Isolation of a Human YAC Contig Encompassing a Cluster of UGT2 Genes and Its Regional Localization to Chromosome 4q13

doi:10.1006/geno.1994.1531

Genomics

Volume 23, Issue 2, 15 September 1994, Pages 496-499

https://doi.org/10.1006/geno.1994.1531 Get rights and content

Abstract

Previously we mapped the gene encoding a human bile acid UDP-glucuronosyltransferase (UGT2B4) to chromosome 4. Here we report the mapping of two additional human UGT2B genes to chromosome 4 utilizing the polymerase chain reaction (PCR) and a panel of human/rodent somatic cell bybrid cell lines. A yeast artificial chromosome contig containing the UGT2B4, UGT2B9, and UGT2B15 genes was isolated, and pulsed-field gel electrophoresis and PCR revealed that several members of the human UGT2B gene subfamily are clustered within a 195-kb region of the YAC contig. These data permitted a provisional ordering of the genes as UGT2B9-UGT2B4-UGT2B15 . Fluorescence in situ hybridization analysis, using the YAC DNA, permitted the regional localization of this gene cluster to chromosome 4q13.

References (0)

Cited by (56)

Eudismic analysis of tricyclic sesquiterpenoid alcohols: Lead structures for the design of potent inhibitors of the human UDP-glucuronosyltransferase 2B7
2007, Bioorganic Chemistry
The epimeric tricyclic sesquiterpenoid alcohols globulol, epiglobulol, cedrol, epicedrol, longifolol, and isolongifolol were investigated in their ability to inhibit the recombinant human UDP-glucuronosyltransferase (UGT) 2B7. The stereoisomers displayed rapidly reversible competitive inhibition, which was substrate-independent. Longifolol and its stereoisomer isolongifolol displayed the lowest competitive inhibition constants (K_ic) of 23 and 26 nM, respectively. The K_ic values of cedrol and its epimer epicedrol were 0.15 and 0.21 μM, those of globulol and epiglobulol were 5.4 and 4.0 μM, respectively. The diastereomeric alcohols exhibited nearly identical affinities toward UGT2B7 indicating that the spatial arrangement of the hydroxy group had no influence on the dissociation of the enzyme–terpenoid complex. The high affinities stemmed presumably from mere hydrophobic interactions between the hydrocarbon scaffold of the terpenoid alcohol and the binding site of the enzyme. Glucuronidation assays revealed that there were large differences in the rates at which the epimeric alcohols were conjugated. Therefore, the spatial arrangement of the hydroxy group controlled the rate of the UGT2B7-catalyzed reaction. The introduction of a methyl group into the side chain of isolongifolol and longifolol increased the steric hindrance. As a result, the rate of the UGT2B7-catalyzed reaction was decreased by more than 88%. The findings indicated that the rate of the UGT2B7-catalyzed glucuronidation is significantly controlled by stereochemical and steric factors. Considering the high inhibition levels exerted by the tricyclic sesquiterpenoid alcohols, these compounds might serve as valuable lead structures for the design of potent inhibitors for UGT2B7.
Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and 2B15 associated with substrate selectivity and autoactivation
2007, Biochemical Pharmacology
Citation Excerpt :
Other reagents and solvents were of analytical reagent grade. The human UGT2B7 and UGT2B15 cDNAs were isolated as reported previously by Jin et al. [14] and by Monaghan et al. [23], respectively. Throughout this paper the designation of nucleotide and amino acid positions for both UGT2B7 and UGT2B15 are referenced relative to the 5′-ATG start codon.
