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<title>Enzyme assay of sulfotransferases for heparan sulfate - Glycoscience Protocols (GlycoPODv2) - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Glycoscience Protocols (GlycoPODv2) [Internet]" /><meta name="citation_title" content="Enzyme assay of sulfotransferases for heparan sulfate" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2023/02/09" /><meta name="citation_author" content="Hiroko Habuchi" /><meta name="citation_author" content="Koji Kimata" /><meta name="citation_pmid" content="37590667" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593928/" /><meta name="citation_keywords" content="heparan sulfate (HS)" /><meta name="citation_keywords" content="heparin" /><meta name="citation_keywords" content="sulfotransferase" /><meta name="citation_keywords" content="heparan sulfate 6-O-sulfotransferase (HS6ST)" /><meta name="citation_keywords" content="heparan sulfate 2-O-sulfotransferase (HS2ST)" /><meta name="citation_keywords" content="heparan sulfate/heparin GlcNAc N-deacetylase/N-sulfotransferase (NDST)" /><meta name="citation_keywords" content="heparan sulfate/heparin 3-O-sulfotransferase (HS3ST)" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Enzyme assay of sulfotransferases for heparan sulfate" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Hiroko Habuchi" /><meta name="DC.Contributor" content="Koji Kimata" /><meta name="DC.Date" content="2023/02/09" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593928/" /><meta name="description" content="Heparan sulfate 2-O-sulfotransferase (HS2ST) transfers sulfate from 3’-phosphoadenosin 5’-phosphosulfate (PAPS) to position 2 of hexuronic acid residues, preferentially to iduronic acid (IdoA) residues and rarely to glucuronic acid (GlcA) residues, in heparan sulfate and heparin, but not to hexuronic acid (HexA) residues neighboring GlcNSO3(6SO4) residues. (1, 2). HS2ST was purified from the detergent-solubilized extracts of cultured CHO cells and cloned (1, 3). The enzymatic properties of HS2ST are as follows: The optimal pH is around 5.5. The enzyme activity is minutely affected by dithiothreitol (DTT) up to 10 mM. Protamine stimulates the activity to the maximum level at a concentration of 0.05 mg/mL." /><meta name="og:title" content="Enzyme assay of sulfotransferases for heparan sulfate" /><meta name="og:type" content="book" /><meta name="og:description" content="Heparan sulfate 2-O-sulfotransferase (HS2ST) transfers sulfate from 3’-phosphoadenosin 5’-phosphosulfate (PAPS) to position 2 of hexuronic acid residues, preferentially to iduronic acid (IdoA) residues and rarely to glucuronic acid (GlcA) residues, in heparan sulfate and heparin, but not to hexuronic acid (HexA) residues neighboring GlcNSO3(6SO4) residues. (1, 2). HS2ST was purified from the detergent-solubilized extracts of cultured CHO cells and cloned (1, 3). The enzymatic properties of HS2ST are as follows: The optimal pH is around 5.5. The enzyme activity is minutely affected by dithiothreitol (DTT) up to 10 mM. Protamine stimulates the activity to the maximum level at a concentration of 0.05 mg/mL." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK593928/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-glycopodv2-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/glycopodv2/g220-enzasssulfohs/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK593928/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} </style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script>
