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Bile acids play essential roles in facilitating the intestinal absorption of lipophilic nutrients as well as regulation of glucose, lipid, and energy homeostasis via activation of some receptors. Bile acids are cytotoxic, and consequently their concentrations are tightly controlled. A critical pathway for bile acid elimination and detoxification is sulfation. The pattern of bile acid sulfation differs by species. Sulfation preferentially occurs at the 3α-OH of bile acids in humans, but at the 7α-OH in mice. A recent study identified mouse cytosolic sulfotransferase 2A8 (mSULT2A8) as the major hepatic 7α-hydroxyl bile acid-sulfating enzyme. To elucidate the 7α-OH specific sulfation mechanism of mSULT2A8, instead of 3α-OH specific sulfation in humans, we determined a crystal structure of mSULT2A8 in complex with cholic acid, a major bile acid, and 3'-phosphoadenosine-5'-phosphate, the sulfate donor product. Our study shows that bile acid-binding mode of mSULT2A8 and how the enzyme holds the 7α-OH group of bile acids at the catalytic center, revealing that the mechanism underlying 7α-OH specific sulfation. The structure shows the substrate binds to mSULT2A8 in an orientation perpendicular to that of human 3α-hydroxyl bile acid-sulfotransferase (hSULT2A1). The structure of the complex provides new insight into species different bile acid metabolism.

Introduction Cytosolic sulfotransferases (SULTs)-mediated sulfation is critically involved in the metabolism of key endogenous compounds such as catecholamines and thyroid/steroid hormones, as well as a variety of drugs and other xenobiotics. Studies performed in the past three decades have yielded a good understanding about the enzymology of the SULTs and their structural biology, phylogenetic relationships, tissue/organ-specific/developmental expression, as well as the regulation of the SULT gene expression. An emerging area is related to the functional impact of the SULT genetic polymorphisms. Areas covered The current review aims to summarize our current knowledge about the above-mentioned aspects of the SULT research. An emphasis is on the information concerning the effects of the polymorphisms of the SULT genes on the functional activity of the SULT allozymes and the associated physiological, pharmacological, and clinical implications. Expert opinion Elucidation of how SULT SNPs may influence the drug-sulfating activity of SULT allozymes will help understand the differential drug metabolism and eventually aid in formulating personalized drug regimens. Moreover, the information concerning the differential sulfating activities of SULT allozymes toward endogenous compounds may allow for the development of strategies for mitigating anomalies in the metabolism of these endogenous compounds in individuals with certain SULT genotypes.

Nevirapine (NVP) is an effective drug for the treatment of HIV infections, but its use is limited by a high incidence of severe skin rash and liver injury. 12-Hydroxynevirapine (12-OH-NVP) is the major metabolite of nevirapine. There is strong evidence that the sulfate of 12-OH-NVP is responsible for the skin rash. While several cytosolic sulfotransferases (SULTs) have been shown to be capable of sulfating 12-OH-NVP, the exact mechanism of sulfation in vivo is unclear. The current study aimed to clarify human SULT(s) and human organs that are capable of sulfating 12-OH-NVP and investigate the metabolic sulfation of 12-OH-NVP using cultured HepG2 human hepatoma cells. Enzymatic assays revealed that of the thirteen human SULTs, SULT1A1 and SULT2A1 displayed strong 12-OH-NVP-sulfating activity. 1-Phenyl-1-hexanol (PHHX), which applied topically prevents the skin rash in rats, inhibited 12-OH-NVP sulfation by SULT1A1 and SULT2A1, implying the involvement of these two enzymes in the sulfation of 12-OH-NVP in vivo. Among five human organ cytosols analyzed, liver cytosol displayed the strongest 12-OH-NVP-sulfating activity, while a low but significant activity was detected with skin cytosol. Cultured HepG2 cells were shown to be capable of sulfating 12-OH-NVP. The effects of genetic polymorphisms of SULT1A1 and SULT2A1 genes on the sulfation of 12-OH-NVP by SULT1A1 and SULT2A1 allozymes were investigated. Two SULT1A1 allozymes, Arg37Asp and Met223Val, showed no detectable 12-OH-NVP-sulfating activity, while a SULT2A1 allozyme, Met57Thr, displayed significantly higher 12-OH-NVP-sulfating activity compared with the wild-type enzyme. Collectively, these results contribute to a better understanding of the involvement of sulfation in NVP-induced skin rash and provide clues to the possible role of SULT genetic polymorphisms in the risk of this adverse reaction.

Cytosolic sulfotransferase SULT1C subfamily is one of the most flexible gene subfamilies during mammalian evolution. The physiological functions of SULT1C enzymes still remain to be fully understood. In this study, common marmoset (Callithrix jacchus), a promising primate animal model, was used to investigate the functional relevance of the SULT1C subfamily. Gene database search revealed 3 intact SULT1C genes and a pseudogene in its genome. These 4 genes were named SULT1C1, SULT1C2, SULT1C3P, and SULT1C5, according to the sequence homology and gene location. Since SULT1C5 is the orthologous gene for human SULT1C2P, we propose, here, to revisit the designation of human SULT1C2P to SULT1C5P. Purified recombinant SULT1C enzymes showed sulfating activities toward a variety of xenobiotic compounds and thyroid hormones. Kinetic analysis revealed high catalytic activities of SULT1C1 and SULT1C5 for 3,3′-T2. It appears therefore that SULT1C isoforms may play a role in the thyroid hormone metabolism in common marmoset.

