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Journal of Industrial Microbiology &... Feb 2023The chemo-enzymatic and enzymatic synthesis of heparan sulfate and heparin are considered as an attractive alternative to the extraction of heparin from animal tissues....
The chemo-enzymatic and enzymatic synthesis of heparan sulfate and heparin are considered as an attractive alternative to the extraction of heparin from animal tissues. Sulfation of the hydroxyl group at position 2 of the deacetylated glucosamine is a prerequisite for subsequent enzymatic modifications. In this study, multiple strategies, including truncation mutagenesis based on B-factor values, site-directed mutagenesis guided by multiple sequence alignment, and structural analysis were performed to improve the stability and activity of human N-sulfotransferase. Eventually, a combined variant Mut02 (MBP-hNST-NΔ599-602/S637P/S741P/E839P/L842P/K779N/R782V) was successfully constructed, whose half-life at 37°C and catalytic activity were increased by 105-fold and 1.35-fold, respectively. After efficient overexpression using the Escherichia coli expression system, the variant Mut02 was applied to N-sulfation of the chemically deacetylated heparosan. The N-sulfation content reached around 82.87% which was nearly 1.88-fold higher than that of the wild-type. The variant Mut02 with high stability and catalytic efficiency has great potential for heparin biomanufacturing.
Topics: Animals; Humans; Sulfates; Sulfotransferases; Heparin
PubMed: 37327079
DOI: 10.1093/jimb/kuad012 -
Gene Jan 2007The xenobiotic-activated nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androstane receptor) and the vitamin D(3)-activated nuclear receptor VDR...
Xenobiotic- and vitamin D-responsive induction of the steroid/bile acid-sulfotransferase Sult2A1 in young and old mice: the role of a gene enhancer in the liver chromatin.
The xenobiotic-activated nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androstane receptor) and the vitamin D(3)-activated nuclear receptor VDR regulate steroid and xenobiotic metabolism by inducing the phase I cytochrome P450 monooxygenases, phase II conjugating transferases, and the phase III transporters, which mediate the efflux of water-soluble lipid metabolites from cells. Metabolic stress due to the deviant expression of steroid- and xenobiotic-metabolizing enzymes is known to have severe health consequences including accelerated aging, and increased expression of these enzymes is associated with extended longevity [Gachon, F, Olela, FF, Schaad, O, Descombes, P and Schibler, U, 2006. The circadian PAR-domain basic leucine zipper transcription factors DBP, TEF, and HLF modulate basal and inducible xenobiotic detoxification. 4, 25-36.; McElwee, JJ, Schuster, E, Blanc, E, Thomas, JH and Gems, D, 2004. Shared Transcriptional Signature in Caenorhabditis elegans Dauer Larvae and Long-lived daf-2 Mutants Implicates Detoxification System in Longevity Assurance. J. Biol. Chem., 279, 44533-43.]. Information on the similarities and dissimilarities in drug metabolism between the young and old, as may be uncovered by studying aging regulation of the genes relevant to steroid and xenobiotic metabolism, is likely to have clinical significance. In this report, we examined the VDR- and PXR-mediated gene induction of the phase II sulfotransferase Sult2A1 in the livers of 4-month- and 20-month-old mice. Sult2A1 converts bile acids, steroids and a number of drugs to the corresponding sulfated metabolites, which are readily eliminated from the body due to increased water solubility. In RT-PCR assay, aging did not change the induction of Sult2A1 mRNAs by the hormonally active vitamin D(3) and the catatoxic synthetic steroid PCN (pregnenolone-16alpha-carbonitrile). Chromatin immunoprecipitation (ChIP) from liver nuclei showed that aging had no effect on the activity of an IR0 enhancer in the Sult2A1 chromatin to recruit VDR, RXR-alpha (retinoid X receptor) and PXR in mice injected with D(3) or PCN. Thus, mice in late life are as competent as those in early life in responding to the hormonal and xenobiotic signaling for Sult2A1 induction. This is the first report describing the role of aging in the functional response of an enhancer in the liver chromatin to the nuclear receptor-dependent signaling.
