cDNA Cloning and Functional Analysis of Minipig Uridine Diphosphate-Glucuronosyltransferase 1A1

Abstract

Uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) plays important roles in the glucuronidation of various drugs and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies, because many of their physiological characteristics are similar to those of humans. In this study, the similarities and differences in enzymatic properties of UGT1A1 between humans and minipigs were precisely identified. Minipig UGT1A1 (mpUGT1A1) cDNA was firstly cloned by the rapid amplification of cDNA ends (RACE) method, and the corresponding protein as well as human UGT1A1 (hUGT1A1) enzyme was expressed in insect cells. Then the kinetics of estradiol at 3-hydroxy position (E-3OH) and 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation by recombinant UGT1A1s as well as human and minipig liver microsomes were analyzed. The homology between mpUGT1A1 and hUGT1A1 at the amino acid level was 80.9%. E-3OH and SN-38 glucuronidation by recombinant hUGT1A1 and mpUGT1A1 showed allosteric sigmoidal kinetics. The CL max value (29.1 µL/min/mg protein) for E-3OH glucuronidation of mpUGT1A1 was significantly higher (1.4-fold) than that of hUGT1A1, whereas the CL max value (0.83 µL/min/mg protein) for SN-38 glucuronidation was significantly lower (27%) than that of hUGT1A1; however, the kinetic models and parameter levels for E-3OH and SN-38 glucuronidation by human and minipig liver microsomes did not parallel those in the respective species. These findings suggest that the enzymatic properties of UGT1A1 are considerably different between humans and minipigs. The information on species differences in UGT1A1 function gained in this study should help with in vivo extrapolation of xenobiotic metabolism and toxicity.Key words uridine diphosphate-glucuronosyltransferase (UGT); UGT1A1; minipig; estradiol at 3-hydroxy position; 7-ethyl-10-hydroxycamptothecin Glucuronidation is important for the detoxification and elimination of various lipophilic endogenous substances (e.g. bile acids, bilirubin and steroids) and xenobiotics (e.g. drugs, environmental chemicals and dietary constituents) in mammals.1) This reaction is catalyzed by a multigenic family of uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) that are localized in the endoplasmic reticulum membrane. 2-4)The human UGT superfamily comprises two families (UGT1 and UGT2) and three subfamilies (UGT1A, UGT2A, and UGT2B).2) The UGT1A gene is localized on chromosome 2q37 and codes proteins with unique N-terminal domains and identical C-terminal domains, which are formed from the alternate mRNA splicing of unique first exons with common exons 2-5. In contrast, UGT2A and UGT2B genes are clustered on chromosome 4q13 and individual UGT proteins are coded by unique genes with six exons. 5,6) In addition, each UGT has been demonstrated to exhibit unique substrate and tissue specificities. 3,4,7) UGT1A1 is expressed in the liver, small intestine and colon, and is exclusively involved in the metabolism of bilirubin. 4,8,9) UGT1A1 has b...