Background Non-opioid and opioid analgesics, as over-the-counter or prescribed medications, are widely used for the management of a diverse array of pathophysiological conditions. Previous studies have demonstrated the involvement of human cytosolic sulfotransferase (SULT) SULT1A1 in the sulfation of acetaminophen, O-desmethylnaproxen (O-DMN), and tapentadol. The current study was designed to investigate the impact of single nucleotide polymorphisms (SNPs) of the human SULT1A1 gene on the sulfation of these analgesic compounds by SULT1A1 allozymes. Methods Human SULT1A1 genotypes were identified by database search. cDNAs corresponding to nine SULT1A1 nonsynonymous missense coding SNPs (cSNPs) were generated by site-directed mutagenesis. Recombinant wild-type and SULT1A1 allozymes were bacterially expressed and affinity-purified. Purified SULT1A1 allozymes were analyzed for sulfation activity using an established assay procedure. Results Compared with the wild-type enzyme, SULT1A1 allozymes were shown to display differential sulfating activities toward three analgesic compounds, acetaminophen, O-desmethylnaproxen (O-DMN), and tapentadol, as well as the prototype substrate 4NP. Conclusion Results obtained indicated clearly the impact of genetic polymorphisms on the drug-sulfation activity of SULT1A1 allozymes. Such information may contribute to a better understanding about the differential metabolism of acetaminophen, O-DMN, and tapentadol in individuals with different SULT1A1 genotypes.

Doxorubicin is a chemotherapeutic drug widely utilized in cancer treatment. An enzyme critical to doxorubicin metabolism is the cytosolic sulfotransferase (SULT) SULT1C4. This study investigated the functional impact of SULT1C4 single nucleotide polymorphisms (SNPs) on the sulfation of doxorubicin by SULT1C4 allozymes. A comprehensive database search was performed to identify various SULT1C4 SNPs. Ten nonsynonymous SULT1C4 SNPs were selected, and the corresponding cDNAs, packaged in pGEX-2TK expression vector, were generated via site-directed mutagenesis. Respective SULT1C4 allozymes were bacterially expressed and purified by affinity chromatography. Purified SULT1C4 allozymes, in comparison with the wild-type enzyme, were analysed for sulphating activities towards doxorubicin and 4-nitrophenol, a prototype substrate. Results obtained showed clearly differential doxorubicin-sulphating activity of SULT1C4 allozymes, implying differential metabolism of doxorubicin through sulfation in individuals with distinct SULT1C4 genotypes.

BACKGROUND AND OBJECTIVES: Previous studies have revealed that sulfation, as mediated by the estrogen-sulfating cytosolic sulfotransferase (SULT) SULT1E1, is involved in the metabolism of 17β-estradiol (E2), 4-hydroxytamoxifen (4OH-tamoxifen) and diethylstilbestrol in humans. It is an interesting question whether the genetic polymorphisms of SULT1E1, the gene that encodes the SULT1E1 enzyme, may impact on the metabolism of E2 and these two drug compounds through sulfation. METHODS: In this study, five missense coding single nucleotide polymorphisms (SNPs) of the SULT1E1 gene were selected for investigating the sulfating activity of the coded SULT1E1 allozymes toward E2, 4OH-tamoxifen and diethylstilbestrol. Corresponding cDNAs were generated by site-directed mutagenesis and recombinant SULT1E1 allozymes were bacterially expressed, affinity-purified, and characterized using enzymatic assays. RESULTS: Purified SULT1E1 allozymes were shown to display differential sulfating activities toward E2, 4OH-tamoxifen and diethylstilbestrol. Kinetic analysis revealed further distinct K(m) (reflecting substrate affinity) and V(max) (reflecting catalytic activity) values of the five SULT1E1 allozymes with E2, 4OH-tamoxifen and diethylstilbestrol as substrates. CONCLUSIONS: Taken together, these findings highlighted the significant differences in E2- as well as drug-sulfating activities of SULT1E1 allozymes, which may have implications in the differential metabolism of E2, 4OH-tamoxifen and diethylstilbestrol in individuals with different SULT1E1 genotypes.

Indoxyl, a derivative of indole originating from tryptophan, may undergo phase-II sulfate-conjugation pathway, thereby forming indoxyl sulfate (IS) in vivo. We previously reported that IS, a well-known uremic toxin, can increase the intracellular oxidation level and decrease the phagocytic activity in a differentiated HL-60 human macrophage cell model. Using the same cell model, the current study aimed to investigate whether indole and indoxyl (the metabolic precursors of indoxyl and IS, respectively) may cause macrophage immune dysfunction. Results obtained indicated that intracellular oxidation level and cytotoxicity markedly increased upon treatment with indole and indoxyl, in comparison with IS. Incubation of the cells with indole and indoxyl also resulted in attenuated phagocytic activity. Human serum albumin (HSA)-binding assay confirmed that tryptophan and IS, but not indole and indoxyl, could selectively bind to the site II in HSA. Collectively, the results indicated that indole and indoxyl may strongly down-regulate the phagocytic immune function of macrophages, whereas IS, formed upon sulfate conjugation of indoxyl, may exhibit enhanced HSA-binding capability, thereby reducing the adverse effects of indoxyl.

