Homodimerization of UDP-glucuronosyltransferase 2B7 (UGT2B7) and identification of a putative dimerization domain by protein homology modeling
Graphical abstract
Homodimerization of UGT2B7 was demonstrated using co-immunoprecipitation. The dimerization domain was identified by protein homology modeling.
Introduction
The UDP-glucuronosyltransferases (UGT) comprise a superfamily of membrane bound enzymes that catalyze the conjugation of glucuronic acid, derived from UDP-glucuronic acid (UDPGA), to typically lipophilic substrates bearing a suitable nucleophilic acceptor functional group. The glucuronidation reaction provides a clearance and detoxification mechanism for drugs from all therapeutic classes, environmental chemicals, and endogenous compounds that include bilirubin, fatty acids, and steroid hormones [1], [2]. Eighteen functional human UGT enzymes that utilize UDPGA as cofactor have been identified to date, and these have been classified in three subfamilies (UGT1A, UGT2A and UGT2B) based on amino acid sequence identity [3]. The individual UGT enzymes exhibit distinct, but frequently overlapping, substrate selectivities [4], [5].
There is accumulating evidence that UGT enzymes exist as oligomers in homodimeric and heterodimeric states [6]. Gel permeation chromatography, cross-linking studies and two-hybrid analysis in yeast and mammalian systems were employed to demonstrate homodimerization of UGT1A1 [7]. In addition to UGT1A1, fluorescence resonance energy transfer (FRET) [8] and co-immunoprecipitation (Co-IP) [8], [9] studies have provided evidence of homodimerization of other UGT1A subfamily proteins (viz. UGT 1A3, 1A4, 1A6, 1A7, 1A8, 1A9 and 1A10). While definitive studies with human UGT2A and UGT2B enzymes are lacking, it has been reported that co-expression of two inactive forms of rat UGT2B1 resulted in restoration of catalytic activity, consistent with functional dimerization [10].
Multiple lines of evidence similarly suggest the occurrence of heterodimerization. FRET and Co-IP data indicated that UGT1A1 may heterodimerize with UGT 1A3, 1A4, 1A6, 1A7, 1A8, 1A9 and 1A10 [8], although two-hybrid analysis did not identify an association between UGT1A1 and UGT1A6 [7]. Consistent with protein–protein interactions, altered enzyme activity and thermal stability have been demonstrated following co-expression of various human UGT1A proteins [11], [12], [13], [14] and co-expression of UGT2B7 and UGT1A proteins [15]. Co-expression of guinea pig UGT2B21 and UGT2B22 similarly resulted in the altered glucuronidation of chloramphenicol and morphine [16], [17], while immunopurification and chemical cross-linking studies are suggestive of interactions between rat UGT2B1 and UGT1A proteins [18].
UGT2B7 is arguably the most important enzyme involved in drug glucuronidation [2], [19]. For example, UGT2B7 contributes to the glucuronidation of opioids, including codeine and morphine [20], [21], [22], anti-cancer agents [23], [24], gemfibrozil [25], valproic acid [26], zidovudine [20], and non-steroidal anti-inflammatory agents [26], [27]. Furthermore, UGT2B7 glucuronidates the mineralocorticoid aldosterone and other C19 and C21 hydroxy-steroids [28], [29], [30]. Data from kinetic and inhibition studies along with the presence of bands corresponding to the molecular weights of oligomeric species on western blots suggests that UGT2B7 may exist as a homodimer [15], [31], [32], [33], but direct evidence for homodimerization is currently lacking.
The aim of the present study was to investigate homodimerization of UGT2B7. Following demonstration of a direct protein–protein interaction by Co-IP, a homology model that identified the dimerisation domain was generated based on the X-ray crystal coordinates of the C-terminal region of human UGT2B7 [34] and several plant glycosyltransferases.
