Human Hydroxysteroid Sulfotransferase Research Articles

Cholesterol and hydroxycholesterol sulfotransferases: identification, distinction from dehydroepiandrosterone sulfotransferase, and differential tissue expression.

In humans, the biotransformation of cholesterol and its hydroxylated metabolites (oxysterols) by sulfonation is a fundamental process of great importance. Nevertheless, the sulfotransferase enzyme(s) that carries out this function has never been clearly identified. Cholesterol is a relatively poor substrate for the previously cloned hydroxysteroid sulfotransferase (HST), i.e. dehydroepiandrosterone (DHEA) sulfotransferase (HST1). Recently, cloning of a single human gene that encodes for two proteins related to HST1 was reported. These newly cloned sulfotransferases (HST2a and HST2b), while exhibiting sequence similarity to other members of the soluble sulfotransferase superfamily, also contain unique structural features. This latter aspect prompted an examination of their substrate specificity for comparison with HST1. Thus, HST1, HST2a, and HST2b were overexpressed as fusion proteins and purified. Furthermore, a novel procedure for the isolation of cholesterol and oxysterol sulfonates was developed that was used in association with HPLC to resolve specific sterol sulfonates. HST1 preferentially sulfonated DHEA and, to a lesser extent, oxysterols; whereas cholesterol was a negligible substrate. The reverse, however, was the case for the HST2 isoforms, particularly HST2b, which preferentially sulfonated cholesterol and oxysterols, in contrast to DHEA, which served as a poor substrate for this enzyme. RT-PCR analysis revealed distinct patterns of HST1, HST2a, and HST2b expression. It was particularly notable that both HST2 isoforms, but not HST1, were expressed in skin, a tissue where cholesterol sulfonation plays an important role in normal development of the skin barrier. In conclusion, substrate specificity and tissue distribution studies strongly suggest that HST2a and HST2b, in contrast to HST1, represent normal human cholesterol and oxysterol sulfotransferases. Furthermore, this study represents the first example of the sulfonation of oxysterols by a specific human HST.

Expression and characterization of the human 3β-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b)

The human hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST), is highly expressed in liver and adrenal cortex and displays reactivity towards a broad range of hydroxysteroids including 3β-hydroxysteroids, 3α-hydroxysteroids, estrogens with a 3-phenolic moiety, and 17-hydroxyl group of androgens. In contrast, characterization of the newly described human hydroxysteroid sulfotransferase SULT2B1 isoforms shows that these enzymes are selective for the sulfation of 3β-hydroxysteroids, such as pregnenolone, epiandrosterone, DHEA, and androstenediol. There was no activity detected towards testosterone, dexamethasone, β-estradiol, androsterone, or p-nitrophenol. The SULT2B1 gene encodes two isoforms, SULT2B1a and SULT2B1b, which are generated by alternate splicing of the first exon; therefore the SULT2B1 isoforms differ at their N-terminals. Northern Blot analysis detected a SULT2B1 message in RNA isolated from the human prostate and placenta. No SULT2B1 message was observed in RNA isolated from human liver, colon, lung, kidney, brain, or testis tissue. Purified SULT2B1a was used to generate a specific rabbit polyclonal anti-SULT2B1 antibody. The anti-SULT2B1 antibody did not react with expressed human EST, P-PST-1, M-PST, DHEA-ST, or ST1B2, during immunoblot analysis. The substrate specificity of the expressed SULT2B1 isoforms suggests that these enzymes are capable of regulating the activity of adrenal androgens in human tissues via their inactivation by sulfation.

Current status of the cytosolic sulfotransferases in the metabolic activation of promutagens and procarcinogens.

Cytosolic sulfotransferases (SULT) catalyze the sulfation of structurally diverse drugs, endogenous compounds and xenobiotics. These reactions involve the transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyl/amino groups of acceptor molecules. Although sulfate conjugation is generally considered as a detoxication pathway producing more water-soluble and often less toxic metabolites, sulfation of certain classes of compounds produce sufficiently electrophilic metabolites that can covalently bind to cellular macromolecules, DNA and RNA. The important roles of electrophilic sulfate ester metabolites in the metabolic activation, mutagenicity and ultimate carcinogenicity of many xenobiotics have been considerably elucidated. Examples include the class of hydroxymethyl polycyclic aromatic hydrocarbons, allylic alcohols, N-hydroxy derivatives of carcinogenic arylamines and heterocyclic amines. Results obtained by many scientists during the last two decade correlate with a hypothesis that electrophilic sulfate esters may be the major ultimate carcinogenic forms of many, if not most, procarcinogens derived from benzylic/allylic alcohols and hydroxy arylamines. Careful analysis of these results suggest that the activities of human hydroxysteroid sulfotransferase (hHST), and a related form in rat liver, rat hydroxysteroid sulfotransferase a (STa), as well as aryl sulfotransferases both from rat and human liver, account for a substantial portion of the activation of benzylic/allylic alcohols in these species. Moreover, aryl sulfotransferases have also been indicated as the responsible SULT family in the bioactivation of hydroxy arylamines in the liver of different species including human. Molecular cloning of the individual sulfotransferases and expression of these individual forms in heterologous expression systems have allowed us to better understand the role of SULTs in the bioactivation of different procarcinogens and the form of sulfotransferase involved in their bioactivation. Additional structure-activity studies with homogeneous forms of rat liver STa and AST IV have also yielded comparative insight into some of the parameters important in recognition of substrates and inhibitors by these enzymes.

