Drug Metab. Pharmacokinet. 29 (3): 259–265 (2014). Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX) Regular Article Extensive Protein-Protein Interactions Involving UDP- glucuronosyltransferase (UGT) 2B7 in Human Liver Microsomes Ryoichi FUJIWARA* and Tomoo ITOH School of Pharmacy, Kitasato University, Tokyo, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: UDP-glucuronosyltransferase (UGT) 2B7 is a membrane protein that catalyzes glucuronidation of endogenous and exogenous substrates. Because UGTs are expressed in the endoplasmic reticulum (ER), their substrates and metabolites need to be transported through the ER membrane. However, insight into the mechanism underlying the transport of substrates/metabolites of UGTs through the ER membrane has not been elucidated. Metabolosome is a functional unit of metabolism consisting of multiple metabolism- related proteins. UGTs might form a metabolosome to facilitate the transport of their substrates and/or metabolites through the ER membrane. In the present study, therefore, extensive protein-protein inter- actions involving UGT2B7 were determined by a shotgun analysis of immunoprecipitate. Our shotgun analysis revealed that 92 proteins were immunoprecipitated with anti-UGT2B7 antibody in human liver microsomes. We further determined that 42 proteins out of the 92 proteins were specifically immuno- precipitated with the anti-UGT2B7 antibody. In addition to UGT2B7, other microsomal enzymes such as UGT1A, CYP3A4, CYP1A2, and a monoamine oxidase, were included in the list of proteins immuno- precipitated with the anti-UGT2B7 antibody, suggesting that these proteins might form a metabolosome to regulate their functions in the liver. Further analyses are needed to elucidate the roles of those proteins in the enzymatic activity of human UGTs. Keywords: UDP-glucuronosyltransferase; protein-protein interactions; metabolosome; UGT2B7; CYP3A4 UGT1A4 is a unique UGT isoform that is responsible for N- Introduction glucuronidation of primary, secondary, and tertiary amine-contain- UDP-glucuronosyltransferases (UGTs; EC 2.4.1.17) are impor- ing xenobiotics such as imipramine and trifluoperazine.6,7) tant membrane-bound enzymes that metabolize drugs by trans- The active site of UGT proteins is located in the luminal side of ferring the glucuronic acid moiety of UDP-glucuronic acid to the the endoplasmic reticulum (ER).8) Therefore, substrates and a co- substrates.1) Human UGTs are divided into two distinct families, substrate of UGTs need to be transported into the ER lumen from UGT1 and UGT2, on the basis of evolutionary divergence and cytoplasm to be metabolized by UGTs. Metabolites also need to homology.2) The UGT1 gene is located on chromosome 2q37 be transported to cytoplasm to be further excreted from the body. and produces nine functional enzymes (UGT1A1, UGT1A3- Because glucuronides are extremely hydrophilic, they are not UGT1A10) by exon sharing.3) The UGT2A and UGT2B genes easily translocated across the ER membrane by passive diffusion. are located on chromosome 4q13, encoding three and seven func- The presence of multiple glucuronide transporters mediating the tional proteins, respectively.2) UGT2A1 and UGT2A2 are formed transport of glucuronides through the ER membrane has been by differential splicing of variable first exons and common exons 2 demonstrated, although those transporters have not been identi- to 6, likely on the UGT1A gene. Meanwhile, UGT2A3 and each fied.9) In contrast, a nucleotide sugar transporter, which is involved UGT2B isoform are encoded by individual genes.2) Each UGT in the transport of UDP-glucuronic acid from cytosol to the luminal enzyme expresses in a tissue-specific manner and exhibits sub- side of ER, has been identified.10) strate specificity.4) Among 19 functional UGT enzymes, UGT2B7 Cytochrome P450s (CYPs) are phase I drug-metabolizing is the most responsible isoform for glucuronidation of clinically enzymes. Similar to UGTs, CYP is a superfamily of enzymes used drugs.5) While most UGT isoforms catalyze the transfer of involved in the metabolism of a wide variety of endogenous and glucuronic acid to hydroxyl- and carboxyl-group of substrates, exogenous compounds. Among all CYP enzymes, CYP3A4 is the Received September 10, 2013; Accepted December 14, 2013 J-STAGE Advance Published Date: December 24, 2013, doi:10.2133/dmpk.DMPK-13-RG-096 *To whom correspondence should be addressed: Ryoichi FUJIWARA, Ph.D., School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Tel. ©81-3-5791-6249, E-mail: [email protected] This work was supported by the Naito Foundation (RF). 