TY - JOUR
T1 - Oligomerization and catalytic parameters of human UDP-glucuronosyltransferase 1A10
T2 - Expression and characterization of the recombinant protein
AU - Kim, Kyungbo
AU - Zheng, Fang
AU - Zhan, Chang Guo
N1 - Publisher Copyright:
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
PY - 2018/10
Y1 - 2018/10
N2 - 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.
AB - 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.
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U2 - 10.1124/dmd.118.082495
DO - 10.1124/dmd.118.082495
M3 - Article
C2 - 30111624
AN - SCOPUS:85052990600
SN - 0090-9556
VL - 46
SP - 1446
EP - 1452
JO - Drug Metabolism and Disposition
JF - Drug Metabolism and Disposition
IS - 10
ER -