108 lines
No EOL
39 KiB
XML
108 lines
No EOL
39 KiB
XML
<?xml version="1.0" encoding="utf-8"?>
|
||
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
|
||
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">
|
||
|
||
<head><meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
|
||
<!-- AppResources meta begin -->
|
||
<meta name="paf-app-resources" content="" />
|
||
<script type="text/javascript">var ncbi_startTime = new Date();</script>
|
||
|
||
<!-- AppResources meta end -->
|
||
|
||
<!-- TemplateResources meta begin -->
|
||
<meta name="paf_template" content="" />
|
||
|
||
<!-- TemplateResources meta end -->
|
||
|
||
<!-- Logger begin -->
|
||
<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK594009" /><meta name="ncbi_domain" content="glycopodv2" /><meta name="ncbi_report" content="printable" /><meta name="ncbi_type" content="fulltext" /><meta name="ncbi_objectid" content="" /><meta name="ncbi_pcid" content="/NBK594009/?report=printable" /><meta name="ncbi_app" content="bookshelf" />
|
||
<!-- Logger end -->
|
||
|
||
<title>Enzyme assay of sulfotransferases for chondroitin/dermatan - Glycoscience Protocols (GlycoPODv2) - NCBI Bookshelf</title>
|
||
|
||
<!-- AppResources external_resources begin -->
|
||
<link rel="stylesheet" href="/core/jig/1.15.2/css/jig.min.css" /><script type="text/javascript" src="/core/jig/1.15.2/js/jig.min.js"></script>
|
||
|
||
<!-- AppResources external_resources end -->
|
||
|
||
<!-- Page meta begin -->
|
||
<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 chondroitin/dermatan" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2023/02/09" /><meta name="citation_author" content="Osami Habuchi" /><meta name="citation_pmid" content="37590734" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK594009/" /><meta name="citation_keywords" content="C4ST" /><meta name="citation_keywords" content="C6ST" /><meta name="citation_keywords" content="D4ST" /><meta name="citation_keywords" content="CHST3" /><meta name="citation_keywords" content="CHST11" /><meta name="citation_keywords" content="CHST14" /><meta name="citation_keywords" content="sulfotransferase" /><meta name="citation_keywords" content="dermatan sulfate" /><meta name="citation_keywords" content="chondroitin sulfate" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Enzyme assay of sulfotransferases for chondroitin/dermatan" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Osami Habuchi" /><meta name="DC.Date" content="2023/02/09" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK594009/" /><meta name="description" content="Chondroitin 4-sulfotransferase (C4ST) and chondroitin 6-sulfotransferase (C6ST) transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to position 4 and 6, respectively, of N-acetylgalactosamine (GalNAc) residues of chondroitin and are involved in the biosynthesis of chondroitin sulfate A (CS-A) and C (1-3). Three isoforms of C4ST: C4ST-1 (CHST11), C4ST-2 (CHST12) and C4ST-3 (CHST13) and two isoforms of C6ST: C6ST-1 (CHST3) and C6ST-2 (CHST7) are known. C4ST-1 and C6ST-1 are able to sulfate dermatan, but these enzymes have distinction in the recognition of uronic acid residues adjacent to the targeted GalNAc residue. C4ST-1 transfers sulfate preferably to GalNAc residues adjacent to the reducing side of the GlcA residue, but C6ST-1 does not show any requirement for the presence of Glucuronic acid (GlcA) residues (4). Dermatan 4-sulfotransferase (D4ST-1) preferentially transfers sulfate to position 4 of GalNAc residues of dermatan and is involved in the biosynthesis of dermatan sulfate (5). In C4ST-1 deficient