Identification and Functional Characterization of a Novel UDP-Glucuronosyltransferase 2A1 Splice Variant: Potential Importance in Tobacco-Related Cancer Susceptibility

Abstract

UDP-glucuronosyltransferase (UGT) 2A1 is a respiratory and aerodigestive tract-expressing phase II detoxifying enzyme that metabolizes various xenobiotics including polycyclic aromatic hydrocarbons (PAHs). In the present study, a novel exon 3 deletion splice variant was identified for UGT2A1 (UGT2A1⌬exon3). As determined by reverse transcription-polymerase chain reaction (PCR), UGT2A1⌬exon3 was shown to be expressed in various tissues including lung, trachea, larynx, tonsil, and colon. The ratio of UGT2A1⌬exon3/wild-type UGT2A1 expression was highest in colon (0.79 Ϯ 0.08) and lung (0.42 Ϯ 0.12) as determined by real-time PCR; an antibody specific to UGT2A1 showed splice variant protein (UGT2A1_i2) to wild-type protein (UGT2A1_i1) ratios in the range of 0.5 to 0.9 in these tissues. Using ultra-pressure liquid chromatography, we found that homogenates prepared from UGT2A1_i2-overexpressing human embryonic kidney 293 cells exhibited no glucuronidation activity against PAHs, including benzo[a]pyrene-7,8-dihydrodiol (B[a]P-7,8-diol). An inducible in vitro system was created to determine the effect of UGT2A1_i2 expression on UGT2A1_i1 activity. Increasing UGT2A1_i2 levels resulted in a significant (p Ͻ 0.01) decrease in the UGT2A1_i1 V max against 1-hydroxy (OH)-pyrene, 3-OH-benzo[a]pyrene, and B[a]P-7,8-diol; no significant changes in K M were observed for any of the three substrates. Coimmunoprecipitation experiments suggested the formation of UGT2A1_i1 and UGT2A1_i2 hetero-oligomers and UGT2A1_i1 homo-oligomers; coexpression of UGT2A1_i1 or UGT2A1_i2 with other UGT1A or UGT2B enzymes caused no change in UGT1A or UGT2B glucuronidation activity. These data suggest that a novel UGT2A1 splice variant regulates UGT2A1-mediated glucuronidation activity via UGT2A1-specific protein-protein interactions, and expression of this variant could play an important role in the detoxification of carcinogens within target tissues for tobacco carcinogenesis.

Modulation of the UGT2B7 Enzyme Activity by C-Terminally Truncated Proteins Derived from Alternative Splicing

“…However, UGT2B7 splice variants were determined to have no effect on the glucuronidation activity of estradiol at position 3 that is performed by UGT1As, namely UGT1A1, suggesting that their modulatory effects could be specific to the UGT2B7 enzyme. This observation is consistent with a previous study showing that coexpression of UGT2A1_i1 or UGT2A1_i2 with other UGT1A or UGT2B enzymes caused no change in UGT1A or UGT2B glucuronidation activity (Bushey and Lazarus, 2012). Additional investigations are needed to better understand the oligomerization of UGT2B7 with its alternative splicing partners as well as with other UGT pathways and its effect on glucuronidation activity in vivo.…”

Unravelling the transcriptomic landscape of the major phase II UDP-glucuronosyltransferase drug metabolizing pathway using targeted RNA sequencing

“…However, we cannot exclude that some of the less frequent exon-exon junctions arise from mapping errors. Consistent with findings of previous laborious targeted approaches, 9,10,14,15,18,27,28 all reported alternative UGT exons and transcripts were identified, validating the robustness of our approach. Overall, this study reports nearly 300 transcribed variants encoded by the ten human genes, well beyond the suspected complexity already hinted by previous studies.…”

“…This exon-skipping event might result in the production of alternative proteins with impaired glucuronidation activity but with regulatory function upon glucuronidation, as recently observed for UGT2A1/UGT2A2_Δex3 variants. 18,28 Alternative splicing may also lead to the inclusion of a novel protein region, as observed for the novel UGT2B7_ex2b variant (Figure 9). This UGT2B7 variant presents a new protein region of 32 amino acids exclusively detected in DM tissues.…”

Quantitative profiling of the UGT transcriptome in human drug-metabolizing tissues

“…Yet, they are able to oligomerize with UGT enzymes to considerably reduce their conjugation activity. 1,3,25,28,29 Moreover, alternative functions of variant UGT1A_i2 proteins in the regulation of redox and glycolytic enzymes via protein-protein interactions have been reported, supporting roles diverging from conjugation reactions. 25,30 This is of particular interest in the context of cancer, where a preferential expression of some UGT alternates is observed.…”