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Analytical and Bioanalytical Chemistry Jan 2014O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently,...
O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [(35)S] 3'-phosphoadenosine-5'-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.
Topics: Biological Assay; Carbohydrate Sequence; Chromatography, Liquid; Colorimetry; Heparin; Heparitin Sulfate; Kinetics; Mass Spectrometry; Molecular Sequence Data; Nitrobenzenes; Substrate Specificity; Sulfotransferases
PubMed: 24271188
DOI: 10.1007/s00216-013-7470-4 -
Neurotoxicology Jan 2013Sulfotransferase catalyzed sulfation regulates the biological activities of various neurotransmitters/hormones and detoxifies xenobiotics. Rat sulfotransferase rSULT1A1...
Sulfotransferase catalyzed sulfation regulates the biological activities of various neurotransmitters/hormones and detoxifies xenobiotics. Rat sulfotransferase rSULT1A1 catalyzes the sulfation of neurotransmitters and xenobiotic phenolic compounds. rSULT2A1 catalyzes the sulfation of hydroxysteroids and xenobiotic alcoholic compounds. In this work, Western blot and real-time RT-PCR were used to investigate the effect of methamphetamine on rSULT1A1 and rSULT2A1 protein and mRNA expression in rat cerebellum, frontal cortex, hippocampus, and striatum. After 1-day treatment, significant induction of rSULT1A1 was observed only in the cerebellum; rSULT2A1 was induced significantly in the cerebellum, frontal cortex, and hippocampus. After 7 days of exposure, rSULT1A1 was induced in the cerebellum, frontal cortex, and hippocampus, while rSULT2A1 was induced significantly in all four regions. Western blot results agreed with the real-time RT-PCR results, suggesting that the induction occurred at the gene transcriptional level. Results indicate that rSULT1A1 and rSULT2A1 are expressed in rat frontal cortex, cerebellum, striatum, and hippocampus. rSULT1A1 and rSULT2A1are inducible by methamphetamine in rat brain sections in a time dependable manner. rSULT2A1 is more inducible than rSULT1A1 by methamphetamine in rat brain sections. Induction activity of methamphetamine is in the order of cerebellum>frontal cortex, hippocampus>striatum. These results suggest that the physiological functions of rSULT1A1 and rSULT2A1 in different brain regions can be affected by methamphetamine.
Topics: Animals; Arylsulfotransferase; Blotting, Western; Brain; Central Nervous System Stimulants; Dose-Response Relationship, Drug; Enzyme Induction; Male; Methamphetamine; RNA, Messenger; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sulfotransferases; Time Factors; Transcription, Genetic
PubMed: 23026138
DOI: 10.1016/j.neuro.2012.09.010 -
Biochemical Pharmacology Nov 2019Drug metabolizing enzymes mediate biotransformation of drugs and play an essential role in drug efficacy and toxicity. Human sulfotransferases are a superfamily of Phase...
Drug metabolizing enzymes mediate biotransformation of drugs and play an essential role in drug efficacy and toxicity. Human sulfotransferases are a superfamily of Phase II detoxification enzymes that metabolize a wide spectrum of endogenous compounds and xenobiotics. SULT2A1 is one of the most abundant hepatic sulfotransferases and it catalyzes the sulfate conjugation of many endogenous substrates, such as bile acids and steroids. In the current study, we utilized a systematic approach by combining a series of computational analyses and in vitro methods to identify miRNAs that repress SULT2A1 expression post-transcriptionally. Our in silico analyses predicted miRNA response elements for hsa-miR-495-3p and hsa-miR-486-5p within the 3'-UTR of SULT2A1 mRNA and the levels of these miRNAs were inversely correlated with that of SULT2A1 mRNA in human liver. Using fluorescence-based RNA electrophoretic mobility shift assays, we found that hsa-miR-495-3p and hsa-miR-486-5p interacted directly with the SULT2A1 3'-UTR. The activity of a luciferase reporter gene construct containing sequences from the SULT2A1 3-UTR was suppressed by hsa-miR-486-5p and hsa-miR-495-3p. Furthermore, gain- and loss-of-function assays demonstrated that hsa-miR-486-5p and hsa-miR-495-3p negatively modulate basal and rifampicin-induced expression of SULT2A1 in HepG2 cells by decreasing mRNA stability.
Topics: Gene Expression Regulation, Enzymologic; Hep G2 Cells; Humans; MicroRNAs; RNA Stability; Rifampin; Sulfotransferases
PubMed: 31445882
DOI: 10.1016/j.bcp.2019.08.019 -
Cells Jun 2022Understanding changes in the expression of genes involved in regulating various components of the neural extracellular matrix (ECM) during aging can provide an insight...