259 260 Ryoichi FUJIWARA and Tomoo ITOH most abundant in both the liver and intestinal tract and is involved Immunoprecipitation: Dynabeads were resuspended in a vial in the metabolism of more than 50% of clinically used drugs.5) for 5 min. Fifty µL (1.5 mg) of Dynabeads was transferred to a tube Although the active site of CYPs is located in the cytoplasmic side and the tube was placed on a magnet to remove the supernatant. of ER, the function of UGTs and CYPs is similar, as they share Two hundred µL of antibodies was added and the tube was rotated substrates such as codeine and acetaminophen. There is also a for 10 min at room temperature. The tube was again placed on functional relationship between UGTs and CYPs in that CYPs a magnet to remove the supernatant, and the Dynabead-antibody catalyze hydroxylation of substrates so that UGTs can transfer complex was washed using 200 µL PBS containing 0.1% Tween glucuronic acid to the hydroxyl group of the substrates. For 20. After the supernatant was removed by placing the tube on a example, UGTs introduce glucuronic acid to the hydroxyl groups magnet, 400 µL of human liver microsomes, which were sus- of trans-3A-hydroxycotinine and trans-4-hydroxyl tamoxifen, which pended in 0.25 M sucrose, was added to the tube. The tube was are oxidative metabolites of nicotine and tamoxifen by CYPs.11,12) then rotated for 20 min at room temperature. The tube was placed This fact led to the hypothesis that UGTs and CYPs interact with on the magnet to remove the supernatant, and the Dynabead- each other to transport substrates and metabolites across the ER antibody-antigen complex was washed three times using 200 µL membrane. To support this hypothesis, protein-protein interactions washing buffer. The supernatant was removed and the beads were between UGTs and CYPs have been demonstrated previously.13,14) resuspended in 100 µL washing buffer. After the bead solution was Furthermore, functional interactions between UGTs and CYPs transferred to a new tube and the supernatant was removed, 30 µL have also been demonstrated by investigating a sequential metabo- of SDS-PAGE buffer was added to elute the immune complex. lism of 7-ethoxycoumarin as a model substrate.15) It has also been Digestion of the immunoprecipitate: Ten µL of reduc- suggested that homo- and hetero-oligomerization of UGT proteins tion buffer (1.5 mg of dithiothreitol dissolved in 1 mL of 100 mM might be involved in the transport of substrates and glucuronides ammonium bicarbonate) was added to the eluted sample from the across the ER membrane.16) Homo- and hetero-oligomers of UGTs Dynabead-antibody-antigen complex. After a 30-min incubation at have been demonstrated by gel permeation chromatography, radia- room temperature, 10 µL of alkylating buffer (10 mg of iodoacet- tion inactivation analyses, cross-linking studies, double-UGT amide dissolved in 1 mL 100 mM ammonium bicarbonate) was isoforms-expression systems, native-PAGE analyses, and fluo- added. After a 30-min incubation at room temperature, 2 µL of rescence resonance energy transfer (FRET) analyses.16–19) How- trypsin solution (10 µg/mL) and 10 µL of 50 mM ammonium ever, insight into the mechanism underlying the transport of bicarbonate were added to the reaction tube. The tube was substrates of UGTs and their metabolites across the ER membrane incubated overnight at 30°C and the solution was evaporated to has not been clarified yet. dryness. Seven µL of 1% formic acid was added to the tube to Metabolosome and transportsome are functional units of dissolve the peptides. The peptide solution was then subjected to metabolism and membrane transport. Metabolosome and trans- nano-LC-MS/MS analysis. portsome are a multi-molecular assembly composed of metaboliz- Nano-LC-MS/MS analysis: Nano-LC-MS/MS analysis was ing enzymes, transport-related proteins (transporters, channels or carried out in a CapLC system (Waters, Milford, MA) with Q-tof pumps), regulatory proteins, scaffold proteins, and other functional microtechnology (Micromass, Manchester, UK). Peptides were cellular components, which are assembled by means of multiple injected into a Symmetry C18 5 µm, 180 µm © 20 mm precolumn protein-protein interactions and/or protein-lipid interactions.20) and subsequently separated using a 1.7 µm BEH 130 C18 100 Formation of metabolosome and/or transportsome assembled by µm © 25 mm separation column maintained at 30°C. Mobile extensive protein-protein interactions involving UGTs might be phases A and B were water with 0.1% formic acid and 0.1% associated with the transport of compounds across the ER mem- formic acid in acetonitrile, respectively. Peptide separation was brane. In the present study, large-scale analysis of protein-protein obtained by a gradient of 1–50% mobile phase B over 120 min at a interaction involving UGTs was carried out by shotgun liquid flow rate of 300 nL/mim, followed by a gradient of 50–85% mobile chromatography–mass spectrometry (LC-MS/MS) proteomic anal- phase B over 1 min and a 25 min rinse with 85% mobile phase B. ysis of immunoprecipitated proteins to identify proteins interacting Data acquisition and processing of the MS/MS analysis were with human UGT2B7 in human liver microsomes. carried out using MassLynx software (Micromass).