Functional characterization of five novel CYP2C8 variants, G171S, R186X, R186G, K247R, and K383N, found in a Japanese population.
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.
Hichiya
Hiroyuki
H
Project Team for Pharmacogenetics, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
Tanaka-Kagawa
Toshiko
T
Soyama
Akiko
A
Jinno
Hideto
H
Koyano
Satoru
S
Katori
Noriko
N
Matsushima
Erika
E
Uchiyama
Shigehisa
S
Tokunaga
Hiroshi
H
Kimura
Hideo
H
Minami
Narihiro
N
Katoh
Masaaki
M
Sugai
Kenji
K
Goto
Yu-ichi
Y
Tamura
Tomohide
T
Yamamoto
Noboru
N
Ohe
Yuichiro
Y
Kunitoh
Hideo
H
Nokihara
Hiroshi
H
Yoshida
Teruhiko
T
Minami
Hironobu
H
Saijo
Nagahiro
N
Ando
Masanori
M
Ozawa
Shogo
S
Saito
Yoshiro
Y
Sawada
Jun-ichi
J
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
D012333:RNA, Messenger; D004247:DNA; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D046988:Proteasome Endopeptidase Complex