Topics: Age Factors; Animals; Chromatin; Enhancer Elements, Genetic; Enzyme Induction; Liver; Mice; RNA, Messenger; Sulfotransferases; Vitamin D; Xenobiotics
PubMed: 17123747
DOI: 10.1016/j.gene.2006.10.006 -
The Journal of Biological Chemistry Feb 2022The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating...
The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid-binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1-3GalNAcα1-O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.
Topics: Glycoproteins; HEK293 Cells; Humans; Kidney; Mucins; Polysaccharides; Sulfates; Sulfotransferases
PubMed: 34954141
DOI: 10.1016/j.jbc.2021.101382 -
The Journal of Biological Chemistry May 2001Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal...
Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydrate sulfotransferases and the presence of mutations of this gene in MCD patients who lack sulfated keratan sulfate in the cornea and serum, hCGn6ST protein is thought to be a sulfotransferase that catalyzes sulfation of GlcNAc in keratan sulfate. In this report, we analyzed the enzymatic activity of hCGn6ST by expressing it in cultured cells. A lysate prepared from HeLa cells transfected with an intact form of hCGn6ST cDNA or culture medium from cells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring sulfate to C-6 of GlcNAc of synthetic oligosaccharide substrates in vitro. When hCGn6ST was expressed together with human keratan sulfate Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly sulfated carbohydrate detected by an anti-keratan sulfate antibody 5D4. These results indicate that hCGn6ST transfers sulfate to C-6 of GlcNAc in keratan sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from missense mutations found in MCD patients abolished enzymatic activity. Moreover, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as hCGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotransferase is the orthologue of hCGn6ST and functions as a sulfotransferase to produce keratan sulfate in the cornea.
Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Cornea; Corneal Dystrophies, Hereditary; DNA, Complementary; HeLa Cells; Humans; Intestines; Keratan Sulfate; Mice; Molecular Sequence Data; Mutation, Missense; Phylogeny; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Sulfotransferases; Transfection; Carbohydrate Sulfotransferases
PubMed: 11278593
DOI: 10.1074/jbc.M009995200 -
Bioorganic Chemistry Nov 2022Sulfation is a common modification of glycans and glycoproteins. Sulfated N-glycans have been identified in various glycoproteins and implicated for biological...
Sulfation is a common modification of glycans and glycoproteins. Sulfated N-glycans have been identified in various glycoproteins and implicated for biological functions, but in vitro synthesis of structurally well-defined full length sulfated N-glycans remains to be described. We report here the first in vitro enzymatic sulfation of biantennary complex type N-glycans by recombinant human CHST2 (GlcNAc-6-O-sulfotransferase 1, GlcNAc6ST-1). We found that the sulfotransferase showed high antennary preference and could selectively sulfate the GlcNAc moiety located on the Manα1,3Man arm of the biantennary N-glycan. The glycan chain was further elongated by bacterial β1,4 galactosyltransferase from Neiserria meningitidis and human β1,4 galactosyltransferase IV(B4GALT4), which led to the formation of different sulfated N-glycans. Using rituximab as a model IgG antibody, we further demonstrated that the sulfated N-glycans could be efficiently transferred to an intact antibody by using a chemoenzymatic Fc glycan remodeling method, providing homogeneous sulfated glycoforms of antibodies. Preliminary binding analysis indicated that sulfation did not affect the apparent affinity of the antibody for FcγIIIa receptor.
Topics: Galactosyltransferases; Glycoproteins; Humans; Immunoglobulin G; Polysaccharides; Sulfates; Sulfotransferases; Carbohydrate Sulfotransferases
PubMed: 35939855
DOI: 10.1016/j.bioorg.2022.106070 -
The Journal of Biological Chemistry Dec 2017Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed drugs worldwide-more than 111 million prescriptions were written in the United...
