Identification and partial characterization of a novel CYP2C9 splicing variant encoding a protein lacking eight amino acid residues.
CYP2C9 is known as an enzyme responsible for the metabolism of various clinically important drugs. Recently, we cloned a cDNA corresponding to a CYP2C9 splicing variant (SV), which seemed to have an open reading frame of a protein with 482 amino acid residues. To investigate whether or not the SV can be translated as a functionally active protein, we expressed the CYP2C9SV in insect cells, and spectrophotometric and enzymatic properties were characterized. The CYP2C9SV protein showed a typical reduced CO-difference spectrum, indicating that the translated protein binds a heme moiety. However, CYP2C9SV did not metabolize tolbutamide or diclofenac at all, suggesting that the SV protein appeared to lack the ability to catalyze reactions mediated by CYP2C9. Although the CYP2C9SV mRNA was detected in all human liver samples examined in this study by real-time PCR, the level was generally low, ranging between 0.7 and 9.6% of the normal CYP2C9 mRNA. These results suggest that the CYP2C9SV protein is unlikely to contribute to CYP2C9 activities, although it appears to be expressed in most individuals.
Ariyoshi
Noritaka
N
Division of Pharmacy, University Hospital, Chiba University School of Medicine, Chiba, Japan. ariyoshi@ho.chiba-u.ac.jp
Shimizu
Yoko
Y
Kobayashi
Yukari
Y
Nakamura
Hiroyoshi
H
Nakasa
Hiromitsu
H
Nakazawa
Kazuyoshi
K
Ishii
Itsuko
I
Kitada
Mitsukazu
M
D000293:Adolescent; D000328:Adult; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D065729:Cytochrome P-450 CYP2C9; D004008:Diclofenac / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry; D018411:Spodoptera; D014044:Tolbutamide / Q000378:metabolism
D007527:Isoenzymes; D012333:RNA, Messenger; D004008:Diclofenac; D014044:Tolbutamide; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases