RESUMO
Indoxyl sulfate (IS), a uremic toxin, is a physiologically active sulfated metabolite, specifically in kidney failure patients. Our previous studies have shown that IS downregulates phagocytic immune function in a differentiated HL-60 human macrophage cell model. However, it remains unclear whether IS exerts similar effects on macrophage function in other cell types or in lipopolysaccharide (LPS)-sensitive immune cell models. Therefore, this study aimed to investigate the effects of IS on intracellular oxidation levels and phagocytic activity in a differentiated U937 human macrophage cell model, both in the absence and presence of LPS. Our results demonstrated that IS significantly increases intracellular oxidation levels and decreases phagocytic activity, particularly in cells activated by LPS. Furthermore, we found that 2-acetylphenothiazine, an NADH oxidase inhibitor, attenuates the effects of IS in LPS-activated macrophage cells. Representative antioxidants, trolox, α-tocopherol, and ascorbic acid, significantly mitigated the effects of IS on the macrophages responding to LPS.
RESUMO
Cytosolic sulfotransferases (SULTs) are cytosolic enzymes that catalyze the transfer of sulfonate group to key endogenous compounds, altering the physiological functions of their substrates. SULT enzymes catalyze the O-sulfonation of hydroxy groups or N-sulfonation of amino groups of substrate compounds. In this study, we report the discovery of C-sulfonation of α,ß-unsaturated carbonyl groups mediated by a new SULT enzyme, SULT7A1, and human SULT1C4. Enzymatic assays revealed that SULT7A1 is capable of transferring the sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate to the α-carbon of α,ß-unsaturated carbonyl-containing compounds, including cyclopentenone prostaglandins as representative endogenous substrates. Structural analyses of SULT7A1 suggest that the C-sulfonation reaction is catalyzed by a novel mechanism mediated by His and Cys residues in the active site. Ligand-activity assays demonstrated that sulfonated 15-deoxy prostaglandin J2 exhibits antagonist activity against the prostaglandin receptor EP2 and the prostacyclin receptor IP. Modification of α,ß-unsaturated carbonyl groups via the new prostaglandin-sulfonating enzyme, SULT7A1, may regulate the physiological function of prostaglandins in the gut. Discovery of C-sulfonation of α,ß-unsaturated carbonyl groups will broaden the spectrum of potential substrates and physiological functions of SULTs.
RESUMO
Organisms have conversion systems for sulfate ion to take advantage of the chemical features. The use of biologically converted sulfonucleotides varies in an evolutionary manner, with the universal use being that of sulfonate donors. Sulfotransferases have the ability to transfer the sulfonate group of 3'-phosphoadenosine 5'-phosphosulfate to a variety of molecules. Cytosolic sulfotransferases (SULTs) play a role in the metabolism of low-molecular-weight compounds in response to the host organism's living environment. This review will address the diverse functions of the SULT in evolution, including recent findings. In addition to the diversity of vertebrate sulfotransferases, the molecular aspects and recent studies on bacterial and plant sulfotransferases are also addressed.
Assuntos
Fosfoadenosina Fosfossulfato , Sulfotransferases , Sulfotransferases/química , Citosol/metabolismo , Fosfoadenosina Fosfossulfato/metabolismo , Sulfatos/metabolismoRESUMO
Acetaminophen (APAP) and p-aminophenol (p-AP) are the analogous simple phenolic compounds that undergo sulfate conjugation (sulfation) by cytosolic sulfotransferases. Sulfation is generally thought to lead to the inactivation and disposal of endogenous as well as xenobiotic compounds. This study aimed to investigate the antioxidative effects of O-sulfated form of APAP and p-AP, i.e., APAPS and p-APS, in comparison with their unsulfated counterparts. Using a 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay, the antioxidant capacity of APAPS was shown to be approximately 126-times lower than that of APAP. In contrast, p-APS displayed comparable activity as unsulfated p-AP. Similar trends concerning the suppressive effects of these chemicals on cellular O2- radical generation were found using an activated granulocytic neutrophil cell model. Collectively, these results indicated that, depending on the presence of an additional "active site", sulfation may not always decrease the antioxidant activities of phenolic compounds.
