A new type of sulfation reaction: C-sulfonation for α,ß-unsaturated carbonyl groups by a novel sulfotransferase SULT7A1.

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.

Development of a plant conveyance system using an AGV and a self-designed plant-handling device: A case study of DIY plant phenotyping.

Plant phenotyping technology has been actively developed in recent years, but the introduction of these technologies into the field of agronomic research has not progressed as expected, in part due to the need for flexibility and low cost. "DIY" (Do It Yourself) methodologies are an efficient way to overcome such obstacles. Devices with modular functionality are critical to DIY experimentation, allowing researchers flexibility of design. In this study, we developed a plant conveyance system using a commercial AGV (Automated Guided Vehicle) as a case study of DIY plant phenotyping. The convey module consists of two devices, a running device and a plant-handling device. The running device was developed based on a commercial AGV Kit. The plant-handling device, plant stands, and pot attachments were originally designed and fabricated by us and our associates. Software was also developed for connecting the devices and operating the system. The run route was set with magnetic tape, which can be easily changed or rerouted. Our plant delivery system was developed with low cost and having high flexibility, as a unit that can contribute to others' DIY' plant research efforts as well as our own. It is expected that the developed devices will contribute to diverse phenotype observations of plants in the greenhouse as well as to other important functions in plant breeding and agricultural production.

Construction of prediction models for growth traits of soybean cultivars based on phenotyping in diverse genotype and environment combinations.

As soybean cultivars are adapted to a relatively narrow range of latitude, the effects of climate changes are estimated to be severe. To address this issue, it is important to improve our understanding of the effects of climate change by applying the simulation model including both genetic and environmental factors with their interactions (G×E). To achieve this goal, we conducted the field experiments for soybean core collections using multiple sowing times in multi-latitudinal fields. Sowing time shifts altered the flowering time (FT) and growth phenotypes, and resulted in increasing the combinations of genotypes and environments. Genome-wide association studies for the obtained phenotypes revealed the effects of field and sowing time to the significance of detected alleles, indicating the presence of G×E. By using accumulated phenotypic and environmental data in 2018 and 2019, we constructed multiple regression models for FT and growth pattern. Applicability of the constructed models was evaluated by the field experiments in 2020 including a novel field, and high correlation between the predicted and measured values was observed, suggesting the robustness of the models. The models presented here would allow us to predict the phenotype of the core collections in a given environment.

Quantitative analysis of seven plant hormones in Lotus japonicus using standard addition method.

Plant hormones have been identified to be versatile signaling molecules essential for plant growth, development, and stress response. Their content levels vary depending on the species, and they also change in response to any external stimuli. Thus, simultaneous quantification of multiple plant hormones is required to understand plant physiology. Sensitive and quantitative analysis using liquid chromatography-linked mass spectrometry (LC-MS/MS) has been used in detecting plant hormones; however, quantification without stable isotopes is yet to be established. In this study, we quantified seven representative plant hormones of Lotus japonicus, which is a model legume for standard addition method. Accurate masses for monoisotopic ions of seven phytohormones were determined for high-resolution mass spectrometry (HR-MS). Selected ion monitoring (SIM) mode based on accurate masses was used in detecting phytohormones in the roots, stems, and leaves. Evaluation of matrix effects showed ion suppression ranging from 10.2% to 87.3%. Both stable isotope dilution and standard addition methods were able to detect plant hormones in the roots, stems, and leaves, with no significant differences in using both approaches and thus a standard addition method can be used to quantify phytohormones in L. japonicus. The method will be effective, especially when stable isotopes are not available to correct for matrix effects.

Varietal differences in flavonoid and antioxidant activity in Japanese soybean accessions.

Soybean seeds contain many antioxidants, including flavonoids and other phytochemicals. Isoflavone is a phytoestrogen that mimics estrogenic effects on target tissues and also exerts antioxidant activity by sequestering free radicals. Despite many cultivars developed to date, varietal differences in flavonoid content and antioxidant activity in Japanese soybean accessions remain less well characterized. Here, we evaluated the seed content of isoflavones, total flavonoids, and total phenolics in 26 soybean accessions. Next, the antioxidant activity of each accession was examined using antioxidant responsive element (ARE) linked to a luciferase reporter in human HepG2 stable cells. The relative ARE luciferase activity rate of all soybean accessions varied up to 4-fold which ranged from 1.00 to 4.02; and 22 accessions exhibited significant antioxidant activities. Correlation analysis indicated that the level of total isoflavone moderately correlated with antioxidant activity.