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK593928_"><span class="title" itemprop="name">Enzyme assay of sulfotransferases for heparan sulfate</span></h1><div class="contrib half_rhythm"><span itemprop="author">Hiroko Habuchi</span>, Doctor of Science<div class="affiliation small">Aichi Medical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.en.tacaidem@ihcubahokorih" class="oemail">pj.en.tacaidem@ihcubahokorih</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Koji Kimata</span>, Doctor of Science<div class="affiliation small">Aichi Medical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-dem-ihcia@atamiK" class="oemail">pj.ca.u-dem-ihcia@atamiK</a></div></div></div><p class="small">Created: <span itemprop="datePublished">October 1, 2021</span>; Last Revision: <span itemprop="dateModified">February 9, 2023</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g220-enzasssulfohs.Introduction"><h2 id="_g220-enzasssulfohs_Introduction_">Introduction</h2><p>Heparan sulfate 2-<i>O</i>-sulfotransferase (HS2ST) transfers sulfate from 3’-phosphoadenosin 5’-phosphosulfate (PAPS) to position 2 of hexuronic acid residues, preferentially to iduronic acid (IdoA) residues and rarely to glucuronic acid (GlcA) residues, in heparan sulfate and heparin, but not to hexuronic acid (HexA) residues neighboring GlcNSO<sub>3</sub>(6SO<sub>4</sub>) residues. (<a class="bk_pop" href="#g220-enzasssulfohs.REF.1">1</a>, <a class="bk_pop" href="#g220-enzasssulfohs.REF.2">2</a>). HS2ST was purified from the detergent-solubilized extracts of cultured CHO cells and cloned (<a class="bk_pop" href="#g220-enzasssulfohs.REF.1">1</a>, <a class="bk_pop" href="#g220-enzasssulfohs.REF.3">3</a>). The enzymatic properties of HS2ST are as follows: The optimal pH is around 5.5. The enzyme activity is minutely affected by dithiothreitol (DTT) up to 10 mM. Protamine stimulates the activity to the maximum level at a concentration of 0.05 mg/mL.</p><p>Heparan sulfate 6-<i>O</i>-sulfotransferase (HS6ST) transfers sulfate from PAPS to position 6 of <i>N</i>-sulfoglucosamine and <i>N</i>-acetylglucosamine residues in heparan sulfate and heparin. HS6ST was purified from the conditioned-medium of cultured CHO cells and cloned (<a class="bk_pop" href="#g220-enzasssulfohs.REF.4">4</a>, <a class="bk_pop" href="#g220-enzasssulfohs.REF.5">5</a>). There are three isoforms: Hs6st-1, -2, -3 and at least 2 spliced forms of Hs6st-2: Hs6st-2S and Hs6st-2L, which are shorter and longer in size than the original form, respectively (<a class="bk_pop" href="#g220-enzasssulfohs.REF.6">6</a>). The substrate specificities of these isoforms greatly overlap, but the individual isoforms reveal characteristic preference for hexuronic acid residue neighboring the <i>N</i>-sulfoglucosamine. HS6ST-1 predominantly sulfates the IdoA-GlcNSO<sub>3</sub> unit. Meanwhile, HS6ST-2 transfers sulfate preferentially to the GlcA-GlcNSO<sub>3</sub> unit and the IdoA(2SO<sub>4</sub>)-GlcNSO<sub>3</sub> unit to produce IdoA(2SO<sub>4</sub>)-GlcNSO<sub>3</sub>(6SO<sub>4</sub>) unit in HS and heparin. The properties of HS6STs are as follows: the optimal pH is around 6.3. Protamine markedly activated the activity. Maximum stimulation by NaCl is around 175 mM. DTT, which has no effect on HS2ST, inhibits HS6ST activity to 19% of the control at a concentration of 10 mM. When CHO cells were cultured, the cell layer retained more than 95% of HS2ST activity, whereas more than 90% of HS6ST activity was secreted into the culture medium. HS2ST activity was rarely detected in mouse serum or human serum, while HS6ST activity was detected.</p><p>Heparan sulfate/heparin GlcNAc <i>N</i>-deacetylase/<i>N</i>-sulfotransferases (NDSTs) catalyzes the initial modification reaction of heparan sulfate and heparin. NDSTs have dual activities that are <i>N</i>-deacetylase and <i>N</i>-sulfotransferase. <i>N</i>-deacetylase activity removes <i>N</i>-acetyl group of GlcNAc residues, and <i>N</i>-sulfotransferase activity transfers sulfate from PAPS to the resulting free amino group of glucosamine. Four isoforms of NDST-1, -2, -3 and -4 are known. The enzymatic properties of these isoforms are markedly different in the ratio of <i>N</i>-deacetylase activity to <i>N</i>-sulfotransferase activity (<a class="bk_pop" href="#g220-enzasssulfohs.REF.7">7</a>, <a class="bk_pop" href="#g220-enzasssulfohs.REF.8">8</a>, <a class="bk_pop" href="#g220-enzasssulfohs.REF.9">9</a>).