This project follows up on the Section's previous description of the UGT1 locus, which has proven critical for the determination of genetic defects in Crigler-Najjar Type I (CN-I) patients. The complex (UGT1A - UGT1G) codes for at least 2 bilirubin (Br), 3 Br-like, and 2 phenol transferases. Seven different exon 1s, each with an upstream promoter and each encoding the amino terminus of an isoform, are arrayed in series with 4 common exons (encoding seven identical carboxyl termini) in the 3~ region of the locus. Predictably, a critical mutation in a common exon inactivates the locus; a deleterious mutation in an exon l as we describe for the UGT1A gene in two CN-I patients affects the amino terminus of that single isoform. The code in patient 1 for the predominant Br isozyme, the HUG- Br1 protein, is missing codon 170 (phe) in exon 1 of UGT1A abolishing a diphenylalanine in a conserved hydrophobic region A encoded by codons 161- 180. Studies with this mutant led to the discovery of the previously unknown major pH optimum (6.4) in contrast to the routinely used pH (7.6) which we show is only 1/3 as effective for Br glucuronidation using either HUG-Br1 protein or human liver microsomes. HUG-Br1-170, inactive at pH 6.4, has normal activity at pH 7.6, a result consistent with a pH- sensitive mutant. The HUG-Br1 protein and microsomes glucuronidate Br best at low ionic strength. The wild-type protein Km value for Br is 2.5 micromoles (pH 6.4 or 7.6); that for HUG-Br1-170 is 5.0 micromoles (pH 7.6). In the genome of CN-I patient 2, a point mutation (G-greater than C, gly to arg) at codon 276 abolishes a diglycine in a conserved region B encoded by codons 270-288 in exon 1 of UGT1A. The HUG-Br1-276, inactive at pH 6.4 and 7.6, defines micro-structure/region B which has 7 hydrophobic-, 7 amide-containing-, and 4 alpha-helix-breaking amino acid residues. Point mutations in common exons 2 and 4 of CN-I patients 3 and 4 with G->A (codon 309) and C->T (codon 376) uncovered conserved region C encoded by codons 298-315 and conserved residue D, ser/ala, respectively. In the common carboxyl terminus micro-regions C and D presumably interact with the common substrate, UDP-glucuronic acid, while hydrophobic regions A and B most likely interact with the lipophile-like Br substrate. Characterization of human transferase encoded by the UDPGTh-3 cDNA, establishes that it specifically glucuronidates the dihydrotestosterone and androstane diol steroids; its mRNA is located in testis and prostate, as well as liver, suggesting a critical role for udpgth-3 in target tissue. The absence of its mRNA, and the presence of that for four other isoforms in the liver of a patient with benign prostate hyperplasia (characterized by depressed glucuronidation of dihydrotestosterone) is supportive evidence for its biochemical importance.