mice, multiple skeletal abnormalities are observed, and the morphology of growth plates is disturbed (6). Deficiency in C4ST-1 in human causes a variety of limb malformations and skeletal defects (7). Deficiency in D4ST-1 (CHST14) in human causes musculocontractural Ehlers-Danlos syndrome (8). C6ST-1 has been implicated in experimental autoimmune encephalomyelitis (9), temporal lobe epilepsy (10), and neuroplasticity and memory in aging (11). Deficiency in C6ST-1 in human causes spondyloepiphyseal dysplasia with severe skeletal abnormality (12)." /><meta name="og:title" content="Enzyme assay of sulfotransferases for chondroitin/dermatan" /><meta name="og:type" content="book" /><meta name="og:description" content="Chondroitin 4-sulfotransferase (C4ST) and chondroitin 6-sulfotransferase (C6ST) transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to position 4 and 6, respectively, of N-acetylgalactosamine (GalNAc) residues of chondroitin and are involved in the biosynthesis of chondroitin sulfate A (CS-A) and C (1-3). Three isoforms of C4ST: C4ST-1 (CHST11), C4ST-2 (CHST12) and C4ST-3 (CHST13) and two isoforms of C6ST: C6ST-1 (CHST3) and C6ST-2 (CHST7) are known. C4ST-1 and C6ST-1 are able to sulfate dermatan, but these enzymes have distinction in the recognition of uronic acid residues adjacent to the targeted GalNAc residue. C4ST-1 transfers sulfate preferably to GalNAc residues adjacent to the reducing side of the GlcA residue, but C6ST-1 does not show any requirement for the presence of Glucuronic acid (GlcA) residues (4). Dermatan 4-sulfotransferase (D4ST-1) preferentially transfers sulfate to position 4 of GalNAc residues of dermatan and is involved in the biosynthesis of dermatan sulfate (5). In C4ST-1 deficient mice, multiple skeletal abnormalities are observed, and the morphology of growth plates is disturbed (6). Deficiency in C4ST-1 in human causes a variety of limb malformations and skeletal defects (7). Deficiency in D4ST-1 (CHST14) in human causes musculocontractural Ehlers-Danlos syndrome (8). C6ST-1 has been implicated in experimental autoimmune encephalomyelitis (9), temporal lobe epilepsy (10), and neuroplasticity and memory in aging (11). Deficiency in C6ST-1 in human causes spondyloepiphyseal dysplasia with severe skeletal abnormality (12)." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK594009/" /><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/g218-enzasssulfocd/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK594009/" /><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>
|
||
|
||
<!-- Page meta end -->
|
||
<link rel="shortcut icon" href="//www.ncbi.nlm.nih.gov/favicon.ico" /><meta name="ncbi_phid" content="CE8CA7FF7D6682710000000000CB00A9.m_5" />
|
||
<meta name='referrer' content='origin-when-cross-origin'/><link type="text/css" rel="stylesheet" href="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/css/3852956/3985586/3808861/4121862/3974050/3917732/251717/4216701/14534/45193/4113719/3849091/3984811/3751656/4033350/3840896/3577051/3852958/3984801/12930/3964959.css" /><link type="text/css" rel="stylesheet" href="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/css/3411343/3882866.css" media="print" /></head>
|
||
<body class="book-part">
|
||
<div class="grid no_max_width">
|
||
<div class="col twelve_col nomargin shadow">
|
||
<!-- System messages like service outage or JS required; this is handled by the TemplateResources portlet -->
|
||
<div class="sysmessages">
|
||
<noscript>
|
||
<p class="nojs">
|
||
<strong>Warning:</strong>
|
||
The NCBI web site requires JavaScript to function.