</p><p>Heparan sulfate/heparin 3-<i>O</i>-sulfotransferase (HS3ST) transfers sulfate from PAPS to position 3 of glucosamine residue and is present in seven isoforms (HS3ST-1, -2, -3A, -3B, -4, -5, -6) in vertebrates. Unlike NSO<sub>3</sub>, 2SO<sub>4</sub> and 6SO<sub>4</sub> 3-<i>O</i>-sulfation is rarely found in HS/heparin, and its occurrence is generally one site or less per chain (<a class="bk_pop" href="#g220-enzasssulfohs.REF.10">10</a>). The 3SO<sub>4</sub> residues transferred by HS3STs are found in various disaccharide units such as HexA-GlcNSO<sub>3</sub>(3SO<sub>4</sub>), HexA(2SO<sub>4</sub>)-GlcNSO<sub>3</sub>(3SO<sub>4</sub>), HexA-GlcNSO<sub>3</sub>(3SO<sub>4</sub>,6SO<sub>4</sub>), and HexA(2SO<sub>4</sub>)-GlcNSO<sub>3</sub>(3SO<sub>4</sub>,6SO<sub>4</sub>). HS3ST-1 produces GlcA-GlcNSO<sub>3</sub>(3SO<sub>4</sub>±6SO<sub>4</sub>) unit, which is the essential component of the antithrombin (AT)-binding domain. In contrast, HS3ST-2, -3, and -4 preferentially produce HexA(2SO<sub>4</sub>)-GlcNSO<sub>3</sub>(3SO<sub>4</sub>) and HexA(2SO<sub>4</sub>)-GlcNSO<sub>3</sub>(3SO<sub>4</sub>,6SO<sub>4</sub>). HS3ST-5 shows activities to form all these disaccharide units (<a class="bk_pop" href="#g220-enzasssulfohs.REF.11">11</a>, <a class="bk_pop" href="#g220-enzasssulfohs.REF.12">12</a>).</p></div><div id="g220-enzasssulfohs.Protocol"><h2 id="_g220-enzasssulfohs_Protocol_">Protocol</h2><p>This protocol is mainly applicable to the cellular extracts from the cultured cells but enzyme activities of the culture media and serum are also detected by this protocol.</p><div id="g220-enzasssulfohs.Materials"><h3>Materials</h3><p>1. [<sup>35</sup>S]PAPS (1x10<sup>4</sup> cpm/pmol)(PerkinElmerA, Waltham, MA.)</p><p>2. Chondrotin sulfate A (Sigma-Aldrich, St. Louis, MO)</p><p>3. Heparan sulfate (Sigma Aldrich, St. Louis, MO)</p><p>4. <i>N</i>-desulfated heparin (Note 1)</p><p>5. Unsaturated heparan/heparin disaccharide mixture (Sigma-Aldrich, St. Louis, MO.)</p><p>6. Heparinase I, Heparinase II,Heparinase III (Sigma-Aldrich, St. Louis, MO.)</p><p>7. Liquid scintillation cocktail (PerkinElmer, Waltham, MA.)</p></div><div id="g220-enzasssulfohs.Instruments"><h3>Instruments</h3><p>1. YMC-pack Polyamine II column (4.6 mm x 25 cm) (YMC Co., Ltd., Kyoto, Japan)</p><p>2. HPLC system</p><p>3. Desalting column (1.0 x 10 cm) filled with Sephadex G-25 superfine (Note 2)</p><p>4. Sephadex G-50 medium (Sigma-Aldrich)</p><p>5. Liquid scintillation counter</p></div><div id="g220-enzasssulfohs.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Enzyme assay of heparan sulfate 2-<i>O</i>-sulfotransferase (HS2ST), heparan sulfate 6-<i>O</i>-sulfotransferases (HS6STs) and heparan sulfate <i>N</i>-sulfotransferases (NDSTs)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare HS-sulfotransferase from cells (Note 3)</p><p>Wash the cell layer with PBS (-) and then scrap the cells off the dish in 2 ml of buffer containing 10 mM Tris-HCl, pH 7.2, 0.5% (w/v) Triton X-100, 10 mM MgCl<sub>2</sub>, 2 mM CaCl<sub>2</sub>, 0.15 M NaCl, 20% (v/v) glycerol, and a mixture of protease inhibitors (5mM <i>N</i>a-<i>p</i>-tosyl-L-lysine chloromethyl ketone, 3 mM <i>N</i>-tosyl-L-phenylalaninechloromethyl ketone, 30 mM phenylmethylsulfonyl fluoride, and 3 mM pepstatin A). Homogenize the cell suspension with a glass homogenizer on ice. After stirring at 4°C for 1h, centrifuged at 4°C for 30 min at 10,000 <i>g</i>. Use the supernatant as the enzyme.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Prepare the reaction mixture (50 μL) containing 2.5 μmol of imidazole-HCl, pH 5.6 (for HS2ST) or pH 6.8 (for HS6STs), 3.75 μg of protamine chloride, 25 nmol (as hexuronic acid) of heparan sulfate, 50 pmol [<sup>35</sup>S]PAPS (about 5 × 10<sup>5</sup> cpm), 5 μL of 1 M NaCl in buffer A (10 mM Tris-HCl, pH 7.2, 0.1% Trton X-100, 10 mM MgCl<sub>2</sub>, 2 mM CaCl<sub>2</sub>, 20% (V/V) glycerol), and enzyme (Note 4). As to NDSTs, the reaction mixture (50 μL) contains 2.5 μmol of HEPES, pH 7.0, 0.1% Triton X-100, 0.5 μmol MgCl<sub>2</sub>, 0.05 μmol MnCl<sub>2</sub>, 25 μg of <i>N</i>-desulfated heparin, 50 pmol [<sup>35</sup>S]PAPS (about 5 × 10<sup>5</sup> cpm), and enzyme (Note 4).