Abstract
Our previous study suggested that hetero-oligomer formation of guinea pig liver UDP-glucuronosyltransferases (UGTs) 2B21 and 2B22 enhances UGT2B21-catalyzed morphine-6-glucuronidation. In this work, further evidence for a functional hetero-oligomer between UGT2B21 and UGT2B22 was provided by studies of the glucuronidation of chloramphenicol with dual expression in COS-7 cells. UGT2B21 expressed in COS cells was capable of glucuronidating the 3-hydroxyl group of morphine, 4-hydroxybiphenyl, borneol, testosterone, androsterone, and estriol, whereas it had some effect on chloramphenicol. On the contrary, UGT2B22 does not exhibit any significant activity toward these typical substrates tested in this study. When UGT2B21 and UGT2B22 were expressed simultaneously, the chloramphenicol glucuronidation was enhanced to 4.5-fold, whereas the activities toward other substrates were little affected except that for the 6-hydroxyl group of morphine. The protein expression level of UGT2B21 was comparable when UGT2B21 was expressed with or without UGT2B22. These results suggest that simultaneous expression of UGT2B21 and UGT2B22 enhances UGT2B21-catalyzed chloramphenicol glucuronidation. Hetero-oligomer formation of UGT2B21 and UGT2B22 may act by fine-tuning the catalytic glucuronidation of chloramphenicol.
Glucuronidation is well known as a major detoxification pathway for both exogenous and endogenous compounds. Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs), members of a superfamily of glycosyltransferases that are found in the endoplasmic reticulum membrane (Mackenzie et al., 1997). The two UGT gene families involved in glucuronidation are divided into two groups, UGT1 and UGT2, on the basis of evolutionary divergence. The N-terminal region of UGT is now believed to be an important determinant of substrate specificity (Mackenzie et al., 1997, references therein).
Experimental evidence suggests that UGTs function as a dimer or tetramer (Gschaidmeier and Bock, 1994; Koiwai et al., 1996; Sato et al., 1996; Ishii et al., 2001). It has been shown that rat UGT2B1, human UGT1A1, and human UGT1A9 form homo-oligomers (Meech and Mackenzie, 1997; Ghosh et al., 2001; Kurkela et al., 2003). Heterodimerization of the UGT2B1 and UGT1A subfamily has also been reported (Ikushiro et al., 1997). It remains to be clarified whether such UGT oligomerizations are functionally relevant, although a change in steroid-UGT activity by coexpression of two catalytically inactive forms of rat liver UGT2B1 has been reported (Meech and Mackenzie, 1997). Our previous study suggested that the hetero-oligomer of guinea pig UGT2B21 and UGT2B22 exhibits a regioselectivity toward morphine glucuronidation different from that of either homo-oligomer, consisting of the respective UGTs (Ishii et al., 2001). However, little is known about the effect of hetero-oligomer formation on the substrate specificity except for morphine glucuronidation. UGT2B22 shows no substantial activity as far as morphine is concerned. However, when UGT2B21 and UGT2B22 were simultaneously expressed in COS-7 cells, extensive glucuronidation at the 6-hydroxyl position of morphine was observed, whereas the 3-hydroxyl position was much less affected. The catalytic activity of UGT2B21 and UGT2B22 hetero-oligomer (Ishii et al., 2001) satisfactorily explains the marked formation of morphine-6-glucuronide (M-6-G), which is a potent metabolite of morphine (Shimomura et al., 1971), in the guinea pig under in vivo and in vitro conditions (Kuo et al., 1991). It is likely that oligomer formation is a general property of the UGT, and so such hetero-oligomer-derived alterations in substrate specificity seem to be important because the physiological function of UGT isoforms may be underestimated in current investigations connecting on polymorphisms and postsequence pharmacogenomics. In this work, further evidence of a functional hetero-oligomer between UGT2B21 and UGT2B22 is provided by studies of the glucuronidation of chloramphenicol.
