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 UGT2B19 protein was established. The activity of UGT2B19 on 3a-hydroxy and 17b-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 mm for testosterone, etiocholanolone and 5b-androstane-3a,17b-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.Keywords: cynomolgus monkey; androgen conjugation; steroid metabolism; extrahepatic tissue; phase II enzymes.To date more than 18 different UDP-glucuronosyltransferase enzymes have been isolated in humans and are classified into two families, UGT1 and UGT2, based on the homology of the amino acid sequence [1]. Less than 50% identity exists between these two families. The isoforms of the UGT1 family are encoded by a single gene locus which is composed of at least 12 different first exons and four common exons (exons 2±5) [2]. The UGT2 family is comprised of separate genes which share the same organization of six exons and five introns [3,4] . The UGT1 family are mainly active on xenobiotics but also demonstrate activity on endogenous compounds such as bilirubin, thyroid hormones and steroids. UGT2B enzymes are active on xenobiotics but the main activity is on steroids and bile acids.In humans, the plasma contains significant levels of steroid metabolites in the form of glucuronide conjugates. It has been suggested that the plasma levels of 5a-reduced C19 steroid glucuronides, androsterone-glucuronide and 5a-androstane3a,17b-diol-glucuronide reflect the peripheral tissue conversion of adrenal and gonadal precursor C19 steroids to active androgens [5]. A recent study suggests...

Molecular Cloning of the Baboon UDP-Glucuronosyltransferase 2B Gene Family and Their Activity in Conjugating Morphine

“…The baboon UGT2Bs fall into three groups: UGT2B43 is the most highly conserved baboon UGT2B with a 92.1 and 90% identity to human UGT2B15 and UGT2B17, respectively; UGT2B41 and UGT2B42 are 88.3 and 88.5% identical to human UGT2B4; and UGT2B39, UGTB240, UGT2B44, UGT2B45, and UGT2B46 are from 88.8 to 90.2% identical to human UGT2B7 (Table 2). These homologies ranging from 88 to 92% are similar to those found in a comparison between monkey and human UGT2B genes (Bélanger et al, , 1999Beaulieu et al, 1998;Barbier et al, 1999a,b;Girard et al, 2002). No baboon UGT2Bs similar to human UGT2B10, UGT2B11, or UGT2B28 are obvious.…”

“…The sequence identity was especially high between the baboon and the monkey UGT2B proteins. The 99.1% identical protein structure between the baboon UGT2B43 and monkey UGT2B20 is the highest reported with any other UGT2B protein (human or primate) characterized to date (Bélanger et al, , 1999Beaulieu et al, 1998;Barbier et al, 1999a,b;Girard et al, 2002). As with UGT2B20, baboon UGT2B43 has an isoleucine substitution of a highly conserved arginine at position 96 not found in any other UGT2B (Barbier et al, 2000b).…”

Discovery of neurosteroid glucuronides in mouse brain

“…The main phase II conjugation reactions of many steroids are sulfation and glucuronidation catalyzed by sulfotransferases (SULT) and glucuronosyltransferases (UGT), respectively. Of these transferases, SULT 2A1 [7,8], SULT 2A2 [9], and SULT 2B1b [7], UGT 1A6 [10,11], UGT 2B7 [10], as well as UGT 2B19 [12] isoenzymes have been found in the rat, human, or monkey brain.…”

Glucuronidation of Anabolic Androgenic Steroids by Recombinant Human Udp-Glucuronosyltransferases

“…The involvement of UGTs of the 2B subfamily in steroid glucuronidation is well documented (Chen et al, 1993;Jin et al, 1993;Bélanger et al, 1999;Turgeon et al, 2001), and evidence of regioand stereoselective conjugation of endogenous androgens and pregnanes for these enzymes has been presented (Jin et al, 1997). The activity of UGT1A isoforms toward steroids was also studied, particularly for aglycones having a C18 structure .…”