Indoxyl sulfate (IS), a uremic toxin, is a sulfate-conjugated metabolite originated from tryptophan. Accumulating uremic toxins may worsen renal diseases and further complicate related disorders including impaired immune functions under oxidative stress conditions. However, it has remained unclear whether or not IS can directly cause the cellular immune dysfunction. We investigated the effects of IS on the intracellular oxidation level and phagocytic activity in a HL-60-differantiated human macrophage cell model. Incubation of the cells in the presence of IS resulted in increasing intracellular oxidation level and decreasing phagocytic activity. In addition to inhibitors for NADH oxidase (NOX), organic anion transporting polypeptide2B1 (OATP2B1), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), a representative antioxidant Trolox, was also shown to significantly relieve the IS-induced oxidation and restore weakened phagocytosis. Collectively, IS may directly down-regulate the phagocytic immune function of macrophages through the oxidation mechanisms including OATP2B1, PKC, PI3K, and NOX pathways. Abbreviations CKD: Chronic kidney disease; IS: Indoxyl sulfate; ROS: Reactive oxygen species; NOX: NADH oxidase; OATP2B1: Organic anion transporting polypeptide2B1; PKC: Protein kinase C; PI3K: Phosphoinositide 3-kinase; 2-APT: 2-acetylphenothiazine

Sulfoconjugation has been shown to be critically involved in the metabolism of acetaminophen (APAP), morphine, tapentadol and O-desmethyl tramadol (O-DMT). The objective of this study was to investigate the effects of single nucleotide polymorphisms (SNPs) of human SULT1A3 and SULT1A4 genes on the sulfating activity of SULT1A3 allozymes toward these analgesic compounds. Twelve non-synonymous coding SNPs (cSNPs) of SULT1A3/SULT1A4 were investigated, and the corresponding cDNAs were generated by site-directed mutagenesis. SULT1A3 allozymes, bacterially expressed and purified, exhibited differential sulfating activity toward each of the four analgesic compounds tested as substrates. Kinetic analyses of SULT1A3 allozymes further revealed significant differences in binding affinity and catalytic activity toward the four analgesic compounds. Collectively, the results derived from the current study showed clearly the impact of cSNPs of the coding genes, SULT1A3 and SULT1A4, on the sulfating activity of the coded SULT1A3 allozymes toward the tested analgesic compounds. These findings may have implications in the pharmacokinetics as well as the toxicity profiles of these analgesics administered in individuals with distinct SULT1A3 and/or SULT1A4 genotypes.

Previous studies have demonstrated the involvement of sulfoconjugation in the metabolism of catecholamines and serotonin. The current study aimed to clarify the effects of single nucleotide polymorphisms (SNPs) of human SULT1A3 and SULT1A4 genes on the enzymatic characteristics of the sulfation of dopamine, epinephrine, norepinephrine and serotonin by SULT1A3 allozymes. Following a comprehensive search of different SULT1A3 and SULT1A4 genotypes, twelve non-synonymous (missense) coding SNPs (cSNPs) of SULT1A3/SULT1A4 were identified. cDNAs encoding the corresponding SULT1A3 allozymes, packaged in pGEX-2T vector were generated by site-directed mutagenesis. SULT1A3 allozymes were expressed, and purified. Purified SULT1A3 allozymes exhibited differential sulfating activity toward catecholamines and serotonin. Kinetic analyses demonstrated differences in both substrate affinity and catalytic efficiency of the SULT1A3 allozymes. Collectively, these findings provide useful information relevant to the differential metabolism of dopamine, epinephrine, norepinephrine and serotonin through sulfoconjugation in individuals having different SULT1A3/SULT1A4 genotypes.

BACKGROUND AND OBJECTIVES: Previous studies have demonstrated the metabolism of tibolone through sulfation, with the cytosolic sulfotransferase (SULT) SULT2A1 as the major responsible enzyme. The current study aimed to investigate how SULT2A1 genetic polymorphisms may affect the dehydroepiandrosterone (DHEA)- and tibolone-sulfating activity of SULT2A1. METHODS: Site-directed mutagenesis was employed to generate cDNAs encoding ten different SULT2A1 allozymes. Recombinant SULT2A1 allozymes were expressed in BL21 E. coli cells, and purified using glutathionine-Sepharose affinity chromatography. An established sulfotransferase assay was used to analyze DHEA- and tibolone-sulfating activity of the purified SULT2A1 allozymes. RESULTS: The nine human SULT2A1 allozymes plus the wild-type SULT2A1 were found to display differential sulfating activity toward DHEA and tibolone. Kinetic analysis revealed that different SULT2A1 allozymes exhibited differential substrate affinity and catalytic efficiency toward the two substrates tested. CONCLUSION: The results obtained provided useful information concerning the differential metabolism of tibolone through sulfation in individuals with different SULT2A1 genotypes.

