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Effects of Coexpression of UGT1A9 on Enzymatic Activities of Human UGT1A Isoforms

Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan (R.F., M.N., H.Y., A.N., M.K., T.Y.); and Food Science and Technology, Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, Toyama, Japan (S.I., T.S.)

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 V_max), imipramine N-glucuronide formation (increased K_m and V_max), and serotonin O-glucuronide formation (decreased V_max) catalyzed by UGT1A1, UGT1A4, and UGT1A6, respectively. On the other hand, the coexpressed UGT1A1 increased K_m and decreased the V_max of the propofol O-glucuronide formation catalyzed by UGT1A9. The coexpressed UGT1A4 and UGT1A6 also increased the V_max of the propofol Oglucuronide formation by UGT1A9. This is the first study showing that human UGT1A isoforms interact with other isoforms to change the enzymatic characteristics.

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