Human Hydroxysteroid Sulfotransferase Research Articles

Endoxifen and other metabolites of tamoxifen inhibit human hydroxysteroid sulfotransferase 2A1 (hSULT2A1).

Although tamoxifen is a successful agent for treatment and prevention of estrogen-dependent breast cancer, its use has been limited by the low incidence of endometrial cancer. Human hydroxysteroid sulfotransferase 2A1 (hSULT2A1) catalyzes the formation of an α-sulfooxy metabolite of tamoxifen that is reactive toward DNA, and this has been implicated in its carcinogenicity. Also, hSULT2A1 functions in the metabolism of steroid hormones such as dehydroepiandrosterone (DHEA) and pregnenolone (PREG). These roles of hSULT2A1 in steroid hormone metabolism and in generating a reactive metabolite of tamoxifen led us to examine its interactions with tamoxifen and several of its major metabolites. We hypothesized that metabolites of tamoxifen may regulate the catalytic activity of hSULT2A1, either through direct inhibition or through serving as alternate substrates for the enzyme. We found that 4-hydroxy-N-desmethyltamoxifen (endoxifen) is a potent inhibitor of hSULT2A1-catalyzed sulfation of PREG and DHEA, with Ki values of 3.5 and 2.8 μM, respectively. In the hSULT2A1-catalyzed sulfation of PREG, 4-hydroxytamoxifen (4-OHTAM) and N-desmethyltamoxifen (N-desTAM) exhibited Ki values of 12.7 and 9.8 μM, respectively, whereas corresponding Ki values of 19.4 and 17.2 μM were observed with DHEA as substrate. A Ki value of 9.1 μM was observed for tamoxifen-N-oxide with DHEA as substrate, and this increased to 16.9 μM for the hSULT2A1-catalyzed sulfation of PREG. Three metabolites were substrates for hSULT2A1, with relative sulfation rates of 4-OHTAM > N-desTAM > > endoxifen. These results may be useful in interpreting ongoing clinical trials of endoxifen and in improving the design of related molecules.

Transcriptional regulation of human hydroxysteroid sulfotransferase SULT2A1 by LXRα.

The nuclear receptor liver X receptor (LXR) plays an important role in the metabolism and homeostasis of cholesterol, lipids, bile acids, and steroid hormones. In this study, we uncovered a function of LXRα (NR1H3) in regulating the human hydroxysteroid sulfotransferase SULT2A1, a phase II conjugating enzyme known to sulfonate bile acids, hydroxysteroid dehydroepiandrosterone, and related androgens. We showed that activation of LXR induced the expression of SULT2A1 at mRNA, protein, and enzymatic levels. A combination of promoter reporter gene and chromatin immunoprecipitation assays showed that LXRα transactivated the SULT2A1 gene promoter through its specific binding to the -500- to -258-base pair region of the SULT2A1 gene promoter. LXR small interfering RNA knockdown experiments suggested that LXRα, but not LXRβ, played a dominant role in regulating SULT2A1. In primary human hepatocytes, we found a positive correlation between the expression of SULT2A1 and LXRα, which further supported the regulation of SULT2A1 by LXRα. In summary, our results established human SULT2A1 as a novel LXRα target gene. The expression of LXRα is a potential predictor for the expression of SULT2A1 in human liver.

Binding interactions of hydroxylated polychlorinated biphenyls (OHPCBs) with human hydroxysteroid sulfotransferase hSULT2A1

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and exposure to PCBs and their hydroxylated metabolites (OHPCBs) has been associated with various adverse health effects. The mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of OHPCBs, and the interaction of OHPCBs with both the SULT1 and SULT2 families of these enzymes has received attention both with respect to metabolic disposition of these molecules and the potential mechanisms for their roles in endocrine disruption. We have previously shown that OHPCBs interact with human hydroxysteroid sulfotransferase hSULT2A1, an enzyme that catalyzes the sulfation of dehydroepiandrosterone (DHEA), other alcohol-containing steroids, bile acids, and many xenobiotics. The objective of our current studies is to investigate the mechanism of inhibition of hSULT2A1 by OHPCBs by combining inhibition kinetics with determination of equilibrium binding constants and molecular modeling of potential interactions. Examination of the effects of fifteen OHPCBs on the sulfation of DHEA catalyzed by hSULT2A1 showed predominantly noncompetitive inhibition patterns. This was observed for OHPCBs that were substrates for sulfation reactions catalyzed by the enzyme as well as those that solely inhibited the sulfation of DHEA. Equilibrium binding experiments and molecular modeling studies indicated that the OHPCBs bind at the binding site for DHEA on the enzyme, and that the observed noncompetitive patterns of inhibition are consistent with binding in more than one orientation to more than one enzyme complex. These results have implications for the roles of SULTs in the toxicology of OHPCBs, while also providing molecular probes of the complexity of substrate/inhibitor interactions with hSULT2A1.

