials: H
authors.lastname: Kunitoh
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nokihara
authors.forename: Teruhiko
authors.initials: T
authors.lastname: Yoshida
authors.forename: Hironobu
authors.initials: H
authors.lastname: Minami
authors.forename: Nagahiro
authors.initials: N
authors.lastname: Saijo
authors.forename: Masanori
authors.initials: M
authors.lastname: Ando
authors.forename: Shogo
authors.initials: S
authors.lastname: Ozawa
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Saito
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sawada
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D065727:Cytochrome P-450 CYP2C8; D004247:DNA / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D007564:Japan; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018411:Spodoptera / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D004247:DNA; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D046988:Proteasome Endopeptidase Complex