Crystal structure of SULT2A3, human hydroxysteroid sulfotransferase

The crystal structure of SULT2A3 human hydroxysteroid sulfotransferase has been solved at 2.4 Å resolution in the presence of 3′-phosphoadenosine 5′-phosphate (PAP). The overall structure is similar to those of SULT1 enzymes such as estrogen sulfotransferase and the PAP binding site is conserved, however, significant differences exist in the positions of loops Pro14–Ser20, Glu79–Ile82 and Tyr234–Gln244 in the substrate binding pocket. Moreover, protein interaction in the crystal structure has revealed a possible dimer-directed conformational alteration that may regulate the SULT activity.

Human Hydroxysteroid Sulfotransferase SULT2B1: Two Enzymes Encoded by a Single Chromosome 19 Gene

We have cloned and characterized cDNAs that encode two human hydroxysteroid sulfotransferase (SULT) enzymes, SULT2B1a and SULT2B1b, as well as the single gene that encodes both of these enzymes. The two cDNAs differed at their 5′-termini and had 1050- and 1095-bp open reading frames that encoded 350 and 365 amino acids, respectively. The amino acid sequences encoded by these cDNAs included “signature sequences” that are conserved in all known cytosolic SULTs. Both cDNAs appeared, on the basis of amino acid sequence analysis, to be members of the hydroxysteroid SULT “family,” SULT2, but they were only 48% identical in amino acid sequence with the single known member of that family in humans, SULT2A1 (also referred to as DHEA ST). Northern blot analysis demonstrated the presence of SULT2B1 mRNA species approximately 1.4 kb in length in human placenta, prostate, and trachea and—faintly—in small intestine and lung. Expression of the two human SULT2B1 cDNAs in COS-1 cells showed that both of the encoded proteins catalyzed sulfation of the prototypic hydroxysteroid SULT substrate, dehydroepiandrosterone, but both failed to catalyze the sulfate conjugation of 4-nitrophenol or 17β-estradiol, prototypic substrates for the phenol and estrogen SULT subfamilies. Both of these cDNAs were encoded by a single gene,SULT2B1.The locations of most exon–intron splice junctions inSULT2B1were identical to those of the only other known human hydroxysteroid SULT geneSULT2A1(previouslySTD). The divergence in 5′-terminal sequences of the two SULT2B1 cDNAs resulted from alternative transcription initiation prior to different 5′ exons, combined with alternative splicing.SULT2B1mapped to human chromosome band 19q13.3, approximately 500 kb telomeric to the location ofSULT2A1.

Sulfation of alpha-hydroxytamoxifen catalyzed by human hydroxysteroid sulfotransferase results in tamoxifen-DNA adducts.

The formation of tamoxifen (TAM)-derived DNA adducts was investigated by incubation of DNA with (E)-alpha-hydroxytamoxifen [(E)-alpha-OHTAM], 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and human recombinant sulfotransferase. Using 32P-post-labeling and HPLC analysis, two TAM-DNA adducts were detected in incubations that included the human hydroxysteroid sulfotransferase SULT2A1 (hHST). When compared with standards of stereoisomers of alpha-(N2-deoxyguanosinyl)tamoxifen 3'-monophosphate (dG3'P-N2-TAM), the major adduct was identified chromatographically as an epimer of the transform of dG-N2-TAM, and the minor adduct was identified as an epimer of the cis-form. The amount of TAM adducts formed by hHST was approximately three times less than that formed by an equivalent amount of rat hydroxysteroid (alcohol) sulfotransferase a. These results indicate that sulfation of alpha-OHTAM catalyzed by hHST results in the formation of dG-N2-TAMs, highly miscoding lesions, in human tissues.

Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture.

