Abstract
Functional protein-protein interactions between UDP-glucuronosyltransferase (UGT)1A isoforms and cytochrome P450 (CYP)3A4 were studied. To this end, UGT1A-catalyzed glucuronidation was assayed in Sf-9 cells that simultaneously expressed UGT and CYP3A4. In the kinetics of UGT1A6-catalyzed glucuronidation of serotonin, both Michaelis constant (Km) and maximal velocity (Vmax) were increased by CYP3A4. When CYP3A4 was coexpressed with either UGT1A1 or 1A7, the Vmax for the glucuronidation of the irinotecan metabolite (SN-38) was significantly increased. S50 and Km both which are the substrate concentration giving 0.5 Vmax were little affected by simultaneous expression of CYP3A4. This study also examined the catalytic properties of the allelic variants of UGT1A1 and 1A7 and their effects on the interaction with CYP3A4. Although the UGT1A1-catalyzing activity of 4-methylumbelliferone glucuronidation was reduced in its variant, UGT1A1*6, the coexpression of CYP3A4 restored the impaired function to a level comparable with the wild type. Similarly, simultaneous expression of CYP3A4 increased the Vmax of UGT1A7*1 (wild type) and *2 (N129K and R131K), whereas the same was not observed in UGT1A7*3 (N129K, R131K, and W208R). In the kinetics involving different concentrations of UDP-glucuronic acid (UDP-GlcUA), the Km for UDP-GlcUA was significantly higher for UGT1A7*2 and *3 than *1. The Km of UGT1A7*1 and *3 was increased by CYP3A4, whereas *2 did not exhibit any such change. These results suggest that (1) CYP3A4 changes the catalytic function of the UGT1A subfamily in a UGT isoform-specific manner and (2) nonsynonymous mutations in UGT1A7*3 reduce not only the ability of UGT to use UDP-GlcUA but also CYP3A4-mediated enhancement of catalytic activity, whereas CYP3A4 is able to restore the UGT1A1*6 function.
Footnotes
- Received September 24, 2013.
- Accepted November 19, 2013.
↵1 Current affiliation: Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Hiroshima, Japan.
↵2 Current affiliation; Faculty of Pharmaceutical Sciences, Toyama University, Toyama, Japan.
This work was supported in part by the Japan Society for Promotion of Science [Grants-in-Aid for Scientific Research (B) Research No. 25293039 and Grants-in-Aid for Scientific Research (C) Research No. 21590164 to Y. I.].
Presented in part at the 25th Annual Meeting of the Japanese Society for the Study of Xenobiotics, Ohmiya, Japan October, 2010 (Koba et al.); the 27th Kyushu Regional Meeting for the Pharmaceutical Society of Japan, Nagasaki, Japan, December 2010 (Koba et al.); the 19th Microsomes and Drug Oxidations (MDO) and 12th European International Society for the Study of Xenobiotics (ISSX) Meeting, Noordwijk aan Zee, The Netherlands, June 2012 (Ishii et al.); the 28th Annual Meeting of the Japanese Society for the Study of Xenobiotics, Tokyo, Japan, October 2013 (Kinoshita et al.)
↵This article has supplemental material available at dmd.aspetjournals.org.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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