Human Udp-Glucuronosyltransferases: Isoform Selectivity and Kinetics of 4-METHYLUMBELLIFERONE and 1-NAPHTHOL Glucuronidation, Effects of Organic Solvents, and Inhibition by Diclofenac and Probenecid (original) (raw)
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Drug Metabolism and Disposition, 2003
UDP-glucuronosyltransferase (UGT) 1A10 is an isoform of UGT1A, which is expressed in extrahepatic, biliary and aerodigestive/gastrointestinal tissues. We have previously reported two nonsynonymous single nucleotide polymorphisms in exon 1 of human UGT1A10 gene; 177G>A and 605C>T resulting in amino acid alterations, M59I and T202I, respectively. In the present study, wildtype (WT) and these variant UGT1A10 cDNAs were transiently expressed in COS-1 cells for functional characterization. Glucuronidation activities in these COS-1 membrane fractions were assayed using 7-hydroxy-4-trifluoromethylcoumarin (HT-FMC) and 17-estradiol (E2) as substrates. WT and variant UGT1A10s catalyzed HTFMC glucuronidation with similar apparent K m values of approximately 5 M, whereas the V max value of T202I normalized by the expressed UGT1A10 protein levels was nearly half of those of WT and M59I. High-performance liquid chromatography analysis of E2 glucuronide revealed that UGT1A10 catalyzed E2 3-O-glucuronidation but not 17-O-glucuronidation. Similarly, the three UGT1A10s catalyzed E2 3-O-glucuronidation with comparable apparent K m values (approximately 2 M), whereas the normalized V max value of T202I was almost half that of WT and M59I. These results suggest that the lowered glucuronidation activity of T202I affects the gastrointestinal glucuronidation of orally administrated chemicals and the enterohepatic circulation of biliary excreted metabolites.
Archives of Biochemistry and Biophysics, 1997
phenols as represented by 4-hydroxybiphenol and octylgallate, as well as 4-hydroxyestrone. In addition, A rabbit liver UDP-glucuronosyltransferase cDNA UGT1A7l possesses catalytic activity toward tertiary that is related to human and rat UGT1A7 has been amines like the tricyclic antidepressant imipramine. identified. The predicted amino acid sequence of the The pattern of UGT1A7l glucuronidation is similar to UGT1A7l displays 80% similarity to that encoded by that observed for human UGT1A9, except tertiary human HP4 (UGT1A9), but 81% to that predicted for amines are not subject to glucuronidation by human human UGT1A7 and 77% to the rat UGT1A7 (UGTA2). UGT1A9. Glucuronidation of tertiary amines is cata-The exons encoding human UGT1A7 and rat UGTA2 lyzed principally by human UGT1A4 as well as rabbit are the seventh of the series of cassette exons that UGT1A4. Although rabbit UGT1A7l catalyzes the flank the 3 common exon series of the UGT1A locus. formation of quarternary ammonium glucuronides, Southern blot analysis demonstrates that the exon sethe V max is considerably less than that observed for quence encoding UGT1A7l is part of a larger cluster of rabbit UGT1A4. Overall, the characterization of rabbit highly related genes. The UGT1A7l RNA is expressed UGT1A7l suggests that this protein represents the orin both neonatal and adult liver, and unlike rat tholog of the human UGT1A7, which to date has not UGT1A2 which is inducible with Ah receptor ligands been identified. ᭧ 1997 Academic Press such as polycyclic aromatic hydrocarbons, rabbit UGT1A7l is not regulated when animals are exposed to these inducers. Following expression of UGT1A7l in COS-1 cells, glucuronidation activity was identified for The UDP-glucuronosyltransferases (UGTs) 3 are a small phenolic molecules like 4-nitrophenol, bulky superfamily of catalytic enzymes involved in the detoxification of endogenous and exogenous compounds. 1 Nomenclature used in this article is based upon recommenda-By converting compounds to water-soluble molecules tions made at the VIIIth International Meeting on Glucuronidation, through the catabolic addition of glucuronic acid, the held May 1996 at the University of Iowa. The human UGT1 locus is UGTs serve to process these substrates for elimination encoded by 12 exon 1 cassettes (Presented by Dr. Owens, NIH), each of which encodes the first 282 amino acids of the UGT1A proteins. in either the bile or urine. The list of potential sub-The proteins encoded by these exons have been characterized as strates is diverse and includes endogenous compounds UGT1A1 through UGT1A12, respectively. In rodents, a similar panel such as hormones and steroids, bilirubin, bile acids of exon 1 sequences (27) flank conserved exons 2-5. These have been and retinoic acids, as well as exogenous agents such 357
Expression and Characterization of Recombinant Human UDP-glucuronosyltransferases (UGTs)
The Journal of Biological Chemistry, 2003
Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of α-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged ...
