Genomic clones of UDP-glucuronosyltransferase family 1 (UGT1) were isolated from wild-type Wistar rats. The UGT1 locus spans > 120 kb and forms a gene complex. In this locus nine unique first exons encoding NH2-terminal portions of each isoform were located at intervals of approximately 10 kb and followed by only one set of commonly used exons (exons II, III, IV, and V) encoding the COOH-terminal portion. From sequence analyses of the unique first exons, the amino acid sequences of the isoforms were deduced and they were divided into two groups: the Bilirubin cluster (B cluster) and the Phenol cluster (A cluster). A and B clusters consisted of four (A1-A4) and five (B1-B5) isoform-specific exons, respectively. A2, A3, B3, and B4 were identified as previously uncharacterized forms, while A4 and B4 were pseudogenes. Isoform B1 was a major component in hepatic microsomes of untreated rats and was induced in clofibrate- and dexamethasone-administered rats. A slight but a significant amount of B1 mRNA was also detected in various tissues such as intestine. mRNAs coding for isoform A1 and isoform A2 were induced in livers of methylcholanthrene (MC)-treated rats. Induction of A1 mRNA was also observed in kidneys of MC-treated rats. A genomic clone containing the commonly used exons was also isolated from Gunn rats and a single base deletion was identified in exon IV. Isoforms of the UGT1 family are made from the complex gene locus by an alternative combination of one of the unique first exons with the commonly used exons.
The interactions between UDP-glucuronosyltransferase (UGT) isozymes, UGT1s and UGT2B1, in rat hepatic microsomes were investigated using an immunopurification technique with anti-peptide antibodies and a chemical cross-linking strategy. A 50 kDa protein coimmunopurified with UGT1s was identified as UGT2B1 by amino-terminal sequencing and immunodetection with anti-peptide antibody against UGT2B1. Evidence for direct interaction of UGT2B1 with UGT1s was obtained by the loss of UGT2B1 adsorption to immunoaffinity column in Gunn rat hepatic microsomes, which lack all UGT1 isozymes. When the microsomes were treated with the chemical cross-linking reagent 1,6-bis(maleimido)-hexane, a cross-linked product with an apparent molecular mass of 120-130 kDa was obtained that immunostained with antibodies against UGT1s and UGT2B1, indicating the formation of a heterodimer containing one of the UGT1 isozymes and UGT2B1. The effects of UGT complex formation on the stimulation of glucuronidation of testosterone and uptake of UDP-glucuronic acid (UDP-GlcUA) by UDP-N-acetylglucosamine (UDP-GlcNAc) were examined. Alkaline pH-induced dissociation of the complexes was associated with the loss of UDP-GlcNAc-dependent stimulation of glucuronidation, suggesting that two functional states of UGTs with different kinetic parameters correspond to the monomer and oligomer form of UGTs in the membranes. The UDP-GlcNAc-dependent stimulation of UDP-GlcUA uptake into the microsomal vesicles also was affected by the extent of complex formation. These results suggest that complex formation of the UGT isozymes affects the UDP-GlcNAc-dependent stimulation of glucuronidation via stimulation of UDP-GlcUA uptake.
We have isolated genomic DNA clones containing rat UDP-glucuronosyltransferase family 1 (UGT1) sequences and have shown drug-responsive and tissue-specific alternative expression of multiple first exons (Emi, Y., Ikushiro, S., and Iyanagi, T. (1995) J. Biochem. (Tokyo) 117, 392-399). The UGT1 locus encodes at least nine UGT1 isoforms. UGT1A1 is a major 3-methylcholanthrene (MC)-inducible form in rat liver. In this report, we have identified a cis-acting element necessary for transcriptional activation of UGT1A1 in hepatocytes. A promoter region was fused to a chloramphenicol acetyltransferase gene, and the resultant construct was transiently transfected into hepatocytes. A DNA fragment carrying 1,100 nucleotides derived from the 5'-flanking region of the UGT1A1 gene was enough for MC induction. Unidirectional deletion of this region revealed that there existed one xenobiotic responsive element (XRE), TGCGTG, between -134 and -129. When a single base substitution was introduced into the XRE, MC-induced expression of the UGT1A1 gene was completely abolished. In addition, an XRE-deleted construct failed to respond to MC. Gel mobility shift assays showed MC-inducible binding of the nuclear aromatic hydrocarbon receptor-ligand complex to this motif. Gel shift-coupled DNase I protection analyses revealed that the GCGTG-core sequence was a target site of the liganded aromatic hydrocarbon receptor. These results suggest that the XRE participates in induction of the rat UGT1A1 gene by MC.
Bilirubin, the oxidative product of heme in mammals, is excreted into the bile after its esterification with glucuronic acid to polar mono- and diconjugated derivatives. The accumulation of unconjugated and conjugated bilirubin in the serum is caused by several types of hereditary disorder. The Crigler-Najjar syndrome is caused by a defect in the gene which encodes bilirubin UDP-glucuronosyltransferase (UGT), whereas the Dubin-Johnson syndrome is characterized by a defect in the gene which encodes the canalicular bilirubin conjugate export pump of hepatocytes. Animal models such as the unconjugated hyperbilirubinemic Gunn rat, the conjugated hyperbilirubinemic GY/TR-, and the Eisai hyperbilirubinemic rat, have contributed to the understanding of the molecular basis of hyperbilirubinemia in humans. Elucidation of both the structure of the UGT1 gene complex, and the Mrp2 (cMoat) gene which encodes the canalicular conjugate export pump, has led to a greater understanding of the genetic basis of hyperbilirubinemia.
