UDP-glucuronosyltransferase (UGT), as an integral membrane protein localized in the endoplasmic reticulum, has the ability to detoxify potentially hazardous xenobiotic substances. Most UGTs are expressed in liver, but UGT1A10 has proven to be an extrahepatic enzyme considerably expressed throughout the gastrointestinal tract. Earlier studies indicated that different UGT isoforms could exist in higher-order homo-oligomers or at least dimers within the membrane, but the formation of intermolecular disulfide bridges between UGT molecules was not often observed. In this study, we expressed recombinant human UGT1A10 in human embryonic kidney (HEK)293 and Chinese hamster ovary (CHO) cells to examine its oligomeric states and characterize its enzymatic activities against two therapeutically interesting substrates, morphine and entacapone, including determination of the catalytic rate constant (kcat) values for the first time. It was observed that a majority of the UGT1A10 protein expressed in HEK293 cells existed in covalently crosslinked higher-order oligomers via formation of intermolecular disulfide bonds, whereas formation of the intermolecular disulfide bonds was not observed in the UGT1A10 protein expressed in CHO cells. Owing to the formation of the covalently crosslinked higher-order oligomers, the UGT1A10 protein expressed in HEK293 cells had much lower catalytic activity (particularly the catalytic rate constant kcat) against both morphine and entacapone, compared with the UGT1A10 protein form expressed in CHO cells against the same substrates.
|Number of pages||7|
|Journal||Drug Metabolism and Disposition|
|State||Published - Oct 2018|
Bibliographical noteFunding Information:
This work was supported in part by the National Institutes of Health National Institute on Drug Abuse Translational Avant-Garde Award (UH2/UH3 DA041115) and Grants R01 DA035552, R01 DA032910, R01 DA013930, and R01 DA025100, and the National Science Foundation Grant CHE-1111761. https://doi.org/10.1124/dmd.118.082495. s This article has supplemental material available at dmd.aspetjournals.org.
Chemicals and Materials. Phusion DNA polymerases, restriction enzymes, and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA). All oligonucleotides were purchased from Eurofins MWG Operon (Louisville, KY). FreeStyle Chinese hamster ovary-S cells (denoted as CHO for convenience), FreeStyle 293 human embryonic kidney cells (denoted as HEK293 for convenience), HEK293FT, FreeStyle CHO Expression Medium, FreeStyle 293 Expression Medium, hypoxanthine/thymidine (HT) supplement, L-glutamine, 4%–12% Tris-glycine Mini Protein Gel, and SimpleBlue SafeStain were purchased from Life Technologies (Carlsbad, CA). Morphine was provided by the National Institute on Drug Abuse (NIDA) Drug Supply Program. Morphine-3-glucuronide, entacapone, entacapone 3-O-glucuronide, UDP-GA, Triton X-100, saccharolactone, b-glucuronidase, phospholipids (phosphatidylcholine type X-E), and the solvents used in high-performance liquid chromatography (HPLC) were purchased from MilliporeSigma (St. Louis, MO). HisPur Cobalt Resin was obtained from Thermo Fisher Scientific (Waltham, MA). Centrifugal filter units were ordered from Millipore (Burlington, MA).
This work was supported in part by the National Institutes of Health National Institute on Drug Abuse Translational Avant-Garde Award (UH2/UH3 DA041115) and Grants R01 DA035552, R01 DA032910, R01 DA013930, and R01 DA025100, and the National Science Foundation Grant CHE-1111761. Chemicals including morphine were kindly provided by the National Institute on Drug Abuse (NIDA) Drug Supply Program. We thank Dr. Zhenyu Jin for laboratory assistance and Drs. Yaxia Yuan and Kuo-Hao Lee for their help on molecular modeling.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
ASJC Scopus subject areas
- Pharmaceutical Science