cDNA Cloning and Characterization of the Human UDP Glucuronosyltransferase, UGT1A3

doi:10.1006/bbrc.1996.1251

Biochemical and Biophysical Research Communications

Volume 225, Issue 3, 23 August 1996, Pages 785-790

https://doi.org/10.1006/bbrc.1996.1251 Get rights and content

Abstract

The cDNA encoding the UDP glucuronosyltransferase, UGT1A3, has been cloned and expressed in cell culture. The deduced amino acid sequence of the cDNA is 90% similar in sequence to that of a previously characterized form, UGT1A4 and to that of a third form, UGT1A5 whose function in unknown. UGT1A3, when expressed in COS cells has a relative molecular mass of 55 kDa and is active in the glucuronidation of estrone and 2-hydroxyestrone. Activity towards other polyhydroxylated estrogens including 4-hydroxyestrogen and estriol and its metabolites was not detected. UGT1A3 was also inactive towards androgens and their metabolites. The enzyme preferentially glucuronidated the N-hydroxy metabolite of 2-acetylaminofluorene and was more active towards the 6- and 12-hydroxylated metabolites of benzo[α]pyrene. RNA encoding UGT1A3 was detected in human liver and colon tissue.

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Cited by (103)

New insights for risks of chlorophenols (CPs) exposure: Inhibition of UDP-glucuronosyltransferases (UGTs)
2018, Chemosphere
Chlorophenols (CPs) are important pollutants extensively utilized in industry, agriculture and forestry. The present study aims to determine the inhibition of CPs on the activity of the important phase II drug-metabolizing enzymes (DMEs) UDP-glucuronosyltransferases (UGTs). 100 μM of fourteen CPs were used for preliminary screening using in vitro incubation. Furthermore, half inhibition concentration (IC₅₀) and inhibition kinetics were determined for CPs with significant inhibition towards UGT isoforms. In silico docking was used to explain the inhibition difference among CPs. Multiple UGT isoforms were inhibited by CPs. In silico docking showed that higher free binding energy due to hydrophobic interactions of 2.4-Dichlorophenol (2.4-DCP) or 4-Chloro-3-methylphenol (4C3MP) with UGT1A9 contributed to stronger inhibition potential of 2.4-Dichlorophenol (2.4-DCP) or 4-Chloro-3-methylphenol (4C3MP) towards UGT1A9 than 4-CP. Pentachlorophenol (PCP) was chosen as the representative CPs to determine the IC₅₀ value towards UGT1A6, UGT1A9 and UGT2B7. IC₅₀ was calculated to be 0.33 μM, 0.24 μM and 31.35 μM for the inhibition of PCP towards UGT1A6, UGT1A9 and UGT2B7. PCP was demonstrated to show competitive inhibition towards UGT1A6, UGT1A9 and UGT2B7, and the inhibition kinetic parameters (Ki) was calculated to be 0.18 μM, 0.01 μM and 5.37 μM for the inhibition of PCP towards UGT1A6, UGT1A9 and UGT2B7. All these information will be beneficial for elucidating the risk of CPs exposure from a new perspective.
UDP-Glycosyltransferases
2018, Comprehensive Toxicology: Third Edition
The UDP-glycosyltransferase enzyme superfamily (UGT) consists of UDP-glucuronosyltransferases (the UGT1 and 2 families), UDP N-acetylglucosaminyltransferase (UGT3A1), UDP-glucosyl/xylosyltransferase (UGT3A2), and UDP-galactosyltransferase (UGT8) that conjugate glucuronic acid, N-acetylglucosamine, glucose, xylose, and galactose sugars to nucleophilic O, N, S, and C atoms on lipophilic compounds via a nucleophilic bimolecular substitution (S_N2) mechanism. The aglycone substrates glycosidated include drugs, environmental chemicals, and endogenous compounds such as bilirubin and steroid hormones. The glycoside products are water-soluble and readily excreted. There are 22 functional human UGTs belonging to four UGT families: UGT1 containing nine members, UGT2 containing seven UGT2B and three UGT2A members, UGT3 containing two members, and UGT8 containing one member. These UGTs reside on the luminal side of the endoplasmic reticulum and consist of two domains, an N-terminal domain involved in substrate selection and catalysis and