|
||
<a href="/guide/browsers/#enablejs" title="Learn how to enable JavaScript" target="_blank">more...</a>
|
||
</p>
|
||
</noscript>
|
||
</div>
|
||
<!--/.sysmessage-->
|
||
<div class="wrap">
|
||
<div class="page">
|
||
<div class="top">
|
||
|
||
<div class="header">
|
||
|
||
|
||
</div>
|
||
|
||
|
||
|
||
<!--<component id="Page" label="headcontent"/>-->
|
||
|
||
</div>
|
||
<div class="content">
|
||
<!-- site messages -->
|
||
<div class="container content">
|
||
<div class="document">
|
||
<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>
|
||
<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK594009_"><span class="title" itemprop="name">Enzyme assay of sulfotransferases for chondroitin/dermatan</span></h1><div class="contrib half_rhythm"><span itemprop="author">Osami Habuchi</span>, Doctor of Science, Ph.D.<div class="affiliation small">Multidisciplinary Pain Center, Aichi Medical Univ<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.ude-ihcia.cceua@ihcubaho" class="oemail">pj.ca.ude-ihcia.cceua@ihcubaho</a></div></div><div class="small">Corresponding author.</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="g218-enzasssulfocd.Introduction"><h2 id="_g218-enzasssulfocd_Introduction_">Introduction</h2><p>Chondroitin 4-sulfotransferase (C4ST) and chondroitin 6-sulfotransferase (C6ST) transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to position 4 and 6, respectively, of <i>N</i>-acetylgalactosamine (GalNAc) residues of chondroitin and are involved in the biosynthesis of chondroitin sulfate A (CS-A) and C (<a class="bk_pop" href="#g218-enzasssulfocd.REF.1">1</a>-<a class="bk_pop" href="#g218-enzasssulfocd.REF.3">3</a>). Three isoforms of C4ST: C4ST-1 (CHST11), C4ST-2 (CHST12) and C4ST-3 (CHST13) and two isoforms of C6ST: C6ST-1 (CHST3) and C6ST-2 (CHST7) are known. C4ST-1 and C6ST-1 are able to sulfate dermatan, but these enzymes have distinction in the recognition of uronic acid residues adjacent to the targeted GalNAc residue. C4ST-1 transfers sulfate preferably to GalNAc residues adjacent to the reducing side of the GlcA residue, but C6ST-1 does not show any requirement for the presence of Glucuronic acid (GlcA) residues (<a class="bk_pop" href="#g218-enzasssulfocd.REF.4">4</a>). Dermatan 4-sulfotransferase (D4ST-1) preferentially transfers sulfate to position 4 of GalNAc residues of dermatan and is involved in the biosynthesis of dermatan sulfate (<a class="bk_pop" href="#g218-enzasssulfocd.REF.5">5</a>). In C4ST-1 deficient mice, multiple skeletal abnormalities are observed, and the morphology of growth plates is disturbed (<a class="bk_pop" href="#g218-enzasssulfocd.REF.6">6</a>). Deficiency in C4ST-1 in human causes a variety of limb malformations and skeletal defects (<a class="bk_pop" href="#g218-enzasssulfocd.REF.7">7</a>). Deficiency in D4ST-1 (CHST14) in human causes musculocontractural Ehlers-Danlos syndrome (<a class="bk_pop" href="#g218-enzasssulfocd.REF.8">8</a>). C6ST-1 has been implicated in experimental autoimmune encephalomyelitis (<a class="bk_pop" href="#g218-enzasssulfocd.REF.9">9</a>), temporal lobe epilepsy (<a class="bk_pop" href="#g218-enzasssulfocd.REF.10">10</a>), and neuroplasticity and memory in aging (<a class="bk_pop" href="#g218-enzasssulfocd.REF.11">11</a>). Deficiency in C6ST-1 in human causes spondyloepiphyseal dysplasia with severe skeletal abnormality (<a class="bk_pop" href="#g218-enzasssulfocd.REF.12">12</a>).</p></div><div id="g218-enzasssulfocd.Protocol"><h2 id="_g218-enzasssulfocd_Protocol_">Protocol</h2><p>This protocol is mainly applicable to the recombinant enzymes. However, enzyme activities of the culture media and cell extracts of the cultured cells, such as chondrocytes, are also detected by this protocol (<a class="bk_pop" href="#g218-enzasssulfocd.REF.1">1</a>, <a class="bk_pop" href="#g218-enzasssulfocd.REF.13">13</a>).</p><div id="g218-enzasssulfocd.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">[<sup>35</sup>S]PAPS (PerkinElmer NEG010, PerkinElmer, Waltham MA)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Chondroitin from squid skin or desulfated chondroitin</p></dd><dt>3.