Introduction of an N-Glycosylation Site into UDP-Glucuronosyltransferase 2B3 Alters Its Sensitivity to Cytochrome P450 3A1-Dependent Modulation

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 V_max of UGT2B3-mediated conjugation. Conversely, for UGT2B3(S316N), the V_max 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.

Keywords: CYP; P450; UDP-glucuronosyltransferase; UGT; cytochrome P450; protein–protein interaction.

FIGURE 1

Immunoblots of the expression of UGT2B3, UGT2B3(S316N), and CYP3A1 in Sf-9 cells. To obtain microsomes simultaneously expressing CYP3A1 and UGT2B3/UGT2B3(S316N), Sf-9 cells were transfected with recombinant baculovirus for CYP3A1 and UGT2B3/UGT2B3(S316N). The lanes labeled “UGT2B3” and “UGT2B3(S316N)” show microsomal samples of single transfected cells. The lanes labeled “UGT2B3+CYP3A1” and “UGT2B3(S316N)+CYP3A1” show double transfected cells. Baculosomes (5 μg protein) from UGT2B3 single-expressing Sf-9 cells were electrophoresed (SDS-PAGE). For baculosomes of CYP3A1-UGT2B3 co-expression systems, protein amounts equivalent to UGT2B3 for the single-expressing system were used. Similarly, baculosomes (3 μg protein) from UGT2B3(S316N) single-expressing Sf-9 cells were electrophoresed. For baculosomes of CYP3A1-UGT2B3(S316N) co-expression systems, protein amounts equivalent to UGT2B3(S316N) for the single-expressing system were used. Mock represents the baculosomes (10 μg protein) from Sf-9 cells infected with baculovirus without passenger DNA which served as controls. The proteins in the gel were electrically transferred to a polyvinilidene difluoride membrane, and blotted with goat anti-mouse low pI form UGT (A-C) and rabbit anti-CYP3A2 (D,E), antibodies, respectively. In right panel of (A), baculosomes (25 μg protein) from UGT2B3(S316N)-expressing Sf-9 cells were treated with Endoglycosidase H (EndoH, 500 U) at 37°C for 17 h. Four microgram protein of the digest was subjected to immunoblotting with goat anti-mouse low pI form UGT antibody as a primary antibody. Black and red arrow heads represent original and deglycosylated UGT2B3(S316N), respectively.

FIGURE 2

Michaelis-Menten plots of 4-MU glucuronidation catalyzed by UGT2B3 in the absence and presence of CYP3A1 co-expression. The plots of UGT2B3 single- and UGT2B3-CYP3A1-dual are shown. The kinetics obtained by varying the 4-MU concentration (A) and UDPGA concentration (B) are shown. In (A), the 4-MU concentration was varied over the range 10-1000 μM while the UDPGA concentration was fixed at 2 mM. In (B), the UDPGA concentration was varied over the range 50 μM–3 mM while the 4-MU concentration was fixed at 1 mM. The total amount of protein added to the assay mixture was standardized at 100 μg. For this, when necessary, control baculosomes were added to the reaction mixture. Kinetic parameters were calculated by fitting the curve to a Michaelis-Menten equation, and they are listed in Table 1.

FIGURE 3

Michaelis-Menten plots of 4-MU glucuronidation catalyzed by UGT2B3(S316N) in the absence and presence of CYP3A1 co-expression. The plots for UGT2B3(S316N) single- and UGT2B3(S316N)-CYP3A1-dual are shown. The kinetics obtained by varying the 4-MU concentration (A) and UDPGA concentration (B) are shown. In (A), the 4-MU concentration was varied over the range 10-1000 μM while the UDPGA concentration was fixed at 2 mM. In (B), the UDPGA concentration was varied over the range 50 μM–7.5 mM while the 4-MU concentration was fixed at 1 mM. The total amount of protein added to the assay mixture was standardized at 100 μg. For this, when necessary, control baculosomes were added to the reaction mixture. The kinetic parameters were calculated by fitting the curve to a Michaelis-Menten equation, and they are listed in Table 2.

FIGURE 4

Effect of EndoH-treatment on the glucuronidation catalyzed by UGT2B3(S316N). Baculosomes (20 μg protein) from UGT2B3(S316N) single- and UGT2B3(S316N)-CYP3A1 dual-expressing Sf-9 cells were treated with Endoglycosidase H (EndoH, 250 U) at 37°C for 1 h. The UGT activity was assayed with 100 or 1000 μM 4-MU and UDPGA fixed at 3 mM. Significantly different from UGT2B3(S316N) single expression (^∗∗p < 0.001; ^∗∗∗p < 0.0001).

FIGURE 5

Detection of an interaction between CYP3A1 and UGT2B3/UGT2B3(S316N) by His-tag pull-down assay. UGT2B3-HA and UGT2B3(S316N)-HA were expressed in the presence and absence of coexpressed His-CYP3A1. The pull-down assay was performed according to methods previously published (Miyauchi et al., 2015) with slight modification. Microsomes (2 mg protein/mL) were solubilized with sodium cholate. Then the solubilized microsomes were used in each assay. His-CYP3A1 and proteins trapped by this P450 were eluted with buffer containing imidazole at a high concentration. Each protein was detected by immunoblotting with a specific antibody: rabbit anti-HA or rabbit anti-His. Solubilized microsomes and His-CYP3A1–trapped samples are indicated as Input (30% of the input) and PD (pull down), respectively. In (A), HA-tagged wild-type UGT2B3 (WT-HA) was used. In (B), HA-tagged UGT2B3(S316N) (MT-HA) was used. WT-HA+CYP-His and MT-HA+CYP-His indicate microsomes coexpressing UGT2B3-HA or UGT2B3(S316N)-HA with His-CYP3A1, respectively. Duplicate assays were carried out. For each pull-down sample, one half of the precipitate was subjected to SDS-PAGE for detection with either HA or His while the lanes shown by “C” represent a negative control sample obtained by the same procedures but using solubilized control microsomes as an input, with microsomes prepared from Sf-9 cells transfected with control baculovirus alone. Details are described in the Section “Materials and Methods”.

FIGURE 6

Comparison of the predicted structure of UGT2B3 wild-type, UGT2B3(S316N), UGT2B2, and UGT2B7: the region spanning from 285 to 450. The models were constructed using the Phyre2 web server (Kelley et al., 2015) with the UGT2B7 (amino acid residue from 285 to 450) as a template. Template: UGT2B7, Miley et al. (2007) (DOI: 10.2210/pdb2o6l/pdb).