Chlorinated Biphenyl Quinones and Phenyl-2,5-benzoquinone Differentially Modify the Catalytic Activity of Human Hydroxysteroid Sulfotransferase hSULT2A1

Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of environmental chemicals, drugs, and other xenobiotics in addition to endogenous compounds that include hydroxysteroids and bile acids. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and oxidized metabolites of PCBs may play significant roles in the etiology of their adverse health effects. Quinones derived from the oxidative metabolism of PCBs (PCB-quinones) react with nucleophilic sites in proteins and also undergo redox cycling to generate reactive oxygen species. This, along with the sensitivity of hSULT2A1 to oxidative modification at cysteine residues, led us to hypothesize that electrophilic PCB-quinones react with hSULT2A1 to alter its catalytic function. Thus, we examined the effects of four phenylbenzoquinones on the ability of hSULT2A1 to catalyze the sulfation of the endogenous substrate, dehydroepiandrosterone (DHEA). The quinones studied were 2'-chlorophenyl-2,5-benzoquinone (2'-Cl-BQ), 4'-chlorophenyl-2,5-benzoquinone (4'-Cl-BQ), 4'-chlorophenyl-3,6-dichloro-2,5-benzoquinone (3,6,4'-triCl-BQ), and phenyl-2,5-benzoquinone (PBQ). At all concentrations examined, pretreatment of hSULT2A1 with the PCB-quinones decreased the catalytic activity of hSULT2A1. Pretreatment with low concentrations of PBQ, however, increased the catalytic activity of the enzyme, while higher concentrations inhibited catalysis. A decrease in substrate inhibition with DHEA was seen following preincubation of hSULT2A1 with all of the quinones. Proteolytic digestion of the enzyme followed by LC/MS analysis indicated PCB-quinone- and PBQ-adducts at Cys55 and Cys199, as well as oxidation products at methionines in the protein. Equilibrium binding experiments and molecular modeling suggested that changes due to these modifications may affect the nucleotide binding site and the entrance to the sulfuryl acceptor binding site of hSULT2A1.

Estrogen‐Related Receptor ERRα Regulation of Human Hydroxysteroid Sulfotransferase (SULT2A1) Gene Expression in Human Caco‐2 Cells

Human hydroxysteroid sulfotransferase, SULT2A1, is important for xenobiotic detoxification and the maintenance of hydroxysteroid homeostasis. Our published report suggested that estrogen-related receptor ERRα downregulates SULT2A1 in Hep G2 cells. The results shown in this study suggest that ERRα upregulates SULT2A1 transcription in Caco-2 cells. The deletion analysis suggested that SULT2A1 promoter region between -65 and -44 is important for this upregulation. Our further investigation suggested that ERRα binding element, ERRE51, mediates ERRα activation of SULT2A1 promoter transcription in Caco-2 cells. The interaction of ERRE51 with ERRα was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Results also suggest that the difference of constitutive androstane receptor transcription levels in Hep G2 and Caco-2 cells at least partially contribute to the cell type dependent ERRα modulation of SULT2A1 promoter transcription. ERRα regulates human SULT2A1 transcription by competing with other nuclear receptors binding to the DNA-promoter region.