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Incubate at 37°C for 20 min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Stop the reaction by heating at 100°C for 1min.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add chondroitin sulfate A (0.1 μmol as GlcA) to the reaction mixture as carrier and vortex well.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add 2.5 vol. of cold 95% ethanol/1.3% (w/v) potassium acetate/0.5 mM EDTA to the reaction mixture, and vortex well. Keep the mixtures at −20ºC for 30 min.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Precipitate <sup>35</sup>S-labeled polysaccharides by centrifugation at 10,000 × <i>g</i> for 30 min and discard the supernatant.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Dissolve the precipitates with 70 μL of distilled water and vortex well.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Inject 50 μL of the solution into a desalting column equilibrated with 0.1 M NH<sub>4</sub>HCO<sub>3</sub> at a flow rate of 2 mL/min. (Note 2)</p></dd><dt>j.</dt><dd><p class="no_top_margin">Collect the void volume to recover <sup>35</sup>S-labeled products and mix with 3 mL of scintillator, and determine the radioactivity by a liquid scintillation counter.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Determination of sulfated positions in the products</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Digest <sup>35</sup>S-labeled products with a mixture of 5 milliunits of heparitinase I, 2.5 milliunits of heparitinase II, and 5 milliunits of heparinase in 50 µL of 50 mM Tris-HCl, pH 7.2, 1 mM CaCl<sub>2</sub>, and 5 µg of bovine serum albumin at 37ºC for 2 h.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Stop the reaction by heating at 100ºC for 1 min.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Inject the digests together with 1 nmole of standard unsaturated disaccharides into a column of YMC-Pack Polyamine II. HPLC is performed and the elution is fractionated into 0.6 mL portions to separate unsaturated disaccharide. (Note 5)</p></dd><dt>d.</dt><dd><p class="no_top_margin">Each fraction is mixed with 3 mL of liquid scintillation cocktail. Determine the radioactivity of the fractions containing ∆HexA(2SO<sub>4</sub>)-GlcNSO<sub>3</sub> (for HS2ST), ∆HexA-GlcNSO<sub>3</sub>(6SO<sub>4</sub>), ∆HexA(2SO<sub>4</sub>)-GlcNSO<sub>3</sub>(6SO<sub>4</sub>) (for HS6STs), and ∆HexA-GlcNSO<sub>3</sub> (for NDSTs), respectively.</p></dd></dl></dd></dl></div><div id="g220-enzasssulfohs.Notes"><h3>Notes</h3><p>1. Chemically <i>N</i>-desulfated heparin is obtained from heparin (Sigma-Aldrich) by solvolysis with DMSO. Solvolysis with 5% (v/v) methanol in DMSO is performed at 50ºC for 1.5 h. (<a class="bk_pop" href="#g220-enzasssulfohs.REF.13">13</a>) or commercially available (Iduron (DSH003/N), ambio (AMS.DSH003-N)</p><p>2. The desalting column (1.0 cm x 10 cm) is prepared by packing Sephadex G-25 superfine suspended in 0.1 M NaCl at a flow rate of 6 mL/min. This column is equilibrated with 0.1 M NH<sub>4</sub>HCO<sub>3</sub> and run at a flow rate of 2 mL/min. HiTrap® Desalting Columns (1.6 x 2.5 cm, GE17-1408-01, Cytiva 17-1408-01) is commercially available. Instead of the desalting column, a spin column can be utilized. The spin column (bed volume 0.9 mL) is made by packing Sephadex G-50 medium suspended in 0.1 M NH<sub>4</sub>HCO<sub>3</sub> into 1 mL syringe under centrifugation at 2000 rpm for 4 min. Samples (100 μL) are loaded to the top of the gel and centrifuged at 2000 rpm for 4 min. [<sup>35</sup>S]Glycosaminoglycans are recovered in the flow through fractions.</p><p>3. Major activities of HS6STs are secreted to the culture medium, but the enzyme activities of the cellular extracts are also detected by this protocol. Culture medium generally contains fetal calf serum, which includes significant HS6ST activity. Therefore, cellular extracts are better enzyme sources for obtaining the more accurate data. As to the sensitivity of this assay, crude extract from 1 × 10<sup>5</sup> fibroblasts (no transfection, endogenous activity) is enough to determine HS2ST and HS6ST activities.