</dt><dd><p class="no_top_margin">Desulfated dermatan</p></dd><dt>4.</dt><dd><p class="no_top_margin">CS-A (Sigma-Aldrich, St. Louis, MO)</p></dd><dt>5.</dt><dd><p class="no_top_margin">pFLAG-CMV-2 plasmid (Sigma-Aldrich, St. Louis, MO)</p></dd><dt>6.</dt><dd><p class="no_top_margin">pcDNA3.1 (Thermo Fisher Scientific, Tokyo)</p></dd><dt>7.</dt><dd><p class="no_top_margin">COS-7 cells</p></dd><dt>8.</dt><dd><p class="no_top_margin">FLAG M2 antibody-conjugated agarose (Sigma-Aldrich)</p></dd><dt>9.</dt><dd><p class="no_top_margin">Clearsol (Nacalai Tesque, Inc., Kyoto, Japan)</p></dd><dt>10.</dt><dd><p class="no_top_margin">Chondroitinase ACII (Commercial chondroitinase ACII is presently unavailable and can be replaced by chondroitinase AC from <i>Flavobacterium heparinum</i> (Sigma-Aldrich))</p></dd><dt>11.</dt><dd><p class="no_top_margin">Chondroitinase ABC (Sigma-Aldrich)</p></dd></dl></div><div id="g218-enzasssulfocd.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Desalting column (1 × 10 cm) filled with Sephadex G-25 superfine (Cytiva, Tokyo)</p></dd><dt>2.</dt><dd><p class="no_top_margin">HPLC system</p></dd><dt>3.</dt><dd><p class="no_top_margin">Partisil-10 SAX column (4.6 mm × 25 cm) (Sigma-Aldrich)</p></dd><dt>4.</dt><dd><p class="no_top_margin">Liquid scintillation counter</p></dd></dl></div><div id="g218-enzasssulfocd.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Prepare the recombinant enzymes (<b>Note 1</b>).</p></dd><dt>2.</dt><dd><p class="no_top_margin">Prepare the acceptor substrate (<b>Note 2</b>).</p></dd><dt>3.</dt><dd><p class="no_top_margin">Prepare the reaction mixture (50 μL). For C4ST and C6ST, the reaction mixtures contain 50 mM imidazole-HCl buffer (pH 6.8), 0.0025% protamine chloride, 2 mM dithiothreitol, 0.5 μmol/mL (as galactosamine) chondroitin, [<sup>35</sup>S]PAPS (about 5.0 × 105 cpm/50 μL), and the recombinant enzyme. For D4ST, the reaction mixtures contain 50 mM imidazole-HCl buffer (pH 6.8), 2 mM dithiothreitol, 5.0 × 105 cpm [<sup>35</sup>S]PAPS, 2 μM PAPS, 0.05% protamine chloride, 50 μg desulfated dermatan sulfate, and the recombinant enzyme (<b>Note 3</b>).</p></dd><dt>4.</dt><dd><p class="no_top_margin">Incubate at 37˚C for 20 min (C4ST and C6ST) or 2 h (D4ST).</p></dd><dt>5.</dt><dd><p class="no_top_margin">Terminate the reaction by immersing the reaction tubes in a boiling water bath for 1 min.</p></dd><dt>6.</dt><dd><p class="no_top_margin">Add 50 nmol CS-A as carrier and three volumes of ethanol containing 1% (w/v) potassium acetate. The mixtures are stirred well and placed on ice for 30 min.</p></dd><dt>7.</dt><dd><p class="no_top_margin">Collect the precipitate with centrifugation at 10,000 rpm for 10 min.</p></dd><dt>8.</dt><dd><p class="no_top_margin">Dissolve the precipitate in 70 μL water and inject 50 μL of the solution into a desalting column equilibrated with 0.1 M NH<sub>4</sub>HCO<sub>3</sub> at the flow rate of 2 mL/min and collect the void fractions (<b>Note 4</b>).</p></dd><dt>9.</dt><dd><p class="no_top_margin">Mix an aliquot of the [<sup>35</sup>S]glycosaminoglycan fraction with Clearsol and determine the radioactivity by a liquid scintillation counter.</p></dd><dt>10.</dt><dd><p class="no_top_margin">Digest the [<sup>35</sup>S]glycosaminoglycan with chondroitinase ACII or chondroitinase ABC (<b>Note 5</b>).</p></dd><dt>11.</dt><dd><p class="no_top_margin">Inject the digests to Partisil-10 SAX column equilibrated with 5 mM KH<sub>2</sub>PO<sub>4</sub> and separate ∆4,5Hexuronic acid (∆HexA)-GalNAc(4SO<sub>4</sub>) and ∆HexA-GalNAc(6SO<sub>4</sub>) (<b>Note 6</b>).</p></dd></dl></div><div id="g218-enzasssulfocd.