Expression, purification and characterization of human cytosolic sulfotransferase (SULT) 1C4

Abstract

Human cytosolic sulfotransferase 1C4 (hSULT1C4) is a dimeric Phase II drug-metabolizing enzyme primarily expressed in the developing fetus. SULTs facilitate the transfer of a hydrophilic sulfonate moiety from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto an acceptor substrate altering the substrate's biological activity and increasing the compound's water solubility. While several of the hSULTs' endogenous and xenobiotic substrates have been identified, the physiological function of hSULT1C4 remains unknown. The fetal expression of hSULT1C4 leads to the hypothesis that the function of this enzyme may be to regulate metabolic and hormonal signaling molecules, such as estrogenic compounds, that may be generated or consumed by the mother during fetal development. Human SULT1C4 has previously been shown to sulfonate estrogenic compounds, such as catechol estrogens; therefore, this study focused on the expression and purification of hSULT1C4 in order to further characterize this enzyme's sulfonation of estrogenic compounds. Molecular modeling of the enzyme's native properties helped to establish a novel purification protocol for hSULT1C4. The optimal activity assay conditions for hSULT1C4 were determined to be pH 7.4 at 37°C for up to 10 min. Kinetic analysis revealed the enzyme's reduced affinity for PAPS compared to PAP. Human SULT1C4 sulfonated all the estrogenic compounds tested, including dietary flavonoids and environmental estrogens; however, the enzyme has a higher affinity for sulfonation of flavonoids. These results suggest hSULT1C4 could be metabolizing and regulating hormone signaling pathways during human fetal development.

Figure 1. Untagged hSULT1C4 binds with a high affinity to Ni-NTA resin

(A) Using MOE, two adjacent histidine (His148 and His274) clusters (blue) were identified near hSULT1C4’s dimerization domain, which functions as an interface for the dimerization of subunit A and subunit B (green). (B) The elution of 1-naphthol (10 μM 1-naphthol, 10 μM PAPS) sulfonation activity from the Ni-NTA column. The volume of each fraction was 0.5 mL. (C) Fractions (5 μL) from the Ni-NTA column were resolved by SDS-PAGE and stained with Coomassie. The molecular weight size markers are indicated to the left of the image.

Figure 2. DEAE-sepharose chromatography results in highly pure, active hSULT1C4

(A) VMD was used to visualize the basic (blue) and acidic (red) charges on the surface of hSULT1C4 (PDB 2GWH) [29]. (B) DEA-sepharose elution profile of hSULT1C4 activity assayed with 10 μM 1-naphthol in the presence of 10 μM PAPS. The volume of each fraction was 2.3 mL; each lane was loaded with 7.5 μL of the fraction. (C) Protein fractions from the DEAE-sepharose column were resolved by SDS-PAGE and stained with Coomassie. The molecular weight size markers are indicated to the left of the image.

Figure 3. Native hSULT1C4 kinetic assay optimization

(A) Sulfonation of 1-naphthol by hSULT1C4 with increasing concentrations of 1-naphthol and 10 μM PAPS. (B) Optimization of pH for hSULT1C4 activity using 1-naphthol as a substrate and varying the pH from 7.0 to 9.0. (C) Human SULT1C4 activity was assessed while the reaction time was varied between 2 and 15 min at 37 °C with 10 μM 1-naphthol and 10 μM PAPS. (D) The optimal BSA concentration in hSULT1C4 activity assays was determined by varying BSA concentrations from 0 to 100 μg/reaction (rxn) in the presence of 10 μM 1-naphthol and 10 μM PAPS.

Figure 4. Comparison of hSULT1C4’s interactions with PAPS and PAP

(A) Specific activity of hSULT1C4 with increasing concentrations of PAPS and 30 μM daidzein. (B) Change in hSULT1C4 (λ_ex = 282 nm, λ_em = 342 nm) intrinsic fluorescence (F) upon PAPS binding. (C) Change in hSULT1C4 intrinsic fluorescence upon PAP binding.

Figure 5

Chemical structures of estrogenic compounds.

Figure 6. Sulfonation of estrogenic compounds by hSULT1C4

Increasing concentrations of (A) genistein, (B) daidzein, (C) apigenin, (D) chrysin, (E) 6,4-dihydroxyflavone, (F) estradiol, and (G) bisphenol A were assayed for sulfonation by hSULT1C4 with 10 μM PAPS.