Molecular cloning, expression and characterization of a monkey steroid UDP-glucuronosyltransferase, UGT2B19, that conjugates testosterone

Although enzymatic processes involved in the formation of active steroids a re well known, less information is available about the enzymes responsible for the metabolism of these hormones. Moreover, the expression of these cat abolic enzymes, which include UDP-glucuronosyltransferases, may play a role in the regulation of the level and action of steroid hormones in steroid t arget tissues. Previous studies have shown that the cynomolgus monkey conta ins high levels of circulating androgen glucuronides, indicating that it re presents the best animal model to study the glucuronidation of steroids in extrahepatic tissues. Two cDNA libraries were constructed from monkey liver and prostate mRNA, and a novel UDP-glucuronosyltransferase UGT2B cDNA, UGT 2B19, was isolated from both libraries. The UGT2B19 cDNA is 2108 bp in leng th and contains an open reading frame of 1584 bp encoding a protein of 528 residues. The UGT2B19 cDNA clone was transfected into HK293 cells and a sta ble cell line expressing UGT2B19 protein was established. The activity of U GT2B19 on 3 alpha-hydroxy and 17 beta-hydroxy positions of steroids was dem onstrated. The enzyme also conjugates xenobiotics including eugenol, l-naph thol and p-nitrophenol. Kinetic analysis revealed that UGT2B19 glucuronidat es steroids with K-m values of 1.6, 2.6 and 4.3 mu M for testosterone, etio cholanolone and 5 beta-androstane-3 alpha,17 beta-diol, respectively. UGT2B 19 transcript was detected, by specific reverse transcriptase-PCR analysis in the liver, ovary, prostate, colon, spleen, kidney, pancreas, brain, cere bellum, mammary gland and epididymis. The molecular characterization of sim ian UGT2B19 demonstrates relevance of using monkey as an animal model to st udy and understand steroid glucuronidation in extrahepatic target tissue.