Determination of the Human Liver UDP-Glucuronosyltransferase 2B4 Domains Involved in the Binding of UDP-Glucuronic Acid Using Photoaffinity Labeling of Fusion Proteins

Pillot, Thierry; Ouzzine, Mohamed; Fournel‐Gigleux, Sylvie; Lafaurie, C.; Tebbi, D.; Treat, Susan; Radominska, Anna; Lester, Roger; Siest, Gérard; Magdalou, Jacques

doi:10.1006/bbrc.1993.2547

Cited by 24 publications

(17 citation statements)

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“…We found that UDP competitively inhibited not only UGT1A9 but also UGT1A1, UGT1A4, and UGT1A6 for the UDPGA binding, although the K i values varied. The carboxyl-terminal domain of UGT proteins is highly conserved and is responsible for the UDPGA binding (Pillot et al, 1993;Tukey and Strassburg, 2000). Therefore, it is conceivable that UDP commonly inhibits UGTs of any isoform, tissue, or species, as supported by previous reports (Gotze et al, 1971;Winsnes, 1972;Hallinan et al, 1979;Koster and Noordhoek, 1983;Yokota et al, 1998).…”

Section: Discussionsupporting

confidence: 53%

Product Inhibition of UDP-Glucuronosyltransferase (UGT) Enzymes by UDP Obfuscates the Inhibitory Effects of UGT Substrates

Fujiwara

Nakajima²,

Yamanaka³

et al. 2007

Drug Metab Dispos

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ABSTRACT:Substrates that are specific for certain UDP-glucuronosyltransferase (UGT) isoforms are usually used as specific inhibitors to identify UGT isoforms responsible for the glucuronidation of drugs. 1-Naphthol and 4-nitrophenol are probe substrates for human UGT1A6. In the present study, we found that UGT1A1-catalyzed estradiol 3-O-glucuronide formation and UGT1A4-catalyzed imipramine N-glucuronide formation in human liver microsomes were prominently decreased in the presence of 1-naphthol, but those by recombinant human UGT1A1 and UGT1A4, respectively, were not. Interestingly, when recombinant UGT1A6 was added in the reaction mixture, these activities by recombinant UGT1A1 and UGT1A4 were diminished in the presence of 1-naphthol. To interpret this phenomenon, the inhibitory effects of 1-naphthol O-glucuronide and UDP, products of the glucuronidation of 1-naphthol, were investigated. We found that UDP strongly inhibited the UGT1A1 (K i ‫؍‬ 7 M) and UGT1A4 (K i ‫؍‬ 47 M) activities in a competitive manner for the 5-diphosphoglucuronic acid binding. These results suggest that UDP produced by UGT1A6-catalyzed 1-naphthol glucuronidation, but not 1-naphthol O-glucuronide and 1-naphthol per se, is the actual inhibition substance. Next, we examined the inhibitory effects of 15 compounds that are substrates of UGTs on estradiol 3-O-glucuronide formation in human liver microsomes compared with those by recombinant UGT1A1. Among them, 4 compounds (1-naphthol, 2-naphthol, 4-nitrophenol, and 4-methylumbelliferone) with high turnover rates (V max /K m value >200 l/ min/mg) showed more potent inhibition of the activity in human liver microsomes compared with that by the recombinant UGT1A1. Thus, we should pay attention to the inhibitory effects of UDP on UGT, which may cause erroneous evaluations in inhibition studies using human liver microsomes.UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes that catalyze the formation of glucuronides by the transfer of glucuronic acid from a cofactor uridine 5Ј-diphosphoglucuronic acid (UDPGA) to hydroxyl, carboxyl, or amine groups of endogenous and exogenous substrates (Dutton, 1980). The hydrophilic glucuronides can readily be excreted from the body via bile and urine. Human UGTs are classified into two subfamilies, UGT1 and UGT2, based on similarities between their amino acid sequences and gene organization (Mackenzie et al., 2005). To date, 19 human UGT isoforms, UGT1A1, -1A3, -1A4, -1A5, -1A6, -1A7, -1A8, -1A9, -1A10, -2A1, -2A2, -2A3, -2B4, -2B7, -2B10, -2B11, -2B15, -2B17, and -2B28, have been identified (Mackenzie et al., 2005). Most UGT enzymes are expressed in liver, but some isoforms such as UGT1A7, -1A8, and -1A10 are exclusively expressed in intestine Burchell et al., 2001).Most pharmacokinetic drug-drug interactions occur at the metabolic level and usually involve changes in the activity of the major drug-metabolizing enzymes such as UGT. Identification of the UGT(s) involved in the metabolism of a given compound allows us to predict potential drug-drug ...

