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EXTL1 and EXTL3 exhibit GlcNAcT activities likely involved in the biosynthesis of HS (Figure 1). EXTL1 exerts only GlcNAcT-II activity, which may be involved in HS chain elongation, whereas EXTL3 is considered to possess activity transferring the first GlcNAc residue to tetrasaccharide-linkage region (so-called GlcNAcT-I activity) along with GlcNAcT-II activities (Figure 1). Unlike EXT1, EXT2, and EXTL3, EXTL2 transfers a GlcNAc residue to the phosphorylated tetrasaccharide-linkage region, terminating glycosaminoglycan (GAG) chain elongation (1). In vitro HS polymerization is induced using tetrasaccharide-linkage analogs as acceptor substrates for the enzyme complex of human EXT1/EXT2 without the aid of EXTL proteins (2). 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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="_NBK594024_"><span class="title" itemprop="name">Enzyme assay of glycosaminoglycan glycosyltransferases for heparan sulfate</span></h1><div class="contrib half_rhythm"><span itemprop="author">Satomi Nadanaka</span>, Ph.D.<div class="affiliation small">Kobe Pharmaceutical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-amrahpebok@kanadans" class="oemail">pj.ca.u-amrahpebok@kanadans</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Hiroshi Kitagawa</span>, Ph.D.<div class="affiliation small">Kobe Pharmaceutical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-amrahpebok@awagatik" class="oemail">pj.ca.u-amrahpebok@awagatik</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 13, 2021</span>; Last Revision: <span itemprop="dateModified">March 20, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g78-gaggths.Introduction"><h2 id="_g78-gaggths_Introduction_">Introduction</h2><p>Exostosin 1 (<i>EXT1</i>) and <i>EXT2</i> genes encode heparan sulfate (HS) co-polymerases, the genetic defects of which result in hereditary multiple exostoses in humans. EXT1 and EXT2 exhibit <i>N</i>-acetylglucosaminyltransferase (GlcNAcT) and glucuronyltransferase (GlcAT) activities required for the HS synthesis (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F1/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF1" rid-ob="figobg78gaggthsF1">Figure 1</a>). EXT1 and EXT2 form a stable complex, which is the biologically relevant form of enzymes. Additionally, three highly homologous <i>EXT</i>-like genes, <i>EXTL1</i>–<i>EXTL3</i>, have been cloned (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F1/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF1" rid-ob="figobg78gaggthsF1">Figure 1</a>). EXTL1 and EXTL3 exhibit GlcNAcT activities likely involved in the biosynthesis of HS (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F1/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF1" rid-ob="figobg78gaggthsF1">Figure 1</a>). EXTL1 exerts only GlcNAcT-II activity, which may be involved in HS chain elongation, whereas EXTL3 is considered to possess activity transferring the first GlcNAc residue to tetrasaccharide-linkage region (so-called GlcNAcT-I activity) along with GlcNAcT-II activities (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F1/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF1" rid-ob="figobg78gaggthsF1">Figure 1</a>). Unlike EXT1, EXT2, and EXTL3, EXTL2 transfers a GlcNAc residue to the phosphorylated tetrasaccharide-linkage region, terminating glycosaminoglycan (GAG) chain elongation (<a class="bk_pop" href="#g78-gaggths.REF.1">1</a>). <i>In vitro</i> HS polymerization is induced using tetrasaccharide-linkage analogs as acceptor substrates for the enzyme complex of human EXT1/EXT2 without the aid of EXTL proteins (<a class="bk_pop" href="#g78-gaggths.REF.2">2</a>). Thus, the mechanism underlying the regulation of GAG biosynthesis by EXTLs remains to be incompletely elucidated.