5α-Cyprinol 27-sulfate is the major biliary bile salt present in cypriniform fish including the zebrafish (Danio rerio). The current study was designed to identify the zebrafish cytosolic sulfotransferase (Sult) enzyme(s) capable of sulfating 5α-cyprinol and to characterize the zebrafish 5α-cyprinol-sulfating Sults in comparison with human SULT2A1. Enzymatic assays using zebrafish homogenates showed 5α-cyprinol-sulfating activity. A systematic analysis, using a panel of recombinant zebrafish Sults, revealed two Sult2 subfamily members, Sult2st2 and Sult2st3, as major 5α-cyprinol-sulfating Sults. Both enzymes showed higher activities using 5α-cyprinol as the substrate, compared to their activity with DHEA, a representative substrate for mammalian SULT2 family members, particularly SULT2A1. pH-Dependence and kinetics experiments indicated that the catalytic properties of zebrafish Sult2 family members in mediating the sulfation of 5α-cyprinol were different from those of either zebrafish Sult3st4 or human SULT2A1. Collectively, these results imply that both Sult2st2 and Sult2st3 have evolved to sulfate specifically C27-bile alcohol, 5α-cyprinol, in Cypriniform fish, whereas the enzymatic characteristics of zebrafish Sult3 members, particularly Sult3st4, correlated with those of human SULT2A1.

Cytosolic sulfotransferase (SULT)-mediated sulfation is generally known to involve the transfer of a sulfonate group from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to a hydroxyl group or an amino group of a substrate compound. We report here that human SULT2A1, in addition to being able to sulfate dehydroepiandrosterone (DHEA) and other hydroxysteroids, could also catalyze the sulfation of Δ4-3-ketosteroids, which carry no hydroxyl groups in their chemical structure. Among a panel of Δ4-3-ketosteroids tested as substrates, 4-androstene-3,17-dione and progesterone were found to be sulfated by SULT2A1. Mass spectrometry analysis and structural modeling supported a reaction mechanism which involves the isomerization of Δ4-3-ketosteroids from the keto form to an enol form, prior to being subjected to sulfation. Results derived from this study suggested a potential role of SULT2A1 as a Δ4-3-ketosteroid sulfotransferase in steroid metabolism.

Previous studies have demonstrated that sulfate conjugation is involved in the metabolism of the active metabolite of tramadol, O-desmethyltramadol (O-DMT). The current study aimed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of mediating the sulfation of O-DMT.The sulfation of O-DMT under metabolic conditions was demonstrated using HepG2 hepatoma cells and Caco-2 human colon carcinoma cells. O-DMT-sulfating activity of thirteen known human SULTs and four human organ specimens was examined using an established sulfotransferase assay. pH-Dependency and kinetic parameters were also analyzed using, respectively, buffers at different pHs and varying O-DMT concentrations in the assays.Of the thirteen human SULTs tested, only SULT1A3 and SULT1C4 were found to display O-DMT-sulfating activity, with different pH-dependency profiles. Kinetic analysis revealed that SULT1C4 was 60 times more catalytically efficient in mediating the sulfation of O-DMT than SULT1A3 at respective optimal pH. Of the four human organ specimens tested, the cytosol prepared from the small intestine showed much higher O-DMT-sulfating activity than cytosols prepared from liver, lung, and kidney. Both cultured HepG2 and Caco-2 cells were shown to be capable of sulfating O-DMT and releasing sulfated O-DMT into cultured media.SULT1A3 and SULT1C4 were the major SULTs responsible for the sulfation of O-DMT. Collectively, the results obtained provided a molecular basis underlying the sulfation of O-DMT and contributed to a better understanding about the pharmacokinetics and pharmacodynamics of tramadol in humans.

Tyrosylprotein sulfotransferases (TPSTs) are enzymes that catalyze post-translational tyrosine sulfation of proteins. In humans, there are only two TPST isoforms, designated TPST1 and TPST2. In a previous study, we reported the crystal structure of TPST2, which revealed the catalytic mechanism of the tyrosine sulfation reaction. However, detailed molecular mechanisms underlying how TPSTs catalyse a variety of substrate proteins with different efficiencies and how TPSTs catalyze the sulfation of multiple tyrosine residues in a substrate protein remain unresolved. Here, we report two crystal structures of the human TPST1 complexed with two substrate peptides that are catalysed by human TPST1 with significantly different efficiencies. The distinct binding modes found in the two complexes provide insight into the sulfation mechanism for these substrates. The present study provides valuable information describing the molecular mechanism of post-translational protein modifications catalysed by TPSTs.

While 25-hydroxyvitamin D3 3-O-sulfate is known to be present in circulation, how it is generated in the body remains unclear. This study aimed to investigate its sulfation in major human organs and to unveil the responsible cytosolic sulfotransferases (SULTs). Of the vitamin D3 -related compounds tested, 25-hydroxyvitamin D3 and 7-dehydrocholesterol are preferentially sulfated by human organ cytosols. Among the 13 human SULTs, SULT2A1 shows sulfating activity toward all vitamin D3 -related compounds, whereas SULT1A1 and SULT2B1a/SULT2B1b show sulfating activity exclusively for, respectively, calcitriol and 7-dehydrocholesterol. These findings suggest that the metabolic pathway leading to the formation of 25-hydroxyvitamin D3 3-O-sulfate may be mediated by the sulfation of 25-hydroxyvitamin D3 or by the conversion of 7-dehydrocholesterol-3-O-sulfate in the skin.