Endoxifen and other metabolites of tamoxifen inhibit human hydroxysteroid sulfotransferase hSULT2A1

Tamoxifen is a breast cancer therapeutic that has been shown to cause endometrial cancer in some women following treatment. hSULT2A1 is an enzyme that catalyzes the formation of an α‐sulfooxy derivative of tamoxifen that is reactive towards DNA. Moreover, hSULT2A1 functions in the metabolism of steroid hormones such as dehydroepiandrosterone (DHEA) and pregnenenolone (PREG). We hypothesize that major metabolites of tamoxifen regulate the catalytic activity of hSULT2A1, either through direct inhibition or through serving as alternate substrates for the enzyme. Our studies show that endoxifen is a potent inhibitor of hSULT2A1‐catalyzed sulfation of PREG and DHEA with Ki values of 3.5 μM and 2.8 μM, respectively. 4‐Hydroxytamoxifen and N‐desmethyltamoxifen had Ki values of 12.7 μM and 9.8 μM, respectively, for sulfation of PREG, whereas Ki values of 19.4 μM and 17.2 μM were observed for these metabolites when determining the sulfation of DHEA. A Ki of 9.1 μM was observed for tamoxifen‐N‐oxide when DHEA was used as a substrate, and this increased to a Ki value of 16.9 μM when PREG was utilized as a substrate. Subsequent assays indicated that N‐desmethyltamoxifen was a substrate for the enzyme. These results may be useful in interpreting ongoing clinical trials of endoxifen and in improving the design of related molecules. [Supported by NIH grant CA038683]

Modification of the catalytic function of human hydroxysteroid sulfotransferase hSULT2A1 by formation of disulfide bonds.

The human cytosolic sulfotransferase hSULT2A1 catalyzes the sulfation of a broad range of xenobiotics, as well as endogenous hydroxysteroids and bile acids. Reversible modulation of the catalytic activity of this enzyme could play important roles in its physiologic functions. Whereas other mammalian sulfotransferases are known to be reversibly altered by changes in their redox environment, this has not been previously shown for hSULT2A1. We have examined the hypothesis that the formation of disulfide bonds in hSULT2A1 can reversibly regulate the catalytic function of the enzyme. Three thiol oxidants were used as model compounds to investigate their effects on homogeneous preparations of hSULT2A1: glutathione disulfide, 5,5'-dithiobis(2-nitrobenzoic acid), and 1,1'-azobis(N,N-dimethylformamide) (diamide). Examination of the effects of disulfide bond formation with these agents indicated that the activity of the enzyme is reversibly altered. Studies on the kinetics of the hSULT2A1-catalyzed sulfation of dehydroepiandrosterone (DHEA) showed the effects of disulfide bond formation on the substrate inhibition characteristics of the enzyme. The effects of these agents on the binding of substrates and products, liquid chromatography-mass spectrometry identification of the disulfides formed, and structural modeling of the modified enzyme were examined. Our results indicate that conformational changes at cysteines near the nucleotide binding site affect the binding of both the nucleotide and DHEA to the enzyme, with the specific effects dependent on the structure of the resulting disulfide. Thus, the formation of disulfide bonds in hSULT2A1 is a potentially important reversible mechanism for alterations in the rates of sulfation of both endogenous and xenobiotic substrates.

Regulation of the human hydroxysteroid sulfotransferase (SULT2A1) by RORα and RORγ and its potential relevance to human liver diseases.

The retinoid-related orphan receptors (RORs) were postulated to have functions in tissue development and circadian rhythm. In this study, we revealed a novel function of RORα (NR1F1) and RORγ (NR1F3) in regulating the human hydroxysteroid sulfotransferase (SULT2A1), a phase II conjugating enzyme known to sulfonate bile acids, hydroxysteroid dehydroepiandrosterone, and related androgens. A combination of promoter reporter gene assay and EMSA and chromatin immunoprecipitation (ChIP) assays showed that both RORα and RORγ transactivated the SULT2A1 gene promoter through their binding to a ROR response element found in the SULT2A1 gene promoter. Interestingly, this ROR response element overlaps with a previously reported constitutive androstane receptor response element on the same promoter. Down-regulation of RORα and/or RORγ by small interfering RNA inhibited the expression of endogenous SULT2A1. In primary human hepatocytes and human livers, we found a positive correlation between the expression of SULT2A1 and RORs, which further supported the regulation of SULT2A1 by RORs. We also found that the expression of RORα and RORγ was impaired in several liver disease conditions, such as steatosis/steatohepatitis, fibrosis, and hepatocellular carcinoma. The positive regulation of human SULT2A1 by RORs is opposite to the negative regulation of Sult2a1 by RORs in rodents. In summary, our results established SULT2A1 as a novel ROR target gene. The expression of RORs is a potential predictor for the expression of SULT2A1 as well as disease conditions.