Understanding changes in the expression of genes involved in regulating various components of the neural extracellular matrix (ECM) during aging can provide an insight into aging-associated decline in synaptic and cognitive functions. Hence, in this study, we compared the expression levels of ECM-related genes in the hippocampus of young, aged and very aged mice. ECM gene expression was downregulated, despite the accumulation of ECM proteoglycans during aging. The most robustly downregulated gene was carbohydrate sulfotransferase 3 (), the enzyme responsible for the chondroitin 6-sulfation (C6S) of proteoglycans. Further analysis of epigenetic mechanisms revealed a decrease in H3K4me3, three methyl groups at the lysine 4 on the histone H3 proteins, associated with the promoter region of the gene, resulting in the downregulation of expression in non-neuronal cells. Cluster analysis revealed that the expression of lecticans-substrates of CHST3-is tightly co-regulated with this enzyme. These changes in ECM-related genes were accompanied by an age-confounded decline in cognitive performance. Despite the co-directional impairment in cognitive function and average expression in the studied age groups, at the individual level we found a negative correlation between mRNA levels of and cognitive performance within the very aged group. An analysis of correlations between the expression of ECM-related genes and cognitive performance in novel object versus novel location recognition tasks revealed an apparent trade-off in the positive gene effects in one task at the expense of another. Further analysis revealed that, despite the reduction in the mRNA, the expression of CHST3 protein is increased in glial cells but not in neurons, which, however, does not lead to changes in the absolute level of C6S and even results in the decrease in C6S in perineuronal, perisynaptic and periaxonal ECM relative to the elevated expression of its protein carrier versican.
Topics: Aging; Animals; Cognition; Epigenesis, Genetic; Mice; Proteoglycans; RNA, Messenger; Sulfotransferases; Carbohydrate Sulfotransferases
PubMed: 35805117
DOI: 10.3390/cells11132033 -
Molecular & Cellular Proteomics : MCP 2021Despite sulfated O-linked glycans being abundant on ovarian cancer (OC) glycoproteins, their regulation during cancer development and involvement in cancer pathogenesis...
Despite sulfated O-linked glycans being abundant on ovarian cancer (OC) glycoproteins, their regulation during cancer development and involvement in cancer pathogenesis remain unexplored. We characterized O-glycans carrying sulfation on galactose residues and compared their expression with defined sulfotransferases regulated during OC development. Desialylated sulfated oligosaccharides were released from acidic glycoproteins in the cyst fluid from one patient with a benign serous cyst and one patient with serous OC. Oligosaccharides characterized by LC-MS were identified as core 1 and core 2 O-glycans up to the size of decamers and with 1 to 4 sulfates linked to GlcNAc residues and to C-3 and/or C-6 of Gal. To study the specificity of the potential ovarian sulfotransferases involved, Gal3ST2 (Gal-3S)-, Gal3ST4 (Gal-3S)-, and CHST1 (Gal-6S)-encoding expression plasmids were transfected individually into CHO cells also expressing the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIg G2b) fusion protein and the human core 2 transferase (GCNT1). Characterization of the PSGL-1/mIg G2b O-glycans showed that Gal3ST2 preferentially sulfated Gal on the C-6 branch of core 2 structures and Gal3ST4 preferred Gal on the C-3 branch independently if core-1 or -2. CHST1 sulfated Gal residues on both the C-3 (core 1/2) and C-6 branches of core 2 structures. Using serous ovarian tissue micro array, Gal3ST2 was found to be decreased in tissue classified as malignant compared with tissues classified as benign or borderline, with the lowest expression in poorly differentiated malignant tissue. Neither Gal3ST4 nor CHST1 was differentially expressed in benign, borderline, or malignant tissue, and there was no correlation between expression level and differentiation stage. The data displays a complex sulfation pattern of O-glycans on OC glycoproteins and that aggressiveness of the cancer is associated with a decreased expression of the Gal3ST2 transferase.
Topics: Adenoma; Animals; CHO Cells; Cricetulus; Cystadenocarcinoma, Serous; Female; Humans; Mucins; Ovarian Neoplasms; Polysaccharides; Sulfates; Sulfotransferases
PubMed: 34555499
DOI: 10.1016/j.mcpro.2021.100150 -
Current Drug Metabolism Oct 2006We hypothesized that aryl acetate- and aryl carboxylate-containing drugs would inhibit human phenol sulfotransferase (SULT1A1), and that selectivity would depend upon...