Tamoxifen increases the risk of human endometrial cancer and is a potent carcinogen in rat liver, in which it produces DNA adducts and cytogenetic damage. Nevertheless its prophylactic use against breast cancer in healthy women is under investigation in several large trials. To investigate whether rat hepatocarcinogenicity predicts human hepatocarcinogenicity we used genetically engineered bacterial and mammalian target cells to investigate how alpha-hydroxy-tamoxifen, a major phase I metabolite of tamoxifen, is further metabolised by rat and human phase II enzymes, sulfotransferases, to mutagenic and DNA-adduct-forming species. We expressed rat hydroxysteroid sulfotransferase a, a liver-specific enzyme, and corresponding human sulfotransferase in bacteria (Salmonella typhimurium) and in a mammalian cell line (Chinese hamster V79 cells) and tested alpha-hydroxytamoxifen for DNA adduct formation and mutagenicity in these systems, using unmodified cells as controls. In cells that expressed rat hydroxysteroid sulfotransferase, alpha-hydroxytamoxifen was mutagenic and formed the same pattern of DNA adducts as that found in the liver of tamoxifen-treated rats. Alpha-hydroxytamoxifen was not activated, or was at least 20 times less active in cells expressing human hydroxysteroid sulfotransferase. All the other six known human xenobiotic-metabolising sulfotransferases were also expressed in S. typhimurium. None activated alpha-hydroxytamoxifen to a mutagen. These results suggest that the risk of DNA adduct formation, and cancer, in the human liver is low and explain why tamoxifen is a powerful carcinogen to the rat liver, and why standard short-term tests fail to detect its mutagenicity.

Chiral Inversion of 1-Hydroxyethylpyrene Enantiomers Mediated by Enantioselective Sulfotransferases

The benzylic alcohol 1-hydroxyethylpyrene (1-HEP) is activated to a mutagen by sulfotransferases. The sulfuric acid ester formed is difficult to detect, as it is rapidly hydrolysed back to the alcohol. Incubation of the individual enantiomers of 1-HEP with human hydroxysteroid sulfotransferase (hHST) or estrogen sulfotransferase (hEST), expressed in bacteria, led to the formation of the other enantiomer. The rates of sulfation were determined from the initial rates of chiral inversion of the alcohol, knowing that hydrolysis follows an SN1 mechanism and therefore produces racemic alcohol. hEST showed high enantioselectivity forS-1-HEP, whereas hHST strongly preferred theR-enantiomer. The rates of sulfation of the preferred enantiomers were high, similar to those for the prototype substrates of hEST (β-estradiol) and hHST (dehydroepiandrosterone). Moreover, after a 30-min incubation ofS-1-HEP with hEST, 95% of the recovered alcohol showed theR-configuration, indicating that several cycles of sulfation and hydrolysis had led to the depletion of one enantiomer and to the enrichment of the other enantiomer.

External Versus Internal Metabolic Activation of Polycyclic Aromatic Compounds in Mutagenicity Tests: A Comparison Using Heterologous Expression Systems

Abstract Benzo[a]pyrene-trans-7,8-dihydrodiol was virtually non-mutagenic to Chinese hamster V79p cells, but was strongly mutagenic to a V79-derived cell line expressing cytochrome P450 1A1, when the cells were exposed separately. In mixed cultures of these two cell types, it showed about 50 % of the mutagenic activity in V79p cells, compared to that observed in the enzyme-proficient cell line, indicating an efficient intercellular transfer of the active metabolite. The benzylic alcohols 1-hydroxymethylpyrene and 6-hydroxymethylbenzo[a]-pyrene were weakly mutagenic to Salmonella typhimurium TA1538 in the absence of a metabolic activation system, but were potent mutagens to a TA1538-derived strain expressing human hydroxysteroid sulfotransferase. 1-Hydroxymethylpyrene also showed strong mutagenicity to TA 1538 in the presence of chloride anions and cofactor-supplemented cytosol of the genetically engineered strain. This was not the case with 6-hydroxymethylbenzo[a]pyrene. It is concluded that some reactive...

Activation of benzylic alcohols to mutagens by rat and human sulfotransferases expressed in Escherichia coli

Human hydroxysteroid sulfotransferase, human phenol-sulfating form of phenol sulfotransferase, rat hydroxysteroid sulfotransferase a and rat phenol sulfotransferase IV were expressed in Escherichia coli. Cytosol preparations of transformed bacteria were used as activating systems in mutagenicity tests with Salmonella typhimurium TA 98. All test compounds, 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 1-(1-pyrenyl)ethanol, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[ a]anthracene and 4 H-cyclopenta l[ def]chrysen-4-ol, were activated by both hydroxysteroid sulfotransferases investigated. However, 1-(1-pyrenyl)ethanol was 67-fold more efficiently activated by the human enzyme, whereas 7-hydroxymethyl-12-methylbenz[ a]anthracene was 27-fold more efficiently activated by the rat enzyme. The phenol sulfotransferases showed relatively low activities with the benzylic alcohols investigated. The only exception was 4 H-cyclopental[ def]chrysen-4-ol, which was activated efficiently by rat phenol sulfotransferase IV. We had previously tested the ability of rat and human hepatic cytosol preparations to activate the same compounds. The results of a statistical analysis suggest that the activities of human hydroxysteroid sulfotransferase, rat hydroxysteroid sulfotransferase a and phenol sulfotransferase IV can account for a substantial portion of the activation of benzylic alcohols in human, female rat and male rat liver, respectively.