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed drugs worldwide-more than 111 million prescriptions were written in the United States in 2014. NSAIDs allosterically inhibit cytosolic sulfotransferases (SULTs) with high specificity and therapeutically relevant affinities. This study focuses on the interactions of SULT1A1 and mefenamic acid (MEF)-a potent, highly specific NSAID inhibitor of 1A1. Here, the first structure of an NSAID allosteric site-the MEF-binding site of SULT1A1-is determined using spin-label triangulation NMR. The structure is confirmed by site-directed mutagenesis and provides a molecular framework for understanding NSAID binding and isoform specificity. The mechanism of NSAID inhibition is explored using molecular dynamics and equilibrium and pre-steady-state ligand-binding studies. MEF inhibits SULT1A1 turnover through an indirect (helix-mediated) stabilization of the closed form of the active-site cap of the enzyme, which traps the nucleotide and slows its release. Using the NSAID-binding site structure of SULT1A1 as a comparative model, it appears that 11 of the 13 human SULT isoforms harbor an NSAID-binding site. We hypothesize that these sites evolved to enable SULT isoforms to respond to metabolites that lie within their metabolic domains. Finally, the NSAID-binding site structure offers a template for developing isozyme-specific allosteric inhibitors that can be used to regulate specific areas of sulfuryl-transfer metabolism.
Topics: Allosteric Site; Anti-Inflammatory Agents, Non-Steroidal; Arylsulfotransferase; Cytosol; Humans; Isoenzymes; Magnetic Resonance Spectroscopy; Mefenamic Acid; Protein Binding; Sulfotransferases
PubMed: 29038294
DOI: 10.1074/jbc.M117.817387 -
Journal of Lipid Research Jun 2017PPARα has been known to play a pivotal role in orchestrating lipid, glucose, and amino acid metabolism via transcriptional regulation of its target gene expression...
PPARα has been known to play a pivotal role in orchestrating lipid, glucose, and amino acid metabolism via transcriptional regulation of its target gene expression during energy deprivation. Recent evidence has also suggested that PPARα is involved in bile acid metabolism, but how PPARα modulates the homeostasis of bile acids during fasting is still not clear. In a mechanistic study aiming to dissect the spectrum of PPARα target genes involved in metabolic response to fasting, we identified a novel mouse gene (herein named mL-STL for mouse liver-sulfotransferase-like) that shared extensive homology with the Sult2a subfamily of a superfamily of cytosolic sulfotransferases, implying its potential function in sulfonation. The mL-STL gene expressed predominantly in liver in fed state, but PPARα was required to sustain its expression during fasting, suggesting a critical role of PPARα in regulating the mL-STL-mediated sulfonation during fasting. Functional studies using recombinant His-tagged mL-STL protein revealed its narrow sulfonating activities toward 7α-hydroxyl primary bile acids, including cholic acid, chenodeoxycholic acid, and α-muricholic acid, and thus suggesting that mL-STL may be the major hepatic bile acid sulfonating enzyme in mice. Together, these studies identified a novel PPARα-dependent gene and uncovered a new role of PPARα as being an essential regulator in bile acid biotransformation via sulfonation during fasting.
Topics: Amino Acid Sequence; Animals; Base Sequence; Bile Acids and Salts; Biocatalysis; Biotransformation; Cloning, Molecular; Cytosol; DNA, Complementary; Down-Regulation; Fasting; Liver; Male; Mice; Organ Specificity; PPAR alpha; RNA, Messenger; Substrate Specificity; Sulfotransferases
PubMed: 28442498
DOI: 10.1194/jlr.M074302 -
Molecules (Basel, Switzerland) Oct 2021The aim of this study was to investigate the chemical space and interactions of natural compounds with sulfotransferases (SULTs) using ligand- and structure-based in...