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">The recombinant human C4ST-1 and C6ST-1 are expressed in COS-7 cells as a fusion protein with a FLAG peptide (<a class="bk_pop" href="#g218-enzasssulfocd.REF.4">4</a>). From the transfected cells, C4ST-1 and C6ST-1 are extracted with 1.5 ml/10-cm dish 10 mM Tris-HCl (pH 7.2), 10 mM MgCl<sub>2</sub>, 2 mM CaCl<sub>2</sub>, 0.5% Triton X-100, and 20% glycerol for 30 min on a rotatory shaker at 4°C. The extracts are centrifuged at 10,000 × <i>g</i> for 10 min. The cellular extracts from ten 10-cm dishes are applied to an anti-FLAG mAb-conjugated agarose column (0.5 mL) (Sigma-Aldrich) equilibrated with buffer A (10 mM Tris-HCl (pH 7.2), 20 mM MgCl<sub>2</sub>, 2 mM CaCl<sub>2</sub>, 10 mM 2-mercaptoethanol, 0.1% Triton X-100, and 20% glycerol) containing 50 mM NaCl for purification of C4ST-1 or with buffer A containing 150 mM NaCl for purification of C6ST-1. The absorbed materials are eluted with 1.5 mL of buffer A containing 118 μg FLAG peptide (Sigma-Aldrich). The recombinant human D4ST-1 is expressed in CHO/Tag cells transfected with pcDNA3.1 containing the ORF of D4ST-1(<a class="bk_pop" href="#g218-enzasssulfocd.REF.5">5</a>). From the transfected cells, D4ST-1 is extracted with 200 μL 20 mM HEPES buffer (pH 7.4), 5 mM MgCl<sub>2</sub>, 175 mM KCl, 2% Triton X-100, and protease inhibitors (23 millitrypsin inhibitor units of aprotinin and 4 μg each of leupeptin, antipain, pepstatin, and chymostatin) per 100-mm diameter culture plate. The homogenate is mixed by rotation for 1 h and sedimented at 12,000 × <i>g</i> for 20 min. The supernatant is designated as the cell extract. The culture medium is pooled and sedimented at 12,000 × <i>g</i> for 20 min. The culture supernatant is adjusted to a final concentration of 20 mM HEPES (pH 7.4), and protease inhibitors are added as noted above.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Chondroitin is obtained from the skin of squid, <i>Todarodes pacificus</i> as described (<a class="bk_pop" href="#g218-enzasssulfocd.REF.14">14</a>,<a class="bk_pop" href="#g218-enzasssulfocd.REF.15">15</a>). Briefly, a crude polysaccharide fraction is obtained from the acetone-dried squid skin by digestion with pronase, deproteinization with 5% trichloroacetic acid and precipitation with 3 volumes of ethanol containing 1% (w/v) potassium acetate. The crude polysaccharide is applied to the DEAE-cellulose column equilibrated with 0.02 M Tris-HCl (pH 7.2), and washed with the same buffer. The column is eluted with 0.4 M NaCl in 0.02 M Tris-HCl (pH 7.2). The fractions containing uronic acid are pooled, dialyzed against distilled water, and precipitated with 2-volumes of ethanol containing 1% (w/v) potassium acetate. Chemically desulfated chondroitin and dermatan are obtained from CS-A from whale cartilage (Seikagaku Corp., Tokyo, Japan) and dermatan sulfate from pig skin (Seikagaku Corp.), respectively, by solvolysis with DMSO (<a class="bk_pop" href="#g218-enzasssulfocd.REF.16">16</a>). Solvolysis with 90% (v/v) DMSO is performed at 100˚C for 60 min.</p></dd><dt>3.</dt><dd><p class="no_top_margin">Enzyme activities of C6ST and C4ST are detectable by using 5 to 10 μL of the affinity-purified preparations.</p></dd><dt>4.</dt><dd><p class="no_top_margin">The desalting column (1.0 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. As a column packed with the same material, 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. Alternatively, dissolve the precipitates in 140 μL water and separate from [<sup>35</sup>S]PAPS and degradation products by Bio-Spin 6 column (Bio-Rad Laboratories, Hercules, CA) (<a class="bk_pop" href="#g218-enzasssulfocd.REF.5">5</a>).</p></dd><dt>5.</dt><dd><p class="no_top_margin">Digestion with chondroitinase ACII (for C6ST and C4ST) or chondroitinase ABC (for D4ST) under the standard conditions is carried out for 4 h at 37°C in the reaction mixture containing, in a final volume of 25 μL, <sup>35</sup>S-labeled glycosaminoglycans, 50 mM Tris-acetate buffer (pH 7.5), 0.1 mg/m of bovine serum albumin and 30 milli units of chondroitinase ACII or chondroitinase ABC (<a class="bk_pop" href="#g218-enzasssulfocd.REF.17">17</a>).</p></dd><dt>6.</dt><dd><p class="no_top_margin">The Partisil-10 SAX column is developed with 5 mM KH<sub>2</sub>PO<sub>4</sub> for 10 min followed by a linear gradient from 5 to 500 mM KH<sub>2</sub>PO<sub>4</sub>. A column temperature is 40°C. Fractions (0.5 mL) are collected at a flow rate of 1 mL/min. Elution time of the standard disaccharides varies with the lot of the column; typically, ∆HexA-GalNAc(6SO<sub>4</sub>) and ∆HexA-GalNAc(4SO<sub>4</sub>) are eluted at 25.5 and 26.5 min, respectively (<a class="bk_pop" href="#g218-enzasssulfocd.REF.18">18</a>).