Abstract
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.
Highlights
Research and the Canada Research Chair Program
This is driven by alternative usage of the first variable coding exon, which is joined to four constant exons [2,3,4,5], encompassing the co-substrate uridine-diphosphate glucuronic acid (UDPGA)-binding4 domain
Upon treatment with probed with a specific monoclonal antibody linked with horse- ponasterone A, cells demonstrated significant levels of radish peroxidase (Invitrogen), as specified in the correspond- UGT1A1_i2, but no significant changes were noted for ing figure legend
Summary
Materials—UDPGA was obtained from Sigma; blasticidin, geneticin (G418), and hygromycin were from Wisent The HEK293 clonal cell lines used for the kinetic studies consisted of clones expressing UGT1A_i1 (pcDNA3.1/tagged with Myc-His epitopes), UGT1A_i2 (pcDNA6/tagged with v5-His epitopes), or both pcDNA3.1-UGT1A_i1/Myc-His and pcDNA6-UGT1A_i2/v5-His, as described previously [3]. To ascertain the level of UGT content in stable UGT1A-HEK293 cell lines, a semiquantitative immunoblot analysis method was performed as described previously [3] using the antibody RC-71 (anti-UGT1A) as the primary antibody and an anti-rabbit IgG horse antibody conjugated with peroxidase as the second antibody (Amersham Biosciences). After centrifugation (13,000 ϫ g for 1 min), 1 mg of supernatant was added with 1 g of specific monoclonal antibody (Invitrogen) in 1 ml of high salt buffer and incubated at 4 °C with 50 l of protein G-Sepharose 4 fast flow (50% slurry) for 15 h. Formation of glucuronides (absolute activity) was corrected by UGT protein content (i1 active enzyme) assessed by Western blot and expressed as relative activity. The difference in glucuronidation rates and kinetic parameters was evaluated for statistical significance by the paired Student’s t test (significant at least at p Ͻ 0.05)
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