Materials and Methods
Materials. UDP-Glucuronic acid (UDP-GlcA) was purchased from Seikagaku Kogyo Co., Ltd. (Tokyo, Japan). UDP-[U-14C]GlcA was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Egg yolk l-α-phosphatidyl choline was obtained from Sigma-Aldrich (St. Louis, MO). Chloramphenicol was purchased from Nacalai Tesque (Kyoto, Japan). An alkaline phosphatase-labeled rabbit antibody to goat IgG was purchased from Cappel Laboratories (Durham, NC). Morphine-3-glucuronide and M-6-G were synthesized in our laboratory (Yoshimura et al., 1968).
Transient Expression of UGT2B21 and UGT2B22 in COS Cells. COS-7 cells (JCRB9127) were obtained from the Japanese Collection of Research Bioresources through the Health Science Research Resources Bank and maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen). The transfection was performed as described previously with expression plasmid for UGT2B21 (pSVL-UGT2B21) (DDBJ/GenBank/EMBL database with accession number AB034987) and UGT2B22 (pTARGET-UGT2B22) (DDBJ/GenBank/EMBL database with accession number AB03988). pSVL-SV40-vector (Amersham Biosciences AB, Uppsala, Sweden) was used for mock-transfection. Cells were harvested and microsomes were prepared.
Assays. UGT activity toward chloramphenicol, borneol, testosterone, androsterone, and estriol was determined according to the method of Bansal and Gessner (1980). UGT activity toward 4-hydroxybiphenyl was measured fluorometrically (Bock et al., 1979). Morphine UGT activity was assayed by a method described elsewhere (Kuo et al., 1991) with slight modifications (Oguri et al., 1996). Protein was measured by the method of Lowry et al. (1951) with bovine serum albumin as a standard.
Immunoblotting. Protein separated by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) was electroblotted on to a polyvinylidine difluoride membrane and reacted with goat anti-mouse low pI form UGT antibody (Mackenzie et al., 1984) as the primary antibody. Immunochemical staining was performed after reaction with alkaline phosphatase-labeled secondary antibody according to the procedure of Blake et al. (1984).
Results
Substrate Specificity of UGT2B21. The substrate specificity of UGT2B21, transiently expressed in COS cells, was examined using UDP-[U-14C]GlcA as a cosubstrate. The results showed that UGT2B21 catalyzed the glucuronidation of 4-hydroxybiphenyl, borneol, androsterone, testosterone, and estriol (Table 1). Although glucuronidation of 3α-hydroxysteroids and 17β-hydroxysteroids is catalyzed by different isoforms in rat liver (Falany et al., 1987), guinea pig liver UGT2B21 was capable of glucuronidating androsterone as well as testosterone, and these features were similar to those of human UGT2B17 (Baulieu et al., 1996). However, the UGT activity toward estriol was higher than that toward both androgens. Although M-6-G formation was not detectable in the assay condition, it was due to the incubation time of 30 min. UGT2B21 catalyzes morphine 3-glucuronidation and is also capable of glucuronidating the 6-hydroxyl group to some extent (Ishii et al., 2001).
Enhancement of Chloramphenicol Glucuronidation by Simultaneous Expression of UGT2B21 and UGT2B22. To examine the chloramphenicol glucuronidation activity of UGT2B21 and UGT2B22, these UGTs were transiently expressed in COS-7 cells. As shown in Figs. 1 and 2, chloramphenicol glucuronidation was catalyzed by UGT2B21-expressed COS-7 cell microsomes to some extent. However, UGT2B22 did not show any glucuronidation activity toward chloramphenicol. In accordance with our previous study describing the functional hetero-oligomer of UGT2B21 and UGT2B22 (Ishii et al., 2001), COS-7 cells were simultaneously transfected with expression plasmids for UGT2B21 and UGT2B22. The immunoblots showed that the UGT2B21 level in COS-7 cell microsomes is similar for single and dual expression (Fig. 3). The 55-kDa and 59-kDa bands were observed when COS-7 cells were transfected with UGT2B21 and UGT2B22 cDNA, respectively. COS-7 cell microsomes, simultaneously expressing UGT2B21 and UGT2B22, were investigated to determine their UGT activity toward chloramphenicol (Figs. 1 and 2). Chloramphenicol glucuronide formation was 4.5-fold higher than that of the UGT2B21 single transfection. Other activities associated with the UGT2B21 isoform (borneol, 4-hydroxybiphenyl, testosterone, androsterone, estriol, and the 3-hydroxyl group of morphine) were not markedly affected by cotransfection of UGT2B22, whereas the activity toward the 6-hydroxyl group of morphine was enhanced (Table 1). These data suggest that the simultaneous expression of UGT2B21 and UGT2B22 in COS-7 cell microsomes enhances UGT2B21-catalyzed chloramphenicol glucuronidation.
