Evaluation of expression and glycosylation status of UGT1A10 in Supersomes and intestinal epithelial cells with a novel specific UGT1A10 monoclonal antibody

UDP-Glucuronosyltransferases (UGTs) are major phase II drug-metabolizing enzymes. Each member of the UGT family exhibits a unique but occasionally overlapping substrate specificity and tissue-specific expression pattern. Earlier studies have reported that human UGT1A10 is expressed in the gastrointe...

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Published in:	Drug metabolism and disposition 2017-09, Vol.45 (9), p.1027-1034
Main Authors:	Oda, Shingo, Kato, Yukiko, Hatakeyama, Masahiko, Iwamura, Atsushi, Fukami, Tatsuki, Kume, Toshiyuki, Yokoi, Tsuyoshi, Nakajima, Miki
Format:	Article
Language:	eng
Subjects:	Adult Aged Amino Acid Sequence Animals Antibodies, Monoclonal - chemistry Antibodies, Monoclonal - immunology Antibody Specificity Crypts Dopamine Duodenum Enzymatic activity Epithelial cells Epithelial Cells - cytology Epithelial Cells - enzymology Evaluation Female Gastrointestinal tract Glucuronosyltransferase - biosynthesis Glucuronosyltransferase - immunology Glucuronosyltransferase - metabolism Glycosylation HEK293 Cells Hep G2 Cells Humans Insect cells Insects Intestines - cytology Intestines - enzymology Isoforms Liver Localization Male MCF-7 Cells Mice Mice, Inbred C57BL Microsomes Middle Aged Monoclonal antibodies mRNA Pharmacology Protein expression Protein folding Proteins Rats Rats, Sprague-Dawley Small intestine Substrate specificity Substrates Variability
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Summary:	UDP-Glucuronosyltransferases (UGTs) are major phase II drug-metabolizing enzymes. Each member of the UGT family exhibits a unique but occasionally overlapping substrate specificity and tissue-specific expression pattern. Earlier studies have reported that human UGT1A10 is expressed in the gastrointestinal tract at the mRNA level, but the evaluation at the protein level, especially tissue or cellular localization, has lagged behind because of the lack of a specific antibody. In this study, we prepared a monoclonal antibody to UGT1A10 to elucidate the tissue/cellular distribution and interindividual variability of UGT1A10 protein expression. Western blot analysis revealed that the prepared antibody does not cross-react with any other human UGTs. Using this specific antibody, we observed that UGT1A10 protein is expressed in the small intestine but not in the liver or kidney. Immunohistochemical analysis revealed the expression of UGT1A10 protein in epithelial cells of the crypts and villi of the duodenum. In the small intestine microsomes from six individuals, the UGT1A10 protein levels exhibited 16-fold variability. Dopamine 3- and 4-glucuronidation, which is mainly catalyzed by UGT1A10 and by other UGT isoforms marginally, exhibited 50- to 65-fold variability, and they were not correlated with the UGT1A10 protein levels. Interestingly, the enzymatic activities of recombinant UGT1A10 in insect cells that were normalized to the UGT1A10 protein level were markedly lower than those in pooled human small intestine microsomes. Thus, the UGT1A10 antibody we generated made it possible to investigate the tissue/cellular distribution and interindividual variability of UGT1A10 protein expression for understanding the pharmacological and toxicological role of UGT1A10.
ISSN:	0090-9556 1521-009X