Human UDP-Glucuronosyltransferase Expression in Insect Cells: Ratio of Active to Inactive Recombinant Proteins and the Effects of a C-Terminal His-Tag on Glucuronidation Kinetics

Zhang, Hongbo; Patana, Anne-Sisko; Mackenzie, Peter I.; Ikushiro, Shinichi; Goldman, Adrian; Finel, Moshe

doi:10.1124/dmd.112.046086

Cited by 28 publications

(18 citation statements)

References 21 publications

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“…Briefly, the zebrafish Ugt open reading frame from each of the 40 pGEM-T plasmids (Huang and Wu, 2010) was subcloned with primers (Supplemental Table 2) into the expression vector pcDNA3 containing a myc tag and confirmed by sequencing. It has been shown that the C-terminal tag does not interfere with the glucuronidating activity of the recombinant human UGT protein (Zhang et al, 2012). For recombinant zebrafish UGT production, human embryonic kidney (HEK)293T cells were transfected at 90% confluency in a 10-cm dish with 12 mg of experimental or mock plasmids using Lipofectamine 2000.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the ZebrafishUgtRepertoire Reveals a New Class of Drug-Metabolizing UDP Glucuronosyltransferases

Wang¹,

Huang²,

Wu³

2014

Mol Pharmacol

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The zebrafish genome contains a gene superfamily of 40 Ugt genes that can be divided into Ugt1, Ugt2, and Ugt5 families. Because the encoded zebrafish UDP glucuronosyltransferase (UGT) proteins do not display orthologous relationships to any of the mammalian and avian UGT enzymes based on molecular phylogeny, it is difficult to predict their substrate specificity. Here, we mapped their tissue-specific expression patterns. We showed that the zebrafish UGT enzymes can be glycosylated. We determined their substrate specificity and catalytic activity toward diverse aglycone substrates. Specifically, we measured mRNA levels of each of the 40 zebrafish Ugt genes in 11 adult tissues and found that they are expressed in a tissue-specific manner. Moreover, functional analyses with the donor of UDP glucuronic acid (UDPGA) for each of the 40 zebrafish UGT proteins revealed their substrate specificity toward 10 important aglycones. In particular, UGT1A1, UGT1A7, and UGT1B1 displayed good glucuronidation activities toward most phenolic aglycones (4-methylumbelliferone, 4-nitrophenol, 1-naphthol, bisphenol A, and mycophenolic acid) and the two carboxylic acids (bilirubin and diclofenac). Importantly, some members of the UGT5, a novel UGT family identified recently, are capable of glucuronidating multiple aglycones with the donor cofactor of UDPGA. In particular, UGT5A5, UGT5B2, and UGT5E1 glucuronidate phenols and steroids with high specificity toward steroid hormones of estradiol and testosterone and estrogenic alkylphenols 4-tert-octylphenol. These results shed new insights into the mechanisms by which fish species defend themselves against vast numbers of xenobiotics via glucuronidation conjugations and may facilitate the establishment of zebrafish as a model vertebrate in toxicological, developmental, and pathologic studies.

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Section: Methodsmentioning

confidence: 99%

Characterization of the ZebrafishUgtRepertoire Reveals a New Class of Drug-Metabolizing UDP Glucuronosyltransferases

Wang¹,

Huang²,

Wu³

2014

Mol Pharmacol

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“…The different recombinant His-tagged dog UGTs were produced in sf9 insect cells, and cell homogenates were used as the study material in the activity assays. Total protein concentrations and relative expression levels of each recombinant dUGT were determined as described previously elsewhere (Zhang et al, 2012a).…”