Section snippets
Chemicals and reagents
4-Methylumbelliferone (4MU), 4-methylumbelliferone β-d-glucuronide (4MUG), and UDP-glucuronic acid trisodium salt (UDPGA) were purchased from Sigma–Aldrich (Sydney, Australia); Pfu Ultra II HS Polymerase from Stratagene (La Jolla, CA, USA); Shrimp Alkaline Phosphatase from Roche Diagnostics GmbH (Penzbeg, Germany); restriction enzymes were from New England Biolabs (Ipswich, MA, USA); Dulbecco's modified Eagle's medium (DMEM), MEM non-essential amino acids solution (10 mM; ×100), and
Expression of UGT2B7 and tagged UGT2B7 proteins
UGT2B7, UGT2B7-HA, UGT2B7-cMYC, and co-expressed UGT2B7-HA + cMYC proteins were expressed in HEK293T cells. Cell lysates were screened for the presence of UGT2B7 protein by immunoblotting using a UGT2B7 specific antibody (Fig. 1A). Expression levels of UGT2B7-HA, UGT2B7-cMYC, and co-expressed UGT2B7-HA + cMYC relative to wild-type were 30.3 ± 1%, 23.8 ± 1.2%, and 18.4 ± 0.6% (mean ± SD of 3 measurements), respectively (Fig. 1A). Lysates probed with anti-HA antibody revealed immunoreactivity with UGT2B7-HA
Discussion
Although homodimerization of UGT1A enzymes has been demonstrated [6], [7], [8], [9], evidence for human UGT2B subfamily enzymes is limited. Furthermore, the mechanism of UGT dimerization and the residues that comprise the dimer interface have not been elucidated. Here, Co-IP experiments with HA- and cMYC-tagged proteins demonstrated that human UGT2B7, which is arguably the most important UGT enzyme involved in drug metabolism, forms catalytically active homodimers with the ability to catalyze
Acknowledgement
This study was supported by a research project grant from the National Health and Medical Research Council of Australia.
References (50)
- et al.
Drug glucuronidation in humans
Pharmacol Ther
(1991) - et al.
UDP-glucuronosyltransferases and clinical drug-drug interactions
Pharmacol Ther
(2005) - et al.
In vitro–in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises
Biochem Pharmacol
(2006) - et al.
Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications
J Biol Chem
(2001) - et al.
Oligomerization of the UDP-glucuronosyltransferase 1A proteins – Homo- and hetero-dimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation
J Biol Chem
(2007) - et al.
Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than the other UGTs and was purified as an active dimeric enzyme
J Biol Chem
(2003) - et al.
UDP-glucuronosyltransferase, the role of the amino terminus in dimerization
J Biol Chem
(1997) - et al.
The interactions between the N-terminal and C-terminal domains of the human UDP-glucuronosyltransferases are partly isoform-specific, and may involve both monomers
Biochem Pharmacol
(2004) - et al.
The regio- and stereo-selectivity of C19 and C21 hydroxysteroid glucuronidation by UGT2B7 and UGT2B11
Arch Biochem Biophys
(1997) - et al.
Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and 2B15 associated with substrate selectivity and autoactivation
Biochem Pharmacol
(2007)
Comparative homology modeling of human cytochrome P4501A1 (CYP1A1) and confirmation of residues involved in 7-ethoxyresorufin O-deethylation by site-directed mutagenesis and enzyme kinetic analysis
Arch Biochem Biophys
Proline brackets and identification of potential functional sites in proteins: toxins to therapeutics
Toxicon
Protein–protein interactions – a review of protein dimer structures
Prog Biophys Mol Biol
The structure of protein–protein recognition sites
J Biol Chem
Nomenclature update for the mammalian UDP-glucuronosyltransferase (UGT) gene family
Pharmacogenet Genom
Predicting human drug glucuronidation: application of in vitro and in silico modeling approaches
Annu Rev Pharmacol Toxicol
UDP-glucuronosyltransferases as oligomeric enzymes
Curr Drug Metab
Interactions between UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities
Drug Metab Dispos
Effects of coexpression of UGT1A9 on enzymatic activities of human UGT1A enzymes
Drug Metab Dispos
Stereoselective glucuronidation of 5-(4′-hydroxyphenyl)-5-phenylhydantoin by human UDP-glucuronosyltransferase (UGT) 1A1, UGT1A9, and UGT2B15: effects of UGT–UGT interactions
