title: Cloning and characterization of a simian UDP-glucuronosyltransferase enzyme UGT2B20, a novel C19 steroid-conjugating protein.
abstract: Steroid glucuronidation by UDP-glucuronosyltransferase (UGT) enzymes is a mechanism leading to catabolism and elimination of steroid hormones. To establish an animal model to investigate the conjugation of steroids by UGT enzymes, previous results revealed that simian and human are unique in having high levels of circulating androsterone glucuronide and androstane-3alpha, 17beta-diol (3d-Diol) glucuronide. A cDNA, UGT2B20, was isolated from cynomolgus monkey liver and prostate libraries. The cDNA was 2075 bp in length and contained an open reading frame of 1590 bp, encoding a protein of 530 amino acid residues. The UGT2B20 clone was transfected and stably expressed in the human embryo kidney HK293 cell line, and the transferase activity of UGT2B20 was tested with 73 compounds. This enzyme was shown to be active with androgens, such as testosterone, dihydrotestosterone (DHT) and 3alpha-Diol, and on catecholoestrogens including 1,3,5,10-oestratriene-3, 4-diol-17-one. Kinetic analysis performed with intact cells yielded apparent Km values of 1.1, 2.3 and 4.6 microM for 3alpha-Diol, DHT and testosterone respectively. Reverse transcriptase-PCR analysis demonstrated that UGT2B20 transcript is expressed in several tissues including the liver, prostate, kidney, epididymis and adrenal of the cynomolgus monkey. Amino acid sequence alignment shows that the UGT2B20 protein is 92% identical with UGT2B15. Both enzymes have similar apparent Km values for DHT and 3alpha-Diol, and demonstrate similar transcript tissue distribution. The characterization of simian UGT2B20 as a structural and functional homologue of human UGT2B15 further demonstrates the similarities of steroid glucuronidation in these two species, and indicates the relevance of using the monkey as an animal model to study and understand steroid glucuronidation in extrahepatic-steroid target tissues.
authors.affiliation: Laboratory of Molecular Endocrinology, CHUL Research Center, Laval University, 2705 Laurier Boulevard, Quebec, Canada G1V 4G2.
authors.forename: O
authors.initials: O
authors.lastname: Barbier
authors.forename: A
authors.initials: A
authors.lastname: Bélanger
authors.forename: D W
authors.initials: DW
authors.lastname: Hum
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005965:Glucuronates / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008252:Macaca fascicularis; D008969:Molecular Sequence Data; D009928:Organ Specificity; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013256:Steroids / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D005965:Glucuronates; D013256:Steroids; D014453:Glucuronosyltransferase