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Section: Discussionsupporting

confidence: 53%

Product Inhibition of UDP-Glucuronosyltransferase (UGT) Enzymes by UDP Obfuscates the Inhibitory Effects of UGT Substrates

Fujiwara

Nakajima²,

Yamanaka³

et al. 2007

Drug Metab Dispos

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“…This region also showed some homology with other proteins that bind sugars, nucleotides, or phosphate groups [165]. Therefore, this region is a good candidate for the UDP binding site.…”

Section: Application Of Photoaffinity Labeling To Characterization Ofmentioning

confidence: 97%

Structural and Functional Studies of Udp-Glucuronosyltransferases*

Radominska‐Pandya

Czernik

Little

et al. 1999

Drug Metabolism Reviews

446

358

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UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum (ER) which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GIcUA) as the sugar donor. Glucuronidation is a major factor in the elimination of lipophilic compounds from the body. In this review, current information on the substrate specificities of UGT1A and 2B family isoforms is discussed. Recent findings with regard to UGT structure and topology are presented, including a dynamic topological model of UGTs in the ER. Evidence from experiments on UGT interactions with inhibitors directed at specific amino acids, photoaffinity labeling, and analysis of amino acid alignments suggest that UDP-GIcUA interacts with residues in both the N- and C-terminal domains, whereas aglycon binding sites are localized in the N-terminal domain. The amino acids identified so far as crucial for substrate binding and catalysis are arginine, lysine, histidine, proline, and residues containing carboxylic acid. Site-directed mutagenesis experiments are critical for unambiguous identification of the active-site architecture.

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“…This forms the signature sequence of UDP-glycosyltransferase that binds the UDP-moiety. Photo-affinity labeling experiments showed that the glucuronic acid moiety of UDP-GlcUA interacts with positively charged residue(s) in the N-terminal domain of UGT2B4 (Pillot et al, 1993). It is reasonable to suppose that these should be the site(s) interacting with the glucuronic acid moiety in the N-terminal domain of 0.048 † † † P , 0.0001 from *1; $$$ P , 0.0001 from single expression.…”

Section: 010mentioning

confidence: 99%

Alteration of the Function of the UDP-Glucuronosyltransferase 1A Subfamily by Cytochrome P450 3A4: Different Susceptibility for UGT Isoforms and UGT1A1/7 Variants

et al. 2013

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Functional protein-protein interactions between UDPglucuronosyltransferase (UGT)1A isoforms and cytochrome P450 (CYP)3A4 were studied. To this end, UGT1A-catalyzed glucuronidation was assayed in Sf-9 cells that simultaneously expressed UGT and CYP3A4. In the kinetics of UGT1A6-catalyzed glucuronidation of serotonin, both Michaelis constant (K m ) and maximal velocity (V max ) were increased by CYP3A4. When CYP3A4 was coexpressed with either UGT1A1 or 1A7, the V max for the glucuronidation of the irinotecan metabolite (SN-38) was significantly increased. S 50 and K m both which are the substrate concentration giving 0.5 V max were little affected by simultaneous expression of CYP3A4. This study also examined the catalytic properties of the allelic variants of UGT1A1 and 1A7 and their effects on the interaction with CYP3A4. Although the UGT1A1-catalyzing activity of 4-methylumbelliferone glucuronidation was reduced in its variant, UGT1A1*6, the coexpression of CYP3A4 restored the impaired function to a level comparable with the wild type. Similarly, simultaneous expression of CYP3A4 increased the V max of UGT1A7*1 (wild type) and *2 (N129K and R131K), whereas the same was not observed in UGT1A7*3 (N129K, R131K, and W208R). In the kinetics involving different concentrations of UDPglucuronic acid (UDP-GlcUA), the K m for UDP-GlcUA was significantly higher for UGT1A7*2 and *3 than *1. The K m of UGT1A7*1 and *3 was increased by CYP3A4, whereas *2 did not exhibit any such change. These results suggest that (1) CYP3A4 changes the catalytic function of the UGT1A subfamily in a UGT isoformspecific manner and (2) nonsynonymous mutations in UGT1A7*3 reduce not only the ability of UGT to use UDP-GlcUA but also CYP3A4-mediated enhancement of catalytic activity, whereas CYP3A4 is able to restore the UGT1A1*6 function.

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Determination of the Human Liver UDP-Glucuronosyltransferase 2B4 Domains Involved in the Binding of UDP-Glucuronic Acid Using Photoaffinity Labeling of Fusion Proteins

Cited by 24 publications

References 0 publications

Product Inhibition of UDP-Glucuronosyltransferase (UGT) Enzymes by UDP Obfuscates the Inhibitory Effects of UGT Substrates

Product Inhibition of UDP-Glucuronosyltransferase (UGT) Enzymes by UDP Obfuscates the Inhibitory Effects of UGT Substrates

Structural and Functional Studies of Udp-Glucuronosyltransferases*

Alteration of the Function of the UDP-Glucuronosyltransferase 1A Subfamily by Cytochrome P450 3A4: Different Susceptibility for UGT Isoforms and UGT1A1/7 Variants

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