We hypothesized that aryl acetate- and aryl carboxylate-containing drugs would inhibit human phenol sulfotransferase (SULT1A1), and that selectivity would depend upon the interaction of the aryl portion of the molecule with the sulfotransferase acceptor binding site. This hypothesis was based on results with the rat orthologue showing that oxidation of phenolic substrates to carboxylate derivatives resulted in competitive inhibition of rat phenol sulfotransferase. We chose nine structurally representative non-steroidal anti-inflammatory agents and determined their inhibitory potency and selectivity toward SULT1A1 and expressed human estrogen sulfotransferase (SULT1E1). The results show that the tested agents reversibly inhibit SULT1A1 activity with IC(50) ranging from 0.1 microM to 3800 microM. These agents also inhibited SULT1E1 (IC(50) = 6 microM to 9000 microM). The agents were clearly isoform selective, with IC(50) ratios (1E1/1A1) ranging from 0.01 to 200. Nimesulide, meclofenamate, and piroxicam were more selective towards SULT1A1 inhibition, while sulindac and ibuprofen were more selective towards SULT1E1 inhibition. Sulfotransferase inhibition was maintained after substituting the carboxylate with enolate (nimesulide) or methylsulfonamide (piroxicam). Kinetic studies determined the type of inhibition of SULT1A1 for three agents (meclofenamate, nimesulide, aspirin) to be non-competitive or partial non-competitive versus both substrate (p-nitrophenol) and cofactor (PAPS). This inhibition mechanism indicates that meclofenamate, nimesulide and aspirin bind near enough to the substrate binding site to prevent catalysis but not affect dissociation of the substrate-enzyme complex. The inhibition of SULT1A1 by meclofenamate, nimesulide, salicylate and aspirin may be clinically relevant based on ratio of inhibition constant to predicted in vivo inhibitor concentration ([I]/IC(50) > 1).
Topics: Anti-Inflammatory Agents, Non-Steroidal; Arylsulfotransferase; Carboxylic Acids; Cytosol; Enzyme Inhibitors; Humans; Liver; Sulfotransferases
PubMed: 17073578
DOI: 10.2174/138920006778520615 -
Glycobiology Jul 2018The HS3ST3A1/B1 genes encode two homologous 3-O-sulfotransferases involved in the late modification step during heparan sulfate (HS) biosynthesis. In addition to the...
The HS3ST3A1/B1 genes encode two homologous 3-O-sulfotransferases involved in the late modification step during heparan sulfate (HS) biosynthesis. In addition to the single nucleotide polymorphisms (SNPs) rs28470223 (C > T) in the promoter region of both HS3ST3A1 and rs62636623 (Gly/Arg) in the stem region of HS3ST3B1, three missense mutations (rs62056073, rs61729712 and rs9906590) located within the catalytic sulfotransferase domain of 3-OST-B1 are linked and associated to Plasmodium falciparum parasitaemia. To ascertain the functional effects of these SNP associations, we investigated the regulatory effect of rs28470223 and characterized the enzymatic activity of the missense SNP rs61729712 (Ser279Asn) localized at proximity of the substrate binding cleft. The SNP rs28470223 results in decreased promoter activity of HS3ST3A1 in K562 cells, suggesting a reduced in vivo transcription activity of the target gene. A comparative kinetic analysis of wt HS3ST3B1 and the Ser269Asn variant (rs61729712) using a HS-derived oligosaccharide substrate reveals a slightly higher catalytic activity for the SNP variant. These genetic and enzymatic studies suggest that genetic variations in enzymes responsible of HS 3-O-sulfation can modulate their promoter and enzymatic activities and may influence P. falciparum parasitaemia.
Topics: Binding Sites; Cell Line, Tumor; Heparitin Sulfate; Humans; Mutation, Missense; Parasitemia; Plasmodium falciparum; Polymorphism, Single Nucleotide; Protein Binding; Sulfotransferases
PubMed: 29718295
DOI: 10.1093/glycob/cwy038 -
The Journal of Biological Chemistry May 2006HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human...
HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAbeta1-3Galbeta1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.
Topics: Animals; CHO Cells; Carbohydrates; Cricetinae; Gene Expression Regulation, Enzymologic; Glucuronosyltransferase; Humans; Protein Binding; Rats; Sulfotransferases
PubMed: 16543228
DOI: 10.1074/jbc.M601453200 -
MSphere Nov 2020Total biosynthesis or whole-cell biocatalytic production of sulfated small molecules relies on the discovery and implementation of appropriate sulfotransferase enzymes....
