Cloning and characterization of a simian UDP-glucuronosyltransferase enzyme UGT2B20, a novel C-19 steroid-conjugating protein

Steroid glucuronidation by UDP-glucuronosyltransferase (UGT) enzymes is a m echanism leading to catabolism and elimination of steroid hormones. To esta blish an animal model to investigate the conjugation of steroids by UGT enz ymes, previous results revealed that simian and human are unique in having high levels of circulating androsterone glucuronide and androstane-3 alpha, 17 beta-diol (3d-Diol) glucuronide. A cDNA, UGT2B20, was isolated from cyno molgus 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 a mino 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 w ith androgens, such as testosterone, dihydrotestosterone (DHT) and 3 alpha- Diol, and on catecholoestrogens including 1,3,5,10-oestratriene-3,4-diol-17 -one. Kinetic analysis performed with intact cells yielded apparent K-m val ues of 1.1, 2.3 and 4.6 mu M for 3 alpha-Diol, DHT and testosterone respect ively. Reverse transcriptase-PCR analysis demonstrated that UGT2B20 transcr ipt is expressed in several tissues including the liver, prostate, kidney, epididymis and adrenal of the cynomolgus monkey. Amino acid sequence alignm ent shows that the UGT2B20 protein is 92% identical with UGT2B15. Both enzy mes have similar apparent K-m values for DHT and 3 alpha-Diol, and demonstr ate similar transcript tissue distribution. The characterization of simian UGT2B20 as a structural and functional homologue of human UGT2B15 further d emonstrates the similarities of steroid glucuronidation in these two specie s, and indicates the relevance of using the monkey as an animal model to st udy and understand steroid glucuronidation in extrahepatic-steroid target t issues.