Three forms of cytochrome P-450, tentatively designated P-450(M-1), P-450(M-2), and P-450(M-3), and one form of cytochrome P-450, P-450(F-1), were purified from the liver microsomes of untreated male and female rats, respectively. Each purified form of the cytochrome showed a single protein band on SDS-polyacrylamide gel electrophoresis, and gave a minimum molecular weight of 51,000 for P-450(M-1), 48,000 for P-450(M-2), 49,000 for P-450(M-3), and 50,000 for P-450(F-1). The carbon monoxide-difference spectra of reduced P-450(M-1), P-450(M-2), P-450(M-3), and P-450(F-1) showed an absorption maximum at 451, 451, 448, and 449 nm, respectively. Judging from the absolute absorption spectra, the four forms of cytochrome P-450 were of low-spin type in the oxidized forms. The antibodies against P-450(M-2) did not crossreact with the other forms in the Ouchterlony double diffusion test, whereas the immunodiffusion test showed immunocrossreactivity between P-450(M-1) and P-450(F-1), P-450(M-1) and P-450(M-3), and P-450(M-3) and P-450(F-1). The NH2-terminal amino acid sequences of the four forms confirmed that they were different molecular species, although significant homology was noticed among P-450(M-1), P-450(M-3), and P-450(F-1). The quantitation of P-450(M-1) and P-450(F-1) in liver microsomes by quantitative immunoprecipitation confirmed that these two forms of cytochrome P-450 were developmentally induced in male and female rats, respectively. P-450(M-2) was also developmentally induced in male rats. In a reconstituted system containing NADPH and NADPH-cytochrome P-450 reductase, P-450(M-1) oxidized benzphetamine at a high rate, whereas the other forms had low activity toward benzphetamine. None of the four forms showed high activity toward benzo(a)pyrene. P-450(M-1) catalyzed the hydroxylation testosterone at the 16 alpha and 2 alpha positions, whereas P-450(M-2) catalyzed the 15 alpha hydroxylation of the same substrate.
ABSTRACT:To clarify the UDP-glucuronosyltransferase (UGT) isoform(s) responsible for the glucuronidation of the thyroid hormone thyroxine (T 4 ) in the human liver, the T 4 glucuronidation activities of recombinant human UGT isoforms and microsomes from seven individual human livers were comparatively examined. Among the 12 recombinant human UGT1A and UGT2B subfamily enzymes examined, UGT1A1, UGT1A3, UGT1A9, and UGT1A10 showed definite activities for T 4 glucuronidation. These UGT1A enzymes, with the exception of UGT1A10, were detected in all of the human liver microsomes examined. Interindividual differences in T 4 glucuronidation activity were observed among the microsomes from the seven individual human livers, and the T 4 glucuronidation activity was closely correlated with -estradiol 3-glucuronidation activity. Furthermore, Spearman correlation analysis for a relationship between the T 4 glucuronidation activity and the level of UGT1A1, UGT1A3, and UGT1A9 in the microsomes revealed that levels of UGT1A1 and UGT1A3, but not that of UGT1A9, were closely correlated with T 4 glucuronidation activity. T 4 glucuronidation activity in human liver microsomes was strongly inhibited by 26,26,26,27,27,27-hexafluoro-1␣,23(S),25-trihydroxyvitamin D 3 (an inhibitor of UGT1A3), moderately inhibited by either bilirubin (an inhibitor of UGT1A1) or -estradiol (an inhibitor of UGT1A1 and UGT1A9), but not inhibited by propofol (an inhibitor of UGT1A9). These findings indicated strongly that glucuronidation of T 4 in the human liver was mediated by UGT1A subfamily enzymes, especially UGT1Al and UGT1A3, and further suggested that the interindividual differences would come from differences in the expression levels of UGT1A1 and UGT1A3 in individual human livers.Thyroid hormone, a thyroxine (T 4 ), is metabolized via deiodination, O-glucuronidation, O-sulfation, ether bond cleavage, and/or oxidative deamination (Visser, 1996;Wu et al., 2005). Among these metabolisms, O-glucuronidation is important, because it is responsible for the metabolism of many endogenous and exogenous chemicals (Radominska-Pandya et al., 1999;Iyanagi, 2007). Visser (1996) had first reported that T 4 glucuronidation was mediated by UDP-glucuronosyltransferase (UGT) 1A subfamily enzymes, in particular UGT1A1 and UGT1A6, in the rat liver. On the other hand, it had been reported that T 4 glucuronidation in the human liver was mediated mainly by UGT1A1 and UGT1A9 (Visser et al., 1993;Findlay et al., 2000). Quite recently, Yamanaka et al. (2007) reported that the T 4 glucuronidation activity in human liver is catalyzed mainly by UGT1A1.However, these previous studies on the contribution of the UGT subfamily enzymes responsible for T 4 glucuronidation in rats and humans were performed using only limited samples and/or techniques. The UGT isoform(s) for the T 4 metabolism has not been clearly determined. In addition, the UGT genes are divided into two families, UGT1 and UGT2, based on a homology of the amino acid sequence (Mackenzie et al., 2005).In the present ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.