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

Abstract

Although enzymatic processes involved in the formation of active steroids are well known, less information is available about the enzymes responsible for the metabolism of these hormones. Moreover, the expression of these catabolic enzymes, which include UDP-glucuronosyltransferases, may play a role in the regulation of the level and action of steroid hormones in steroid target tissues. Previous studies have shown that the cynomolgus monkey contains high levels of circulating androgen glucuronides, indicating that it represents 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, UGT2B19, was isolated from both libraries. The UGT2B19 cDNA is 2108 bp in length 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 stable cell line expressing UGT2B 19 protein was established. The activity of UGT2B 19 on 3α-hydroxy and 17β-hydroxy positions of steroids was demonstrated. The enzyme also conjugates xenobiotics including eugenol, 1- naphthol and p-nitrophenol. Kinetic analysis revealed that UGT2B19 glucuronidates steroids with K(m) values of 1.6, 2.6 and 4.3 μM for testosterone, etiocholanolone and 5β-androstane-3α,17β-diol, respectively. UGT2B19 transcript was detected, by specific reverse transcriptase-PCR analysis in the liver, ovary, prostate, colon, spleen, kidney, pancreas, brain, cerebellum, mammary gland and epididymis. The molecular characterization of simian UGT2B19 demonstrates relevance of using monkey as an animal model to study and understand steroid glucuronidation in extrahepatic target tissue.