Disulfide bond-mediated stabilization of the oligomers of UDP-glucuronosyltransferase 2B7.

UDP-glucuronosyltransferase (UGT) is an important drug-metabolizing enzyme involved in the detoxification of hydrophobic chemicals by conjugating them with hydrophilic glucuronic acid. Previous studies have revealed that UGTs form oligomers between or among the same or other isoforms, but the molecular mechanism underlying this formation remains unclear. In this study, we used optimized electrophoretic techniques to analyze UGT2B7 homo-oligomer formation. UGT2B7 was expressed in COS-1 cells, and lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). By omitting the heating and reducing steps during SDS-PAGE sample preparation, bands corresponding to dimer, tetramer, and higher-order oligomers were detected in addition to monomeric bands. Since these SDS-stable UGT2B7 oligomer bands disappeared with the addition of reducing agents, we hypothesized that intermolecular disulfide bonds are involved in the formation of UGT oligomers. The cysteine residues important for this oligomer formation were investigated. Analyses using alanine substitution and deletion mutants suggested that three cysteine residues of UGT2B7, Cys127, Cys156, and Cys282, are important not only for oligomer formation but also for glucuronidation ability. We further investigated the oligomerization of UGT2B7 in intact living cells using two membrane-permeable cross-linkers, disuccinimidyl suberate and dithiobis(succinimidyl propionate). UGT2B7-expressing cells were treated with these reagents and analyzed by western blot. This cross-linker treatment markedly reduced the UGT2B7 monomer band and increased the formation of higher-molecular-mass species. These results indicated that the majority of UGT2B7 is present within cells as oligomers, maintaining its enzymatic function, rather than as a monomer.