Proceedings of the National Academy of Sciences, 1988
A rat kidney phenol UDP-glucuronosyltransferase cDNA was used to isolate a human liver phenol UDPglucuronosyltransferase cDNA by screening of a human liver cDNA library in the expression vector Agtll. The 2.4-kilobase cDNA contained an open reading frame of 1593 base pairs coding for a protein of 531 residues. The human liver cDNA was subcloned into the vector pKCRH2. Transfection of this recombinant plasmid into COS-7 cells allowed the expression of a protein of-55 kDa. The enzyme synthesized was a glycoprotein, as indicated by a reduction in molecular mass of :3 kDa after biosynthesis in the presence of tunicamycin. The expressed enzyme rapidly catalyzed the glucuronidation of 1-naphthol, 4-methylumbelliferone, and 4-nitrophenol. The use of a related series of simple phenols provided an outline description of the substituent restrictions imposed upon the phenolic structures accepted as substrates. The glucuronidation of testosterone, androsterone, and estrone was not catalyzed by this cloned UDP-glucuronosyltransferase. Human hepatic microsomal UDP-glucuronosyltransferase [UDPGT; UDPglucuronate l-D-glucuronosyltransferase (acceptor-unspecific); EC 2.4.1.17] has a major role in the conjugation and production of more hydrophilic glucuronides for excretion of drugs, xenobiotics, and endogenous compounds (1). Overwhelming evidence indicates that in rat liver these glucuronidation reactions are catalyzed by a family of isoenzymes. The existence of UDPGT isoenzymes that would specifically glucuronidate bilirubin, bile acids, 5hydroxytryptamines, and drugs (such as morphine, clofibrate, and chloramphenicol) has been suggested from kinetic analysis of in vitro enzyme assays of microsomes prepared from adult (2-6) and developing (7) liver. Inherited defects of
Journal of Biological Chemistry, 2007
The human UDP-glucuronosyltransferase UGT1A6 is the primary phenol-metabolizing UDP-glucuronosyltransferase isoform. It catalyzes the nucleophilic attack of phenolic xenobiotics on UDP-glucuronic acid, leading to the formation of water-soluble glucuronides. The catalytic mechanism proposed for this reaction is an acid-base mechanism that involves an aspartic/glutamic acid and/or histidine residue. Here, we investigated the role of 14 highly conserved aspartic/glutamic acid residues over the entire sequence of human UGT1A6 by sitedirected mutagenesis. We showed that except for aspartic residues Asp-150 and Asp-488, the substitution of carboxylic residues by alanine led to active mutants but with decreased enzyme activity and lower affinity for acceptor and/or donor substrate. Further analysis including mutation of the corresponding residue in other UGT1A isoforms suggests that Asp-150 plays a major catalytic role. In this report we also identified a single active site residue important for glucuronidation of phenols and carboxylic acid substrates by UGT1A enzyme family. Replacing Pro-40 of UGT1A4 by histidine expanded the glucuronidation activity of the enzyme to phenolic and carboxylic compounds, therefore, leading to UGT1A3-type isoform in terms of substrate specificity. Conversely, when His-40 residue of UGT1A3 was replaced with proline, the substrate specificity shifted toward that of UGT1A4 with loss of glucuronidation of phenolic substrates. Furthermore, mutation of His-39 residue of UGT1A1 (His-40 in UGT1A4) to proline led to loss of glucuronidation of phenols but not of estrogens. This study provides a step forward to better understand the glucuronidation mechanism and substrate recognition, which is invaluable for a better prediction of drug metabolism and toxicity in human. * This work was supported by grants from the Agence Nationale de la Recherche and the Ligue Ré gionale contre le Cancer.
