Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A6 enzymes.
UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major isoform in the human liver that glucuronidates numerous drugs, environmental chemicals and endogenous substrates. In this study, human and cynomolgus monkey UGT1A6 cDNAs (humUGT1A6 and monUGT1A6, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate UGT1A6s. The enzymatic properties of UGT1A6 proteins were characterized by the kinetic analysis of serotonin (5-hydroxytryptamine, 5-HT) and 4-methylumbelliferone (4-MU) glucuronidation. humUGT1A6 and monUGT1A6 showed 96% identity in their nucleotide and amino acid sequences. Immunoblotting analysis using an antibody raised against human UGT1A6 showed that protein staining intensities were different between human and cynomolgus monkey UGT1A6 enzymes in microsomal fractions from livers and yeast cells, although both enzymes were detectable. The apparent K(m) value (15 mM) for 5-HT glucuronidation of cynomolgus monkey liver microsomes was significantly higher than that (8.6mM) of human liver microsomes, whereas V(max) values were lower in cynomolgus monkeys (2.8 nmol/min/mg protein) than in humans (8.6 nmol/min/mg protein). No significant species difference was observed in K(m) (approximately 90 microM) or V(max) (approximately 25 nmol/min/mg protein) values for liver microsomal 4-MU glucuronidation. In yeast cell microsomes, K(m) values (approximately 6mM) for 5-HT glucuronidation by recombinant UGT1A6s were similar, while a V(max) value (0.1nmol/min/mg protein) of monUGT1A6 was significantly lower than that (0.7 nmol/min/mg protein) of humUGT1A6. In 4-MU glucuronidation, both K(m) (210 microM) and V(max) (3.5 nmol/min/mg protein) values of monUGT1A6 were significantly higher than those of humUGT1A6 (K(m), 110 microM; V(max), 1.5nmol/min/mg protein). These findings suggest that the enzymatic properties of UGT1A6 were extensively different between humans and cynomolgus monkeys, although humUGT1A6 and monUGT1A6 showed high homology at the amino acid level. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.
Hanioka
Nobumitsu
N
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan.
Takeda
Yuri
Y
Jinno
Hideto
H
Tanaka-Kagawa
Toshiko
T
Naito
Shinsaku
S
Koeda
Akiko
A
Shimizu
Takefumi
T
Nomura
Mamoru
M
Narimatsu
Shizuo
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008252:Macaca fascicularis; D008861:Microsomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D012701:Serotonin / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D015262:Xenobiotics / Q000032:analysis / Q000633:toxicity
D018076:DNA, Complementary; D020719:Glucuronides; D011994:Recombinant Proteins; D015262:Xenobiotics; D012701:Serotonin; D006923:Hymecromone; C110655:UDP-glucuronosyltransferase, UGT1A6; D014453:Glucuronosyltransferase