Effects of coexpression of UGT1A9 on enzymatic activities of human UGT1A isoforms.
We established stable HEK293 cell lines expressing double isoforms, UGT1A1 and UGT1A9, UGT1A4 and UGT1A9, or UGT1A6 and UGT1A9, as well as stable cell lines expressing each single isoform. To analyze the protein-protein interaction between the UGT1As, we investigated the thermal stability and resistance to detergent. UGT1A9 uniquely demonstrated thermal stability, which was enhanced in the presence of UDP-glucuronic acid (>90% of control), and resistance to detergent. Interestingly, UGT1A1, UGT1A4, and UGT1A6 acquired thermal stability and resistance to detergent by the coexpression of UGT1A9. An immunoprecipitation assay revealed that UGT1A6 and UGT1A9 interact in the double expression system. Using the single expression systems, it was confirmed that estradiol 3-O-glucuronide, imipramine N-glucuronide, serotonin O-glucuronide, and propofol O-glucuronide formations are specific for UGT1A1, UGT1A4, UGT1A6, and UGT1A9, respectively. By kinetic analyses, we found that the coexpressed UGT1A9 significantly affected the kinetics of estradiol 3-O-glucuronide formation (decreased Vmax), imipramine N-glucuronide formation (increased Km and Vmax), and serotonin O-glucuronide formation (decreased Vmax) catalyzed by UGT1A1, UGT1A4, and UGT1A6, respectively. On the other hand, the coexpressed UGT1A1 increased Km and decreased the Vmax of the propofol O-glucuronide formation catalyzed by UGT1A9. The coexpressed UGT1A4 and UGT1A6 also increased the Vmax of the propofol O-glucuronide formation by UGT1A9. This is the first study showing that human UGT1A isoforms interact with other isoforms to change the enzymatic characteristics.
Fujiwara
Ryoichi
R
Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
Nakajima
Miki
M
Yamanaka
Hiroyuki
H
Nakamura
Akiko
A
Katoh
Miki
M
Ikushiro
Shin-ichi
S
Sakaki
Toshiyuki
T
Yokoi
Tsuyoshi
T
D002384:Catalysis / Q000187:drug effects; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004958:Estradiol / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007099:Imipramine / Q000031:analogs & derivatives / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D017830:Octoxynol / Q000494:pharmacology; D015742:Propofol / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D013696:Temperature; D014162:Transfection; D000090264:UDP-Glucuronosyltransferase 1A9
D003902:Detergents; D020719:Glucuronides; D007527:Isoenzymes; C000715591:UGT1A9 protein, human; C514870:imipramine N-glucuronide; D012701:Serotonin; D004958:Estradiol; D017830:Octoxynol; D014453:Glucuronosyltransferase; D000090264:UDP-Glucuronosyltransferase 1A9; D007099:Imipramine; D015742:Propofol