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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 -
The Journal of Biological Chemistry Jun 2011Heparan sulfate (HS) is a highly sulfated polysaccharide participated in essential physiological functions from regulating cell growth to blood coagulation. HS contains...
Heparan sulfate (HS) is a highly sulfated polysaccharide participated in essential physiological functions from regulating cell growth to blood coagulation. HS contains sulfated domains known as N-S domains and low sulfate domains known as N-Ac domains. The distribution of the domain structures is likely governed by the action of glucosaminyl N-deacetylase/N-sulfotransferase (NDST). Here, we sought to determine the substrate specificity of NDST using model substrates and recombinant NDST protein. We discovered that NDST-1 carries out the modification in a highly ordered fashion. The enzyme sulfates the substrate from the nonreducing end toward the reducing end consecutively, leading to the product with a cluster of N-sulfo glucosamine residues. Furthermore, a preexisting N-sulfo glucosamine residue prevents the action of NDST-1 at the residues immediately located at the nonreducing end, allowing the formation of an N-Ac domain. Our results provide the long sought evidence for understanding the formation of sulfated versus nonsulfated domains in the HS isolated from cells and tissues. The study demonstrates the regulating role of NDST-1 in mapping the sulfation patterns of HS.
Topics: Animals; Carbohydrate Conformation; Heparitin Sulfate; Protein Structure, Tertiary; Rats; Sulfotransferases
PubMed: 21454625
DOI: 10.1074/jbc.M111.224311 -
Biochemical Society Transactions Apr 2003The glycoprotein hormones lutropin (LH) and thyrotropin and a limited number of additional glycoproteins bear carbohydrate structures terminating with the unique... (Review)
Review
The glycoprotein hormones lutropin (LH) and thyrotropin and a limited number of additional glycoproteins bear carbohydrate structures terminating with the unique sequence SO(4)-4-GalNAcbeta1,4GlcNAcbeta that has been conserved in the glycoprotein hormones of all vertebrate species. Synthesis of these structures is mediated by a protein-specific beta1,4GalNAc-transferase and a GalNAc-4-sulphotransferase (GalNAc-4-ST1). GalNAc-4-ST1 is a member of a family of sulphotransferases that includes HNK-1 sulphotransferase, chondroitin-4-sulphotransferases-1-3 and dermatan-4-sulphotransferase-1. With the exception of HNK-1-ST, these sulphotransferases add sulphate to the C-4 hydroxy group of either terminal or non-terminal beta1,4-linked GalNAc. GalNAc-4-ST1 is most highly expressed in pituitary, cerebellum and other regions of the brain. The terminal GalNAcSO(4) on LH is recognized by the cysteine-rich domain of the mannose/GalNAc-4-SO(4) receptor located in hepatic endothelial cells. Each cysteine-rich domain binds a single terminal GalNAc-4-SO(4), and the receptor must form non-covalently associated homodimers in order to simultaneously engage two GalNAc-4-SO(4) moieties on separate oligosaccharides with sufficient affinity to form stable complexes. The receptor mediates the clearance of LH from the blood. This clearance, in conjunction with the stimulated release of hormone from dense core granules in pituitary gonadotroph cells, is required to produce the episodic rise and fall in LH levels needed for optimal oestrogen production during the implantation of embryos in the uterus.
Topics: Acetylgalactosamine; Animals; Glycoproteins; Hormones; Humans; Oligosaccharides; Sulfotransferases; Tissue Distribution
PubMed: 12653629
DOI: 10.1042/bst0310326 -
Glycoconjugate Journal Oct 2018Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated...
Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-β-GalNAc derivatives and 4-azido-β-GalNAc derivatives were synthesized for their potential application as inhibitors of GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed that 4-pivaloylamino- and 4-azido-β-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC value ranging from 0.800 to 0.828 mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.
Topics: Amides; Animals; Enzyme Inhibitors; Galactosamine; Humans; Sulfotransferases
PubMed: 30173355
DOI: 10.1007/s10719-018-9839-2 -
European Journal of Drug Metabolism and... Aug 2018Previous studies have demonstrated the metabolism of tibolone through sulfation, with the cytosolic sulfotransferase (SULT) SULT2A1 as the major responsible enzyme. The...