Discussion
Further evidence of a functional hetero-oligomer of UGT2B21 and UGT2B22, which are involved in the extensive formation of chloramphenicol glucuronide, has been obtained. UGT2B21 shows glucuronidation activity toward chloramphenicol to some extent, whereas UGT2B21 catalyzes glucuronidation of the 3-hydroxyl group of morphine. However, its activity toward the 6-hydroxyl group is much less (Ishii et al., 2001). It is interesting that UGT2B22 did not show any glucuronidation activity toward chloramphenicol as well as morphine (Ishii et al., 2001). Our previous study showed that simultaneous expression of UGT2B21 and UGT2B22 in COS-7 cell microsomes resulted in enhanced M-6-G formation. Therefore, cotransfection experiments with UGT2B21 and UGT2B22 were carried out to see whether chloramphenicol glucuronidation could also be affected. The chloramphenicol glucuronide formation was markedly enhanced by coexpression of UGT2B21 and UGT2B22.
It is suggested that a new substrate, chloramphenicol, has been identified, which is more extensively glucuronidated by the hetero-oligomer of UGT2B21 and UGT2B22 than by the homo-oligomer of UGT2B21. Previous work showed that detergent treatment to the mixture of independently expressed UGT2B21 and UGT2B22 did not alter UGT2B21-catalyzed morphine glucuronidation (Ishii et al., 2001). It seems reasonable to conclude that UGT hetero-oligomer formation alters the substrate specificity, rendering it different from that of the homo-oligomers.
Steroids such as estrogen, and 3α-hydroxy and 17β-hydroxy androgen are substrates of UGT2B21. 4-Hydroxybiphenyl and a terpenoid, borneol, are also substrates of UGT2B21. UGT2B21 exhibited relatively low chloramphenicol glucuronidation activity, whereas rat UGT2B1 is the only isoform that has been reported to catalyze chloramphenicol glucuronidation (Mackenzie, 1987). As far as we know, there have been no reports claiming that a UGT isoform like UGT2B22 is involved in the enhancement of chloramphenicol glucuronidation. Furthermore, the human UGT(s) involved in chloramphenicol glucuronidation remains to be identified. It is possible that similar phenomena will be observed in humans. Cotransfection of UGT2B22 did not result in a large magnitude of the activity of UGT2B21 toward the substrates other than morphine and chloramphenicol. Thus, hetero-oligomer of UGT2B21 and UGT2B22 may fine-tune this activity to be capable of glucuronidating chloramphenicol and the 6-hydroxyl group of morphine.
Although we have tested a limited range of substrates, no detectable activity of UGT2B22 was observed toward typical UGT substrates in our assay condition. Like UGT2B22, there have been reports of orphan UGT isoforms such as human UGT2B10 (Jin et al., 1993) and UGT2B11 (Baulieu et al., 1998), the substrates of which have not yet been identified. However, little attention has been paid to the active hetero-oligomers of human UGT isoforms. Thus, it is possible that the physiological functions of UGT isoforms are not fully elucidated when only one isoform expression system is used. Further investigations are necessary to investigate the impact of UGT active hetero-oligomers on polymorphisms and postsequence pharmacogenomics.
Acknowledgments
We are grateful to Prof. P. I. Mackenzie for providing anti-UGT antibody.
Footnotes
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Part of this research was supported by a grant-in-aid from the Ministry of Education, Sports, Culture, Science and Technology, Japan (Research No. 08557088).
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ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; M-6-G, morphine-6-glucuronide; UDP-GlcA, UDP-glucuronic acid; TLC, thin-layer chromatography.
- Received February 26, 2004.
- Accepted June 29, 2004.
- The American Society for Pharmacology and Experimental Therapeutics