In this study, we aimed to obtain a comprehensive account of the human cytosolic sulfotransferases (SULTs) that are capable of sulfating 6-O-desmethylnaproxen (O-DMN), a major metabolite of naproxen. Of the 13 known human SULTs tested, 7 (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, and SULT1E1) displayed O-DMN-sulfating activity, when analyzed using an elevated substrate concentration (500 μmol·L−1) together with 14 μmol·L−1 of the sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate (PAPS). At 10 μmol·L−1 O-DMN concentration, however, only SULT1A1 and SULT1A3 displayed detectable activity, with the former being nearly 2 orders of magnitude more active than the latter. A pH-dependence study indicated that SULT1A1 exhibited a broad pH optimum spanning pH 5.5–7. Kinetic parameters of the sulfation of O-DMN by SULT1A1 were determined. The production and release of sulfated O-DMN was demonstrated using cultured human HepG2 hepatoma cells and Caco-2 colon carcinoma cells. Moreover, assays using human organ specimens revealed that the O-DMN-sulfating activities present in the cytosols of liver and small intestine (at 502.5 and 497.2 pmol·min−1·(mg protein)−1, respectively) were much higher than those detected for the cytosols of lung and kidney. Taken together, these results provided relevant information concerning the sulfation of O-DMN both in vitro and in vivo.

Previous studies reported that tapentadol-sulfate represented one of the major metabolites of tapentadol excreted in urine. The current study aimed to identify the human cytosolic sulfotransferases (SULTs) that is(are) capable of sulfating tapentadol and to examine whether human cells and human organ specimens are capable of sulfating tapentadol. Thirteen human SULTs, previously expressed and purified, as well as human organ cytosols, were analyzed for tapentadol-sulfating activity using an established sulfotransferase assay. Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [35S]sulfate in the presence of different concentrations of tapentadol. Three of the thirteen human SULTs, SULT1A1, SULT1A3, and SULT1C4, were found to display sulfating activity toward tapentadol. Kinetic analysis revealed that SULT1A3 displayed the highest catalytic efficiency in mediating the sulfation of tapentadol, followed by SULT1A1 and SULT1C4. Using cultured HepG2 and Caco-2 cells, the generation and release of sulfated tapentadol under metabolic conditions was demonstrated. Moreover, of the four human organ specimens (kidney, liver, lung, and small intestine) tested, the cytosols prepared from small intestine and liver showed significant tapentadol-sulfating capacity (at 0.0203 and 0.0054 nmol/min/mg, respectively). Taken together, the results derived from the current study provided a molecular basis underlying the sulfation of tapentadol in humans.

The cytosolic sulfotransferases (SULTs) are Phase II detoxifying enzymes that mediate the sulfate conjugation of numerous xenobiotic molecules. While the research on the SULTs has lagged behind the research on Phase I cytochrome P-450 enzymes and other Phase II conjugating enzymes, it has gained more momentum in recent years. This review aims to summarize information obtained in several fronts of the research on the SULTs, including the range of the SULTs in different life forms, concerted actions of the SULTs and other Phase II enzymes, insights into the structure–function relationships of the SULTs, regulation of SULT expression and activity, developmental expression of SULTs, as well as the use of a zebrafish model for studying the developmental pharmacology/toxicology.

Sulfoconjugation plays a vital role in the detoxification of xenobiotics and in the metabolism of endogenous compounds. In this study, we aimed to identify new members of the sulfotransferase (SULT) superfamily in the silkworm Bombyx mori. Based on amino acid sequence and phylogenetic analyses, two new enzymes, swSULT ST1 and swSULT ST2, were identified that appear to belong to a distinct group of SULTs including several other insect SULTs. We expressed, purified, and characterized recombinant SULTs. While swSULT ST1 sulfated xanthurenic acid and pentachlorophenol, swSULT ST2 exclusively utilized xanthurenic acid as a substrate. Based on these results, and those concerning the tissue distribution and substrate specificity toward pentachlorophenol analyses, we hypothesize that swSULT ST1 plays a role in the detoxification of xenobiotics, including insecticides, in the silkworm midgut and in the induction of gametogenesis in silkworm ovary and testis. Collectively, the data obtained herein contribute to a better understanding of SULT enzymatic functions in insects.

Objectives Phenylephrine and salbutamol are drugs that are used widely to treat diseases/disorders, such as nasal congestion, hypotension, and asthma, in individuals of different age groups. Human cytosolic sulfotransferase (SULT) SULT1A3 has been shown to be critically involved in the metabolism of these therapeutic agents. This study was carried out to investigate the effects of single nucleotide polymorphisms of human SULT1A3 and SULT1A4 genes on the sulfation of phenylephrine and salbutamol by SULT1A3 allozymes. Materials and methods Wild-type and SULT1A3 allozymes, prepared previously by site-directed mutagenesis in conjunction with bacterial expression and affinity purification, were analyzed for sulfating activity using an established assay procedure. Results Purified SULT1A3 allozymes, in comparison with the wild-type enzyme, showed differential sulfating activities toward phenylephrine and salbutamol. Kinetic studies showed further significant variations in their substrate-binding affinity and catalytic activity toward phenylephrine and salbutamol. Conclusion The results obtained showed clearly the differential enzymatic characteristics of SULT1A3 allozymes in mediating the sulfation of phenylephrine and salbutamol. This information may contribute toward a better understanding of the pharmacokinetics of these two drugs in individuals with distinct SULT1A3 and/or SULT1A4 genotypes.

