Interactions between Human UGT 1 A 1 , UGT 1 A 4 , and UGT 1 A 6 Affect Their Enzymatic Activities

Protein-protein interactions between human UDP-glucuronosyltransferase (UGT) 1A1, UGT1A4, and UGT1A6 were investigated using double expression systems in HEK293 cells (UGT1A1/ UGT1A4, UGT1A1/UGT1A6, and UGT1A4/UGT1A6). The substrates specific for UGT1A1 (estradiol and bilirubin), UGT1A4 (imipramine and trifluoperazine), and UGT1A6 (serotonin and diclofenac) were used to determine the effects of the coexpression of the other UGT1A isoforms on the enzymatic activity. The coexpression of UGT1A4 and UGT1A6 decreased the S50 and Vmax values of UGT1A1-catalyzed estradiol 3-O-glucuronide formation and increased the Vmax value of UGT1A1-catalyzed bilirubin O-glucuronide formation. The coexpression of UGT1A1 decreased the Vmax value of UGT1A4-catalyzed imipramine N-glucuronide formation but had no effect on UGT1A4-catalyzed trifluoperazine N-glucuronide formation. The coexpression of UGT1A6 had no effect on UGT1A4-catalyzed imipramine N-glucuronide formation but increased the Km and Vmax of UGT1A4-catalyzed trifluoperazine Nglucuronide formation. The coexpression of both UGT1A1 and UGT1A4 increased the Vmax values of UGT1A6-catalyzed serotonin and diclofenac O-glucuronide formation. Thus, the effects of the coexpression of other UGT1A isoforms on the kinetics of specific activities were different depending on the UGT1A isoforms and substrates. Native polyacrylamide gel electrophoresis analysis of the double expression systems showed multiple bands at approximately 110 kDa, indicating the existence of heterodimers as well as homodimers of UGTs. In conclusion, we found that human UGT1A1, UGT1A4, and UGT1A6 interact with each other, possibly by heterodimerization, and that their effects on the enzymatic activities are complex depending on the isoforms and substrates. Human UDP-glucuronosyltransferases (UGTs; EC 2.4.1.17) are a family of membrane-bound enzymes of the endoplasmic reticulum. They catalyze the glucuronidation of various endogenous and exogenous compounds, converting them into more polar glucuronides (Tukey and Strassburg, 2000). The human genome encodes at least 19 different UGTs, and they are divided into three subfamilies, UGT1A, 2A, and 2B, according to the degree of nucleotide sequence identities and genomic organization (Mackenzie et al., 2005). UGT1A isoforms are encoded by a single gene that has multiple unique exons 1 and common exons 2 to 5. Five of the human UGT1A isoforms, UGT1A1, UGT1A3, UGT1A4, UGT1A6, and UGT1A9, are expressed in the liver and characterized by individual catalytic activity profiles (Tukey and Strassburg, 2000). UGTs have been reported to form homoand hetero-oligomers. Matern et al. (1982) proposed UGT homo-oligomerization for the first time based on gel permeation chromatography of rat liver microsomes. The homo-oligomer formation of rat UGT2B1 (Meech and Mackenzie, 1997), human UGT1A1 (Ghosh et al., 2001), and human UGT1A9 (Kurkela et al., 2003) was subsequently demonstrated by SDS-PAGE, two-hybrid analysis, and immobilized meta-chelating chromatography, respectively. In addition, radiation inactivation analysis revealed that dimers and tetramers of UGT have different catalytic roles (Peters et al., 1984; Gschaidmeier and Bock, 1994). Ishii et al. (2001, 2004) showed that the interaction between guinea pig UGT2B21 and UGT2B22 influenced UGT2B21-catalyzed morphine 3-glucuronide and 6-glucuronide formation in a heterologous expression system in COS-7 cells. It has also been reported that coexpression of human UGT1A4 affects the activity of human UGT1A9 (Kurkela et al., 2004). Unfortunately, a limitation of these studies was that the activities were evaluated by transient expression systems. The expression levels and the expression ratios of the two UGT isoforms differ in each transient transfection. To overcome the issue, we recently established stable cell lines expressing double human UGT isoforms, UGT1A1/UGT1A9, UGT1A4/UGT1A9, and UGT1A6/UGT1A9 in HEK293 cells (Fujiwara et al., 2007). In that study, the interaction between UGT1A6 and UGT1A9 was suggested by the immunoprecipitation analysis, and the heterodimerization of UGT1A1, UGT1A4, and UGT1A6 with UGT1A9 was supported by thermal stability assay. Moreover, it was demonstrated that coexpression of UGT1A9 affected the kinetics of UGT1A1, UGT1A4, and UGT1A6 activities, and vice versa. The interactions between UGT1A1, UGT1A4, and UGT1A6 have remained to be elucidated. In the present extended study, we investigated the effects of the interactions between UGT1A1, UGT1A4, and UGT1A6 on their catalytic activities using the stable Article, publication date, and citation information can be found at http://dmd.aspetjournals. org. doi:10.1124/dmd.107. 016402. ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; PAGE, polyacrylamide gel electrophoresis; UDPGA, UDP-glucuronic acid; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline. 0090-9556/07/3510-1781–1787$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 10 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 16402/3252851 DMD 35:1781–1787, 2007 Printed in U.S.A. 1781 at A PE T Jornals on Jne 1, 2017 dm d.aspurnals. org D ow nladed from double expression systems. Furthermore, native PAGE analysis was carried out to determine the heterodimerization between these iso-