Sulfotransferase Activity Research Articles

Novel topical booster enhances follicular sulfotransferase activity in patients with androgenetic alopecia: a new strategy to improve minoxidil response

Topical minoxidil has been used as a topical treatment for androgenetic alopecia (AGA) for more than 30 years. Approximately 60 to 70% of patients do not achieve hair growth. Minoxidil is a pro-drug. In order to exert biological activity, minoxidil requires conversion to minoxidil sulfate by sulfotransferase enzymes (SULT1A1). We have reported extensively that SULT1A1 activity in the outer root sheath (ORS) of the hair follicle correlates directly with topical minoxidil response. We have demonstrated the clinical utility and validity of a colorimetric test to measure the follicular SULT1A1 activity in plucked hair as a method to predict minoxidil responders. This same test also predicts clinical response to oral minoxidil.

Abstract 2151: Monitoring AMP Concentration,the universal product of diverse enzymatic reactions

Abstract Many enzymatic reactions use Adenosine triphosphate (ATP) as a substrate and produce adenosine monophosphate (AMP) as the common product of these reactions. AMP also functions as a modulator of many enzymes and a key cellular metabolite regulating energy homeostasis and signal transduction. For example, by activating AMP-Dependent protein kinase (AMPK), it maintains the energy charge ratio at optimal level during metabolic activities; and its concentration is altered during disregulated disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates in a homogenous and high through-put formatted form. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by first depleting the remaining residual ATP substrate and converting AMP produced into ATP, the later can be quantified using luciferase/luciferin and generating light. We also tested this assay to measure direct activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP-Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono- and poly-ADP-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system we can measure the activities of sulfotransferases. In summary, we have developed a very strong platform to monitor AMP concentrations that is applicable to diverse enzymatic reactions. Citation Format: Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. Monitoring AMP Concentration,the universal product of diverse enzymatic reactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2151.

In silico study for inhibiting thyroid hormone sulfotransferase activity by halogenated phenolic chemicals

Thyroid hormones (THs) are essential to proper growth and development of human bodies. Inhibiting the sulfation metabolism of THs has been demonstrated to be an important way for some environmental pollutants, such as halogenated phenolic compounds, to interfere THs homeostasis, thereby causing health problems. However, the important property characteristics that govern the sulfation inhibition of these chemicals are not well understood, and the experimental data on inhibition potential is limited. In this work, an in silico approach was developed to investigate the structure-activity relationship for their sulfotransferases (SULTs) inhibition. A series of quantum chemical descriptors that quantify the electronic and energy properties of 22 halogenated phenolic compounds have been calculated to establish a predictive model and analyzed their corresponding contributions to SULTs inhibition. Density functional theory (DFT) B3LYP/6−31G** has been employed to optimize molecular geometries to obtain a total of 15 descriptors for every compound. The implementation of linear regression shows three descriptors that represent molecular mass, positive charges on hydrogen atoms, and energy of frontier orbitals strongly correlate with SULTs inhibition potential. This indicates molecular size, hydrogen-bond strength, and nucleophilic-electrophilic reactivity may play important roles in SULTs inhibition. The derived regression model has good statistical performance (r2 = 0.84, rms = 0.35), and different validation strategies indicate it can serve as an efficient predictive tool for other chemicals in application domain but with no experimental data, consequently assisting in their THs sulfation inhibition and health risk assessment.

Cadmium-induced oxidative stress, histopathology, and transcriptome changes in the hepatopancreas of freshwater crayfish (Procambarus clarkii)