Carbonylation, an oxidative modification of the amino group of arginine and lysine residues caused by reactive oxygen species, has emerged as a new type of oxidative damage. Protein carbonylation has been shown to exert adverse effects on various protein functions. Recently, the role of food components in the attenuation of oxidative stress has been the focus of many studies. Most of these studies focused on the chemical properties of food components. However, it is also important to determine their effects on protein functions via post-translational modifications. In this study, we developed a novel procedure for evaluating the antioxidant capacity of food components. Hydrogen peroxide (H2O2)-induced protein carbonylation in HL-60 cells was quantitatively analyzed by using fluorescent dyes (Cy5–hydrazide dye and IC3–OSu dye), followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and fluorescence determination. Among a panel of food components tested, quinic acid, kaempferol, saponin, squalene, trigonelline, and mangiferin were shown to be capable of suppressing protein carbonylation in HL-60 cells. Our results demonstrated that this fluorescence labeling/SDS–PAGE procedure allows for the detection of oxidative stress-induced protein carbonylation with high sensitivity and quantitative accuracy. This method should be useful for the screening of new antioxidant food components as well as the analysis of their suppression mechanism.

Sulfation has been reported to be a major pathway for the metabolism and inactivation of rotigotine in vivo. The current study aimed to identify the human cytosolic sulfotransferase (SULT) enzyme(s) capable of mediating the sulfation of rotigotine. Of the 13 known human SULTs examined, 6 of them (SULT1A1, 1A2, 1A3, 1B1, 1C4, 1E1) displayed significant sulfating activities toward rotigotine. pH dependence and kinetic parameters of the sulfation of rotigotine by relevant human SULTs were determined. Of the 6 human organ samples tested, small intestine and liver cytosols displayed considerably higher rotigotine-sulfating activity than did brain, lung, and kidney. Moreover, sulfation of rotigotine was shown to occur in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under metabolic conditions. Collectively, the results obtained provided a molecular basis underlying the previous finding of the excretion of sulfated rotigotine by patients undergoing treatment with rotigotine.

The aim of the present study was to identify human cytosolic sulfotransferases (SULTs) that are capable of sulfating benzyl alcohol and to examine whether benzyl alcohol sulfation may occur in cultured human cells as well as in human organ homogenates. A systematic analysis revealed that of the 13 known human SULTs, SULT1A1 SULT1A2, SULTA3, and SULT1B1 are capable of mediating the sulfation of benzyl alcohol. The kinetic parameters of SULT1A1 that showed the strongest benzyl alcohol-sulfating activity were determined. HepG2 human hepatoma cells were used to demonstrate the generation and release of sulfated benzyl alcohol under the metabolic settings. Moreover, the cytosol or S9 fractions of human liver, lung, kidney and small intestine were examined to verify the presence of benzyl alcohol sulfating activity in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

Steroid sulfatase (STS) plays an important role in the regulation of steroid hormones. Metabolism of steroid hormones in zebrafish has been investigated, but the action of steroid sulfatase remains unknown. In this study, a zebrafish sts was cloned, expressed, purified, and characterized in comparison with the orthologous human enzyme. Enzymatic assays demonstrated that similar to human STS, zebrafish Sts was most active in catalyzing the hydrolysis of estrone-sulfate and estradiol-sulfate, among five steroid sulfates tested as substrates. Kinetic analyses revealed that the K(m) values of zebrafish Sts and human STS differed with respective substrates, but the catalytic efficiency as reflected by the V(max)/K(m) appeared comparable, except for DHEA-sulfate with which zebrafish Sts appeared less efficient. While zebrafish Sts was catalytically active at 28°C, the enzyme appeared more active at 37°C and with similar K(m) values to those determined at 28°C. Assays performed in the presence of different divalent cations showed that the activities of both zebrafish and human STSs were stimulated by Ca(2+), Mg(2+), and Mn(2+), and inhibited by Zn(+2) and Fe(2+). EMATE and STX64, two known mammalian steroid sulafatase inhibitors, were shown to be capable of inhibiting the activity of zebrafish Sts. Collectively, the results obtained indicated that zebrafish Sts exhibited enzymatic characteristics comparable to the human STS, suggesting that the physiological function of STS may be conserved between zebrafish and humans.

Members of the cytosolic sulfotransferase (SULT) SULT2A subfamily are known to be critically involved in the homeostasis of steroids and bile acids. SULT2A8, a 7α-hydroxyl bile acid-preferring mouse SULT, has been identified as the major enzyme responsible for the mouse-specific 7-O-sulfation of bile acids. Interestingly, SULT2A8 lacks a conservative catalytic His residue at position 99th. The catalytic mechanism underlying the SULT2A8-mediated 7-O-sulfation of bile acids thus remained unclear. In this study, we performed a mutational analysis in order to gain insight into this yet-unresolved issue. Results obtained revealed two amino acid residues, His48 and Leu99, that are unique to the mouse SULT2A8, but not other SULTs, are essential for its 7-O-sulfating activity toward bile acids. These findings suggested that substitutions of two amino acids, which might have occurred during the evolution of the mouse SULT2A8 gene, endowed mouse SULT2A8 the capacity to catalyze the 7-O-sulfation of bile acids.