BACKGROUND AND OBJECTIVES
Previous studies have demonstrated the metabolism of tibolone through sulfation, with the cytosolic sulfotransferase (SULT) SULT2A1 as the major responsible enzyme. The current study aimed to investigate how SULT2A1 genetic polymorphisms may affect the dehydroepiandrosterone (DHEA)- and tibolone-sulfating activity of SULT2A1.
METHODS
Site-directed mutagenesis was employed to generate cDNAs encoding ten different SULT2A1 allozymes. Recombinant SULT2A1 allozymes were expressed in BL21 E. coli cells, and purified using glutathione-sepharose affinity chromatography. An established sulfotransferase assay was used to analyze DHEA- and tibolone-sulfating activity of the purified SULT2A1 allozymes.
RESULTS
The nine human SULT2A1 allozymes plus the wild-type SULT2A1 were found to display differential sulfating activity toward DHEA and tibolone. Kinetic analysis revealed that different SULT2A1 allozymes exhibited differential substrate affinity and catalytic efficiency toward the two substrates tested.
CONCLUSION
The results obtained provided useful information concerning the differential metabolism of tibolone through sulfation in individuals with different SULT2A1 genotypes.
Topics: Cells, Cultured; Dehydroepiandrosterone; Humans; Isoenzymes; Kinetics; Mutagenesis, Site-Directed; Norpregnenes; Polymorphism, Genetic; Sulfotransferases
PubMed: 29392568
DOI: 10.1007/s13318-017-0458-2 -
The Journal of Biological Chemistry Dec 2000Based on sequence homology with a previously cloned human GlcNAc 6-O-sulfotransferase, we have identified an open reading frame (ORF) encoding a novel member of the...
Based on sequence homology with a previously cloned human GlcNAc 6-O-sulfotransferase, we have identified an open reading frame (ORF) encoding a novel member of the Gal/GalNAc/GlcNAc 6-O-sulfotransferase (GST) family termed GST-5 on the human X chromosome (band Xp11). GST-5 has recently been characterized as a novel GalNAc 6-O-sulfotransferase termed chondroitin 6-sulfotransferase-2 (Kitagawa, H., Fujita, M., Itio, N., and Sugahara K. (2000) J. Biol. Chem. 275, 21075-21080). We have coexpressed a human GST-5 cDNA with a GlyCAM-1/IgG fusion protein in COS-7 cells and observed four-fold enhanced [(35)S]sulfate incorporation into this mucin acceptor. All mucin-associated [(35)S]sulfate was incorporated as GlcNAc-6-sulfate or Galbeta1-->4GlcNAc-6-sulfate. GST-5 was also expressed in soluble epitope-tagged form and found to catalyze 6-O-sulfation of GlcNAc residues in synthetic acceptor structures. In particular, GST-5 was found to catalyze 6-O-sulfation of beta-benzyl GlcNAc but not alpha- or beta-benzyl GalNAc. In the mouse genome we have found a homologous ORF that predicts a novel murine GlcNAc 6-O-sulfotransferase with 88% identity to the human enzyme. This gene was mapped to mouse chromosome X at band XA3.1-3.2. GST-5 is the newest member of an emerging family of carbohydrate 6-O-sulfotransferases that includes chondroitin 6-sulfotransferase (GST-0), keratan-sulfate galactose 6-O-sulfotransferase (GST-1), the ubiquitously expressed GlcNAc 6-O-sulfotransferase (GST-2), high endothelial cell GlcNAc 6-O-sulfotransferase (GST-3), and intestinal GlcNAc 6-O-sulfotransferase (GST-4).
Topics: Acetylglucosamine; Amino Acid Sequence; Animals; Base Sequence; Catalysis; Chromosome Mapping; DNA Primers; DNA, Complementary; Isoenzymes; Mice; Molecular Sequence Data; Open Reading Frames; Sulfates; Sulfotransferases; X Chromosome; Carbohydrate Sulfotransferases
PubMed: 10956661
DOI: 10.1074/jbc.M006414200 -
Molecules (Basel, Switzerland) Jan 2015Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein... (Review)
Review
Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS) is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST) through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.
