UDPGT cDNA expression and UDPGT1 in human liver.

Abstract

Following expression of UDPGTh1 and UDPGTh2 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 and 17-epiestriol, and hyodeoxycholic acid (HDCA), but the UDPGTh2 isozyme was 100-fold more efficient than UDPGTh1. UDPGTh1 and UDPGTh2 are 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 confers isoform substrate specificity. The data indicate a high level of conservation in the amino terminus is not required for the preservation of substrate specificity. Analysis of glucuronidation activity encoded by UDPGTh1/UDPGTh2 chimeric cDNAs constructed at their common restriction sites, Sac I (codon 279), Nco I (codon 385), and Hha I (codon 469), showed that nine amino acids between residues 385 and 469 are important for catalytic efficiency, suggesting that this region represents a domain which is critical for catalysis but distinct from that responsible for aglycon selection. Screening of leukocyte DNA cosmid library with human UDPGT-Br1 (1-470 bps) or UDPGT-Br2 (1-450 bps) resulted in three overlapping clones, which were isolated and mapped by endonucleases. Construction of subclones and DNA sequencing, Southern blot analysis revealed that a cluster of 4 exons (132, 88, 220, 1032 bps in one clone) encodes the entire region of 3' identity shared between human UDPGT-phenol, human UDPGT-Br1 and human UDPGT-Br2. A similar strategy but using probes which correspond to the unique regions of human UDPGT-Br1 and human UDPGT-Br2 showed that the exon 1 of UGT1A and UGT1D encodes the unique region of human UDPGT-Br1, and human UDPGT-Br2 and is located 5.6 and 49 Kb, respectively, upstream of the 4 common exons.