Activation of benzylic alcohols to mutagens by rat and human sulfotransferases expressed in

Human hydroxysteroid sulfotransferase, human phenol-sulfating form of phenol sulfotransferase, rat hydroxysteroid sulfotransferase a and rat phenol sulfotransferase IV were expressed in Escherichia coli . Cytosol preparations of transformed bacteria were used as activating systems in mutagenicity tests with Salmonella typhimurium TA 98. All test compounds, 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 1-(1-pyrenyl)ethanol, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[ a ]anthracene and 4 H -cyclopenta l [ def ]chrysen-4-ol, were activated by both hydroxysteroid sulfotransferases investigated. However, 1-(1-pyrenyl)ethanol was 67-fold more efficiently activated by the human enzyme, whereas 7-hydroxymethyl-12-methylbenz[ a ]anthracene was 27-fold more efficiently activated by the rat enzyme. The phenol sulfotransferases showed relatively low activities with the benzylic alcohols investigated. The only exception was 4 H -cyclopental[ def ]chrysen-4-ol, which was activated efficiently by rat phenol sulfotransferase IV. We had previously tested the ability of rat and human hepatic cytosol preparations to activate the same compounds. The results of a statistical analysis suggest that the activities of human hydroxysteroid sulfotransferase, rat hydroxysteroid sulfotransferase a and phenol sulfotransferase IV can account for a substantial portion of the activation of benzylic alcohols in human, female rat and male rat liver, respectively.

Molecular cloning of a chiral-specific 3 alpha-hydroxysteroid sulfotransferase.

A novel stereoselective hydroxysteroid sulfotransferase (HST) that acts on neutral steroids having the 3-hydroxyl group in the alpha orientation but not on steroids where the 3-hydroxyl group is oriented in the beta position has been cloned and expressed. Primary screening of a guinea pig adrenal cDNA library was performed by colony hybridization using an oligonucleotide probe based on a highly homologous region among known steroid sulfotransferases. Selected clones consisted of overlapping sequences but were incomplete. The rapid amplification of cDNA ends procedure was used to construct a full-length guinea pig HST cDNA. The guinea pig HST cDNA transiently transfected into Chinese hamster ovary K1 cells expressed a protein identical in size to that of purified guinea pig HST specific for 3 alpha-hydroxylated neutral steroids that was recently reported (Driscoll, W. J., Martin, B. M., Chen, H.-C., and Strott, C. A. (1993) J. Biol. Chem. 268, 23496-23503). The expressed HST likewise exhibited sulfotransferase activity that was directed specifically toward steroid substrates containing a 3-hydroxyl group in the alpha orientation; on the other hand, steroids with a 3 beta-hydroxyl group were not sulfonated by the expressed HST. Thus, the cloned HST cDNA clearly coded for a steroid sulfotransferase with chiral specificity for 3 alpha-hydroxylated neutral steroids and was, therefore, given the designation of guinea pig 3 alpha-hydroxysteroid sulfotransferase (gp3 alpha-HST). The full-length gp3 alpha-HST cDNA consisted of 1182 base pairs and coded for a protein containing 287 amino acids. The deduced amino acid sequence of the protein shares 65/79, 65/80, and 62/76% identity/similarity with rat, mouse, and human HST, respectively. Northern blot analysis of guinea pig tissues revealed no apparent gender differences in either mRNA species or distribution; a single 1.4-kilobase HST mRNA species was present in adrenal and liver tissue. Interestingly, the adrenal mRNA content was considerably more abundant than that found in the liver. Evidence for 3 alpha-HST mRNA was not detected in kidney, heart, lung, muscle, spleen, or uterus.

Identification of a New Adult Human Liver Sulfotransferase with Specificity for Endogenous and Xenobiotic Estrogens

We present evidence for the existence of a previously undescribed sulfotransferase isoenzyme in adult human liver which sulfates endogenous and xenobiotic estrogens (estrone and 17α-ethinylestradiol). The enzyme was resolved from other sulfotransferases using anion exchange chromatography and further purified by gel filtration and affinity chromatography. Using antibodies against rat liver estrogen and hydroxysteroid sulfotransferases, it was shown that the adult human estrogen sulfotransferase has a subunit molecular weight of 33kDa, is immunologically related to its rat counterpart and also to the human phenolsulfotransferase enzyme family, but not to human hydroxysteroid sulfotransferase.

Morality of the Ambulance Ride

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 α (PPARα)-activating concentration of ciprofibrate (10^-⁴ M) increased (HUMAN)SULT2A1 mRNA, immunoreactive protein, and enzymatic activity levels by ∼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 PPARα. 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 PPARα 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.