Despite the important role of UDP-glucuronosyltransferases (UGT) in the metabolism of drugs, environmental chemicals and endogenous compounds, the structural features of these enzymes responsible for substrate binding and selectivity remain poorly understood. Since UGT2B7 and UGT2B15 exhibit distinct, but overlapping, substrate selectivities, UGT2B7–UGT2B15 chimeras were constructed here to identify substrate binding domains. A UGT2B7-15-7 chimera that incorporated amino acids 61–194 of UGT2B15 glucuronidated the UGT2B15 substrates testosterone and phenolphthalein, but not the UGT2B7 substrates zidovudine and 11α-hydroxyprogesterone. Derived apparent K_m values for testosterone and phenolphthalein glucuronidation by UGT2B7-15_(61–194)-7 were similar in magnitude to those determined for UGT2B15. Moreover, glucuronidation of the non-selective substrate 4-methylumbelliferone (4MU) by UGT2B7-15_(61–194)-7 and UGT2B15 followed Michaelis–Menten and weak substrate inhibition kinetics, respectively, whereas 4MU glucuronidation by UGT2B7 exhibited sigmoidal kinetics characteristic of autoactivation. Six UGT2B7-15-7 chimeras that incorporated smaller domains of UGT2B15 were subsequently generated. Of these, UGT2B7-15_(61–157)-7, UGT2B7-15_(91–157)-7 and UGT2B7-15_(61–91)-7 glucuronidated 4MU, but activity towards the other substrates investigated here was not detected. Like UGT2B7, the UGT2B7-15_(61–157)-7, UGT2B7-15_(91–157)-7 and UGT2B7-15_(61–91)-7 chimeras exhibited sigmoidal 4MU glucuronidation kinetics. The sigmoidal 4MU kinetic data were well modelled using both the Hill equation and the expression for a two-site model that assumes the simultaneous binding of two substrate molecules at equivalent sites. It may be concluded that residues 61–194 of UGT2B15 are responsible for substrate binding and for conferring the unique substrate selectivity of UGT2B15, while residues 158–194 of UGT2B7 appear to facilitate the binding of multiple 4MU molecules within the active site.
Oligomerization of the UDP-glucuronosyltransferase 1A proteins: Homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation
2007, Journal of Biological Chemistry
UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of β-d-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.
Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A6 enzymes
2006, Chemico-Biological Interactions
Citation Excerpt :
The UGT1A gene is localized on chromosome 2q37 and encodes proteins with unique N-terminal domains and identical C-terminal domains, which are formed from the alternate mRNA splicing of unique first exons with common exons 2–5. In contrast, UGT2B genes are clustered on chromosome 4q13 and individual UGT proteins are encoded by unique genes with six exons [2,4–6]. Each UGT exhibits unique substrate and tissue specificities [2,3].
UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major isoform in the human liver that glucuronidates numerous drugs, environmental chemicals and endogenous substrates. In this study, human and cynomolgus monkey UGT1A6 cDNAs (humUGT1A6 and monUGT1A6, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate UGT1A6s. The enzymatic properties of UGT1A6 proteins were characterized by the kinetic analysis of serotonin (5-hydroxytryptamine, 5-HT) and 4-methylumbelliferone (4-MU) glucuronidation. humUGT1A6 and monUGT1A6 showed 96% identity in their nucleotide and amino acid sequences. Immunoblotting analysis using an antibody raised against human UGT1A6 showed that protein staining intensities were different between human and cynomolgus monkey UGT1A6 enzymes in microsomal fractions from livers and yeast cells, although both enzymes were detectable. The apparent K_m value (15 mM) for 5-HT glucuronidation of cynomolgus monkey liver microsomes was significantly higher than that (8.6 mM) of human liver microsomes, whereas V_max values were lower in cynomolgus monkeys (2.8 nmol/min/mg protein) than in humans (8.6 nmol/min/mg protein). No significant species difference was observed in K_m (approximately 90 μM) or V_max (approximately 25 nmol/min/mg protein) values for liver microsomal 4-MU glucuronidation. In yeast cell microsomes, K_m values (approximately 6 mM) for 5-HT glucuronidation by recombinant UGT1A6s were similar, while a V_max value (0.1 nmol/min/mg protein) of monUGT1A6 was significantly lower than that (0.7 nmol/min/mg protein) of humUGT1A6. In 4-MU glucuronidation, both K_m (210 μM) and V_max (3.5 nmol/min/mg protein) values of monUGT1A6 were significantly higher than those of humUGT1A6 (K_m, 110 μM; V_max, 1.5 nmol/min/mg protein). These findings suggest that the enzymatic properties of UGT1A6 were extensively different between humans and cynomolgus monkeys, although humUGT1A6 and monUGT1A6 showed high homology at the amino acid level. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.