Structure-activity relationships for hydroxylated polychlorinated biphenyls as inhibitors of the sulfation of dehydroepiandrosterone catalyzed by human hydroxysteroid sulfotransferase SULT2A1.

Polychlorinated biphenyls (PCBs) are persistent worldwide pollutants that are of concern due to their bioaccumulation and health effects. Metabolic oxidation of PCBs results in the formation of hydroxylated metabolites (OHPCBs). Among their biological effects, OHPCBs have been shown to alter the metabolism of endocrine hormones, including inhibition of mammalian cytosolic sulfotransferases (SULTs) that are responsible for the inactivation of thyroid hormones and phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1). OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of 1 μM [(3)H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited the sulfation of DHEA, with IC(50) values ranging from 0.6 μM to 96 μM, and eight of these OHPCBs were also substrates for the enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q(2) value of 0.697 and an r(2) value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.

Estrogen-related receptor ERRα-mediated downregulation of human hydroxysteroid sulfotransferase (SULT2A1) in Hep G2 cells

Hydroxysteroid sulfotransferase SULT2A1 catalyzes the sulfation of hydroxysteroids and xenobiotics. It plays an important role in the detoxification of hydroxyl-containing xenobiotics and in the regulation of the biological activities of hydroxysteroids. ERRα is an orphan member of the nuclear receptor superfamily that is closely related to estrogen receptor alpha (ERα). Here we report that the mRNA expression of human SULT2A1 was suppressed by ERRα in Hep G2 cells. To investigate the mechanisms of this regulation, the effects of ERRα on human SULT2A1 promoter transcription in Hep G2 cells were investigated. Reporter luciferase assay results showed that ERRα significantly represses human SULT2A1 promoter transcription in Hep G2 cells. Deletion analysis indicated that human SULT2A1 promoter region between positions −188 and −130 is necessary for its repression by ERRα in Hep G2 cells. The 5′ DNA −188 to −130 region of human SULT2A1 contains IR2 and DR4 hormone response elements and two putative ERRα response elements (ERREs) (ERRE188: GCAAGCTCA and ERRE155: ATAAGTTCA). Interestingly, ERRE188 overlaps with the IR2 element and ERRE155 overlaps with the DR4 element. Our further investigation demonstrated that ERRα represses human SULT2A1 promoter transcription by competing with other nuclear receptors for binding to IR2 or DR4 elements. The interaction of ERRE188 and ERRE155 elements with ERRα was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. Our results suggest that ERRα may play an important role in regulating the metabolism of drugs and xenobiotics and in regulating endogenous hydroxysteroid activities via the regulation of SULT2A1.

Substrate inhibition in human hydroxysteroid sulfotransferase SULT2A1: Studies on the formation of catalytically non-productive enzyme complexes

The cytosolic sulfotransferase hSULT2A1 is the major hydroxysteroid (alcohol) sulfotransferase in human liver, and it catalyzes the 3′-phosphoadenosine-5′-phosphosulfate (PAPS)-dependent sulfation of various endogenous hydroxysteroids as well as many xenobiotics that contain alcohol and phenol functional groups. The hSULT2A1 often displays substrate inhibition, and we have hypothesized that a key element in this response to increasing substrate concentration is the formation of non-productive ternary dead-end enzyme complexes involving the nucleotide product, adenosine 3′,5′-diphosphate (PAP). One of these substrates for hSULT2A1 is dehydroepiandrosterone (DHEA), a major circulating steroid hormone in humans that serves as precursor to both androgens and estrogens. We have utilized DHEA in both initial velocity studies and equilibrium binding experiments in order to evaluate the potential role of ternary complexes in substrate inhibition of the enzyme. Our results indicate that hSULT2A1 forms non-productive ternary complexes that involve either DHEA or dehydroepiandrosterone sulfate, and the formation of these ternary complexes displays negative cooperativity in the binding of DHEA.