</p><p><b>Monosaccharide Abbreviations used in this chapter</b>: Gal: galactose; GlcNAc: <i>N</i>-acetylglcosamine; GlcA: glucuronic acid; Xyl: xylose</p></div><div id="g78-gaggths.Protocol"><h2 id="_g78-gaggths_Protocol_">Protocol</h2><p>Ext gene family in HS biosynthesis is involved in four kinds of glycosyltransferase activities including GlcNAcT-I, GlcNAcT-II, HS-GlcAT-II, and polymerization activities. In this chapter, the assay protocols for measuring the four enzymatic activities in HS biosynthesis are described.</p><div id="g78-gaggths.Materials"><h3>Materials</h3><p>1. GlcAβ1-3Galβ1-<i>O</i>-C<sub>2</sub>H<sub>4</sub>NH-benzyloxycarbonyl (chemically synthesized linkage region analog) (<b>Note 1</b>)</p><p>2. <i>N</i>-Acetylheparosan oligosaccharides derived from the capsular polysaccharide of Escherichia coli K5, GlcAβ1-4GlcNAcα1-(4GlcAβ1-4GlcAcα1)n with the nonreducing terminal GlcA</p><p>3. <i>N</i>-Acetylheparosan oligosaccharides derived from the capsular polysaccharide of Escherichia coli K5, GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n with the nonreducing terminal GlcNAc</p><p>4. GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-<i>O</i>-Ser-Gly-Trp-Pro-Asp-Gly (chemically synthesized) (<a class="bk_pop" href="#g78-gaggths.REF.2">2</a>)</p><p>5. UDP-[<sup>3</sup>H]GlcNAc (1,000–2,000 GBq/mmol) (NET434050UC, PerkinElmer Life Sciences, Waltham, MA)</p><p>6. UDP-[<i>U</i>-<sup>14</sup>C]GlcA (10 GBq/mmol) (NEC414050UC, PerkinElmer Life Sciences)</p><p>7. MES-NaOH (pH 6.5)</p><p>8. MnCl<sub>2</sub></p><p>9. ATP-2Na</p><p>10. UDP-GlcA</p><p>11. MeOH</p><p>12. NH<sub>4</sub>HCO<sub>3</sub></p><p>13. Heparinase III (EC4.2.2.8) (IBEX or Sigma-Aldrich)</p><p>14. Sodium acetate buffer</p><p>15. Calcium acetate</p></div><div id="g78-gaggths.Instruments"><h3>Instruments</h3><p>1. Nova-Pak C18 column (3.9 × 150 mm) (Waters, Milford, MA)</p><p>2. Chromatography system (FPLC, ÄKTA, and so on)</p><p>3. 2300TR liquid scintillation counter (PerkinElmer, Waltham, MA)</p><p>4. Syringe column (TERUMO SS-01T) packed with Superdex G-25 superfine (Cytiva, Tokyo, Japan) (<a class="bk_pop" href="#g78-gaggths.REF.3">3</a>)</p><p>5. Superdex peptide HR 10/30 column (Cytiva)</p><p>6. Superdex 200 10/300 GL (Cytiva)</p><p>7. High-performance liquid chromatography (HPLC)</p><p>8. SpeedVac vacuum concentrator (Thermo Fisher Scientific, Waltham, MA)</p></div><div id="g78-gaggths.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Preparation of recombinant soluble enzyme proteins is mentioned in the methods described in the chapter “Enzyme assay of glycosaminoglycan glycosyltransferases for chondroitin sulfate”.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Assay for GlcNAcT-I activity (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F2/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF2" rid-ob="figobg78gaggthsF2">Figure 2</a>) (<a class="bk_pop" href="#g78-gaggths.REF.4">4</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">The reaction mixture for GlcNAcT-I activity (a total volume of 20 μL) contains 10 μL of enzyme source (<b>Note 2</b>), 100 mM of MES-NaOH (pH 6.5), 10 mM of MnCl<sub>2</sub>, 1 mM of ATP-2Na, 250 nmol GlcAβ1-3Galβ1-<i>O</i>-C<sub>2</sub>H<sub>4</sub>NH-benzyloxycarbonyl, and 250 μM of UDP-[<sup>3</sup>H]GlcNAc (approximately 1.1 × 10<sup>6</sup> dpm).</p></dd><dt>b.</dt><dd><p class="no_top_margin">The enzyme reaction is performed at 37°C for 4 h.</p></dd><dt>c.</dt><dd><p class="no_top_margin">The 3H-labeled GlcNAcT-I reaction products are separated by HPLC analysis on a Nova-Pak C18 column using MeOH as the eluent at a flow rate of 1.0 mL/min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">The collected fractions (2 mL each) are analyzed using a liquid scintillation counter.