a C-terminal domain responsible for binding the sugar donor, UDP glucuronic acid, UDP-glucose, UDP-xylose, UDP-N-acetylglucosamine, or UDP-galactose depending on UGT isoform. The UGT protein is integrated into the endoplasmic reticulum membrane by a hydrophobic series of amino acids near the C-terminus and appears to form homo- or heterodimers with other UGT proteins and complexes with other drug-metabolizing enzymes. UGT genes are regulated by several liver-enriched transcription factors (LETFs) such as HNF1 and HNF4α and by ligand-activated transcription factors (TFs) such as AhR, Nrf2, PXR, CAR, PPAR, FXR, LXR, ER, and AR. Regulation by these TFs ensures that each tissue or organ has a distinct complement of UGTs. Several polymorphisms in UGT genes and their regulatory regions have been shown to alter the glycosidation capacity of tissues and organs and to be risk factors for disease and adverse drug events.
Glucuronidation of estrone and 16α-hydroxyestrone by human UGT enzymes: The key roles of UGT1A10 and UGT2B7
2015, Journal of Steroid Biochemistry and Molecular Biology
The glucuronidation of estrone and 16α-hydroxyestrone by recombinant human UDP-glucuronosyltransferase enzymes (UGTs) of subfamilies 1A, 2A and 2B was studied. Microsomes from human liver and small intestine were also tested for the glucuronidation of these two estrogens. The results revealed that UGT1A10 is by far the most active enzyme in estrone glucuronidation. UGT1A10 also exhibited high rate of 16α-hydroxyestrone conjugation at the 3-OH, whereas UGT2B7 catalyzed its glucuronidation at high rates at the 16-OH. Human liver microsomes exhibited high rates of 16α-hydroxyestrone-16-glucuronide formation, but very low formation rates of either 16α-hydroxyestrone-3-glucuronide or estrone glucuronide. On the other hand, human intestine microsomes catalyzed the formation of all these 3 different glucuronides at high rates. Kinetic analyses revealed very low K_m value for 16α-hydroxyestrone glucuronidation by UGT2B7, below 4 μM, suggesting higher affinity than commonly found among UGTs and their substrates. In further studies with UGT1A10, mutant F93G exhibited increased glucuronidation rates of 16α-hydroxyestrone, but not estrone, whereas mutations in F90 did not reveal any activity with either estrogen. Taken together, the results of this study significantly expand our understanding on the metabolism of estrogens and their interactions with the human UGTs.
A comprehensive review of UDP-glucuronosyltransferase and esterases for drug development
2015, Drug Metabolism and Pharmacokinetics
Citation Excerpt :
As for inhibitors, atazanavir and erlotinib are known to specifically inhibit this enzyme [119,120]. UGT1A3: UGT1A3 metabolizes endobiotics such as bile acid (CDCA, LCA, HDCA) [48,121] and xenobiotics such as polyaromatic hydrocarbons [122], amines [123], non-steroidal anti-inflammatory drugs (NSAIDs) [124], and statins [125]. Despite the high sequence similarity (amino acid homology of 93%) with UGT1A4 in particular, UGT1A3 and UGT1A4 differ in terms of substrate selectivity, i.e., UGT1A3 catalyzes the acyl glucuronidation of carboxylic acid-containing drugs but UGT1A4 does so only minimally [126].
UDP-glucuronosyltransferase (UGT) and esterases are recognized as the most important non-P450 enzymes because of their high contribution to drug metabolism. UGTs catalyze the transfer of glucuronic acid to hydroxyl, carboxyl, or amine groups of compounds, whereas esterases hydrolyze compounds that contain ester, amide, and thioester bonds. These enzymes, in most cases, convert hydrophobic compounds to water-soluble metabolites to facilitate the elimination of compounds from the body. Information about these enzymes is steadily increasing, although our knowledge is still behind our understanding of P450. This review gives an overview of recent findings in UGT and esterases studies focusing on tissue distribution, gene regulation, substrate and inhibitor specificity, and species differences. In particular, the absolute protein content of UGT isoforms and esterases in human tissues could be available. In the field of esterases, it is becoming clear that enzymes other than carboxylesterase are involved in drug hydrolysis. In addition, there is an interesting interplay between UGTs and esterases in the formation and hydrolytic deglucuronidation of acyl-glucuronide, which is considered to be a reactive metabolite. With the growing awareness of the importance of non-P450 enzymes in drug development, issues that should be resolved are discussed.
