The role of genetic polymorphisms in the sulfation of pregnenolone by human cytosolic sulfotransferase SULT2B1a.

Pregnenolone is a key intermediate in the biosynthesis of many steroid hormones and neuroprotective steroids. Sulfotransferase family cytosolic 2B member 1 (SULT2B1a) has been reported to be highly selective to sulfate pregnenolone. This study aimed to clarify the effect of missense single nucleotide polymorphisms (SNPs) of the human SULT2B1 gene on the sulfating activity of coded SULT2B1a allozymes toward Pregnenolone. To investigate the effects of single nucleotide polymorphisms of the SULT2B1 gene on the sulfation of pregnenolone by SULT2B1a allozymes, 13 recombinant SULT2B1a allozymes were generated, expressed, and purified using established procedures. Human SULT2B1a SNPs were identified by a comprehensive database search. 13 SULT2B1a nonsynonymous missense coding SNPs (cSNPs) were selected, and site-directed mutagenesis was used to generate the corresponding cDNAs, packaged in pGEX-2TK expression vector, encoding these 13 SULT2B1a allozymes, which were bacterially expressed in BL21 E. coli cells and purified by glutathione-Sepharose affinity chromatography. Purified SULT2B1a allozymes were analyzed for sulfating activities towards pregnenolone. In comparison with the wild-type SULT2B1a, of the 13 allozymes, 11 showed reduced activity toward pregnenolone at 0.1 µM. Specifically, P134L and R259Q allozymes, reported to be involved in autosomal-recessive congenital ichthyosis, displayed low activity (1-10%) toward pregnenolone. The findings of this study may demonstrate the impact of genetic polymorphism on the sulfation of pregnenolone in individuals with different SULT2B1 genotypes.

SULT genetic polymorphisms: physiological, pharmacological and clinical implications.

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.

Effects of genetic polymorphisms on the sulfation of doxorubicin by human SULT1C4 allozymes.

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.

Impact of Human SULT1E1 Polymorphisms on the Sulfation of 17ß-Estradiol, 4-Hydroxytamoxifen, and Diethylstilbestrol by SULT1E1 Allozymes.

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 17ß-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 of the SULT1E1 gene were selected to investigate 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 Km (reflecting substrate affinity) and Vmax (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 the 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.

Functional analysis of novel sulfotransferases in the silkworm Bombyx mori.

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.

Effect of SULT2B1 genetic polymorphisms on the sulfation of dehydroepiandrosterone and pregnenolone by SULT2B1b allozymes.

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.

Impact of SULT1A3/SULT1A4 genetic polymorphisms on the sulfation of phenylephrine and salbutamol by human SULT1A3 allozymes.

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.

Effects of the human SULT1A1 polymorphisms on the sulfation of acetaminophen,O-desmethylnaproxen, and tapentadol.

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.

Effects of human SULT1A3/SULT1A4 genetic polymorphisms on the sulfation of acetaminophen and opioid drugs by the cytosolic sulfotransferase SULT1A3.

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.

Effects of genetic polymorphisms on the sulfation of dehydroepiandrosterone and pregnenolone by human cytosolic sulfotransferase SULT2A1.

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.

On the role of genetic polymorphisms in the sulfation of cholesterol by human cytosolic sulphotransferase SULT2B1b.

Sulphated cholesterol, like its unsulphated counterpart, is known to be biologically active and serves a myriad of biochemical/physiological functions. Of the 13 human cytosolic sulphotransferases (SULTs), SULT2B1b has been reported as the main enzyme responsible for the sulphation of cholesterol. As such, SULT2B1b may play the role as a key regulator of cholesterol metabolism. Variations in the sulphating activity of SULT2B1b may affect the sulphation of cholesterol and, consequently, the related physiological events. This study was designed to evaluate the impact of the genetic polymorphisms on the sulphation of cholesterol by SULT2B1b. Ten recombinant SULT2B1b allozymes were generated, expressed, and purified. Purified SULT2B1b allozymes were shown to display differential cholesterol-sulphating activities, compared with the wild-type enzyme. Kinetic studies revealed further their distinct substrate affinity and catalytic efficiency toward cholesterol. These findings showed clearly the impact of genetic polymorphisms on the cholesterol-sulphating activity of SULT2B1b allozymes, which may underscore the differential metabolism of cholesterol in individuals with different SULT2B1b genotypes.

Sulfation of catecholamines and serotonin by SULT1A3 allozymes.

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.

On the sulfation of O-desmethyltramadol by human cytosolic sulfotransferases.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

On the Molecular Basis Underlying the Metabolism of Tapentadol Through Sulfation.

BACKGROUND AND OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSION: Taken together, the results derived from the current study provided a molecular basis underlying the sulfation of tapentadol in humans.