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Assay for GlcNAcT-II activity (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F3/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF3" rid-ob="figobg78gaggthsF3">Figure 3</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">The reaction mixture for GlcNAcT-II activity (a total volume of 20 μL) contains 10 μL of enzyme source, 100 mM of MES-NaOH (pH 6.5), 10 mM of MnCl<sub>2</sub>, 1 mM of ATP-2Na, 20 μg GlcAβ1-4GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n (<b>Note 3</b>), and 250 μM of UDP-[<sup>3</sup>H]GlcNAc (approximately 1.1 × 10<sup>6</sup> dpm).</p></dd><dt>b.</dt><dd><p class="no_top_margin">The enzyme reaction is performed at 37°C for 4 h.</p></dd><dt>c.</dt><dd><p class="no_top_margin">The <sup>3</sup>H-labeled GlcNAcT-II reaction products are analyzed by syringe columns (TERUMO SS-01T)</p><dl class="temp-labeled-list"><dt>i.</dt><dd><p class="no_top_margin">Pack with Sephadex G-25 (superfine) equilibrated with 0.2 M NH<sub>4</sub>HCO<sub>3</sub>. Centrifuge the column at 800 rpm for 5 min, followed by the second centrifuge at 2,000 rpm for 5 min.</p></dd><dt>ii.</dt><dd><p class="no_top_margin">Add 30 μL of 0.2 M NH<sub>4</sub>HCO<sub>3</sub> to the reaction products. Apply the samples to the packed syringe column and centrifuge at 2,000 rpm for 5 min.</p></dd><dt>iii.</dt><dd><p class="no_top_margin">Add 50 μL of 0.2 M NH<sub>4</sub>HCO<sub>3</sub> to the top of the syringe columns and centrifuge at 2,000 rpm for 5 min.</p></dd><dt>iv.</dt><dd><p class="no_top_margin">Measure radioactivity of the eluates using a scintillation counter.</p></dd></dl></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">Assay for HS-GlcAT-II activity (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F3/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF3" rid-ob="figobg78gaggthsF3">Figure 3</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">The reaction mixture for HS-GlcAT-II activity (a total volume of 20 μL) contains 10 μL of enzyme source, 100 mM of MES-NaOH (pH 6.5), 10 mM of MnCl<sub>2</sub>, 1 mM of ATP-2Na, 20 μg GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n (<b>Note 3</b>), and 250 μM of UDP-[<i>U</i>-<sup>14</sup>C]GlcA (approximately 1.6 × 10<sup>5</sup> dpm).</p></dd><dt>b.</dt><dd><p class="no_top_margin">The enzyme reaction is performed at 37°C for 4 h.</p></dd><dt>c.</dt><dd><p class="no_top_margin">The <sup>14</sup>C-labeled HS-GlcAT-II reaction products are analyzed by syringe columns as described in Method 3-c.</p></dd></dl></dd><dt>5.</dt><dd><p class="no_top_margin">Assay for polymerization activity (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F4/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF4" rid-ob="figobg78gaggthsF4">Figure 4</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">The reaction mixture for polymerization activity (a total volume of 20 μL) contains 10 μL of enzyme source, 100 mM of MES-NaOH (pH 6.5), 10 mM of MnCl<sub>2</sub>, 1 mM of ATP-2Na, 100 nmol GlcAβ1-3Galβ1-<i>O</i>-C<sub>2</sub>H<sub>4</sub>NH-benzyloxycarbonyl, 250 μM of UDP-[<sup>3</sup>H]GlcNAc (approximately 5.5 × 10<sup>5</sup> dpm), and 250 μM of UDP-GlcA.</p></dd><dt>b.</dt><dd><p class="no_top_margin">The enzyme reaction is performed at 37°C for overnight.</p></dd><dt>c.</dt><dd><p class="no_top_margin">The <sup>3</sup>H-labeled polymerization products are separated by gel filtration using a Superdex Peptide HR 10/30 column (<b>Note 4</b>) using NH<sub>4</sub>HCO<sub>3</sub> gradient as the eluent at a flow rate of 0.4 mL/min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">The collected fractions (0.4 mL each) are analyzed using a liquid scintillation counter.</p></dd></dl></dd><dt>6.</dt><dd><p class="no_top_margin">The reaction products are characterized taking advantage of the substrate specificities of respective glycosidases</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">GlcNAcT-I-reaction products (<a class="bk_pop" href="#g78-gaggths.REF.3">3</a>) (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F2/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF2" rid-ob="figobg78gaggthsF2">Figure 2</a>)</p><dl class="temp-labeled-list"><dt>i.