PT  - JOURNAL ARTICLE
AU  - Ryan T. Bushey
AU  - Philip Lazarus
TI  - Identification and functional characterization of a novel UGT2A1 splice variant: Potential importance in tobacco-related cancer susceptibility
AID  - 10.1124/jpet.112.198770
DP  - 2012 Jan 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - jpet.112.198770
4099  - http://jpet.aspetjournals.org/content/early/2012/09/13/jpet.112.198770.short
4100  - http://jpet.aspetjournals.org/content/early/2012/09/13/jpet.112.198770.full
AB  - UGT2A1 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 (termed "UGT2A1Δexon3"). As determined by reverse-transcription polymerase chain reaction, UGT2A1Δexon3 was shown to be expressed in various tissues including lung, trachea, larynx, tonsil, and colon. The ratio of UGT2A1Δexon3:wild-type UGT2A1 expression was highest in colon (0.79±0.08) and lung (0.42±0.12) as determined by real-time PCR; an antibody specific to UGT2A1 showed splice variant protein (termed "UGT2A1_i2") to wild-type protein (termed "UGT2A1_i1") ratios in the range of 0.5-0.9 in these tissues. Using ultra-pressure liquid chromatography, homogenates prepared from UGT2A1_i2-over-expressing HEK293 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<0.01) decrease in the UGT2A1_i1 Vmax against 1-hydroxy (OH)-pyrene, 3-OH-B(a)P and B(a)P-7,8-diol; no significant changes in KM were observed for any of the three substrates. Co-immunoprecipitation experiments suggested the formation of UGT2A1_i1 and UGT2A1_i2 hetero-oligomers and UGT2A1_i1 homo-oligomers; co-expression 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 that expression of this variant could play an important role in the detoxification of carcinogens within target tissues for tobacco carcinogenesis.