Assuntos
Acetaminofen , Sulfatos , Aminofenóis , Antioxidantes/farmacologia , Fenóis , Sulfotransferases , XenobióticosRESUMO
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.
Assuntos
Exantema , Infecções por HIV , Sulfotransferases/metabolismo , Animais , Arilsulfotransferase/genética , Arilsulfotransferase/metabolismo , Citosol/metabolismo , Exantema/metabolismo , Infecções por HIV/metabolismo , Humanos , Isoenzimas/metabolismo , Nevirapina/metabolismo , Polimorfismo Genético , Ratos , Sulfatos/metabolismo , Sulfotransferases/genéticaRESUMO
Polyphenols in plants are important for defense responses against microorganisms, insect herbivory, and control of feeding. Owing to their antioxidant, anti-cancer, and anti-inflammatory activities, their importance in human nutrition has been acknowledged. However, metabolism of polyphenols derived from mulberry leaves in silkworms (Bombyx mori) remains unclear. Sulfotransferases (SULT) are involved in the metabolism of xenobiotics and endogenous compounds. The purpose of this study is to investigate the metabolic mechanism of polyphenols mediated by B. mori SULT. Here, we identified a novel SULT in silkworms (herein, swSULT ST3). Recombinant swSULT ST3 overexpressed in Escherichia coli effectively sulfated polyphenols present in mulberry leaves. swSULT ST3 showed high specific activity toward genistein among the polyphenols. Genistein-7-sulfate was produced by the activity of swSULT ST3. Higher expression of swSULT ST3 mRNA was observed in the midgut and fat body than in the hemocytes, testis, ovary, and silk gland. Polyphenols inhibited the aldo-keto reductase detoxification of reactive aldehydes from mulberry leaves, and the most noticeable inhibition was observed with genistein. Our results suggest that swSULT ST3 plays a role in the detoxification of polyphenols, including genistein, and contributes to the effects of aldo-keto reductase in the midgut of silkworms. This study provides new insight into the functions of SULTs and the molecular mechanism responsible for host plant selection in lepidopteran insects.
Assuntos
Bombyx , Morus , Aldo-Ceto Redutases/metabolismo , Animais , Bombyx/genética , Feminino , Genisteína/metabolismo , Insetos , Larva/genética , Masculino , Morus/metabolismo , Polifenóis/metabolismo , Polifenóis/farmacologia , Sulfotransferases/metabolismoRESUMO
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.
Assuntos
Callithrix , Animais , Clonagem Molecular , Humanos , CinéticaRESUMO
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.
Assuntos
Preparações Farmacêuticas/metabolismo , Sulfotransferases/genética , Animais , Citosol/metabolismo , Regulação Enzimológica da Expressão Gênica , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , Sulfatos/metabolismo , Sulfotransferases/metabolismoRESUMO
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.
Assuntos
Doxorrubicina/metabolismo , Polimorfismo de Nucleotídeo Único , Sulfotransferases/genética , Sulfotransferases/metabolismo , Citosol/metabolismo , Genótipo , Humanos , Isoenzimas/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Nitrofenóis/metabolismo , Sulfatos/metabolismoRESUMO
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.
Assuntos
Ácidos e Sais Biliares/metabolismo , Sulfatos/metabolismo , Sulfotransferases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Humanos , Cinética , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Especificidade por Substrato , Sulfotransferases/metabolismoRESUMO
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.