Human Hydroxysteroid Sulfotransferase 2A1 is Down Regulated by Nitric Oxide in Human Hep G2 Cells

Human Hydroxysteroid Sulfotransferase 2A1 is Down Regulated by Nitric Oxide in Human Hep G2 Cells

Pentachlorophenol and other chlorinated phenols are substrates for human hydroxysteroid sulfotransferase hSULT2A1.

Pentachlorophenol (PCP) is a persistent chemical contaminant that has been extensively investigated in terms of its toxicology and metabolism. Similar to PCP, other chlorinated phenol derivatives are also widely present in the environment from various sources. Even though some of the chlorine-substituted phenols, and particularly PCP, are well-known inhibitors of phenol sulfotransferases (SULTs), these compounds have been shown to undergo sulfation in humans. To investigate the enzymatic basis for sulfation of PCP in humans, we have studied the potential for PCP as well as the mono-, di-, tri-, and tetra-chlorinated phenols to serve as substrates for human hydroxysteroid sulfotransferase, hSULT2A1. Our results show that all of these compounds are substrates for this isoform of sulfotransferase, and the highest rates of sulfation are obtained with PCP, trichlorophenols, and tetrachlorophenols. Much lower rates of sulfation were obtained with isomers of monochlorophenol and dichlorophenol as substrates for hSULT2A1. Thus, the sulfation of polychlorinated phenols catalyzed by hSULT2A1 may be a significant component of their metabolism in humans.

Human Hydroxysteroid Sulfotransferase SULT2B1 Pharmacogenomics: Gene Sequence Variation and Functional Genomics

The human hydroxysteroid sulfotransferase (SULT) 2B1 gene is a member of the cytosolic SULT gene superfamily. The two SULT2B1 isoforms, SULT2B1a and SULT2B1b, are encoded by a single gene as a result of alternative transcription initiation and alternative splicing. SULT2B1b catalyzes the sulfonation of 3beta-hydroxysteroid hormones and cholesterol, whereas SULT2B1a preferentially catalyzes pregnenolone sulfonation. We used a genotype-to-phenotype approach to identify and characterize common sequence variation in SULT2B1. Specifically, we resequenced all exons, splice junctions, and approximately 2.5 kb of the 5'-flanking regions (FRs) for each isoform using 60 DNA samples each from African-American and Caucasian-American subjects. We observed 100 polymorphisms, including four nonsynonymous coding single nucleotide polymorphisms and one 6-base pair deletion-all within the "shared" region of the open reading frame. Functional genomic studies of the wild type (WT) and five variant allozymes for each isoform performed with a mammalian expression system showed that variant allozyme activities ranged from 64 to 88% of WT for SULT2B1a and from 76 to 98% for SULT2B1b. Relative levels of immunoreactive protein were similar to those for enzyme activity. Luciferase reporter gene constructs for 2.5 kb of the SULT2B1b 5'-FR displayed a cell line-dependent pattern of variation in activity. Finally, deletion of the proline-rich SULT2B1 carboxyl terminus resulted in intracellular protein aggregate formation and accelerated degradation of the truncated protein. These studies resulted in the identification of common SULT2B1 gene sequence variation, as well as insight into the effects of that variation on the function of this important steroid-metabolizing enzyme.

Regulation of human hepatic hydroxysteroid sulfotransferase (SULT2A1) expression by rifampicin.

Human hydroxysteroid sulfotransferase (SULT2A1) catalyzes the sulfonation and detoxication of endogenous and xenobiotic substrates. The effects of rifampicin (RIF), a ligand of the human pregnane X receptor (PXR), on SULT2A1 expression were evaluated in primary cultured human hepatocytes. SULT2A1 mRNA content was increased by RIF treatment in seven hepatocyte preparations, but was reduced in three others. Transient transfection of HepG2 cells with reporter constructs localized two RIF-responsive regions in the SULT2A1 promoter (one at -6160 and another at −54), which contained hepatocyte nuclear factor 4α (HNF4α) binding sites, as confirmed by EMSA and ChIP. Co-transfection of HepG2 cells with PXR suppressed reporter expression from constructs containing the HNF4α sites, and suppression was augmented by rifampicin treatment. Transfection with a DNA binding-defective PXR mutant failed to ablate the suppressive effects of PXR and rifampicin. These results suggest that rifampicin-liganded PXR suppresses human hepatic SULT2A1 expression through a mechanism that involves interference with HNF4α, possibly through a protein-protein interaction. Supported by ES05823 (M.R.M.), HL50710 (T.A.K.) and ES06636.