Cadmium (Cd) is a common contaminant in environment. Crayfish are considered suitable for indicating the impact of heavy metals on the environment. However, there is limited information on the mechanisms causing damage to the hepatopancreas of Procambarus clarkii exposed to Cd. We exposed adult male P. clarkii to 2.0, 5.0, and 10.0 mg/L Cd for 24, 48, and 72 h to explore Cd toxicity. Afterwards, we measured bioaccumulations in the hepatopancreas and determined malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST). Additionally, the hepatopancreas histopathology was analyzed and the transcriptome analysis of the P. clarkii hepatopancreas under Cd stress was conducted. The results revealed that hepatopancreas could accumulate Cd in a time- and dose-dependent manner. Cd induced significant changes in MDA content and antioxidant enzyme activity. Severe histological alterations were observed in crayfish hepatopancreas. After 72 h exposure to 2.0, 5.0, and 10.0 mg/L Cd, transcriptome analysis identified 1061, 747, and 1086 differentially expressed genes (DEGs), respectively. Exposure to 5.0 mg/L Cd inhibited heme binding, tetrapyrrole binding, iron ion binding and activity of oxidoreductase and sulfotransferase, while exposure to 10.0 mg/L Cd enhanced the export of matters from nucleus. In the hepatopancreas treated with 10.0 mg/L Cd, pathways related to diseases and immune system were significantly enriched. Meanwhile, 31, 31, 24, 7, and 12 identified DEGs were associated with the oxidation-reduction process, immune system, ion homeostasis, digestion and absorption, and ATPases, respectively. Our study provides comprehensive information for exploring the toxic mechanisms of Cd and candidate biomarkers for aquatic Cd risk evaluation.

Potential for Heightened Sulfur-Metabolic Capacity in Coastal Subtropical Microalgae

SummaryThe activities of microalgae support nutrient cycling that helps to sustain aquatic and terrestrial ecosystems. Most microalgal species, especially those from the subtropics, are genomically uncharacterized. Here we report the isolation and genomic characterization of 22 microalgal species from subtropical coastal regions belonging to multiple clades and three from temperate areas. Halotolerant strains including Halamphora, Dunaliella, Nannochloris, and Chloroidium comprised the majority of these isolates. The subtropical-based microalgae contained arrays of methyltransferase, pyridine nucleotide-disulfide oxidoreductase, abhydrolase, cystathionine synthase, and small-molecule transporter domains present at high relative abundance. We found that genes for sulfate transport, sulfotransferase, and glutathione S-transferase activities were especially abundant in subtropical, coastal microalgal species and halophytic species in general. Our metabolomics analyses indicate lineage- and habitat-specific sets of biomolecules implicated in niche-specific biological processes. This work effectively expands the collection of available microalgal genomes by ∼50%, and the generated resources provide perspectives for studying halophyte adaptive traits.

Effects of inhibition of hepatic sulfotransferase activity on renal genotoxicity induced by lucidin-3-O-primeveroside.

Sulfotransferase 1A (SULT1A) expression is lower in the liver of humans than that of rodents. Therefore, species differences should be taken into consideration when assessing the risk of rodent hepatocarcinogens metabolically activated by SULT1A in humans. Although some renal carcinogens require SULT1A-mediated activation, it is unclear how SULT1A activity in the liver affects renal carcinogens. To explore the effects of SULT1A activity in the liver on genotoxicity induced by SULT1A-activated renal carcinogens, B6C3F1 mice or gpt delta mice of the same strain background were given lucidin-3-O-primeveroside (LuP), a hepatic and renal carcinogen of rodents, for 4 or 13weeks, respectively, and pentachlorophenol (PCP) as a liver-specific SULT inhibitor, was given from 1week before LuP treatment to the end of the experiment. A 4week exposure of LuP induced lucidin-specific DNA adduct formation. The suppression of Sult1a expression was observed only in the liver but not in the kidneys of PCP-treated mice, but co-administration of PCP suppressed LuP-induced DNA adduct formation in both organs. Thirteen-week exposure of LuP increased mutation frequencies and cotreatment with PCP suppressed these increases in both organs. Given that intact levels of SULT activity in the liver were much higher than in the kidneys of rodents, SULT1A may predominantly activate LuP in the liver, consequently leading to genotoxicity not only in the liver but also in the kidney. Thus, species differences should be considered in human risk assessment of renal carcinogens activated by SULT1A as in the case of the corresponding liver carcinogens.

