24-hydroxycholesterol sulfation by human cytosolic sulfotransferases: formation of monosulfates and disulfates, molecular modeling, sulfatase sensitivity, and inhibition of liver x receptor activation

Abstract

24-Hydroxycholesterol (24-OHChol) is a major cholesterol metabolite and the form in which cholesterol is secreted from the brain. 24-OHChol is transported by apolipoprotein E to the liver and converted into bile acids or excreted. In both brain and liver, 24-OHChol is a liver X receptor (LXR) agonist and has an important role in cholesterol homeostasis. 24-OHChol sulfation was examined to understand its role in 24-OHChol metabolism and its effect on LXR activation. 24-OHChol was conjugated by three isoforms of human cytosolic sulfotransferase (SULT). SULT2A1 and SULT1E1 sulfated both the 3- and 24-hydroxyls to form the 24-OHChol-3, 24-disulfate. SULT2B1b formed only 24-OHChol-3-sulfate. The 3-sulfate as a monosulfate or as the disulfate was hydrolyzed by human placental steroid sulfatase, whereas the 24-sulfate was resistant. At physiological 24-OHChol concentrations, SULT2A1 formed the 3-monosulfate and the 3, 24-disulfate as a result of a high affinity for sulfation of the 3-OH in 24-OHChol-24-sulfate. Molecular docking simulations indicate that 24-OHChol-24-sulfate binds in an active configuration in the SULT2A1 substrate binding site with high affinity only when the SULT2A1 homodimer structure was used. 24-OHChol is an LXR activator. In contrast, the 24-OHChol monosulfates were not LXR agonists in a fluorescence resonance energy transfer coactivator recruitment assay. However, both the 24-OHChol-3-sulfate and 24-sulfate were antagonists of LXR activation by N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trif-luoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317) with an IC(50) of 0.15 and 0.31 muM, respectively. Inhibition of LXR activation by the 24-OHChol monosulfates at low nanomolar concentrations indicates that sulfation has a role in LXR regulation by oxysterols.

Figures

Fig. 1.

Sulfated products of 24-OHChol generated by SULT2A1 and SULT2B1b, and sensitivity to hydrolysis by STS. A, the structure of 24-OHChol is shown, and the arrows identify the 3- and 24-hydroxyl groups that are sulfated. B, the formation of 24-OHChol monosulfate and disulfate is shown, as well as their sensitivity to STS hydrolysis. 24-OHChol (20 μM) was incubated with SULT2A1 or SULT2B1b in the presence of 10 μM [35S]PAPS for 30 min at 37°C. The reactions were terminated by extraction with chloroform, and an aliquot was applied to a silica gel TLC plate. For treatment with STS, an aliquot of the reaction after chloroform extraction was incubated with 1 μg of STS for 30 min at 37°C. The STS reaction was extracted with chloroform, and an aliquot of the aqueous phase was spotted on the TLC plate. The plates were developed as described under Materials and Methods and exposed to autoradiograph film to identify 35S-containing bands. [35S]PAPS does not move from the loading zone.

Fig. 2.

Sulfation of 24-OHChol by SULT2A1. Increasing concentrations of 24-OHChol were sulfated by SULT2A1 (2.6 ng) in the presence of 10 μM [35S]PAPS. Reactions were run for 10 min at 37°C and then terminated by extraction with chloroform. The aqueous phase of duplicate reactions was then treated with STS for 30 min and stopped by chloroform extraction. Aliquots of the reactions were loaded onto silica gel TLC plates and developed as described under Materials and Methods. The plates were then exposed to autoradiograph film, and the radioactive bands were scraped into vials for scintillation counting. A, the formation of 24-OHChol monosulfate and disulfate at low 24-OHChol concentrations. B, the levels of 24-OHChol monosulfate and disulfate formation with increasing 24-OHChol concentrations, as well as the residual 24-OHChol monosulfate present after STS treatment.

Fig. 3.

Sulfation of 24-OHChol by SULT1E1. Increasing concentrations of 24-OHChol were sulfated by SULT1E1 in the presence of 10 μM [35S]PAPS. Reactions were run for 20 min at 37°C and then terminated by extraction with chloroform. The aqueous phase of duplicate reactions was then treated with STS for 30 min and stopped by chloroform extraction. Aliquots of the reactions were loaded onto silica gel TLC plates and developed as described under Materials and Methods. The levels of 24-OHChol monosulfate and disulfate formation with increasing 24-OHChol concentrations, as well as the residual 24-OHChol monosulfate present after STS treatment, are shown.