Drug Metab Dispos
Interactions between human UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A enzymes
J Pharm Sci
Simultaneous expression of guinea pig UDP-glucuronosyltransferase 2B21 and 2B22 in COS-7 cells enhances UDP-glucuronosyltransferase 2B21-catalyzed morphine 6-glucuronide formation
Mol Pharmacol
Simultaneous expression of guinea pig UDP-glucuronosyltransferase 2B21 (UGT2B21) and 2B22 in COS-7 cells enhances UGT2B21-catalyzed chloramphenicol glucuronidation
Drug Metab Dispos
Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes
Biochemistry
The prediction of drug glucuronidation parameters in humans: UDP-glucuronosyltransferase enzyme-selective substrate and inhibitor probes for reaction phenotyping and in vitro–in vivo extrapolation of drug clearance and drug-drug interaction potential
Drug Metab Rev
Cited by (29)
The UGTome: The expanding diversity of UDP glycosyltransferases and its impact on small molecule metabolism
2019, Pharmacology and TherapeuticsCitation Excerpt :In the absence of a defined structure, one useful approach is the application of homology models to the investigation of UGT-UGT structure–function; however, this remains at an early stage. Lewis et al. (2011) developed a homology model of UGT2B7 from plant and human template structures to elucidate a putative UGT2B7 dimerization domain. In this model, the proposed dimerization domain was comprised of eighteen amino acids with a putative dimerization signature motif (FPPSYVPVVMS), which is conserved in all UGT2B enzymes.
UDP-Glycosyltransferases
2018, Comprehensive Toxicology: Third EditionAdvances in drug metabolism and pharmacogenetics research in Australia
2017, Pharmacological ResearchCitation Excerpt :However, studies with UGT2B7-2B15 hybrid proteins showed residues 158–194 of UGTB7 appear to facilitate the binding of multiple ligand molecules in the active site [26]. Subsequent studies demonstrated that the dimerisation domain of UGT2B7 falls within this region suggesting that the two catalytic sites result from homodimerization [27]. Experimental approaches for IV-IVE, both reaction phenotyping and quantitative IV-IVE, has been a major area of research at Flinders University.
Homo- and hetero-dimerization of human UDP-glucuronosyltransferase 2B7 (UGT2B7) wild type and its allelic variants affect zidovudine glucuronidation activity
2015, Biochemical PharmacologyCitation Excerpt :In the 136 Japanese subjects with arrhythmia, diplotypes UGT2B7*1/*1, UGT2B7*1/*2, UGT2B7*1/*71S, UGT2B7*2/*2, UGT2B7*2/*71S, UGT2B7*71S/*71S and UGT2B7*71S/*5 were reported with frequencies of 0.324, 0.331, 0.162, 0.044, 0.088, 0.044 and 0.007, respectively [7]. Despite the existence of homo-dimeric UGT2B7 wild type [22,23] and the existence of hetero-dimers formed by UGT1As, it is still unclear whether these three UGT2B7 allelic variants A71S, H268Y and D398N could form homo- or hetero-dimers with each other or with wild type, and how their activities are affected by different dimeric states. In the present study, UGT2B7 allozymes WT, A71S, H268Y, and D398N were expressed in insect cells individually and in pairs using the Bac-to-Bac expression system.
The UDP-glucuronosyltransferases: Their role in drug metabolism and detoxification
2013, International Journal of Biochemistry and Cell BiologyEvaluation of UGT protein interactions in human hepatocytes: Effect of siRNA down regulation of UGT1A9 and UGT2B7 on propofol glucuronidation in human hepatocytes
2013, Archives of Biochemistry and BiophysicsCitation Excerpt :These metabolites can then be further detoxified through conjugation reactions, carried out by the UGTs or other Phase II drug metabolizing enzymes, including the N-acetyltransferases, sulfotransferases, and glutathione S-transferases. There is accumulating evidence suggesting that protein–protein interactions occur between these enzymes and that these interactions play a significant role in modulating enzyme activity [3–12]. Co-localization and protein–protein interactions between drug metabolizing enzymes allows concerted metabolism to occur more efficiently [13].