</p><p>4. When crude extracts are used as enzyme, 1 μmole NaF and 0.1 μmole AMP are added to the reaction mixture to prevent degradation of 3’-phosphoadenosin 5’-phosphosulfate (PAPS).</p><p>5. Elute by a linear gradient from 40 mM- to 550 mM-KH<sub>2</sub>PO<sub>4</sub>, and a subsequent elution with 550 mm-KH<sub>2</sub>PO<sub>4</sub> at a flow rate of 1.2 ml/min at 40°C. The elution is monitored by UV absorption at 232 nm. Each peak is identified by comparison of its retention time with those of standard unsaturated disaccharides.</p></div></div><div id="g220-enzasssulfohs.References"><h2 id="_g220-enzasssulfohs_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.1">Kobayashi M, Habuchi H, Habuchi O, Saito M, Kimata K. Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells. <span><span class="ref-journal">J Biol Chem. </span>1996 Mar 29;<span class="ref-vol">271</span>(13):7645–53.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8631801" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8631801</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.271.13.7645" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.2">Liu C, Sheng J, Krahn JM, Perera L, Xu Y, Hsieh PH, Dou W, Liu J, Pedersen LC. Molecular mechanism of substrate specificity for heparan sulfate 2-O-sulfotransferase. <span><span class="ref-journal">J Biol Chem. </span>2014 May 9;<span class="ref-vol">289</span>(19):13407–18.</span> Epub 2014 Mar 20. PMID. [<a href="/pmc/articles/PMC4036349/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4036349</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/24652287" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24652287</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M113.530535" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.3">Kobayashi M, Habuchi H, Yoneda M, Habuchi O, Kimata K. Molecular cloning and expression of Chinese hamster ovary cell heparan-sulfate 2-sulfotransferase. <span><span class="ref-journal">J Biol Chem. </span>1997 May 23;<span class="ref-vol">272</span>(21):13980–5.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9153262" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9153262</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.272.21.13980" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.4">Habuchi H, Habuchi O, Kimata K. Purification and characterization of heparan sulfate 6-sulfotransferase from the culture medium of Chinese hamster ovary cells. <span><span class="ref-journal">J Biol Chem. </span>1995 Feb 24;<span class="ref-vol">270</span>(8):4172–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7876170" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7876170</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.270.8.4172" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.5">Habuchi H, Kobayashi M, Kimata K. Molecular characterization and expression of heparan-sulfate 6-sulfotransferase. Complete cDNA cloning in human and partial cloning in Chinese hamster ovary cells. <span><span class="ref-journal">J Biol Chem. </span>1998 Apr 10;<span class="ref-vol">273</span>(15):9208–13.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9535912" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9535912</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.273.15.9208" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.6">Habuchi H, Tanaka M, Habuchi O, Yoshida K, Suzuki H, Ban K, Kimata K. The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine. <span><span class="ref-journal">J Biol Chem. </span>2000 Jan 28;<span class="ref-vol">275</span>(4):2859–68.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10644753" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10644753</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.275.4.2859" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.7">Aikawa J, Grobe K, Tsujimoto M, Esko JD. Multiple isozymes of heparan sulfate/heparin GlcNAc N-deacetylase/GlcN N-sulfotransferase. Structure and activity of the fourth member, NDST4. <span><span class="ref-journal">J Biol Chem. </span>2001 Feb 23;<span class="ref-vol">276</span>(8):5876–82.