Fig. 4.

Formation of 24-OHChol sulfate by SULT2B1b. Increasing concentrations of 24-OHChol were converted to a monosulfate by SULT2B1b in the presence of 10 μM [35S]PAPS. Each point represents the mean of three assays ± S.D. The error bars are essentially contained within the data points.

Fig. 5.

LC/MS/MS analysis of the 24-OHChol sulfates formed by SULT2A1 and SULT2B1b. A, the monosulfated products generated by sulfation of 24-OHChol with SULT2A1. MS/MS analysis of the peaks identified the more abundant peak as the 3-sulfate (9.0 min) and the smaller peak (8.3 min) as the 24-sulfate. B, the 24-OHChol disulfate eluted at 7.3 min, consistent with its more charged nature. C, STS treatment of the disulfate generates only the STS-resistant 24-sulfate eluting at 8.3 min. D, sulfation of 24-OHChol with SULT2B1b results in the formation of only the 3-monosulfate.

Fig. 6.

Identification of 24-OHChol-24-sulfate by LC/MS/MS analysis. The site of sulfate conjugation in the putative 24-OHChol-24-sulfate was analyzed by LC/MS/MS after fragmentation with increasing energies as described under Materials and Methods. After fragmentation at 60 eV, the major ions detected were the parent ion 24-OHChol (481 Da), the sulfate moiety (96.9 Da), and a fragment derived from the A-ring containing the 3-OH (59.1 Da). The presence of the 3-OH indicates the sulfate is conjugated at the 24-position.

Fig. 7.

Conversion of the 24-OHChol monosulfates to the disulfate by SULT2A1. The two 24-OHChol monosulfates were synthesized as described under Materials and Methods and used in reactions to assay their conversion to 24-OHChol disulfate by SULT2A1. The reactions used nonradiolabeled monosulfates and [35S]PAPS to monitor formation of the disulfate using the TLC assay described under Materials and Methods. A, the conversion of 24-OHChol-3-sulfate to the disulfate. B, the sulfation of 24-OHChol-24-sulfate to the disulfate. The data represent three sets of reactions done in duplicate.

Fig. 8.

Molecular simulation of the orientation of the 24-OHChol monosulfates in the active site of SULT2A1. The two monosulfates of 24-OHChol were molecularly oriented into the active site of SULT2A1 (Protein Data Bank code 1efh) using the Autodock program to allow sulfonation of the free hydroxyl to allow disulfate formation. A, the orientation of 24-OHChol-3-sulfate in the substrate binding site in the SULT2A1 monomer. The BFE is −3.5 Kcal/mol, and the 24-hydroxyl group is close to Ser97 (0.35 nm) and His99 (0.60 nm). B, in the SULT2A1 monomer structure, 24-OHChol-24-sulfate does not readily bind in the pocket but prefers to bind to Lys138 and Ser80 in an apparent noncatalytic orientation. The BFE of the 24-OHChol-24-sulfate in this orientation is −10.0 Kcal/mol. C, the orientation of 24-OHChol-24-sulfate in the SULT2A1 dimer structure. In the SULT2A1 dimer structure, Glu73 from the other monomer inhibits access of the 24-sulfate to Lys138 and allows for docking in the active site in an apparent catalytically active conformation. The BFE is −7.9 Kcal/mol, and the 3-hydroxyl group is 0.4 nm from His99.

Fig. 9.

In vitro interactions of 24-OHChol sulfate metabolites with LXRα. The LanthaScreen TR-FRET LXRα coactivator assay was used to evaluate the abilities of 24-OHChol-3-sulfate and 24-OHChol-24-sulfate to interact with LXRα, either as agonists (A) or antagonists (B). T0901317 and 24-OH-Chol were included as positive control LXRα agonists, and antagonist activities were determined in the presence of 100 nM T0901317. Binding curves were generated using Prism 5. The EC50s for T091317 and 24-OHChol were 0.030 and 1.99 μM, respectively, and there was no detectable activation for the sulfated metabolites. The IC50s of the sulfated metabolites were 0.149 μM for 24-OHChol-3-sulfate and 0.309 μM for 24-OHChol-24-sulfate.