</p></dd></dl></div></div><div id="g218-enzasssulfocd.References"><h2 id="_g218-enzasssulfocd_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.1">Habuchi O, Matsui Y, Kotoya Y, Aoyama Y, Yasuda Y, Noda M. Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes. <span><span class="ref-journal">J Biol Chem. </span>1993 Oct 15;<span class="ref-vol">268</span>(29):21968–74.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8408053" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8408053</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.2">Fukuta M, Uchimura K, Nakashima K, Kato M, Kimata K, Shinomura T, Habuchi O. Molecular cloning and expression of chick chondrocyte chondroitin 6-sulfotransferase. <span><span class="ref-journal">J Biol Chem. </span>1995 Aug 4;<span class="ref-vol">270</span>(31):18575–80.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7629189" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7629189</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.270.31.18575" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.3">Yamauchi S, Mita S, Matsubara T, Fukuta M, Habuchi H, Kimata K, Habuchi O. Molecular cloning and expression of chondroitin 4-sulfotransferase. <span><span class="ref-journal">J Biol Chem. </span>2000 Mar 24;<span class="ref-vol">275</span>(12):8975–81.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10722746" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10722746</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.275.12.8975" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.4">Yamada T, Ohtake S, Sato M, Habuchi O. Chondroitin 4-sulphotransferase-1 and chondroitin 6-sulphotransferase-1 are affected differently by uronic acid residues neighbouring the acceptor GalNAc residues. <span><span class="ref-journal">Biochem J. </span>2004 Dec 15;<span class="ref-vol">384</span>(Pt 3):567–75.</span> [<a href="/pmc/articles/PMC1134142/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1134142</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15324304" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15324304</span></a>] [<a href="http://dx.crossref.org/10.1042/BJ20040965" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.5">Evers MR, Xia G, Kang HG, Schachner M, Baenziger JU. Molecular cloning and characterization of a dermatan-specific N-acetylgalactosamine 4-O-sulfotransferase. <span><span class="ref-journal">J Biol Chem. </span>2001 Sep 28;<span class="ref-vol">276</span>(39):36344–53.</span> Epub 2001 Jul 24. PMID. [<a href="https://pubmed.ncbi.nlm.nih.gov/11470797" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11470797</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M105848200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.6">Klüppel M, Wight TN, Chan C, Hinek A, Wrana JL. Maintenance of chondroitin sulfation balance by chondroitin-4-sulfotransferase 1 is required for chondrocyte development and growth factor signaling during cartilage morphogenesis. <span><span class="ref-journal">Development. </span>2005 Sep;<span class="ref-vol">132</span>(17):3989–4003.</span> Epub 2005 Aug 3. PMID. [<a href="https://pubmed.ncbi.nlm.nih.gov/16079159" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16079159</span></a>] [<a href="http://dx.crossref.org/10.1242/dev.01948" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.7">Shabbir RMK, Nalbant G, Ahmad N, Malik S, Tolun A. Homozygous CHST11 mutation in chondrodysplasia, brachydactyly, overriding digits, clino-symphalangism and synpolydactyly. <span><span class="ref-journal">J Med Genet. </span>2018 Jul;<span class="ref-vol">55</span>(7):489–496.</span> Epub 2018 Mar 7. PMID. [<a href="https://pubmed.ncbi.nlm.nih.gov/29514872" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 29514872</span></a>] [<a href="http://dx.crossref.org/10.1136/jmedgenet-2017-105003" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.8">Kosho T. CHST14/D4ST1 deficiency: New form of Ehlers-Danlos syndrome. <span><span class="ref-journal">Pediatr Int. </span>2016 Feb;<span class="ref-vol">58</span>(2):88–99.