Abstract
Our previous studies have demonstrated functional protein–protein interactions between cytochrome P450 (CYP) 3A and UDP-glucuronosyltransferase (UGT). However, the role of carbohydrate chains of UGTs in the interaction with CYP is not well understood. To address this issue, we examined whether CYP3A1 modulates the function of UGT2B3 which lacks potential glycosylation sites. We also examined whether the introduction of N-glycosylation to UGT2B3 affects CYP3A-dependent modulation of UGT function. To introduce a potential glycosylation site into UGT2B3, Ser 316 of UGT2B3 was substituted with Asn by site-directed mutagenesis. A baculovirus-Sf-9 cell system for expressing CYP3A1 and UGT2B3/UGT2B3(S316N) was established using a Bac-to-Bac system. Glycosylation of UGT2B3(S316N) was demonstrated in this expression system. The microsomal activity of recombinant UGT was determined using 4-methylumbelliferone as a substrate. The effect of CYP3A1 co-expression on UGT function was examined by comparing the kinetic profiles between single (UGT alone) and double expression (UGT plus CYP) systems. The kinetics of the two expression systems fitted a Michaelis-Menten equation. When the 4-MU concentration was varied, co-expression of CYP3A1 lowered the Vmax of UGT2B3-mediated conjugation. Conversely, for UGT2B3(S316N), the Vmax in the dual expression system was higher than that in the single expression system. The data obtained demonstrate that the introduction of N-glycosylation to UGT2B3 alters its sensitivity to CYP3A1-dependent modulation while CYP3A1 enhanced UGT2B3(S316N) activity, and wild-type UGT2B3 was suppressed by CYP3A1. These data suggest that N-glycosylation of UGT is one of the determinants regulating the interaction between CYP3A and UGT.
Highlights
Introduction of an NGlycosylation Site into UDP-Glucuronosyltransferase 2B3 Alters Its Sensitivity to Cytochrome P450 3A1-Dependent ModulationEdited by: Vita Dolzan, University of Ljubljana, SloveniaReviewed by: Todd D
This study demonstrates that the introduction of N-glycosylation to UGT2B3 alters UGT sensitivity to a functional protein−protein interaction with CYP3A: wild-type UGT2B3 which lacks
N-glycosylation sites is suppressed by CYP3A1, whereas this P450 increases UGT2B3(S316N) activity
Summary
The potential for drug metabolism varies from one individual to another. drug metabolism capacity is a factor determining whether a drug produces pharmacological or adverse effects. Earlier studies have reported that N-glycosylation affects the enzyme activity of UGTs (Mackenzie, 1990a,b; Barbier et al, 2000; Nakajima et al, 2010; Nagaoka et al, 2012). We have reported that CYP3A4 interacts with UGT1A1, 1A6, 1A7, and 2B7 (Takeda et al, 2005; Ishii et al, 2014) All of these UGT isoforms possess potential N-glycosylation sites. Because of the important roles of N-glycosylation in UGT folding and function, we hypothesized that the N-glycosylation of UGT affects the interaction between CYP3A and UGT To address this issue, we focused on rat UGT2B3 (Mackenzie, 1987) which does not possess potential glycosylation sites. We examined whether CYP3A1 modulates UGT2B3 and whether the introduction of N-glycosylation to UGT2B3 affects the CYP3A-dependent modulation of the UGT function
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