Topics: Amino Acid Sequence; Animals; Enzyme Assays; Humans; Kinetics; Molecular Sequence Data; Protein Processing, Post-Translational; Sulfotransferases; Tyrosine
PubMed: 25635379
DOI: 10.3390/molecules20022138 -
Proceedings of the Japan Academy.... 2013Sulfation confers negative charge on glycolipids and the attached sulfate group presents a part of determinants for the molecular interactions. Mammalian... (Review)
Review
Sulfation confers negative charge on glycolipids and the attached sulfate group presents a part of determinants for the molecular interactions. Mammalian sulfoglycolipids are comprised of two major members, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol). Sulfatide is abundant in the myelin sheath and seminolipid is unique to the spermatogenic cells. The carbohydrate moiety of sulfatide and seminolipid is biosynthesized via sequential reactions catalyzed by common enzymes: ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST). To elucidate the biological function of sulfoglycolipids, we have purified CST, cloned the CST gene, and generated CST-knockout mice. CST-null mice completely lack sulfoglycolipids all over the body. CST-null mice manifest some neurological disorders due to myelin dysfunction, an aberrant enhancement of oligodendrocyte terminal differentiation, and an arrest of spermatogenesis. CST-deficiency ameliorates L-selectin-dependent monocyte infiltration in the renal interstitial inflammation, indicating that sulfatide is an endogenous ligand of L-selectin. Studies on the molecular mechanisms underlying the biological events for which sulfoglycolipids are essential are ongoing
Topics: Animals; Cloning, Molecular; Gene Expression Regulation, Enzymologic; Gene Targeting; Glycolipids; Humans; Sulfotransferases
PubMed: 23574804
DOI: 10.2183/pjab.89.129 -
Drug Metabolism and Disposition: the... Feb 2008The Caco-2 cell line and its subclone TC7 are frequently used for studying human intestinal transport and metabolism of xenobiotics. We have investigated the expression... (Comparative Study)
Comparative Study
The Caco-2 cell line and its subclone TC7 are frequently used for studying human intestinal transport and metabolism of xenobiotics. We have investigated the expression of soluble sulfotransferases (SULT) in parental Caco-2 and TC7 cells by immunoblotting. SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C1, SULT1C2, and SULT2A1 were expressed in both cell lines. SULT2B1a, SULT2B1b, and SULT4A1 were absent. SULT1E1 protein was found in TC7 but not in Caco-2 cells. Other differences in SULT between the cell lines were minor. More important was the influence of differentiation. Expression of the various SULT forms was low or not detectable in cultures just reaching confluence but then increased strongly. Likewise, the rate of sulfation of the model substrate 3-hydroxybenzo[a]pyrene was increased with increasing culture duration. Benzo[a]pyrene-1-sulfate and -3-sulfate were formed in both cell lines when benzo[a]pyrene was used as a substrate. A further metabolite, 3-hydroxybenzo[a]pyrene-glucuronide, was detected in TC7 but not in parental Caco-2 cells. Cytochrome P450 inducers enhanced the conversion of benzo[a]pyrene to these metabolites without altering mRNA levels of major phenol-conjugating SULT forms (SULT1A1, SULT1A3, and SULT1B1). Overall, differentiated Caco-2 and TC7 cells are rich sources of SULT, as is human intestinal mucosa. The SULT pattern is most similar to that found in small intestine, although levels of SULT1A1 and SULT1B1 are lower, and those of SULT1C1 are higher in Caco-2 and TC7 cells than previously found in intestinal samples. The differentiation-dependent expression of SULT in the cultured cells reflects the in vivo situation, where SULT expression is focused to differentiated enterocytes.
Topics: Caco-2 Cells; Cell Differentiation; Cell Line, Tumor; Genotype; Humans; Polycyclic Aromatic Hydrocarbons; RNA, Messenger; Sulfotransferases
PubMed: 17967930
DOI: 10.1124/dmd.107.018036 -
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