Optimized expression of heparin sulfotransferases and their application in sulfation of animal derived heparin

Heparin is a very important anticoagulant drug. Currently, heparin is mainly extracted from porcine mucosa. However, animal-derived heparin shows low anticoagulant activity due to the low proportion of the anticoagulant active unit, the GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S pentasaccharide. In this study we proposed an enzymatic strategy to sulfate the animal-sourced heparin to increase the proportion of anticoagulant pentasaccharide and the anticoagulant activity. First, three sulfotransferases HS2ST, HS6ST, and HS3ST were expressed tentatively in Escherichia coli and Pichia pastoris. After measuring the sulfotransferase activity, we confirmed P. pastoris GS115 is the better host for sulfotransferases production. Then, the maltose binding protein (MBP) and thioredoxin (TrxA) were fused separately to the N-terminal of sulfotransferases to increase enzyme solubility. As a result, the yields of HS2ST and HS6ST were increased to (839±14) U/L and (792±23) U/L, respectively. Subsequent sulfation of the animal-sourced heparin with the recombinant HS2ST, HS6ST and HS3ST increased the anticoagulant activity from (76±2) IU/mg to (189±17) IU/mg.

Comparative analysis of root transcriptome profiles between low- and high-cadmium-accumulating genotypes of wheat in response to cadmium stress.

Wheat, one of the most broadly cultivated and consumed food crops worldwide, can accumulate high Cd contents in their edible parts, which poses a major hazard to human health. Cd accumulation ability differs among varieties in wheat, but the underlying molecular mechanism is largely unknown. Here, key genes responsible for Cd accumulation between two contrasting wheat genotypes (low-Cd accumulation one L17, high-Cd accumulation one H17) were investigated. Total 1269 were differentially expressed genes (DEGs) in L17 after Cd treatment, whereas, 399 Cd-induced DEGs were found in H17. GO-GO network analysis showed that heme binding was the most active GO, and metal binding was the second one that associated with other GOs in response to Cd stress in both genotypes. Pathway-pathway network analysis showed that phenylpronanoid biosynthesis and glutathione metabolism were the top pathways in response to Cd stress in both genotypes. Furthermore, we found that DEGs related to ion binding, antioxidant defense mechanisms, sulfotransferase activity, and cysteine biosynthetic process were more enriched in L17. In conclusion, our results not only provide the foundation for further exploring the molecular mechanism of Cd accumulation in wheat but also supply new strategies for improving phytoremediation ability of wheat by genetic engineering.

Characterization of follicular minoxidil sulfotransferase activity in a cohort of pattern hair loss patients from the Indian Subcontinent.

Several studies have established that sulfotransferase enzyme activity in the outer root sheath of plucked hair follicles predicts response to topical minoxidil in the treatment of pattern hair loss. However, the prevalence of this enzyme activity among Indian patients has not been studied. Additionally, no reports in the literature characterize sulfotransferase activity based on sex, age, duration of hair loss, grade of hair loss, and family history. In this study we utilized a sulfotransferase activity assay first reported by Goren et al. We characterize the follicular sulfotransferase activity of 120 pattern hair loss patients visiting a dermatology outpatient clinic in India. Overall, 40.8% of patients with pattern hair loss had low levels of sulfotransferase. Surprisingly, 49.3% of men had low levels of sulfotransferase compared to 26.6% of women. No correlation was found between sulfotransferase activity and age, duration of hair loss, grade of hair loss, or family history. A sub-analysis of patient reported outcomes (PRO) validated previous findings that sulfotransferase enzyme activity is a predictive marker for minoxidil response in pattern hair loss patients.

Low-dose daily aspirin reduces topical minoxidil efficacy in androgenetic alopecia patients.

Topical minoxidil is the only US FDA approved OTC drug for the treatment of androgenetic alopecia (AGA). Minoxidil is a pro-drug converted into its active form, minoxidil sulfate, by the sulfotransferase enzymes in the outer root sheath of hair follicles. Previously, we demonstrated that sulfotransferase activity in hair follicles predicts response to topical minoxidil in the treatment of AGA. In the human liver, sulfotransferase activity is significantly inhibited by salicylic acid. Low-dose OTC aspirin (75-81 mg), a derivative of salicylic acid, is used by millions of people daily for the prevention of coronary heart disease and cancer. It is not known whether oral aspirin inhibits sulfotransferase activity in hair follicles, potentially affecting minoxidil response in AGA patients. In the present study, we determined the follicular sulfotransferase enzymatic activity following 14 days of oral aspirin administration. In our cohort of 24 subjects, 50% were initially predicted to be responders to minoxidil. However, following 14 days of aspirin administration, only 27% of the subjects were predicted to respond to topical minoxidil. To the best of our knowledge, this is the first study to report the effect of low-dose daily aspirin use on the efficacy of topical minoxidil.