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/26646600" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 26646600</span></a>] [<a href="http://dx.crossref.org/10.1111/ped.12878" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.9">Miyamoto K, Tanaka N, Moriguchi K, Ueno R, Kadomatsu K, Kitagawa H, Kusunoki S. Chondroitin 6-O-sulfate ameliorates experimental autoimmune encephalomyelitis. <span><span class="ref-journal">Glycobiology. </span>2014 May;<span class="ref-vol">24</span>(5):469–75.</span> Epub 2014 Feb 28. PMID. [<a href="https://pubmed.ncbi.nlm.nih.gov/24584141" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24584141</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwu014" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.10">Yutsudo N, Kitagawa H. Involvement of chondroitin 6-sulfation in temporal lobe epilepsy. Exp Neurol. 2015 Dec;274(Pt B):126-33. doi: 10.1016/j.expneurol.2015.07.009. Epub 2015 Jul 29. PMID: 26231575. [<a href="https://pubmed.ncbi.nlm.nih.gov/26231575" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 26231575</span></a>] [<a href="http://dx.crossref.org/10.1016/j.expneurol.2015.07.009" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.11">Yang S, Gigout S, Molinaro A, Naito-Matsui Y, Hilton S, Foscarin S, Nieuwenhuis B, Tan CL, Verhaagen J, Pizzorusso T, Saksida LM, Bussey TM, Kitagawa H, Kwok JCF, Fawcett JW. Chondroitin 6-sulphate is required for neuroplasticity and memory in ageing. <span><span class="ref-journal">Mol Psychiatry. </span>2021 Jul 16;</span> [<a href="/pmc/articles/PMC8758471/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC8758471</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/34272488" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 34272488</span></a>] [<a href="http://dx.crossref.org/10.1038/s41380-021-01208-9" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.12">van Roij MH, Mizumoto S, Yamada S, Morgan T, Tan-Sindhunata MB, Meijers-Heijboer H, Verbeke JI, Markie D, Sugahara K, Robertson SP. Spondyloepiphyseal dysplasia, Omani type: further definition of the phenotype. <span><span class="ref-journal">Am J Med Genet A. </span>2008 Sep 15;<span class="ref-vol">146A</span>(18):2376–84.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18698629" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18698629</span></a>] [<a href="http://dx.crossref.org/10.1002/ajmg.a.32482" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.13">Yamauchi S, Hirahara Y, Usui H, Takeda Y, Hoshino M, Fukuta M, Kimura JH, Habuchi O. Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line. <span><span class="ref-journal">J Biol Chem. </span>1999 Jan 22;<span class="ref-vol">274</span>(4):2456–63.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9891016" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9891016</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.274.4.2456" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.14">Anno K, Kawai Y, Seno N. Isolation of chondroitin from squid skin. <span><span class="ref-journal">Biochim Biophys Acta. </span>1964 Nov 1;<span class="ref-vol">83</span>:348–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14236709" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14236709</span></a>] [<a href="http://dx.crossref.org/10.1016/0926-6526(64)90013-8" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>15.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.15">Habuchi O, Miyata K. Stimulation of glycosaminoglycan sulfotransferase from chick embryo cartilage by basic proteins and polyamines. <span><span class="ref-journal">Biochim Biophys Acta. </span>1980 Dec 4;<span class="ref-vol">616</span>(2):208–17.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/6938246" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 6938246</span></a>] [<a href="http://dx.crossref.org/10.1016/0005-2744(80)90139-4" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>16.