Identification and functional characterization of UDP-glucuronosyltransferases UGT1A8*1, UGT1A8*2 and UGT1A8*3

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Abstract
UDP-glucuronosyltransferase (UGT) 1A8 is part of the UGT1 locus and is expressed exclusively in extrahepatic tissues. Analysis of UGT1A8 exon 1 sequence has identified four genotypes from a population of 69 individuals. While there are four alleles, one of the single base pair changes leads to a silent mutation at T255, while the other mutations lead to amino acid substitutions at positions 173 and 277, creating three allelic variants. UGT1A8*1 (A173C277), UGT1A8*1a (T255A>G), UGT1A8*2 (G173C277) and UGT1A8*3 (A173Y277). The allelic frequencies of UGT1A8*1, UGT1A8*1a, UGT1A8*2 and UGT1A8*3 are 0.551, 0.282, 0.145 and 0.022, respectively. To examine the properties of the UGT1A8 proteins, UGT1A8*1 and UGT1A8*2 were cloned from a human colon cDNA library and UGT1A8*3 generated by mutagenesis using UGT1A8*1 as template. The cDNAs were expressed in HK293 cells to examine catalytic function as well as abundance as observed by analysis of UGT1A8-GFP (green fluorescent protein) expression. The single amino acid change that identifies UGT1A8*1 (A173) and UGT1A8*2 (G173) has little impact on function, while the UGT1A8*3 (Y277) is a conserved amino acid alteration represented by a dramatic reduction in catalytic activity. Protein abundance, as determined by Western blot analysis following transient transfection, is not altered. In addition, functional UGT1A8-GFP variants displayed staining in the cytoplasmic region, indicating that each protein is expressed in similar cellular compartments. Together, these data suggest that the null UGT1A8*3 results from structural changes and not a lack of protein expression. Allelic variation leading to singular codon changes could potentially alter drug metabolism in extrahepatic tissues.