Total biosynthesis or whole-cell biocatalytic production of sulfated small molecules relies on the discovery and implementation of appropriate sulfotransferase enzymes. Although fungi are prominent biocatalysts and have been used to sulfate drug-like phenolics, no gene encoding a sulfotransferase enzyme has been functionally characterized from these organisms. Here, we identify a phenolic sulfotransferase, FgSULT1, by genome mining from the plant-pathogenic fungus PH-1. We expressed FgSULT1 in a chassis to modify a broad range of benzenediol lactones and their nonmacrocyclic congeners, together with an anthraquinone, with the resulting unnatural natural product (uNP) sulfates displaying increased solubility. FgSULT1 shares low similarity with known animal and plant sulfotransferases. Instead, it forms a sulfotransferase family with putative bacterial and fungal enzymes for phase II detoxification of xenobiotics and allelochemicals. Among fungi, putative FgSULT1 homologues are encoded in the genomes of spp. and a few other genera in nonsyntenic regions, some of which may be related to catabolic sulfur recycling. Computational structure modeling combined with site-directed mutagenesis revealed that FgSULT1 retains the key catalytic residues and the typical fold of characterized animal and plant sulfotransferases. Our work opens the way for the discovery of hitherto unknown fungal sulfotransferases and provides a synthetic biological and enzymatic platform that can be adapted to produce bioactive sulfates, together with sulfate ester standards and probes for masked mycotoxins, precarcinogenic toxins, and xenobiotics. Sulfation is an expedient strategy to increase the solubility, bioavailability, and bioactivity of nutraceuticals and clinically important drugs. However, chemical or biological synthesis of sulfoconjugates is challenging. Genome mining, heterologous expression, homology structural modeling, and site-directed mutagenesis identified FgSULT1 of PH-1 as a cytosolic sulfotransferase with the typical fold and active site architecture of characterized animal and plant sulfotransferases, despite low sequence similarity. FgSULT1 homologues are sparse in fungi but form a distinct clade with bacterial sulfotransferases. This study extends the functionally characterized sulfotransferase superfamily to the kingdom Fungi and demonstrates total biosynthetic and biocatalytic synthetic biological platforms to produce unnatural natural product (uNP) sulfoconjugates. Such uNP sulfates may be utilized for drug discovery in human and veterinary medicine and crop protection. Our synthetic biological methods may also be adapted to generate masked mycotoxin standards for food safety and environmental monitoring applications and to expose precarcinogenic xenobiotics.
Topics: Animals; Cell Line, Tumor; Chlorocebus aethiops; Fungal Proteins; Fusarium; Humans; Mutagenesis, Site-Directed; Sulfotransferases; Vero Cells
PubMed: 33239367
DOI: 10.1128/mSphere.00949-20 -
Journal of Applied Toxicology : JAT Oct 2012Sudden cardiac death is a significant health issue, causing millions of deaths worldwide annually. Studies have found that the likelihood of such death is higher in... (Review)
Review
Sudden cardiac death is a significant health issue, causing millions of deaths worldwide annually. Studies have found that the likelihood of such death is higher in winter. Further studies identified that the highest likelihood occurs on Christmas Day and New Years Day, but not the interim period. Thanksgiving, Independence Day and the Islamic holiday Eid Al-Fitr also show significant increases in the rate of cardiac events or death. A number of mechanisms have been proposed, but none have satisfactorily explained the evidence. This article reviews the data supporting the existence of a holiday cardiac death phenomenon, the involvement of catecholamines and the normal modes of human catecholamine deactivation. Further evidence is reviewed that supports a hypothesized mechanism whereby critical SULT1A catecholamine deactivation enzymes can in some patients be inhibited by naturally-occurring phenols and polyphenols in foods and alcohols. If deactivation is inhibited by holiday consumption excesses, holiday stress or excitement could lead to a buildup of catecholamines that can cause fatal arrhythmias. Awareness of this mechanism could reduce deaths, both through doctor/patient education leading to a moderation in consumption and through the potential identification of patients with a predisposition to SULT1A inhibition. This hypothesis also raises parallels between sudden cardiac death in adults and Sudden Infant Death Syndrome (SIDS). The possible involvement of SULT1A inhibition in SIDS is discussed.
Topics: Adult; Aged; Alcohol Drinking; Alleles; Animals; Arylsulfotransferase; Catecholamines; Death, Sudden, Cardiac; Diet; Disease Susceptibility; Holidays; Humans; Infant; Isoenzymes; Polymorphism, Single Nucleotide; Sudden Infant Death; Sulfotransferases
PubMed: 22678655
DOI: 10.1002/jat.2764