Phylogenetic origins for severe acetaminophen toxicity in snake species compared to other vertebrate taxa

While it has been known for a while that some snake species are extremely sensitive to acetaminophen, the underlying mechanism for this toxicity has not been reported. To investigate if essential detoxification enzymes are missing in snake species that are responsible for biotransformation of acetaminophen in other vertebrate species, livers were collected from a variety of snake species, together with samples from alligator, snapping turtle, cat, rat, and cattle. Subcellular fractions were analyzed for enzymatic activities of phenol-type sulfotransferase and UDP‑glucuronosyltransferase, total glutathione S‑transferase, and N‑acetyltransferase. The results showed that none of the snake species, together with the cat samples, had any phenol-type glucuronidation activity, and that this activity was much lower in alligator and turtle samples than in the mammalian species. Combined with the lack of N‑acetyltransferase activity in snakes and cats, this would explain the accumulation of the aminophenol metabolite, which induces methemoglobinemia and subsequent suffocation of snakes and cats after acetaminophen exposure. While previous investigations have concluded that in cats the gene for the phenol-type glucuronosyltransferase isoform has turned into a pseudogene because of several point mutations, evaluation of genomic information for snake species revealed that they have only 2 genes that may code for glucuronosyltransferase isoforms. Similarity of these genes with mammalian genes is <50%, and suggests that the expressed enzymes may act on other types of substrates than aromatic amines. This indicates that the extreme sensitivity for acetaminophen in snakes is based on a different phylogenetic origin than the sensitivity observed in cats.

HS6ST1 Insufficiency Causes Self-Limited Delayed Puberty in Contrast With Other GnRH Deficiency Genes.

ContextSelf-limited delayed puberty (DP) segregates in an autosomal-dominant pattern, but the genetic basis is largely unknown. Although DP is sometimes seen in relatives of patients with hypogonadotropic hypogonadism (HH), mutations in genes known to cause HH that segregate with the trait of familial self-limited DP have not yet been identified.ObjectiveTo assess the contribution of mutations in genes known to cause HH to the phenotype of self-limited DP.Design, Patients, and SettingWe performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited DP, validated the pathogenicity of the identified gene variant in vitro, and examined the tissue expression and functional requirement of the mouse homolog in vivo.ResultsA potentially pathogenic gene variant segregating with DP was identified in 1 of 28 known HH genes examined. This pathogenic variant occurred in HS6ST1 in one pedigree and segregated with the trait in the six affected members with heterozygous transmission (P = 3.01 × 10−5). Biochemical analysis showed that this mutation reduced sulfotransferase activity in vitro. Hs6st1 mRNA was expressed in peripubertal wild-type mouse hypothalamus. GnRH neuron counts were similar in Hs6st1+/− and Hs6st1+/+ mice, but vaginal opening was delayed in Hs6st1+/− mice despite normal postnatal growth.ConclusionsWe have linked a deleterious mutation in HS6ST1 to familial self-limited DP and show that heterozygous Hs6st1 loss causes DP in mice. In this study, the observed overlap in potentially pathogenic mutations contributing to the phenotypes of self-limited DP and HH was limited to this one gene.

