Amino acid positions 69–132 of UGT1A9 are involved in the C-glucuronidation of phenylbutazone

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Abstract

Phenylbutazone (PB) is known to be biotransformed to its O- and C-glucuronide. Recently, we reported that PB C-glucuronide formation is catalyzed by UGT1A9. Interestingly, despite UGT1A8 sharing high amino acid sequence identity with UGT1A9, UGT1A8 had no PB C-glucuronidating activity. In the present study, we constructed eight UGT1A9/UGT1A8 chimeras and evaluated which region is important for PB C-glucuronide formation. All of the chimeras and UGT1A8 and UGT1A9 had 7-hydroxy-(4-trifluoromethyl)coumarin (HFC) O-glucuronidating activity. The Km values for HFC glucuronidation of UGT1A8, UGT1A9 and their chimeras were divided into two types, UGT1A8 type (high Km) and UGT1A9 type (low Km), and these types were determined according to whether their amino acids at positions 69–132 were those of UGT1A8 or UGT1A9. Likewise, PB O-glucuronidating activity was also detected by all of the chimeras, and their Km values were divided into two types. On the contrary, PB C-glucuronidating activity was detected by UGT1A9(1–132)/1A8(133–286), UGT1A9(1–212)/1A8(213–286), UGT1A8(1–68)/1A9(69–286), and UGT1A8(1–68)/1A9(69–132)/1A8(133–286) chimeras. The region 1A9(69–132) was common among chimeras having PB C-glucuronidating activity. Of interest is that UGT1A9(1–68)/1A8(69–132)/1A9(133–286) had lost PB C-glucuronidation activity, but retained activities of PB and HFC O-glucuronidation. These results strongly suggested that amino acid positions 69–132 of UGT1A9 are responsible for chemoselectivity for PB and affinity to substrates such as PB and HFC.

Section snippets

Chemicals

7-Hydroxy-(4-trifluoromethyl) coumarin (HFC) glucuronide, PB and UDPGA were purchased from Sigma–Aldrich (St. Louis, MO). HFC was purchased from Kanto Chemicals Co. (Tokyo, Japan). Bac-to-Bac baculovirus expression system, Cellfectin transfection reagent, SF900 II and Sf9 cells were purchased from Invitrogen (Carlsbad, CA). Anti-UGT1A antibody (WB-UGT1A) was purchased from BD Gentest (Woburn, MA). ECLTM advance Western blotting detection kit was purchased from GE Healthcare (Piscataway, NJ).

Expression of chimeric and wild type UGT proteins

Expression level of each protein in Sf9 microsomes was determined (Fig. 2). Relative expression levels of UGT1A9, UGT1A9(1–68)/1A8(69–286), UGT1A9(1–132)/1A8(133–286), UGT1A9(1–212)/1A8(213–286), UGT1A8(1–68)/1A9(69–286), UGT1A8(1–132)/1A9(133–286), UGT1A8(1–212)/1A9(213–286), UGT1A8(1–68)/1A9(69–132)/1A8(133–286), UGT1A9(1–68)/1A8(69–132)/1A9(133–286) and UGT1A8 were 1, 1.2, 1.5, 3, 1.5, 1.6, 1.8, 1.7, 0.9 and 2.5, respectively. All enzymatic activities expressed in this paper have been

Discussion

We recently reported that UGT1A9 catalyzes PB C-glucuronidation [10]. After that report, Kerdpin et al. [22] also revealed that sulfinpyrazone, a derivative of PB, is C-glucuronidated by mainly UGT1A9 and, to a minor extent, by UGTs 1A7 and 1A10. From these results, the question arose as to why despite UGTs 1A7, 1A8, 1A9 and 1A10 having high amino acid sequence identity with each other, only UGT1A8 did not show PB and sulfinpyrazone C-glucuronidating activity. In the present study, to

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