Ser100-Phosphorylated RORα Orchestrates CAR and HNF4α to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6.

We have previously shown that the retinoid-related orphan receptor alpha (RORα) phosphorylation plays a pivotal role in sulfotransferase 1E1 gene regulation within mouse liver. Here, we found serine 100-phosphorylated RORα orchestrates constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α) to induce CYP2B6 by phenobarbital (PB) in human primary hepatocytes (HPHs). RORα knockdown using small interfering RNAs suppressed CYP2B6 mRNAs in HPH, whereas transient expression of RORα in COS-1 cells activated CYP2B6 promoter activity in reporter assays. Through chromatin immunoprecipitation (IP) and gel shift assays, we found that RORα in the form of phosphorylated (p-) S100 directly bound to a newly identified RORα response element (RORα response element on CYP2B6 promoter, -660/-649) within the CYP2B6 promoter in untreated or treated HPH. In PB-treated HPH, p-Ser100 RORα was both enriched in the distal phenobarbital response element module (PBREM) and the proximal okadaic acid response element (OARE), a known HNF4α binding site. Chromatin conformation capture assay revealed direct contact between the PBREM and OARE only in PB-treated HPH. Moreover, CAR preferably interacted with phosphomimetically mutated RORα at Ser100 residue in co-IP assay. A gel shift assay with a radiolabeled OARE module and nuclear extracts prepared from PB-treated mouse liver confirmed that HNF4α formed a complex with Ser 100-phosphorylated RORα, as shown by supershifted complexes with anti-p-Ser100 RORα and anti-HNF4α antibodies. Altogether, the results established that p-Ser100 RORα bridging the PBREM and OARE orchestrates CAR and HNF4α to form active chromatin complex during PB-induced CYP2B6 expression in human primary hepatocytes. SIGNIFICANCE STATEMENT: CYP2B6 is a vital enzyme for the metabolic elimination of xenobiotics, and it is prone to induction by xenobiotics, including phenobarbital via constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α). Here, we show that retinoid-related orphan receptor alpha (RORα), through phosphorylated S100 residue, orchestrated CAR-HNF4α interaction on the CYP2B6 promoter in human primary hepatocyte cultures. These results signify not only the role of RORα in the molecular process of CYP2B6 induction, but it also reveals the importance of conserved phosphorylation sites within the DNA-binding domain of the receptor.

Phenobarbital-induced phosphorylation converts nuclear receptor RORα from a repressor to an activator of the estrogen sulfotransferase gene Sult1e1 in mouse livers.

The estrogen sulfotransferase SULT1E1 sulfates and inactivates estrogen, which is reactivated via desulfation by steroid sulfatase, thus regulating estrogen homeostasis. Phenobarbital (PB), a clinical sedative, activates Sult1e1 gene transcription in mouse livers. Here, the molecular mechanism by which the nuclear receptors CAR, which is targeted by PB, and RORα communicate through phosphorylation to regulate Sult1e1 activation has been studied. RORα, a basal activity repressor of the Sult1e1 promoter, becomes phosphorylated at serine 100 and converts to an activator of the Sult1e1 promoter in response to PB. CAR regulates both the RORα phosphorylation and conversion. Our findings suggest that PB signals CAR to communicate with RORα via serine 100 phosphorylation, converting RORα from transcription repressor to activator of the Sult1e1 gene and inducing SULT1E1 expression in mouse livers.

Sulfotransferase 4A1 Increases Its Expression in Mouse Neurons as They Mature.

Cytosolic sulfotransferases (SULTs) catalyze sulfation and play essential roles in detoxification of xenobiotics as well as inactivation of endobiotics. SULT4A1, which was originally isolated as a brain-specific sulfotransferase, is the most highly conserved isoform among SULTs in vertebrates. Here, expression of SULT4A1 was examined neuron enriched and neuron-glia mixed cells derived from mouse embryo brains at day 14 gestation and mixed glia from 2-day-old neonate brains. Western blots showed an increase of SULT4A1 expression as neurons maturated. Reverse-transcription polymerase chain reaction and agarose gel analysis found two different forms (variant and wild type) of SULT4A1 mRNA in neurons; the level of wild type correlated with the protein level of SULT4A1. SULT1E1 was not expressed in mouse brains, neuron-enriched cells, or mixed glia cells. SULT1A1 protein was only detected in adult brains. Immunofluorescence staining of neuron-glia mixed cells confirmed selective expression of SULT4A1 in neurons, including dopaminergic neurons, but not in either astrocytes or microglia. Thus, SULT4A1 is a neuron-specific sulfotransferase and may play a role in neuronal development.

