The Human UGT2B7 Nanodisc

Abstract

The 20 uridine diphosphate glycosyl-transferases (UGTs) encoded in the human genome form an essential homeostatic network of overlapping catalytic functions that surveil and regulate the activity and clearance of scores of small molecule metabolites. Biochemical and biophysical UGT studies have been hampered by the inability to purify these membrane-bound proteins. Here, using cell-free expression and nanodisc technology, we assemble and purify to homogeneity the first UGT nanodisc-the human UGT2B7•nanodisc. The complex is readily isolated in milligram quantities. It is stable and its initial-rate parameters are identical within error to those associated with UGT2B7 in microsomal preparations (i.e., Supersomes). The high purity of the nanodisc preparation simplifies UGT assays, which allows complexities traditionally associated with microsomal assays (latency and the albumin effect) to be circumvented. Each nanodisc is shown to harbor a single UGT2B7 monomer. The methods described herein should be widely applicable to UGTs, and these findings are expected to set the stage for experimentalists to more freely explore the structure, function, and biology of this important area of phase II metabolism. SIGNIFICANCE STATEMENT: Lack of access to pure, catalytically competent human uridine diphosphate glucuronosyl-transferases (UGTs) has long been an impediment to biochemical and biophysical studies of this disease-relevant enzyme family. Here, we demonstrate this barrier can be removed using nanodisc technology-a human UGT2B7•nanodisc is assembled, purified to homogeneity, and shown to have activity comparable to microsomal UGT2B7.

Fig. 1.

Purity and homogeneity of the UGT2B7•nanodisc. (A) SDS-PAGE analysis. Lane 1, molecular weight standards; lane 2, purified UGT2B7•nanodiscs (1.0 µg). Proteins were visualized via protein fluorescence in TCE-infused gels (see Results and Discussion). (B) Negative stain, EM image of the purified UGT2B7•nanodisc (original magnification, 50,000×). (C) Size-exclusion chromatography. Each color-coded peak represents the chromatographic profile of a purified component: UGT2B7•nanodisc (0.14 mg, black), empty nanodisc (0.11 mg, red), and free MSP1D1 (0.11 mg, blue). Protein was monitored at 280 nm, and the peaks were normalized to the highest measured absorbance.

Fig. 2.

Standard curve to determine UGT2B7:nanodisc stoichiometry. The normalized fluorescent intensity of protein-standard bands in TCE-infused SDS-PAGE gels is plotted vs. picomole of Trp calculated from known amino acid compositions and extinction coefficients. Intensities are normalized relative to the maximum measured intensity. The standard curve was constructed using five purified proteins [MSP1D1 (synthetic), SULT2A1 (human), SULT1E1 (human), hexokinase (yeast), and GST4A (human)], each of which was loaded at three different concentrations. Fluorescence intensity (λ_ex = 310 nm, λ_em ≥ 500 nm) was quantitated using Image Laboratory Software.

Fig. 3.

Albumin effects on nanodisc and microsomal UGT2B7 turnover. (A) Microsomal UGT2B7. Supersomes (5.0 mg/ml) were incubated with alamethicin (25 µM) and saccharolactone (5.0 mM) for 60 min in an ice-water bath (∼0°C) prior to reaction. Reaction conditions: Supersomes (25 µg protein), BSA (0, black dots, or 1.0% wt/vol, red dots), 4-MU (20 μM, 0.12 × K_m), alamethicin (12.5 nM), saccharolactone (2.5 μM), UDPGA (5.0 mM, 20 × K_m), MgCl₂ (5.0 mM), KPO₄ buffer (50 mM), pH 7.5, 25°C. Reactions were initiated by adding UDPGA to a final volume of 500 μl, and reaction progress was monitored via the fluorescence decrease associated with 4-MU glucuronidation (λ_ex = 370 nm, λ_em = 451 nm). (B) The UGT2B7•nanodisc. Assays were identical to (A) except that Supersomes were replaced with the UGT2B7•nanodisc (50 nM), and alamethicin and saccharolactone were absent.

Fig. 4.

Initial-rate behavior of UGT2B7 in nanodiscs and microsomes. (A) 1/Turnover vs. 1/[UDPGA]. Reaction conditions: UGT2B7-nanodisc (100 nM active sites, black dots) or UGT2B7 Supersomes (110 μg, blue dots), UDPGA (60–1250 µM, 0.2–5 × K_m), 4-MU (3.5 mM free, 20 × K_m), BSA (1.0% wt/vol, Supersome assay only), MgCl₂ (5.0 mM), KPO₄ (50 mM), pH 7.5, 25 ± 2°C. Supersomes were incubated (0°C, 60 min) with alamethicin (25 μM) and saccharolactone (5.0 mM) prior to assay. Reactions were initiated by addition of UDPGA to a final volume of 500 μl. Reaction progress was monitored via the fluorescence decrease associated with 4-MU glucuronidation (λ_ex = 370 nm, λ_em = 451 nm). (B) 1/Turnover vs. 1/[4-MU]. Assays were identical to (A) except [UDPGA] was fixed (5.0 mM, 21 × K_m) and [4-MU] was varied from 35 to 900 µM (0.2–5 × K_m). (C) 4-MU substrate inhibition. Assays were identical to (B) except [4-MU] was varied from 0.035 to 11 mM (0.03–10 × K_i). Initial-rate parameters were obtained by weighted-least-squares fitting to either a simple Michaelis-Menten (A and B) or a partial substrate inhibition model (C). Initial-rate parameters are compiled in Table 1.

Fig. 5.

Morphine glucuronides produced by nanodisc and microsomal UGT2B7. Reverse phase HPLC chromatograms of the morphine conjugates produced by the nanodisc (black dots) and microsomal (red dots) UGT2B7 are superposed. The 3- and 6-hydroxyl glucuronide peaks of morphine along with a partial peak of unconjugated morphine are labeled M3G, M6G and M, respectively. The concentrations of 2B7 in the nanodisc and Supersome assays were matched (see main text). Reaction conditions: UGT2B7 nanodisc (100 nM, UGT active site) or UGT2B7 Supersomes (100 µg ml⁻¹), morphine (100 μM), UDPGA (5.0 mM, 21 × K_m), BSA (1.0% wt/vol, Supersomes only), alamethicin (25 nM, Supersomes only), saccharolactone (5.0 μM, Supersomes only), MgCl₂ (5.0 mM), KPO₄ (50 mM), pH 7.5, 25 ± 2°C. Reactions were initiated by addition of UDGPA. Less than 5% of the morphine consumed at the end point of the reaction was formed during the measurement. Reaction were quenched and chromatographically separated as described in Materials and Methods, subsection Rate Studies Using Morphine.