Administration of low dose triclosan to pregnant ewes results in placental uptake and reduced estradiol sulfotransferase activity in fetal liver and placenta

Sulfonation is a major pathway of estrogen biotransformation with a role in regulating estrogen homeostasis in humans and sheep. Previous in vitro studies found that triclosan is an especially potent competitive inhibitor of ovine placental estrogen sulfotransferase, with Kic of <0.1 nM. As the placenta is the main organ responsible for estrogen synthesis in pregnancy in both women and sheep, and the liver is another site of estrogen biotransformation, this study examined the effects of triclosan exposure of pregnant ewes on placental and hepatic sulfotransferase activity. Triclosan, 0.1 mg/kg/day, or saline vehicle was administered to late gestation fetal sheep for two days either by direct infusion into the fetal circulation or infusion into the maternal blood. On the third day, fetal liver and placenta were harvested and analyzed for triclosan and for cytosolic estradiol sulfotransferase activity. Placenta contained higher concentrations of triclosan than liver in each individual sheep in both treatment groups. There was a negative correlation between triclosan tissue concentration (pmol/g tissue) and cytosolic sulfotransferase activity (pmol/min/mg protein) towards estradiol. These findings demonstrated that in the sheep exposed to very low concentrations of triclosan, this substance is taken up into placenta and reduces estrogen sulfonation.

The sulfoconjugation of androstenone and dehydroepiandrosterone by human and porcine sulfotransferase enzymes

Porcine sulfotransferase 2A1 (pSULT2A1) is a key enzyme involved in the testicular and hepatic sulfoconjugation of steroids such as dehydroepiandrosterone (DHEA) and potentially androstenone. This latter steroid is a major cause of boar taint, which is an unpleasant off-odour and off-flavour in pork from male pigs. Sulfotransferase 2B1 (pSULT2B1) may also be important, although no direct evidence exists for its involvement in sulfoconjugation of steroids. The purpose of this study was to investigate the sulfoconjugation activity of human and porcine sulfotransferases towards DHEA and androstenone. pcDNA 3.1 vectors expressing porcine (p) SULT2A1, pSULT2B1, human (h) SULT2A1, hSULT2B1a, and hSULT2B1b enzymes were transfected into human embryonic kidney cells. Transfected cells were then incubated with either androstenone or dehydroepiandrosterone (DHEA) in both time-course and enzyme kinetics studies. The production of sulfonates of androstenone metabolites and DHEA sulfonate increased over time for all enzymes with the exception of pSULT2B1. Enzyme kinetics analysis showed that androstenone and DHEA were poor substrates for the human orthologs, hSULT2B1a and hSULT2B1b. Human and porcine SULT2A1 showed substantially different substrate affinities for androstenone (Km 5.8 ± 0.6 µM and 74.1 ± 15.9 µM, respectively) and DHEA (Km 9.4 ± 2.5 µM and 3.3 ± 1.9 µM, respectively). However, these enzymes did show relatively similar sulfonation efficiencies for DHEA (Vmax/Km 50.5 and 72.9 for hSULT2A1 and pSULT2A1, respectively). These results highlight the species differences in sulfonation activity and provide direct evidence, for the first time, suggesting that pSULT2B1 is not involved in sulfonation of either androstenone metabolites or DHEA.

Inter-individual Variability in Activity of the Major Drug Metabolizing Enzymes in Liver Homogenates of 20 Individuals

Inter-individual variability in hepatic drug metabolizing enzyme (DME) activity is a major contributor to heterogeneity in drug clearance and safety. Accurate data on expression levels and activities of DMEs is an important prerequisite for in vitro-in vivo extrapolation and in silico based predictions. Characterization and assessment of inter-correlations of the major DMEs cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs) have been extensively documented, but simultaneous quantification including other major DMEs has been lacking. Assessment of inter-donor variability and inter-correlations of CYPs, UGTs, sulfotransferases (SULTs), glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH: quinone oxidoreductase 2 (NQO2) in a set of 20 individual liver homogenates. The main drug metabolizing isoforms of CYP and UGT have been reaction phenotype in individual liver microsomes and NQO1, NQO2, GSTT1 and GSTT2 in corresponding cytosol. In addition, we assessed overall SULT activity in liver cytosol using acetaminophen and 7-hydroxycoumarin as non-selective substrates and cytosolic GST activity using the non-selective substrate 1-chloro-2,4-dinitrobenzene (CDNB). Expression of GST isoforms was also assessed. While hepatic NQO1 activity was highly variable, NQO2 activity was more conserved. In addition, we found that of the hepatic GST isoforms, the variation in GSTM3 levels, which is poorly studied, was highest. The majority of significant correlations were found amongst CYP and UGT enzyme activities. The dataset presented provides the absolute quantification of the largest number of hepatic DME activities so far and constitute an essential resource for in silico toxicokinetic and metabolic modelling studies.

