ALTERED CODING FOR A STRICTLY CONSERVED DI-GLYCINE IN THE MAJOR BILIRUBIN UDP-GLUCURONOSYLTRANSFERASE OF A CRIGLER-NAJJAR TYPE-I PATIENT

The characterization (Ritter, J.K., Chen, F., Sheen, Y. Y., Tran, H. M ., Kimura, S., Yeatman, M. T., and Owens, I. S. (1992) J. Biol. Chem. 267, 3257-3261) of the single copy UGT1 gene complex locus encoding bo th bilirubin and phenol UDP-glucuronosyltransferases (transferase) has been critical to the determination of genetic defects in Crigler-Najj ar patients. The complex (UGT1A-UGT1M) codes for at least two bilirubi n, three bilirubin-like, and eight phenol transferase isozymes. In the 5' region, a minimum of 13 different exons 1, each with an upstream p romoter, are arrayed in series with 4 common exons in the 3' region of the locus. Each exon 1 encodes the amino terminus of a transferase, a nd the common exons encode the common carboxyl terminus of each isofor m. Although a deleterious mutation in a common exon inactivates the en tire locus, a deleterious mutation in an exon 1, as we report here for the UGT1A gene in a Crigler-Najjar Type I patient, affects the amino terminus of that s ingle isoform. Recessively inherited mutant alleles for the predominant bilirubin isozyme, the HUG-Br1 protein, substitut ed Arg for Gly at codon 276 (G276R) in exon 1 of UGT1A abolishing a co nserved di-glycine. The mutant HUG-Br1-G276R protein expressed in COS- 1 cells had no detectable bilirubin glucuronidating activity at either pH 7.6 or 6.4. Although each of the bilirubin-type isozymes contains a conserved peptide between residues 270 and 288, all UDP-glucuronosyl transferases contain a di-glycine at approximately position 276-277, m aking it strictly conserved. Structure-func tion relationship was stud ied by site-directed mutations of the HUG-Br1 cDNA; G276A, G276Q, G276 E, G276I, and P270G mutants were inactive, and V275I- and P285G-altere d transferases expressed normal activity. Conservation of residues bet ween the related baculoviral ecdysone UDP-glucosyltransferase and the UDP-glucuronosyltransferases confirms the critical role of the Gly-276 as well as other residues.