Assuntos
Dietilestilbestrol/metabolismo , Estradiol/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Sulfotransferases/genética , Sulfotransferases/metabolismo , Tamoxifeno/análogos & derivados , Dietilestilbestrol/farmacologia , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Antagonistas de Estrogênios/metabolismo , Antagonistas de Estrogênios/farmacologia , Estrogênios/metabolismo , Estrogênios/farmacologia , Estrogênios não Esteroides/metabolismo , Estrogênios não Esteroides/farmacologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Estrutura Secundária de Proteína , Sulfotransferases/química , Tamoxifeno/metabolismo , Tamoxifeno/farmacologiaRESUMO
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.
Assuntos
Indóis/efeitos adversos , Macrófagos/imunologia , Macrófagos/metabolismo , Oxirredução/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células HL-60 , Humanos , Indicã/metabolismo , Macrófagos/efeitos dos fármacos , Ligação Proteica , Albumina Sérica/metabolismo , Triptofano/metabolismoRESUMO
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.
Assuntos
Bombyx/enzimologia , Inativação Metabólica , Sulfotransferases/química , Sequência de Aminoácidos , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Feminino , Gametogênese , Trato Gastrointestinal/enzimologia , Proteínas de Insetos , Larva/enzimologia , Masculino , Ovário , Pentaclorofenol/metabolismo , Filogenia , Sulfotransferases/metabolismo , Testículo , Xanturenatos/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Indicã/farmacologia , Espaço Intracelular/metabolismo , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Toxinas Biológicas/farmacologia , Antioxidantes/farmacologia , Cromanos/farmacologia , Células HL-60 , Humanos , Macrófagos/metabolismo , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Fagocitose/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Desidroepiandrosterona/química , Polimorfismo de Nucleotídeo Único , Pregnenolona/química , Sulfotransferases/química , Humanos , Isoenzimas , Sulfotransferases/genéticaRESUMO
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.
Assuntos
Albuterol/metabolismo , Arilsulfotransferase/genética , Fenilefrina/metabolismo , Sulfotransferases/genética , Albuterol/uso terapêutico , Arilsulfotransferase/química , Arilsulfotransferase/metabolismo , Asma/tratamento farmacológico , Asma/genética , Genótipo , Humanos , Hipotensão/tratamento farmacológico , Hipotensão/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Fenilefrina/uso terapêutico , Polimorfismo de Nucleotídeo Único/genética , Sulfatos/metabolismo , Sulfotransferases/química , Sulfotransferases/metabolismoRESUMO
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.
Assuntos
Acetaminofen/metabolismo , Arilsulfotransferase/genética , Naproxeno/análogos & derivados , Tapentadol/metabolismo , Analgésicos não Narcóticos/metabolismo , Analgésicos Opioides/metabolismo , Citosol/metabolismo , Escherichia coli/citologia , Genótipo , Humanos , Isoenzimas , Mutagênese Sítio-Dirigida , Naproxeno/metabolismo , Polimorfismo de Nucleotídeo Único , Sulfatos/metabolismoRESUMO
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 Km values of zebrafish Sts and human STS differed with respective substrates, but the catalytic efficiency as reflected by the Vmax/Km 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 Km 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 Ca2+, Mg2+, and Mn2+, and inhibited by Zn+2 and Fe2+. 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.
Assuntos
Sulfato de Desidroepiandrosterona/metabolismo , Estradiol/análogos & derivados , Estrona/análogos & derivados , Esteril-Sulfatase/genética , Esteril-Sulfatase/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Cátions/metabolismo , Clonagem Molecular/métodos , Inibidores Enzimáticos/farmacologia , Estradiol/metabolismo , Estrona/metabolismo , Humanos , Esteril-Sulfatase/antagonistas & inibidores , Peixe-ZebraRESUMO
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.
Assuntos
Ácidos e Sais Biliares/metabolismo , Citosol/enzimologia , Histidina/metabolismo , Sulfatos/metabolismo , Sulfotransferases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Catálise , Domínio Catalítico , Clonagem Molecular , Histidina/genética , Humanos , Camundongos , Mutação , Filogenia , Homologia de Sequência de Aminoácidos , Sulfotransferases/química , Sulfotransferases/genéticaRESUMO
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.