Alternatively spliced products of the UGT1A gene interact with the enzymatically active proteins to inhibit glucuronosyltransferase activity in vitro.
UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.
Bellemare
Judith
J
Pharmacogenomics Laboratory, Centre Hospitalier de l'Université Laval Research Center and Faculty of Pharmacy, Laval University, Boulevard Laurier, Québec, QC, Canada.
Rouleau
Mélanie
M
Girard
Hugo
H
Harvey
Mario
M
Guillemette
Chantal
C
D017398:Alternative Splicing; D015153:Blotting, Western; D002460:Cell Line; D003106:Colon / Q000201:enzymology; D004947:Esophagus / Q000201:enzymology; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics*; D006801:Humans; D047468:Immunoprecipitation; D007421:Intestine, Small / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D009097:Multienzyme Complexes / Q000378:metabolism*; D020033:Protein Isoforms; D055503:Protein Multimerization; D014162:Transfection; D000099148:UGT1A1 Enzyme
D014453:Glucuronosyltransferase; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D000099148:UGT1A1 Enzyme