Simultaneous Expression of Guinea Pig UDP-Glucuronosyltransferase 2B21 and 2B22 in COS-7 Cells enhances UDP-Glucuronosyltransferase 2B21-Catalyzed Morphine-6-Glucuronide Formation
- Yuji Ishii1,1,
- Aya Miyoshi1,
- Ryoichi Watanabe1,
- Kazuoki Tsuruda1,
- Minoru Tsuda1,1,
- Yuki Yamaguchi-Nagamatsu1,
- Kunihiro Yoshisue1,
- Mitsuko Tanaka,
- Daisuke Maji1,
- Satoru Ohgiya2 and
- Kazuta Oguri1
- 1Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan (Y.I, A.M., R.W., K.T., M.T., Y.Y.-N., K.Y, M.T., D.M., K.O.); and 2Hokkaido National Industrial Research Institute, Sapporo, Japan (S.O.)
- Prof. Kazuta Oguri, Ph.D., Graduate School of Pharmaceutical Sciences, Kyushu University 62, 3–1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail:oguri{at}xenoba.phar.kyushu-u.ac.jp
Abstract
Although UDP-glucuronosyltransferases (UGTs) act as an important detoxification system for many endogenous and exogenous compounds, they are also involved in the metabolic activation of morphine to form morphine-6-glucuronide (M-6-G). The cDNAs encoding guinea pig liver UGT2B21 and UGT2B22, which are intimately involved in M-6-G formation, have been cloned and characterized. Although some evidence suggests that UGTs may function as oligomers, it is not known whether hetero-oligomer formation leads to differences in substrate specificity. In this work, evidence for a functional hetero-oligomer between UGT2B21 and UGT2B22 is provided by studies on the glucuronidation of morphine in transfected COS-7 cells. Cells transfected with UGT2B21 cDNA catalyzed mainly morphine-3-glucuronide formation although M-6-G was also formed to some extent. In contrast, cells transfected with UGT2B22 cDNA did not show any significant activity toward morphine. When UGT2B21 and UGT2B22 were expressed simultaneously in different ratios in COS-7 cells, extensive M-6-G formation was observed. This stimulation of M-6-G formation was not observed, however, when microsomes containing UGT2B21were mixed with those containing UGT2B22 in the presence of detergent. Furthermore, this effect was not very marked when human UGT1A1 and UGT2B21 were coexpressed in COS-7 cells. This is the first report suggesting that UGT hetero-oligomer formation leads to altered substrate specificity.
Footnotes
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↵1 Present address: Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan.
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Part of this research was supported by a grant-in-aid from the Ministry of Education, Sports and Culture, Japan.
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Presented in part at 15th Annual Meeting of Japanese Society for Study of Xenobiotics, Fukuoka, Japan, October 2000 (Ishii et al.); the IXth International Workshop on Glucuronidation and the UDP-Glucuronosyltransferases, Brisbane, Australia, October 1998 (Miyoshi et al.); 116th Annual Meeting of the Japanese Society of Pharmaceutical Sciences, Kanazawa, Japan, March 1996 (Tsuruda et al.); and the 12th Kyushu Regional Meeting of Japanese Society of Pharmaceutical Science, Fukuoka, Japan, December 1995 (Tsuda et al.).
- Abbreviations:
- M-6-G
- morphine-6-glucuronide
- UGT
- UDP-glucuronosyltransferase
- M-3-G
- morphine-3-glucuronide
- UDP-GlcA
- UDP-glucuronic acid
- Ptd-choline
- l-α-phosphatidyl choline
- PVDF
- polyvinylidene difluoride
- PAGE
- polyacrylamide gel electrophoresis
- PCR
- polymerase chain reaction
- bp
- base pair(s)
- RACE
- rapid amplification of cDNA ends
- ORF
- open reading frame
- AP1
- adapter primer 1
- CMV
- cytomegalovirus
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- Received January 2, 2001.
- Accepted July 25, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
