Isolation and characterization of a simian UDP-glucuronosyltransferase UGT2B18 active on 3-hydroxyandrogens

Abstract

A monkey cDNA, UGT2B18, encoding a UDP-glucuronosyltransferase (UGT) active on 3-hydroxyandrogens, has been isolated and characterized. Previous results suggested that the monkey represents the most appropriate animal model for studying the physiologic relevance of steroid UGTs. UGT2B18 was isolated from a cynomolgus monkey prostate cDNA library using human UGT2B7, UGT2B10 and UGT2B15 cDNA as probes. The cDNA is 1748 bp in length and contains an open reading frame of 1587 bp encoding a protein of 529 residues. The UGT2B18 cDNA clone was transfected into HK293 cells and a stable cell line expressing UGT2B18 protein was established. Western blot analysis of the UGT2B18-HK293 cell line using a human UGT2B17 polyclonal antibody (EL-93) revealed high expression of a 53 kDa UGT2B protein. The transferase activity of UGT2B18 was tested with over 60 compounds and was demonstrated to be principally active on C19 steroids having an hydroxyl group at position 3alpha of the steroid molecule. UGT2B18 was also active on planar phenols and bile acids. Kinetic analysis revealed that UGT2B18 glucuronidates 3-hydroxyandrogens with high velocity and affinity. Using cell homogenates, Km values of 5.1, 7.8 and 23 microM for androsterone (ADT), etiocholanolone and androstane-3alpha, 17beta diol (3alpha-diol) were obtained, respectively. Specific RT-PCR analysis demonstrated the expression of UGT2B18 transcripts in several tissues including liver, prostate, kidney, testis, adrenal, bile duct, bladder, colon, small intestine, cerebellum and pancreas suggesting a contribution of this isoenzyme to the high plasma levels of glucuronidated ADT and 3alpha-diol found in the cynomolgus monkey.