UDP-Glucuronosyltransferases
2010, Comprehensive Toxicology, Second Edition
The UDP-glucuronosyltransferases (UGT) are enzymes that conjugate glucuronic acid to nucleophilic O, N, S, and C atoms on lipophilic compounds via a nucleophilic bimolecular (S_N2) mechanism. The aglycone substrates glucuronidated include drugs, environmental toxins, and endogenous compounds such as bilirubin and steroid hormones. The glucuronide products are water-soluble and readily excreted. There are 19 functional human UGTs belonging to two UGT families: UGT1 containing nine members and UGT2 containing seven UGT2B and three UGT2A members. These UGTs reside on the luminal side of the endoplasmic reticulum and consist of two domains, an N-terminal domain involved in substrate selection and catalysis and a C-terminal domain responsible for binding the glucuronic acid donor, UDP glucuronic acid. The UGT protein is integrated into the endoplasmic reticulum membrane by a hydrophobic series of amino acids near the C-terminus and appears to form homo- or hetero-dimers with other UGT proteins and complexes with other drug metabolizing enzymes. UGT genes are regulated by several liver-enriched transcription factors such as HNF1 and HNF4α and by ligand-activated transcription factors such as AhR, Nrf2, PXR, CAR, PPAR, FXR, and LXR. Regulation by these transcription factors ensures that each tissue or organ has a distinct complement of UGTs. Several polymorphisms in UGT genes and their regulatory regions have been shown to alter the glucuronidation capacity of tissues and organs and to be risk factors for disease and adverse drug events.
Regulation of the human bile acid UDP-glucuronosyltransferase 1A3 by the farnesoid X receptor and bile acids
2010, Journal of Hepatology
Cholestasis is a serious complication of many liver diseases leading to increased serum bile acids (BA) and their conjugates. Chenodeoxycholic (CDCA) acid is a substrate of the human hepatic UDP-glucuronosyltransferase (UGT) 1A3. UGT1A3 may, therefore, be a BA-inducible gene relevant to BA regulation.
BA and human bile were used to induce UGT1A3 in HepG2 cells. Genomic DNA was analyzed by PCR amplification and sequencing. Transcriptional regulation was studied by DNA mutagenesis, RT-PCR, luciferase reporter gene constructs and electrophoretic mobility shift assays (EMSA).
CDCA differentially induced UGT1A3 but not UGT1A4 expression. Bile from ursodeoxycholic acid (UDCA)-treated and untreated patients differentially induced UGT1A3. A farnesoid X receptor (FXR) half-site DNA motif was identified in the UGT1A3 5′ upstream region. The FXR inducer GW4064 activated UGT1A3 transcription, and electrophoretic mobility shift assays identified UGT1A3 as a FXR target gene.
Transcriptional regulation of the human bile acid and xenobiotic UGT1A3 by its substrate CDCA and FXR is shown. CDCA glucuronidation can be controlled by feed back inhibition proceeding via the glucuronidation of CDCA. UDCA does not induce UGT1A3 transcription. Since UGT1A3 is significantly induced by xenobiotics this physiologically links xenobiotic and bile acid metabolism to cholestasis.

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Regular ArticlecDNA Cloning and Characterization of the Human UDP Glucuronosyltransferase, UGT1A3

Abstract

Regular Article
cDNA Cloning and Characterization of the Human UDP Glucuronosyltransferase, UGT1A3