The cytosolic sulfotransferase (SULT) SULT2A1 is known to mediate the sulfation of DHEA as well as some other hydroxysteroids such as pregnenolone. The present study was designed to investigate how genetic polymorphisms of the human SULT2A1 gene may affect the sulfation of DHEA and pregnenolone. Online databases were systematically searched to identify human SULT2A1 single nucleotide polymorphisms (SNPs). Of the 98 SULT2A1 non-synonymous coding SNPs identified, seven were selected for further investigation. Site-directed mutagenesis was used to generate cDNAs encoding these seven SULT2A1 allozymes, which were expressed in BL21 Escherichia coli cells and purified by glutathione-Sepharose affinity chromatography. Enzymatic assays revealed that purified SULT2A1 allozymes displayed differential sulfating activity toward both DHEA and pregnenolone. Kinetic analyses showed further differential catalytic efficiency and substrate affinity of the SULT2A1 allozymes, in comparison with wild-type SULT2A1. These findings provided useful information concerning the effects of genetic polymorphisms on the sulfating activity of SULT2A1 allozymes.

1-Naphthol (1-Nap) and 2-naphthol (2-Nap) are phenolic isomers that may be subjected to sulfate conjugation in vivo. Phase-II sulfate conjugation of phenolic compounds is generally thought to result in their inactivation. This study aimed to investigate the antioxidative effects of 1-NapS and 2-NapS, in comparison with their unsulfated counterparts, using established free radical scavenging assays. Based on the calculated EC50 values, 1-NapS resulted in 5.60 to 7.35-times lower antioxidative activity than 1-Nap. In contrast, 2-NapS showed comparable activities as did the unsulfated 2-Nap. Collectively, the results obtained indicated that sulfate conjugation of the Nap isomers did not always result in the decrease of their antioxidant activity, and the antioxidant activity that remained appeared to depend on the position of sulfation.

Previous studies have demonstrated the presence of the sulfated form of 6-gingerol, a major pharmacologically active component of ginger, in plasma samples of normal human subjects who were administered 6-gingerol. The current study was designed to systematically identify the major human cytosolic sulfotransferase enzyme(s) capable of mediating the sulfation of 6-gingerol. Of the 13 known human cytosolic sulfotransferases examined, six (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C4, SULT1E1) displayed significant sulfating activity toward 6-gingerol. Kinetic parameters of SULT1A1, SULT1A3, SULT1C4, and SULT1E1 that showed stronger 6-gingerol-sulfating activity were determined. Of the four human organ samples tested, small intestine and liver cytosols displayed considerably higher 6-gingerol-sulfating activity than those of the lung and kidney. Moreover, sulfation of 6-gingerol was shown to occur in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under the metabolic setting. Collectively, these results provided useful information relevant to the metabolism of 6-gingerol through sulfation both in vitro and in vivo.

Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity.

Ethnopharmacological relevance In Turkey, daphnetin-containing Daphne oleoides is used as a folk medicine for treating rheumatic pain and lumbago. A daphnetin-containing traditional Chinese medicine tablet, named Zushima-Pian, is available in China for treating rheumatoid arthritis. The present study aimed to investigate the metabolism of daphnetin through sulfation in cultured human cells and to identify the human cytosolic sulfotransferase(s) (SULT(s)) that is(are) capable of mediating the sulfation of daphnetin. Materials and methods Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [35S]sulfate in the presence of different concentrations of daphnetin. Thirteen known human SULTs, previously expressed and purified, as well as cytosols of human kidney, liver, lung, and small intestine, were examined for daphnetin-sulfating activity using an established sulfotransferase assay. Results [35S]sulfated daphnetin was found to be generated and released by HepG2 cells and Caco-2 cells labeled with [35S] sulfate in the presence of daphnetin. Among the 13 known human SULTs, SULT1A1, SULT1A2, SULT1A3, SULT1B1, and SULT1C4 displayed significant sulfating activity toward daphnetin. Of the four human organ samples later tested, small intestine and liver cytosols displayed considerably higher daphnetin-sulfating activity than those of lung and kidney. Conclusion The results derived from the present study showed unequivocally that daphnetin could be sulfated in cultured human cells and by purified human SULT enzymes as well as human organ cytosols. The information obtained provided a basis for further studies on the metabolism of daphnetin through sulfation in vivo.

The cytosolic sulphotransferase SULT1C3 remained the most poorly understood human SULT. The SULT1C3 gene has been shown to contain alternative exons 7 and 8, raising the question concerning their evolutionary origin and implying the generation of multiple SULT1C3 variants. Two SULT1C3 splice variants, SULT1C3a and SULT1C3d, were investigated to verify the impact of alternative C-terminal sequences on their sulphating activity. Sequence homology and gene location analyses were performed to verify the orthology of the SULT1C3 gene. The SULT1C3 gene appears to be present only in humans and other primates, but alternative exons 7b and 8b share high degrees of homology with corresponding regions of rodent SULT1C1 genes, implying their evolutionary origin being from a defunct human SULT1C1 gene. Purified recombinant SULT1C3a and SULT1C3d were analyzed for sulphating activities toward a variety of endogenous and xenobiotic compounds. While SULT1C3a displayed weaker activities and strict substrate specificity toward hydroxyl-chlorinated biphenyls, SULT1C3d exhibited broader substrate specificity toward bile acids and thyroid hormones as well as hydroxyl-chlorinated biphenyls. Molecular docking simulation suggested that Tyr249 and Met257 may play an important role in substrate recognition by SULT1C3d. Alternative splicing of exons 7 and 8 sequences resulted in differential catalytic properties of SULT1C3 variants.

