Identification and functional characterization of UDP-glucuronosyltransferases UGT1A8*1, UGT1A8*2 and UGT1A8*3.
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.
Huang
Yue-Hua
YH
Laboratory of Environmental Toxicology, Department of Chemistry, Cancer Center, University of California, San Diego, La Jolla, California 92093-0636, USA.
Galijatovic
Alema
A
Nguyen
Nghia
N
Geske
Donald
D
Beaton
Deirdre
D
Green
Judith
J
Green
Mark
M
Peters
Wilbert H
WH
Tukey
Robert H
RH
D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D003106:Colon / Q000201:enzymology; D004247:DNA / Q000097:blood / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000302:isolation & purification; D005091:Exons; D005838:Genotype; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D004247:DNA; D014453:Glucuronosyltransferase; C454320:UDP-glucuronosyltransferase, UGT1A8