Hydroxylated Polychlorinated Biphenyls Are Substrates and Inhibitors of Human Hydroxysteroid Sulfotransferase SULT2A1

Polychlorinated biphenyls (PCBs) are important persistent environmental contaminants. PCBs can be metabolically converted to their hydroxylated metabolites (OHPCBs), and in recent years, these OHPCBs have been observed to inhibit human sulfotransferases (SULTs) such as the phenol SULTs (SULT family-1) involved in the metabolism of estrogen and various other endogenous and xenobiotic phenols. In the present study, we have investigated the hypothesis that OHPCBs interact with family 2 hydroxysteroid (alcohol) SULTs (e.g., human SULT2A1), enzymes that are physiologically important for the metabolic transformations of several key endogenous hydroxysteroids as well as xenobiotic alcohols. We have examined the interactions of three OHPCBs with purified recombinant human SULT2A1 (also known as either human DHEA-ST or ST2A3). These studies with SULT2A1 were carried out on 4'-hydroxy-2,5-dichlorobiphenyl (4'-OH PCB 9), 4-hydroxy-2',3,5-trichlorobiphenyl (4-OH PCB 34), and 4'-hydroxy-2,3',4,5'-tetrachlorobiphenyl (4'-OH PCB 68). Our results showed that 4-OH PCB 34 and 4'-OH PCB 68 were substrates for SULT2A1, and 4-OH PCB 34 exhibited substrate inhibition similar to that seen with the physiological substrate dehydroepiandrosterone (DHEA). Although the sulfation of 4-OH PCB 34 and 4'-OH PCB 68 represents a potential metabolic route for these compounds, these OHPCBs may also compete with other xenobiotic substrates as well as endogenous substrates for SULT2A1. The third OHPCB studied, 4'-OH PCB 9, was not a substrate for SULT2A1 but was an inhibitor of the enzyme. Thus, the interactions of OHPCBs with human SULT2A1 represent both a potential route of metabolism and a possible source of interference with sulfation reactions catalyzed by this enzyme.

Regulation of Human Hepatic Hydroxysteroid Sulfotransferase Gene Expression by the Peroxisome Proliferator-Activated Receptor α Transcription Factor

Human hydroxysteroid sulfotransferase or (HUMAN)SULT2A1 catalyzes the sulfonation of procarcinogen xenobiotics, hydroxysteroids, and bile acids and plays a dynamic role in hepatic cholesterol homeostasis. The treatment of primary cultured human hepatocytes with a peroxisome proliferator-activated receptor alpha (PPARalpha)-activating concentration of ciprofibrate (10(-) (4) M) increased (HUMAN)SULT2A1 mRNA, immunoreactive protein, and enzymatic activity levels by approximately 2-fold. By contrast, expression of (RAT)SULT2A3, the rat counterpart to (HUMAN)SULT2A1, was induced by treatment of primary hepatocyte cultures with an activator of the pregnane X receptor, but not PPARalpha. In HepG2 cells, transient transfection analyses of luciferase reporter constructs containing upstream regions of the (HUMAN)SULT2A1 gene implicated a candidate peroxisome proliferator response element (PPRE) at nucleotides (nt) -5949 to -5929 relative to the transcription start site. Site-directed mutagenesis and electrophoretic mobility shift assay studies confirmed that this distal PPRE (dPPRE), a direct repeat nuclear receptor motif containing one intervening nt, represented a functional PPRE. Chromatin immunoprecipitation analysis indicated that the (HUMAN)SULT2A1 dPPRE was also a functional element in the context of the human genome. These data support a major role for the PPARalpha transcription factor in the regulation of hepatic (HUMAN)SULT2A1. Results also indicate that important species differences govern the transactivation of SULT2A gene transcription by nuclear receptors.

