The relative protein abundance of UGT1A alternative splice variants as a key determinant of glucuronidation activity in vitro.
Alternative splicing (AS) is one of the most significant components of the functional complexity of human UDP-glucuronosyltransferase enzymes (UGTs), particularly for the UGT1A gene, which represents one of the best examples of a drug-metabolizing gene regulated by AS. Shorter UGT1A isoforms [isoform 2 (i2)] are deficient in glucuronic acid transferase activity but function as negative regulators of enzyme activity through protein-protein interaction. Their abundance, relative to active UGT1A enzymes, is expected to be a determinant of the global transferase activity of cells and tissues. Here we tested whether i2-mediated inhibition increases with greater abundance of the i2 protein relative to the isoform 1 (i1) enzyme, using the extrahepatic UGT1A7 as a model and a series of 23 human embryonic kidney 293 clonal cell lines expressing variable contents of i1 and i2 proteins. Upon normalization for i1, a significant reduction of 7-ethyl-10-hydroxycamptothecin glucuronide formation was observed for i1+i2 clones (mean of 53%) compared with the reference i1 cell line. In these clones, the i2 protein content varied greatly (38-263% relative to i1) and revealed two groups: 17 clones with i2 < i1 (60% ± 3%) and 6 clones with i2 ≥ i1 (153% ± 24%). The inhibition induced by i2 was more substantial for clones displaying i2 ≥ i1 (74.5%; P = 0.001) compared with those with i2 < i1 (45.5%). Coimmunoprecipitation supports a more substantial i1-i2 complex formation when i2 exceeds i1. We conclude that the relative abundance of regulatory i2 proteins has the potential to drastically alter the local drug metabolism in the cells, particularly when i2 surpasses the protein content of i1.
Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec Research Center and Faculty of Pharmacy, Laval University, Québec City, Québec, Canada.
Mélanie
M
Rouleau
Joannie
J
Roberge
Sarah-Ann
SA
Falardeau
Lyne
L
Villeneuve
Chantal
C
Guillemette
D017398:Alternative Splicing; D002166:Camptothecin / Q000031:analogs & derivatives / Q000378:metabolism; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics / Q000378:metabolism
C441475:7-ethyl-10-hydroxycamptothecin glucuronide; D020719:Glucuronides; D007527:Isoenzymes; D014453:Glucuronosyltransferase; D002166:Camptothecin