Sulfotransferase-catalyzed biotransformation of liguzinediol and comparison of its metabolism in different species using UFLC-QTOF-MS

Liguzinediol (2,5-dihydroxymethyl-3,6-dimethylpyrazine, LZDO) is a potential agent for the low-risk treatment of heart failure. 2-N-acetylcysteine-LZDO (2-NAC-LZDO) and 2-cysteine-LZDO (2-Cys-LZDO) are major LZDO metabolites found in the pharmacokinetic studies of rats and beagle dogs. To elucidate the biotransformation pathway and related enzymes, an incubation system with 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as a cofactor and N-acetylcysteine (NAC) as a trapping agent was established using liver cytosol. An ultra-flow liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UFLC-QTOF-MS) method was used to identify the major metabolites. 2-NAC-LZDO could be detected among four species (humans, monkeys, dogs, and rats) and is the dominant metabolite in human liver cytosol (HLC). The sulfotransferase (SULT) inhibitors 2,6-dichloro-4-nitrophenol (DCNP) and quercetin at a concentration of 1 μM, suppressed 2-NAC-LZDO formation in HLC by 87 and 46%, respectively. This result suggested that sulfotransferase was involved in 2-NAC-LZDO formation. The metabolism of LZDO in different species indicated that SULT activity in dogs, rats, and monkeys was higher than that in humans. Further SULT phenotyping revealed that SULT1A1 is the predominant enzyme involved in the sulfation of LZDO. The underlying mechanism for the biotransformation of LZDO was demonstrated. The potential pathway is via the sulfation of LZDO to form sulfate, and the spontaneous cleavage of the sulfate group to generate highly reactive electrophilic cations, which can bind to NAC to form the major metabolites.

Utility of AMP Detection System for Monitoring the Activities of Diverse Enzyme Reactions

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are disregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP‐Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono‐ and poly‐ADP‐ribosyltransferases. Sulfotransferases use 3′‐Phosphoadenosine‐5′‐phosphosulfate (PAPS) as the universal sulfo‐group donor and Phosphoadenosine‐5′‐phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgiresident PAP‐specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP‐Glo system we can measure the activities of sulfotransferases. Thus by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP‐Glo assay.Support or Funding InformationThe research was supported by Promega Corp.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Utilizing SULT4A1 Mutant Mouse Models to Characterize SULT4A1

Identified by our lab in 2000, sulfotransferase 4A1 (SULT4A1) belongs to the cytosolic sulfotransferase (SULT) gene superfamily of phase II conjugation enzymes based on key sequence and structural similarities. Broadly, SULTs catalyze the transfer of a sulfonate moiety (SO3−) to a diverse set of endogenous and exogenous substrates in tandem with the obligate cofactor 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS). Expression studies have shown SULT4A1 to be localized to neuronal tissues, and SULT4A1 orthologs have been found in all vertebrate species examined. Remarkably, human SULT4A1 shares a high degree of sequence homology with other vertebrate SULT4A1 orthologs, more than any other human SULT. Despite the conserved nature of SULT4A1 among vertebrates, all attempts to demonstrate sulfotransferase activity have failed and the neuronal function of this orphan enzyme remains unclear. To elucidate an understanding of SULT4A1, CRISPR‐Cas9 gene editing technology was performed to disrupt SULT4A1 function in a C57BL/6J mouse background and two mutations of interest were pursued. A 28 bp deletion (Δ28) in exon 1 resulted in a frameshift mutation and a premature stop codon at amino acid (AA) 62. A second mutation of interest was a 12 bp (Δ12) in‐frame deletion in exon 3 of four AA immediately adjacent to the active site histidine. Dynamic modeling studies of the Δ12 mutation suggested a structural disruption to the substrate binding pocket. Interestingly, homozygous mutant pups of both lines displayed a severe and progressive neurological pathology beginning 8–10 days post‐natal, until death at day 21–25. Neurological symptoms included persistent tremor, ataxia, absence seizures, and decreased weight gain. Homozygous mutant mice of both strains were confirmed to lack detectable SULT4A1, while heterozygous mice of both strains showed a modest decrease compared to wild‐type (WT) mice. Since little is known about SULT4A1 function, subcellular fractionation was performed to determine if SULT4A1 localizes to other cellular compartments. Indeed, SULT4A1 was detected in mitochondrial, cytosolic, and microsomal fractions, a previously unreported finding. Due to the early onset of neurological symptoms, SULT4A1 expression was assessed in primary cortical neurons (PCNs) harvested from gestational day 15 mice. PCNs were cultured in vitro to day 12 and robust expression of SULT4A1 was observed in these cells by immunostaining. In summary, SULT4A1 is a critical protein for neurofunction and normal development, as mice lacking functional SULT4A1 displayed a progressively severe and lethal neurological pathology. These mutant mouse models will be instrumental into characterizing the function of SULT4A1.Support or Funding InformationThis work was supported by GM113980 to CNF and NS08695301 to SAA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SULFATION PATHWAYS: Expression of SULT2A1, SULT2B1 and HSD3B1 in the porcine testis and epididymis.