Section: Methodsmentioning

confidence: 99%

Dog UDP-Glucuronosyltransferase Enzymes of Subfamily 1A: Cloning, Expression, and Activity

et al. 2014

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Understanding drug glucuronidation in the dog, a preclinical animal, is important but currently poorly characterized at the level of individual enzymes. We have constructed cDNAs for the 10 dog UDP-glucuronosyltransferases of subfamily 1A (dUGT1As), expressed them in insect cells, and assayed their activity as well as the activity of the nine human UGT1As, toward 14 compounds. The goal was to find out whether individual dUGT1As and individual human UGT1As have similar substrate specificities. The results revealed similarities but also many differences. For example, similarly to the human UGT1A10, dUGT1A11 exhibited high glucuronidation activity toward the 3-OH of 17-b-estradiol, 17-a-estradiol, and ethinylestradiol, and also conjugated the drug entacapone. Unlike the human UGT1A10, however, it failed to catalyze considerable rates of R-propranolol, diclofenac, and indomethacin glucuronidation.The estrogen glucuronidation assays revealed that dUGT1A8 and dUGT1A10 have a capacity to catalyze the formation of (linked) diglucuronides, an activity no human UGT1A exhibited. dUGT1A2-dUGT1A4 are homologs of the human UGT1A4, but none of them catalyzed N-glucuronidation of dexmedetomidine. Contrary to the human UGT1A4, however, dUGT1A2-dUGT1A4 catalyzed indomethacin and diclofenac glucuronidation. It may be concluded that, perhaps with the exception of UGT1A6, high similarities in substrate specificity between individual dog and human UGTs of subfamily 1A are rare or partial. Activity assays with liver and intestine microsomes of both dog and human further revealed interspecies differences, particularly in glucuronidation rates. In the dog, the microsomes assays also strongly suggested important roles for dUGTs of other subfamilies, mainly in the liver.

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“…30.6% coefficient of variation for rUGT1A4), which may hinder IVIVE-based prediction of both hepatic and renal glucuronidation clearance (14). In addition, UGTs expressed in insect cells may have a substantial amount of inactive protein present that can be reduced by lowering the amount of baculovirus used to infect cells (21). Therefore, the use of metabolic rate data from recombinant UGTs in a quantitative IVIVE setting may require correction for presence of inactive enzyme.…”

Section: Use Of In Vitro Systems To Understand Renal Drug Eliminationmentioning

confidence: 99%

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

Scotcher

Jones²,

Posada

et al. 2016

AAPS J

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ABSTRACT.The programme for the 2015 AAPS Annual Meeting and Exhibition (Orlando, FL; 25-29 October 2015) included a sunrise session presenting an overview of the state-of-the-art tools for in vitro-in vivo extrapolation (IVIVE) and mechanistic prediction of renal drug disposition. These concepts are based on approaches developed for prediction of hepatic clearance, with consideration of scaling factors physiologically relevant to kidney and the unique and complex structural organisation of this organ. Physiologically relevant kidney models require a number of parameters for mechanistic description of processes, supported by quantitative information on renal physiology (system parameters) and in vitro/in silico drug-related data. This review expands upon the themes raised during the session and highlights the importance of high quality in vitro drug data generated in appropriate experimental setup and robust system-related information for successful IVIVE of renal drug disposition. The different in vitro systems available for studying renal drug metabolism and transport are summarised and recent developments involving state-of-the-art technologies highlighted. Current gaps and uncertainties associated with system parameters related to human kidney for the development of physiologically based pharmacokinetic (PBPK) model and quantitative prediction of renal drug disposition, excretion, and/or metabolism are identified.

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Human UDP-Glucuronosyltransferase Expression in Insect Cells: Ratio of Active to Inactive Recombinant Proteins and the Effects of a C-Terminal His-Tag on Glucuronidation Kinetics

Cited by 28 publications

References 21 publications

Characterization of the ZebrafishUgtRepertoire Reveals a New Class of Drug-Metabolizing UDP Glucuronosyltransferases

Characterization of the ZebrafishUgtRepertoire Reveals a New Class of Drug-Metabolizing UDP Glucuronosyltransferases

Dog UDP-Glucuronosyltransferase Enzymes of Subfamily 1A: Cloning, Expression, and Activity

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

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