Comparison of glucuronidating activity of two human cDNAs, UDPGTh2 and UDPGTH2

Abstract

Two human liver UDP-glucuronosyltransferase cDNA clones, HLUG25 and UDPGTh2 were previously shown to encode isozymes active in the glucuronidation of hyodeoxycholic acid (HDCA) and certain estrogen derivatives (e.g., estriol and 3,4-catechol estrogens), respectively. In this study we have found that the UDPGTh2-encoded isoform (UDPGTh2) and HLUG25-encoded isoform (UDPGTh1) have parallel aglycone specificities. When expressed in COS 1 cells, each isoform metabolized three types of dihydroxy- or trihydroxy-substituted ring structures, including the 3,4-catechol estrogen (4-hydroxyestrone), estriol, 17-epiestriol, and HDCA, but the UDPGTh2 isozyme was 100-fold more efficient than UDPGTh1. UDPGTh1 and UDPGTh2 were 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terminus, a domain which conferred the substrate specificity. The data indicated that a high level of conservation in the amino terminus was not required for the preservation of substrate selectivity. Analysis of glucuronidation activity encoded by UDPGTh1/UDPGTh2 chimeric cDNA constructed at their common restriction sites, Sac I (codon 297), Nco I (codon 385), and Hha I (codon 469), showed that nine amino acids between residues 385 and 469 were important for catalytic efficiency, suggesting that this region represented a domain which was critical for the catalysis but distinct from that responsible for aglycone selection. These data indicate that UDPGTh2 is a primary isoform responsible for the detoxification of the bile salt intermediate as well as the active estrogen intermediates.