Isolation and characterization of the monkey UDP-glucuronosyltransferase cDNA clone monUGT1A01 active on bilirubin and estrogens

Although enzymes that catalyze the formation of steroids are well known, le ss information is available about the enzymes involved in the metabolism of these hormones. Steroid glucuronidation, by UDP-glucuronosyltransferase en zymes, is one mechanism by which steroid hormones can be metabolized and el iminated from a tissue. Previous results suggest that the monkey represents the most appropriate animal model for studying the physiologic relevance o f steroid glucuronidating enzymes. The monkey UGT1A01 cDNA clone was isolat ed by RT-PCR amplification of the liver RNA. The cDNA contains an open read ing frame of 1599 bp encoding a protein of 533 residues. The primary struct ure of monkey UGT1A01 is 95% identical to human UGT1A1 To compare monkey an d human UGT1A1 enzymes, both cDNA clones were transfected into HK293 cells an stable cell lines expressing each UGT1A1 protein were established. Weste rn blot analysis of the monUGT1A01-HK293 and hUGT1A1-HK293 cell lines using a anti-UGT1A polyclonal antibody (RC-71) revealed expression of exogenous 55 kDa UGT1 proteins. The transferase activities of monkey and human UGT1A1 proteins were tested with over 60 compounds and were demonstrated to be ac tive on the same compounds, For endogenous compounds only bilirubin and CIS steroids were glucuronidated by these enzymes. Using microsome preparation (from HK293 cell expressing monkey UGT1A01), the apparent K-m values were 13, 5 and 6 muM for the conjugation of estradiol, 2-hydroxyestradiol and 2- hydroxyestrone, respectively, and were very similar to the values obtained with human UGT1A1 Specific RT-PCR analysis demonstrated the expression of m onkey and human UGT1A1 transcripts in several tissues including liver, kidn ey, intestine, prostate, testis and ovary suggesting a contribution of this isoenzyme to estrogen metabolism in the cynomolgus monkey as in human. (C) 2001 Elsevier Science Ltd. All rights reserved.