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.16">Nagasawa K, Inoue Y, Tokuyasu T. An improved method for the preparation of chondroitin by solvolytic desulfation of chondroitin sulfates. <span><span class="ref-journal">J Biochem. </span>1979 Nov;<span class="ref-vol">86</span>(5):1323–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/521436" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 521436</span></a>] [<a href="http://dx.crossref.org/10.1093/oxfordjournals.jbchem.a132648" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>17.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.17">Ohtake S, Kimata K, Habuchi O. A unique nonreducing terminal modification of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase. <span><span class="ref-journal">J Biol Chem. </span>2003 Oct 3;<span class="ref-vol">278</span>(40):38443–52.</span> Epub 2003 Jul 21. PMID. [<a href="https://pubmed.ncbi.nlm.nih.gov/12874280" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12874280</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M306132200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>18.</dt><dd><div class="bk_ref" id="g218-enzasssulfocd.REF.18">Ohtake S, Kimata K, Habuchi O. Recognition of sulfation pattern of chondroitin sulfate by uronosyl 2-O-sulfotransferase. <span><span class="ref-journal">J Biol Chem. </span>2005 Nov 25;<span class="ref-vol">280</span>(47):39115–23.</span> Epub 2005 Sep 27. PMID. [<a href="https://pubmed.ncbi.nlm.nih.gov/16192264" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16192264</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M508816200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594009_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g218-enzasssulfocd.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>
|
||
<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a><p class="small">Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view a copy of this license, visit <a href="http://creativecommons.org/licenses/by-nc-nd/4.0/" ref="pagearea=meta&targetsite=external&targetcat=link&targettype=uri">http://creativecommons.org/licenses/by-nc-nd/4.0/</a>.</p></div><div class="small"><span class="label">Bookshelf ID: NBK594009</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/37590734" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">37590734</a></span></div><div style="margin-top:2em" class="bk_noprnt"><a class="bk_cntns" href="/books/n/glycopodv2/">Contents</a><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/glycopodv2/g84-assaychst10/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/glycopodv2/g219-enzasssucsds/" title="Next page in this title">Next ></a></div></div></div></div>
|
||
|
||
</div>
|
||
</div>
|
||
</div>
|
||
<div class="bottom">
|
||
|
||
<div id="NCBIFooter_dynamic">
|
||
<!--<component id="Breadcrumbs" label="breadcrumbs"/>
|
||
<component id="Breadcrumbs" label="helpdesk"/>-->
|
||
|
||
</div>
|
||
|
||
<script type="text/javascript" src="/portal/portal3rc.fcgi/rlib/js/InstrumentNCBIBaseJS/InstrumentPageStarterJS.js"> </script>
|
||
</div>
|
||
</div>
|
||
<!--/.page-->
|
||
</div>
|
||
<!--/.wrap-->
|
||
</div><!-- /.twelve_col -->
|
||
</div>
|
||
<!-- /.grid -->
|
||
|
||
<span class="PAFAppResources"></span>
|
||
|
||
<!-- BESelector tab -->
|
||
|
||
|
||
|
||
<noscript><img alt="statistics" src="/stat?jsdisabled=true&ncbi_db=books&ncbi_pdid=book-part&ncbi_acc=NBK594009&ncbi_domain=glycopodv2&ncbi_report=printable&ncbi_type=fulltext&ncbi_objectid=&ncbi_pcid=/NBK594009/?report=printable&ncbi_app=bookshelf" /></noscript>
|
||
|
||
|
||
<!-- usually for JS scripts at page bottom -->
|
||
<!--<component id="PageFixtures" label="styles"></component>-->
|
||
|
||
|
||
<!-- CE8B5AF87C7FFCB1_0191SID /projects/books/PBooks@9.11 portal105 v4.1.r689238 Tue, Oct 22 2024 16:10:51 -->
|
||
<span id="portal-csrf-token" style="display:none" data-token="CE8B5AF87C7FFCB1_0191SID"></span>
|
||
|
||
<script type="text/javascript" src="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/js/3879255/4121861/3501987/4008961/3893018/3821238/3400083/3426610.js" snapshot="books"></script></body>
|
||
</html> |