In the porcine testis, in addition to estrogen sulfates, the formation of numerous sulfonated neutral hydroxysteroids has been observed. However, their functions and the underlying synthetic pathways are still widely unclear. To obtain further information on their formation in postpubertal boars, the expression of sulfotransferases considered relevant for neutral hydroxysteroids (SULT2A1, SULT2B1) was investigated in the testis and defined segments of the epididymis applying real-time RT-qPCR, Western blot and immunohistochemistry (IHC). Sulfotransferase activities were assessed in tissue homogenates or cytosolic preparations applying dehydroepiandrosterone and pregnenolone as substrates. A high SULT2A1 expression was confirmed in the testis and localized in Leydig cells by IHC. In the epididymis, SULT2A1 expression was virtually confined to the body. SULT2B1 expression was absent or low in the testis but increased significantly along the epididymis. Immunohistochemical observations indicate that both enzymes are secreted into the ductal lumen via an apocrine mechanism. The results from the characterization of expression patterns and activity measurements suggest that SULT2A1 is the prevailing enzyme for the sulfonation of hydroxysteroids in the testis, whereas SULT2B1 may catalyze the formation of sterol sulfates in the epididymis. In order to obtain information on the overall steroidogenic capacity of the porcine epididymis, the expression of important steroidogenic enzymes (CYP11A1, CYP17A1, CYP19, HSD3B1, HSD17B3, SRD5A2) was monitored in the defined epididymal segments applying real-time RT-qPCR. Surprisingly, in addition to a high expression of SRD5A2 in the epididymal head, a substantial expression of HSD3B1 was detected, which increased along the organ.

Expression and activities of sulfotransferase in rat brain

Sulfotransferase (SULT) has been found in the brain; however, the details of its function remain unclear. The present study aimed to elucidate the regional differences in the expression of SULT1 and SULT2 mRNA and SULT activities in the eight functional regions of the rat brain (cerebellum, cortex, hippocampus, medulla oblongata, midbrain, olfactory bulb, striatum, and thalamus).All SULT1 isoforms were detected in the medulla oblongata and thalamus. SULT2A1 mRNA was not observed in any of the eight regions, whereas SULT2B1a and SULT2B1b were found in all regions. The SULT2B1b mRNA expression level in the medulla oblongata was 1.7-fold higher than that in the liver. The sulfonation of p-nitrophenol and pregnenolone was detected in all regions. The kinetics of p-nitrophenol sulfonation in the cerebellum fitted to the substrate inhibition model (Km = 37.6 nM, Vmax = 2.72 pmol/min/mg, Vinh = 1.60 pmol/min/mg, and Ki = 0.87 μM). The pregnenolone sulfonation also exhibited substrate inhibition kinetics (Km = 0.99 μM, Vmax = 1.53 pmol/min/mg, and Ki = 54.67 μM).We clarified that SULT1 and SULT2 were expressed and had metabolizing capacities in the rat brain, suggesting that brain SULTs may be involved in metabolism of endogenous compounds and drugs.