The aim of this study was to investigate the chemical space and interactions of natural compounds with sulfotransferases (SULTs) using ligand- and structure-based in silico methods. An in-house library of natural ligands (hormones, neurotransmitters, plant-derived compounds and their metabolites) reported to interact with SULTs was created. Their chemical structures and properties were compared to those of compounds of non-natural (synthetic) origin, known to interact with SULTs. The natural ligands interacting with SULTs were further compared to other natural products for which interactions with SULTs were not known. Various descriptors of the molecular structures were calculated and analyzed. Statistical methods (ANOVA, PCA, and clustering) were used to explore the chemical space of the studied compounds. Similarity search between the compounds in the different groups was performed with the ROCS software. The interactions with SULTs were additionally analyzed by docking into different experimental and modeled conformations of SULT1A1. Natural products with potentially strong interactions with SULTs were outlined. Our results contribute to a better understanding of chemical space and interactions of natural compounds with SULT enzymes and help to outline new potential ligands of these enzymes.
Topics: Biological Products; Cluster Analysis; Flavonoids; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Structure; Polyphenols; Structure-Activity Relationship; Sulfotransferases
PubMed: 34770768
DOI: 10.3390/molecules26216360 -
International Journal of Biological... 2005Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary...
Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96). Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl(2) for its activity. Maximum TPST activity was observed at 20 mM MnCl(2). The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.
Topics: Chlorides; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Kinetics; Manganese Compounds; Parotid Gland; Reference Values; Saliva; Sulfotransferases; Tyrosine
PubMed: 16244708
DOI: 10.7150/ijbs.1.141 -
The Journal of Biological Chemistry Sep 2001We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated dermatan-4-sulfotransferase-1 (D4ST-1) (GenBank(TM) accession number...
We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated dermatan-4-sulfotransferase-1 (D4ST-1) (GenBank(TM) accession number AF401222) based on its homology to HNK-1 sulfotransferase. The cDNA predicts an open reading frame encoding a type II membrane protein of 376 amino acids with a 43-amino acid cytoplasmic domain and a 316-amino acid luminal domain containing two potential N-linked glycosylation sites. D4ST-1 has significant amino acid identity with HNK-1 sulfotransferase (21.4%), N-acetylgalactosamine-4-O-sulfotransferase 1 (GalNAc-4-ST1) (24.7%), N-acetylgalactosamine-4-O-sulfotransferase 2 (GalNAc-4-ST2) (21.0%), chondroitin-4-O-sulfotransferase 1 (27.3%), and chondroitin-4-O-sulfotransferase 2 (22.8%). D4ST-1 transfers sulfate to the C-4 hydroxyl of beta1,4-linked GalNAc that is substituted with an alpha-linked iduronic acid (IdoUA) at the C-3 hydroxyl. D4ST-1 shows a strong preference in vitro for sulfate transfer to IdoUAalpha1,3GalNAcbeta1,4 that is flanked by GlcUAbeta1,3GalNAcbeta1,4 as compared with IdoUAalpha1,3GalNAcbeta1,4 flanked by IdoUAalpha1,3GalNAcbeta1,4. The specificity of D4ST-1 when assayed in vitro suggests that the addition of sulfate to GalNAc occurs immediately after epimerization of GlcUA to IdoUA. The open reading frame of D4ST-1 is encoded by a single exon located on human chromosome 15q14. Northern blot analysis reveals a single 2.4-kilobase transcript. D4ST-1 message is expressed in virtually all tissues at some level but is most highly expressed in pituitary, placenta, uterus, and thyroid. The properties of D4ST-1 indicate that sulfation of the GalNAc moieties in dermatan is mediated by a distinct GalNAc-4-O-sulfotransferase and occurs following epimerization of GlcUA to IdoUA.
Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; CHO Cells; Carbohydrate Sequence; Chromatography, Gel; Chromosomes, Human, Pair 15; Cloning, Molecular; Cricetinae; DNA, Complementary; Dermatan Sulfate; Dose-Response Relationship, Drug; Exons; Humans; Models, Chemical; Models, Genetic; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Open Reading Frames; Protein Binding; Protein Structure, Tertiary; RNA, Messenger; Sequence Homology, Amino Acid; Sulfotransferases; Time Factors; Tissue Distribution; Transfection
PubMed: 11470797
DOI: 10.1074/jbc.M105848200