While 25-hydroxyvitamin D

Cytosolic sulfotransferase (SULT)-mediated sulfation is generally known to involve the transfer of a sulfonate group from the active sulfate, 3’-phosphoadenosine 5’-phosphosulfate (PAPS), to a hydroxyl group or an amino group of a substrate compound. We report here that human SULT2A1, in addition to being able to sulfate dehydroepiandrosterone (DHEA) and other hydroxysteroids, could also catalyze the sulfation of Δ4-3-ketosteroids, which carry no hydroxyl groups in their chemical structure. Among a panel of Δ4-3-ketosteroids tested as substrates, 4-androstene-3,17-dione and progesterone were found to be sulfated by SULT2A1. Mass spectrometry analysis and structural modeling supported a reaction mechanism which involves the isomerization of Δ4-3-ketosteroids from the keto form to an enol form, prior to being subjected to sulfation. Results derived from this study suggested a potential role of SULT2A1 as a Δ4-3-ketosteroid sulfotransferase in steroid metabolism.

The current study was designed to examine the sulfation of eight opioid drugs, morphine, hydromorphone, oxymorphone, butorphanol, nalbuphine, levorphanol, nalorphine, and naltrexone, in HepG2 human hepatoma cells and human organ samples (lung, liver, kidney, and small intestine) and to identify the human SULT(s) responsible for their sulfation. Analysis of the spent media of HepG2 cells, metabolically labeled with [35S]sulfate in the presence of each of the eight opioid drugs, showed the generation and release of corresponding [35S]sulfated derivatives. Five of the eight opioid drugs, hydromorphone, oxymorphone, butorphanol, nalorphine, and naltrexone, appeared to be more strongly sulfated in HepG2 cells than were the other three, morphine, nalbuphine, and levorphanol. Differential sulfating activities toward the opioid drugs were detected in cytosol or S9 fractions of human lung, liver, small intestine, and kidney, with the highest activities being found for the liver sample. A systematic analysis using eleven known human SULTs and kinetic experiment revealed SULT1A1 as the major responsible SULTs for the sulfation of oxymorphone, nalbuphine, nalorphine, and naltrexone, SULT1A3 for the sulfation of morphine and hydromorphone, and SULT2A1 for the sulfation of butorphanol and levorphanol. Collectively, the results obtained imply that sulfation may play a significant role in the metabolism of the tested opioid drugs in vivo.

Pregnenolone and dehydroepiandrosterone (DHEA) are hydroxysteroids that serve as biosynthetic precursors for steroid hormones in human body. SULT2B1b has been reported to be critically involved in the sulfation of pregnenolone and DHEA, particularly in the sex steroid-responsive tissues. The current study was designed to investigate the impact of the genetic polymorphisms of SULT2B1 on the sulfation of DHEA and pregnenolone by SULT2B1b allozymes. Ten SULT2B1b allozymes previously prepared were shown to exhibit differential sulfating activities toward DHEA and pregnenolone in comparison to the wild-type enzyme. Kinetic studies revealed further significant changes in their substrate-binding affinity and catalytic activity toward DHEA and pregnenolone. Taken together, these results indicated clearly a profound effect of SULT2B1 genetic polymorphisms on the sulfating activity of SULT2B1b allozymes toward DHEA and pregnenolone, which may have implications in inter-individual variations in the homeostasis of these two important steroid precursors.

In plants, flavonoids have been shown to be subjected to conjugation modifications such as glycosylation, methylation, and sulfation. Among these modifications, sulfation is known as an important pathway in the regulation of the levels of endogenous compounds such as steroids. Although a large variety of flavonoid sulfates also exist in plants, the detailed biochemical characterization of Arabidopsis thaliana sulfotransferases (AtSULTs) remains to be fully clarified. We report here that uncharacterized AtSULT202E1 (AGI code: At2g03770), a SULT202E subfamily member, shows the sulfating activity toward flavonoids. The general characteristics of the enzyme were studied on the optimum temperature and pH, the effect of divalent cations, and the thermal stability with kaempferol as substrate. A comparative analysis of the sulfation of flavonoids by AtSULT202E1, AtSULT202B1 and AtSULT202A1 revealed that three AtSULTs have differential substrate specificities. Surprisingly, 3-hydroxyflavone was sulfated only by AtSULT202A1 while 7-hydroxyflavone was highly sulfated by AtSULT202E1 and AtSULT202B1. These results indicate that flavonols might be sulfated in a position specific manner. In conclusion, our studies indicate that a novel AtSULT202E1 has the sulfating activity toward flavonoids together with AtSULT202B1 and AtSULT202A1. The existence of three flavonoid sulfotransferases in A. thaliana suggests that sulfation of flavonoids have an important role in regulation of their functions.