</span> Epub 2000 Nov 21. PMID. [<a href="https://pubmed.ncbi.nlm.nih.gov/11087757" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11087757</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M009606200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.8">Kjellén L. Glucosaminyl N-deacetylase/N-sulphotransferases in heparan sulphate biosynthesis and biology. <span><span class="ref-journal">Biochem Soc Trans. </span>2003 Apr;<span class="ref-vol">31</span>(2):340–2.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12653633" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12653633</span></a>] [<a href="http://dx.crossref.org/10.1042/bst0310340" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.9">Carlsson P, Presto J, Spillmann D, Lindahl U, Kjellén L. Heparin/heparan sulfate biosynthesis: processive formation of N-sulfated domains. <span><span class="ref-journal">J Biol Chem. </span>2008 Jul 18;<span class="ref-vol">283</span>(29):20008–14.</span> Epub 2008 May 16. PMID. [<a href="https://pubmed.ncbi.nlm.nih.gov/18487608" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18487608</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M801652200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.10">Thacker BE, Xu D, Lawrence R, Esko JD. Heparan sulfate 3-O-sulfation: a rare modification in search of a function. <span><span class="ref-journal">Matrix Biol. </span>2014 Apr;<span class="ref-vol">35</span>:60–72.</span> Epub 2013 Dec 19. PMIDReview. [<a href="/pmc/articles/PMC4039620/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4039620</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/24361527" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24361527</span></a>] [<a href="http://dx.crossref.org/10.1016/j.matbio.2013.12.001" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.11">Mochizuki H, Yoshida K, Shibata Y, Kimata K. Tetrasulfated disaccharide unit in heparan sulfate: enzymatic formation and tissue distribution. <span><span class="ref-journal">J Biol Chem. </span>2008 Nov 7;<span class="ref-vol">283</span>(45):31237–45.</span> Epub 2008 Aug 28. PMID. [<a href="/pmc/articles/PMC2662186/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2662186</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18757372" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18757372</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M801586200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.12">Mochizuki H, Futatsumori H, Suzuki E, Kimata K. A quantitative method to detect non-antithrombin-binding 3-O-sulfated units in heparan sulfate. <span><span class="ref-journal">J Biol Chem. </span>2021 Jan-Jun;<span class="ref-vol">296</span>:100115. </span> Epub 2020 Dec 3. PMID. [<a href="/pmc/articles/PMC7948761/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC7948761</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/33234593" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 33234593</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.RA120.015864" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="g220-enzasssulfohs.REF.13">Inoue Y, Nagasawa K. Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol. <span><span class="ref-journal">Carbohydr Res. </span>1976 Jan;<span class="ref-vol">46</span>(1):87–95.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1248016" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1248016</span></a>] [<a href="http://dx.crossref.org/10.1016/s0008-6215(00)83533-8" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593928_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g220-enzasssulfohs.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div id="bk_toc_contnr"></div></div></div>
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