Inducibility of UDP-glucuronosyltransferase 1As by β-naphthoflavone in HepG2 cells
2006, Food and Chemical Toxicology
UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms (UGT1A1, UGT1A6 and UGT1A9), mainly expressed in the human liver, and examined the inducibility of UGT1As by β-naphthoflavone (BNF) in human hepatoma HepG2 cells. The cells were pretreated for 72 h with BNF at concentrations of 25, 50 and 100 μM. 7-Ethyl-10-hydroxycamptothecin (SN-38) glucuronidation, used as a probe for UGT1A1, showed sigmoidal kinetics with a Hill coefficient (n) of 1.2–1.3 in control and BNF-pretreated HepG2 cells. The V_max values were significantly increased 3.6- to 4.3-fold by BNF, whereas there was no significant change in the S₅₀ values by BNF at any concentration examined. On the other hand, 4-methylumbelliferone (4-MU) glucuronidation as a probe for UGT1A6 and UGT1A9 in the control and BNF-pretreated HepG2 cells exhibited a biphasic kinetic pattern. Although K_m1 values for the low-K_m phase were similar between the control and BNF-pretreated HepG2 cells, K_m2 values for the high-K_m phase of BNF-pretreated HepG2 cells were reduced to 54–69% of control HepG2 cells. The values of V_max1 and V_max2 for the low- and high-K_m phases, respectively, were significantly increased 1.9- to 2.6-fold by BNF at 25 and/or 50 μM but not 100 μM. With respect to V_max (V_max1 and V_max2) and V_max/K_m (V_max1/K_m1 and V_max2/K_m2), the values were significantly increased 2.0- to 3.2-fold by BNF at all concentrations examined. Furthermore, real-time reverse transcription polymerase chain reaction using TaqMan probes demonstrated that BNF concentration-dependently induced mRNA levels of UGT1A1 but not UGT1A6 or UGT1A9 in HepG2 cells (1.3- to 6.0-fold). These results suggest that the inducibility of UGT1A isoforms in HepG2 cells by BNF is different from other aryl hydrocarbon receptor agonists previously reported, and should provide useful information for the prediction of drug–drug interactions and toxicological assessment of environmental chemicals.
Integration of hepatic drug transporters and phase II metabolizing enzymes: Mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites
2006, European Journal of Pharmaceutical Sciences
The liver is the primary site of drug metabolism in the body. Typically, metabolic conversion of a drug results in inactivation, detoxification, and enhanced likelihood for excretion in urine or feces. Sulfation, glucuronidation, and glutathione conjugation represent the three most prevalent classes of phase II metabolism, which may occur directly on the parent compounds that contain appropriate structural motifs, or, as is usually the case, on functional groups added or exposed by phase I oxidation. These three conjugation reactions increase the molecular weight and water solubility of the compound, in addition to adding a negative charge to the molecule. As a result of these changes in the physicochemical properties, phase II conjugates tend to have very poor membrane permeability, and necessitate carrier-mediated transport for biliary or hepatic basolateral excretion into sinusoidal blood for eventual excretion into urine. This review summarizes sulfation, glucuronidation, and glutathione conjugation reactions, as well as recent progress in elucidating the hepatic transport mechanisms responsible for the excretion of these conjugates from the liver. The discussion focuses on alterations of metabolism and transport by chemical modulators, and disease states, as well as pharmacodynamic and toxicological implications of hepatic metabolism and/or transport modulation for certain active phase II conjugates. A brief discussion of issues that must be considered in the design and interpretation of phase II metabolite transport studies follows.

View all citing articles on Scopus

View full text

Short CommunicationIsolation of a Human YAC Contig Encompassing a Cluster of UGT2 Genes and Its Regional Localization to Chromosome 4q13

Abstract

Short Communication
Isolation of a Human YAC Contig Encompassing a Cluster of UGT2 Genes and Its Regional Localization to Chromosome 4q13