Interactions with other human UDP-glucuronosyltransferases attenuate the consequences of the Y485D mutation on the activity and substrate affinity of UGT1A6

Objectives To explore the possible role of hetero-oligomerization among the human UDP-glucuronosyltransferases in attenuating the consequences of the pathological Y486D mutation (UGT1A1 numbering) that often causes hyperbilirubinaemia. Owing to exon sharing in the human UGT1A gene, the equivalent mutation is present in all other UGT1As of the affected individuals. It is unknown, however, if this mutation results in clinical conditions, other than impaired bilirubin conjugation by UGT1A1. Methods The main experimental approach in this study was to try and form hetero-oligomers of selected UDP-gl ucuronosyltransferases by coinfecting insect cells with recombinant baculoviruses that encode different human UDP-glucuronosyltransferases and mutants thereof. The infected cells were analysed for both relative expression levels and catalytic activity in each case, the combination of which yielded normalized activity. Kinetic analyses and copurification by affinity chromatography were also performed. Results Coinfections with UGT1A4 increased the normalized scopoletin glucuronidation of 6YD (the Y485D mutant of UGT1A6) much more than it affected 1 YD (the Y486D mutant of UGT1A1). Serotonin glucuronidation analyses revealed that coexpression of 6YD with most other human UDP-glucuronosyltransferases significantly increased the normalized activity of this mutant. Using 1-naphthol as the aglycone substrate, the K-m of 6YD for the cosubstrate UDP-glucuronic acid was about 50 times higher than in UGT1A6. Yet, coexpression of 6YD with UGT1A4 lowered the K-m for UDP-glucuronic acid to the level of UGT1A6. Coexpression also influenced wild-type UGT1A6 and UGT2B7, increasing the normalized activity of UGT1A6, but decreasing it for UGT2B7. Conclusion Hetero-oligomerization may play an important role in UDP-glucuronosyltransferases activity.