Crystal Structure of Human Cholesterol Sulfotransferase (SULT2B1b) in the Presence of Pregnenolone and 3′-Phosphoadenosine 5′-Phosphate: RATIONALE FOR SPECIFICITY DIFFERENCES BETWEEN PROTOTYPICAL SULT2A1 AND THE SULT2B1 ISOFORMS

The gene for human hydroxysteroid sulfotransferase (SULT2B1) encodes two peptides, SULT2B1a and SULT2B1b, that differ only at their amino termini. SULT2B1b has a predilection for cholesterol but is also capable of sulfonating pregnenolone, whereas SULT2B1a preferentially sulfonates pregnenolone and only minimally sulfonates cholesterol. We have determined the crystal structure of SULT2B1a and SULT2B1b bound to the substrate donor product 3'-phosphoadenosine 5'-phosphate at 2.9 and 2.4 A, respectively, as well as SULT2B1b in the presence of the acceptor substrate pregnenolone at 2.3 A. These structures reveal a different catalytic binding orientation for the substrate from a previously determined structure of hydroxysteroid sulfotransferase (SULT2A1) binding dehydroepiandrosterone. In addition, the amino-terminal helix comprising residues Asp19 to Lys26, which determines the specificity difference between the SULT2B1 isoforms, becomes ordered upon pregnenolone binding, covering the substrate binding pocket.

Mutational Analysis of Human Hydroxysteroid Sulfotransferase SULT2B1 Isoforms Reveals That Exon 1B of the SULT2B1 Gene Produces Cholesterol Sulfotransferase, whereas Exon 1A Yields Pregnenolone Sulfotransferase

As a result of an alternative exon 1, the gene for human hydroxysteroid sulfotransferase (SULTB1) encodes for two peptides differing only at their amino termini. The SULT2B1b isoform preferentially sulfonates cholesterol. Conversely, the SULT2B1a isoform avidly sulfonates pregnenolone but not cholesterol. The outstanding structural feature that distinguishes the SULT2B1 isoforms from the prototypical SULT2A1 isozyme is the presence of extended amino- and carboxyl-terminal ends in the former. Investigating the functional significance of this unique characteristic reveals that removal of 53 amino acids from the relatively long carboxyl-terminal end that is common to both SULT2B1 isoforms has no effect on the catalytic activity of either isoform. On the other hand, removal of 23 amino acids from the amino-terminal end that is unique to SULT2B1b results in loss of cholesterol sulfotransferase activity, whereas removal of 8 amino acids from the amino-terminal end that is unique to SULT2B1a has no effect on pregnenolone sulfotransferase activity. Deletion analysis along with site-directed mutagenesis of SULT2B1b reveal that the amino acid segment 19-23 residues from the amino terminus and particularly isoleucines at positions 21 and 23 are crucial for cholesterol catalysis. In the gene for SULT2B1, exon 1B encodes for only the unique amino-terminal region of SULT2B1b; however, exon 1A encodes for the unique amino-terminal end of SULT2B1a plus an additional 48 amino acids. Thus, if the gene for SULT2B1 employs exon 1B, cholesterol sulfotransferase is synthesized, whereas if exon 1A is used, pregnenolone sulfotransferase is produced.

Biochemical Characterization and Tissue Distribution of Human SULT2B1

The human hydroxysteroid sulfotransferase (SULT) family is comprised of two subfamilies, SULT2A1 and SULT2B1. We characterized the substrate specificity, in vitro biochemical properties, and tissue distribution patterns of human SULT2B1a and SULT2B1b. In contrast to the wide substrate specificity of SULT2A1, SULT2B1a and SULT2B1b specifically catalyzed the sulfonation of 3β-hydroxysteroids with high catalytic efficiency. Both SULT2B1 enzymes also sulfonated dihydrotestosterone. In vitro studies revealed that the biochemical properties of SULT2B1a and SULT2B1b were not significantly different from each other. However, tissue expression analysis suggested that they are differentially regulated. In contrast to the limited tissue distribution of SULT2A1, SULT2B1 was detected in a variety of hormone-responsive tissues including placenta, ovary, uterus, and prostate. The catalytic activity toward dehydroepiandrosterone and dihydrotestosterone, biologically important androgens, coupled with expression in prostate suggests that SULT2B1 may play a novel regulatory role that protects against the mitogenic effects of androgens.