Pharmacogenetics, 2002
We identified four novel polymorphisms in the CACNA1S gene that encodes the ␣ 1 -subunit of the dihydropyridine receptor. Mutations in this gene are associated with two genetic diseases: malignant hyperthermia and hypokalemic periodic paralysis. The nucleotide substitutions c2403T¡C and c5398T¡C did not result in amino acid replacement, the nucleotide substitution c4475C¡A caused the replacement of the Ala1492 with an Asp residue and an A insertion was identified in intron 36. By using methods based on digestion with restriction enzymes we calculated the frequencies of these novel polymorphisms, as well as heterozygosity, in normal subjects from southern Italy. Clin Chem Lab Med 2003; 41(1):20 -22
Journal of Pharmacology and Experimental Therapeutics, 2007
There is currently little information regarding the localization of UDP-glucuronosyltransferases (UGTs) in human renal cortex and medulla, and the functional contribution of renal UGTs to drug glucuronidation remains poorly defined. Using human kidney sections and human kidney cortical microsomes (HKCM) and human kidney medullary microsomes (HKMM), we combined immunohistochemistry to investigate UGT1A and UGT2B7 expression with in vitro microsomal studies to determine the kinetics of S-naproxen acyl glucuronidation. With the exception of the glomerulus, Bowman's capsule, and renal vasculature, UGT1A proteins and UGT2B7 were expressed throughout the proximal and distal convoluted tubules, the loops of Henle, and the collecting ducts. Additionally, UGT1A and UGT2B7 expression was demonstrated in the macula densa, supporting a potential role of UGTs in regulating aldosterone. Consistent with the immunohistochemical data, S-naproxen acyl glucuronidation was catalyzed by HKCM and HKMM. Kinetic data were well described by the two-enzyme Michaelis-Menten equation. K m values for the high-affinity components were 34 Ϯ 14 M (HKCM) and 45 Ϯ 14 M (HKMM). Fluconazole inhibited the high-affinity component establishing UGT2B7 as the enzyme responsible for S-naproxen glucuronidation in cortex and medulla. The low-affinity component was relatively unaffected by fluconazole (Ͻ15% inhibition), supporting the presence of other UGTs with S-naproxen glucuronidation capacity (e.g., UGT1A6 and UGT1A9) in cortex and medulla. We postulate that the ubiquitous distribution of UGTs in mammalian kidney may buffer physiological responses to endogenous mediators, but at the same time competitive xenobiotic-endobiotic interactions may provide an explanation for the adverse renal effects of drugs, including nonsteroidal anti-inflammatory drugs. Enzymes of the UDP-glucuronosyltransferase (UGT) superfamily catalyze the covalent linkage of glucuronic acid, derived from UDP-glucuronic acid (UDPGA), to typically lipophilic substrates containing a carboxylic acid, hydroxyl or amine functional group (Miners and Mackenzie, 1991). Thus, glucuronidation is an important elimination mechanism for numerous drugs, environmental chemicals, and endogenous compounds (e.g., bilirubin, fatty acids, eicosanoids, and hydroxysteroids) in humans (Miners and Mackenzie, 1991; Tsoutsikos et al., 2004). UGTs have been classified into two families, UGT1 and UGT2, and of the human UGT proteins identified to date, 16 have the capacity to catalyze the glucuronidation of endogenous compounds and/or xenobiotics: UGT 1A1,
Glucuronidation and the UDP-glucuronosyltransferases in health and disease
Drug metabolism and …, 2004
For most xenobiotics and many endobiotics, glucuronidation constitutes a major route of elimination and thereby may substantially modulate substrate concentrations and effects. In some cases, glucuronidation forms the biologically active molecule. Recent studies have ...
A Novel Method for the Immunoquantification of UDP-Glucuronosyltransferases in Human Tissue
Drug Metabolism and Disposition, 2011
Glucuronidation is a major pathway of drug and xenobiotic metabolism, catalysed by members of the UDP-glucuronosyltransferase (UGT) family. Predicting the contribution of individual UGTs to drug metabolism would be of considerable value in drug development, and would be greatly aided by the availability of detailed absolute expression levels of these proteins; this is hampered by the lack of purified protein standards due to the hydrophobic membrane-associated nature of UGTs and the consequential difficulties in expression and purification. Here we describe a novel solution to this problem by expressing UGTs in E. coli as fusion proteins with ribonuclease S-peptide, targeted to the periplasm with the pelB leader sequence. Following addition of ribonuclease S-protein to membrane extracts, a functional ribonuclease is reconstituted that provides a direct and absolute quantification of the amount of UGT fusion protein; this is subsequently used to generate standard curves for immunoquantification by immunoblotting. To illustrate the value of the method, we have quantified the expression of UGTs 1A1 and 1A6 in human liver and kidney microsomes using new isoform-specific antibodies developed against peptides from these proteins. Expression levels of both proteins in liver were highly variable (28-fold and 20-fold, respectively), and correlated strongly with UGT enzyme activity towards probe substrates bilirubin and 1-naphthol, respectively. The method is broadly applicable and provides a straightforward means of determining absolute, as opposed to relative, quantities of UGT proteins present in human tissues.
Archives of biochemistry and biophysics, 2002
An isoform (rhesus UGT1A01) orthologus to the human UGT1A1 was cloned and sequenced from female rhesus monkey liver cDNA using primers designed from the human nucleotide sequences. Open reading frame analysis of the PCR-generated product encodes a 533-amino acid protein with a proposed 27-residue signal peptide. Nucleotide sequence comparison of rhesus UGT1A01 to other rhesus UGT1A isoforms detected a single-transition mutation at nucleotide 1520 (T ! C), resulting in a neutral F to S substitution at position 507. Rhesus UGT1A01 was greater than 99 and 95% identical to cynomolgus UGT1A01 and human UGT1A1, respectively. The rhesus UGT1A01 was expressed in HK-293 cells for functional analysis. Catalytic activity of UGT1A01 was determined with 7-hydroxy-4-(trifluoromethyl)-coumarin and more specific human UGT1A1 substrates (1-naphthol, b-estradiol, 17a-ethinylestradiol, and bilirubin). Expression of UGT1A01 protein was also detected by a Western blot utilizing a polyclonal antibody developed against the human UGT1A family. Ó