Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A6 enzymes
Introduction
In mammals, glucuronidation is important for the detoxification and elimination of a large number of endogenous compounds (e.g. bile acids, bilirubin and steroids) and exogenous compounds (e.g. drugs, environmental chemicals and dietary constituents). This reaction is catalyzed by a multigenic family of UDP-glucuronosyltransferases (UGTs), which are typical membrane proteins of the endoplasmic reticulum and the nuclear envelope [1], [2], [3]. So far, two UGT gene families have been identified in humans, UGT1 and UGT2[2], [4]. The UGT1A gene is localized on chromosome 2q37 and encodes proteins with unique N-terminal domains and identical C-terminal domains, which are formed from the alternate mRNA splicing of unique first exons with common exons 2–5. In contrast, UGT2B genes are clustered on chromosome 4q13 and individual UGT proteins are encoded by unique genes with six exons [2], [4], [5], [6]. Each UGT exhibits unique substrate and tissue specificities [2], [3].
UGT1A6 is expressed in the liver, bile duct, stomach, colon, kidney and brain [3]. It plays important roles in the glucuronidation of planar and small aromatic molecules, including drugs such as acetaminophen and potential carcinogens chemically related to hydroxylated polycyclic aromatic hydrocarbons [3], [7]. In addition, serotonin (5-hydroxytryptamine, 5-HT) was reported as an endogenous substrate for UGT1A6 [8]. Interindividual differences in UGT1A6-mediated glucuronidation may have important pharmacological, physiological and toxicological consequences. Krishnaswamy et al. [9] have reported that individual variability in UGT1A6 expression and function in human liver is considerable, with more than 120-fold variability in UGT1A6 protein content and more than 13-fold variability in 5-HT glucuronidation activities. Although the mechanism for this variability is unclear at present, it seems likely to involve both genetic and environmental causes [3], [10].
Many UGT enzymes have been suggested to be expressed in hepatic and/or extrahepatic tissues of mammals including humans, monkeys, rats and mice [3], [11], and the cDNAs of several isoforms were cloned (http://som.flinders.edu.au/FUSA/ClinPharm/UGT/udgpa.html). Compared with rats and mice, monkeys are phylogenetically closer to humans, and various monkey species such as rhesus monkeys (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis) and marmosets (Callithrix jacchus) have been used in studies on safety evaluation and biotransformation for drug development. cDNA encoding an ortholog of human UGT1A6 has been cloned in cynomolgus monkeys; however, the functions of protein encoded by cDNA have not been characterized. Furthermore, examination of the functional characterization of monkey UGT enzymes is an important aspect of drug metabolism research.
The purpose of this study was to precisely identify differences in the enzymatic properties of UGT1A6 between humans and cynomolgus monkeys. To achieve this, human and cynomolgus monkey UGT1A6 cDNAs were cloned, and the corresponding UGT1A6 proteins were heterologously expressed in yeast cells. The enzymatic properties of UGT1A6 proteins were examined by kinetic analysis using 5-HT and 4-methylumbelliferone (4-MU) as substrates.
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Materials
Human liver total RNA, individual human liver microsomes (one male, 77-year-old; two females, 54 and 56-year-old) and rabbit anti-human UGT1A6 antibody were purchased from BD Biosciences (San Jose, CA). Three male cynomolgus monkey livers (4-year-old, 2.7–2.9 kg) were supplied by Ina Research Inc. (Ina, Japan). The use of human and cynomolgus monkey livers for this study was approved by the ethical review boards of Okayama University. The RNeasy mini kit and QIAshredder were purchased from
Sequence analysis
Sequence analysis determined that humUGT1A6 and monUGT1A6 cDNAs have an open reading frame of 1599 bp, and the cDNAs encode the respective proteins of 532 amino acids. Fig. 1 shows the alignment of the deduced amino acid sequences between humUGT1A6 and monUGT1A6. humUGT1A6 and monUGT1A6 showed 96% identity at both nucleotide and amino acid levels. The nucleotide sequence of humUGT1A6 was identical to that of cDNA registered in GenBank (Accession No. NM_001072). The amino acids at 25, 134 and 214
Discussion
UGT1A6, a member of the UGT superfamily, plays a key role in the glucuronidation of medicines and environmental chemicals [3], [7]. UGT1A6 has also been shown to be regulated by aryl hydrocarbon receptor agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and/or antioxidant-type inducers such as t-butylhydroquinone in mammalian cell lines [20], [21]. Although monkeys, including cynomolgus monkeys, are commonly used as an animal model for the development of medicines, and particularly for the
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research (18590116) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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2013, European Journal of Pharmaceutical SciencesFunctional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A1 enzymes
2010, Life SciencesCitation Excerpt :Three male cynomolgus monkey livers (4 years old, 2.7–2.9 kg) were supplied by Ina Research Inc. (Nagano, Japan). Cynomolgus monkey liver microsomes were prepared as described previously (Hanioka et al. 2006). The use of human and cynomolgus monkey livers for this study was approved by the ethics review boards of Okayama University.
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These authors contributed equally and should be considered joint first authors.