Novel polymorphic human UDP-glucuronosyltransferase (UGT) 2A3: Cloning functional characterization of enzyme variants comparative tissue expression and gene induction

摘要

UDP-glucuronosyltransferases (UGTs) are critical to the detoxification of numerous drugs, environmental pollutants, and endogenous molecules. However as yet not all of the human UGTs have been cloned and characterized. cDNA clones from the UGT2A3 gene (located on chromosome 4q13) were isolated using pooled human liver RNA. Approximately 10% of clones contained a c.1489A>G nucleotide substitution yielding proteins with a residue 497 alanine (UGT2A3.2) instead of a threonine (UGT2A3.1). The allele frequency of this polymorphism (rs13128286) was 0.13 in a European-American population as determined by direct DNA sequencing. Of 81 structurally diverse glucuronidation substrates tested, UGT2A3 expressed by a baculovirus system selectively glucuronidated bile acids – particularly hyodeoxycholic acid at the 6-hydroxy position. Apparent Km values of UGT2A3.1 and UGT2A3.2 for hyodeoxycholic acid 6-glucuronidation were 69±7 and 44±12 µM, respectively. Of 29 different extrahepatic tissues evaluated by real-time PCR, UGT2A3 mRNA was most highly expressed in small intestine (160% of liver), colon (78% of liver) and adipose tissue (91% of liver). An in silico scan of the proximal UGT2A3 promoter/5’-regulatory region identified transcription factor consensus elements consistent with tissue selective expression in liver (HNF1), and intestine (CDX2), as well as induction by rifampicin (PXR). In LS180 human intestinal cells, rifampicin increased UGT2A3 mRNA by more than 4.5-fold compared with vehicle while levels were not significantly affected by the AhR ligand β-naphthoflavone. This is the first report establishing UGT2A3 as a functional enzyme, and represents significant progress toward the goal of having a complete set of recombinant human UGTs for comparative functional analyses.