Δ4-3-ketosteroids as a new class of substrates for the cytosolic sulfotransferases.

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

Regioselective production of sulfated polyphenols using human cytosolic sulfotransferase-expressing Escherichia coli cells.

Dietary polyphenols present in fruits and vegetables have been reported to manifest beneficial health effects on humans. Polyphenol metabolites including their sulfated derivatives have been shown to be biologically active. Primarily due to the difficulty in preparing regiospecific sulfated polyphenols for detailed investigations, the exact functions of sulfated polyphenols, however, remain unclear. The current study aimed to develop a procedure for the regioselective production of sulfated polyphenols using Escherichia coli cells expressing human cytosolic sulfotransferases (SULTs). Two regioisomers of sulfated genistein were produced by E. coli cells expressing human SULT1A3, SULT1C4, or SULT1E1, and purified using Diaion HP20 resin, followed by high pressure liquid chromatography (HPLC). Structural analysis using mass spectrometry (MS) and nuclear magnetic resonance (NMR) revealed that E. coli cells expressing SULT1A3 preferentially produced genistein 4'-sulfate, whereas E. coli cells expressing SULT1C4 preferentially produced genistein 7-sulfate. To improve the bioproductivity, the effects of several factors including the concentrations of glucose and SO42-, and growth temperature were investigated. The bioproduction procedure established in this study will be valuable for the production of regioselective sulfated polyphenols for use in future studies on their biological functions.

Updated perspectives on the cytosolic sulfotransferases (SULTs) and SULT-mediated sulfation.

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

Phosphorylation of Farnesoid X Receptor at Serine 154 Links Ligand Activation With Degradation.

Comparison of 11 human nuclear receptor amino acid sequences revealed a conserved phosphorylation motif within their DNA-binding domains as an intramolecular signal that regulates proteolytic degradation. Nuclear receptors use this signal to either degrade or proscribe degradation through either the proteasome or nonproteasome pathways. A phosphomimetic farnesoid X receptor (FXR) S154D mutant neither bound to nor trans-activated an FXR-response element-driven reporter gene and was rapidly degraded in COS-1 cells. Ectopically expressed FXR had increased Ser154 phosphorylation in COS-1 cells after ligand treatment, and knock-down of the nuclear vaccinia-related kinase 1 (VRK1) greatly reduced this phosphorylation. FXR was phosphorylated at Ser154 in the nucleus of centrilobular hepatocytes only in ligand-treated mice. Thus, FXR Ser154 phosphorylation is a rheostat for activation and subsequent degradation that controls receptor levels and activity.

Identification of a novel flavonoid glycoside sulfotransferase in Arabidopsis thaliana.

The discovery of sulfated flavonoids in plants suggests that sulfation may play a regulatory role in the physiological functions of flavonoids. Sulfation of flavonoids is mediated by cytosolic sulfotransferases (SULTs), which utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. A novel SULT from Arabidopsis thaliana, designated AtSULT202B7 (AGI code: At1g13420), was cloned and expressed in Escherichia coli. Using various compounds as potential substrates, we demonstrated, for the first time, that AtSULT202B7 displayed sulfating activity specific for flavonoids. Intriguingly, the recombinant enzyme preferred flavonoid glycosides (e.g. kaempferol-3-glucoside and quercetin-3-glucoside) rather than their aglycone counterparts. Among a series of hydroxyflavones tested, AtSULT202B7 showed the enzymatic activity only for 7-hydroxyflavone. pH-dependency study showed that the optimum pH was relatively low (pH 5.5) compared with those (pH 6.0-8.5) previously reported for other isoforms. Based on the comparison of high performance (pressure) liquid chromatography (HPLC) retention times between sulfated kaempferol and the deglycosylated product of sulfated kaempferol-3-glucoside, the sulfation site in sulfated kaempferol-3-glucoside appeared to be the hydroxyl group of the flavonoid skeleton. In addition, by using direct infusion mass spectrometry, it was found that the sulfated product had one sulfonate group within the molecule. These results indicated that AtSULT202B7 functions as a flavonoid glycoside 7-sulfotransferase.

Identification and characterization of a novel kaempferol sulfotransferase from Arabidopsis thaliana.

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

Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases.

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.