</dt><dd><p class="no_top_margin">Pool and concentrate the reaction products by SpeedVac</p></dd><dt>ii.</dt><dd><p class="no_top_margin">Digest with 10 mUnits of heparinase III (EC4.2.2.8) (IBEX or Sigma-Aldrich) (<b>Note 5</b>) in a total volume of 50 μL of 20 mM of sodium acetate buffer (pH 7.0) containing 2 mM of calcium acetate at 37°C overnight.</p></dd><dt>iii.</dt><dd><p class="no_top_margin">Analyze the digested or undigested samples using HPLC on a Nova-Pak C18 column.</p></dd></dl><p>The radioactivity of the GlcNAcT-I-reaction products is released by digestion with heparinase III and eluted at the elution position of free [<sup>3</sup>H]GlcNAc.</p></dd><dt>b.</dt><dd><p class="no_top_margin">GlcNAcT-II-reaction products (<a class="bk_pop" href="#g78-gaggths.REF.3">3</a>)</p><dl class="temp-labeled-list"><dt>i.</dt><dd><p class="no_top_margin">Pool and concentrate the reaction products by SpeedVac.</p></dd><dt>ii.</dt><dd><p class="no_top_margin">Digest the reaction products with 10 mUnits of heparinase III (EC4.2.2.8) (IBEX or Sigma-Aldrich) in a total volume of 100 μL of 20 mM of sodium acetate buffer (pH 7.0) containing 2 mM of calcium acetate at 37°C overnight.</p></dd><dt>iii.</dt><dd><p class="no_top_margin">Analyze the digested samples by gel filtration on a Superdex peptide HR 10/30 column.</p></dd></dl><p>The radioactivity peak of the GlcNAcT-II-reaction products eluted near the void volume is released by digestion with heparinase III and shifted to the free [<sup>3</sup>H]GlcNAc position.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Polymerization products (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F4/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF4" rid-ob="figobg78gaggthsF4">Figure 4</a>)</p><dl class="temp-labeled-list"><dt>i.</dt><dd><p class="no_top_margin">Pool and concentrate the polymerized products by SpeedVac.</p></dd><dt>ii.</dt><dd><p class="no_top_margin">Digest with 6 mUnits of heparinase III (EC4.2.2.8) in a total volume of 100 μL of 20 mM of sodium acetate buffer (pH 7.0) containing 2 mM of calcium acetate at 37°C overnight.</p></dd><dt>iii.</dt><dd><p class="no_top_margin">Analyze the digested samples by gel filtration using a Superdex peptide HR 10/30 column.</p></dd></dl><p>The radioactivity peak corresponding to the polymerized products is observed near the void volume (<a class="figpopup" href="/books/NBK594024/figure/g78-gaggths.F4/?report=objectonly" target="object" rid-figpopup="figg78gaggthsF4" rid-ob="figobg78gaggthsF4">Figure 4</a>). This peak is shifted to the elution position of unsaturated disaccharides by digestion with heparitinase III, when heparosan polymer is synthesized by polymerization reactions.</p></dd></dl></dd></dl></div><div id="g78-gaggths.Notes"><h3>Notes</h3><p>1. Acceptor substrates for GlcNAcT-I and GlcAβ1-3Galβ1-<i>O</i>-C<sub>2</sub>H<sub>4</sub>NH-benzyloxycarbonyl were kindly provided by Prof. Jun-ichi Tamura (Tottori University).</p><p>2. Each EXT protein is expressed in COS-1 or COS-7 cells as a soluble form fused to Protein A. Forty-eight h after transfection, the secreted Protein A-tagged proteins are purified with IgG-Sepharose beads (Cytiva). The enzyme-bound beads are used for enzyme assays. Additionally, co-expression of EXT1 and EXT2 is required for the polymerization reaction (<a class="bk_pop" href="#g78-gaggths.REF.2">2</a>).</p><p>GlcNAcT and GlcAT assays using crude cell lysates as an enzyme source have been reported (<a class="bk_pop" href="#g78-gaggths.REF.5">5</a>).</p><p>3. Oligosaccharide acceptors for GlcNAcT-II and HS-GIcAT-II can be prepared as described previously (<a class="bk_pop" href="#g78-gaggths.REF.6">6</a>). K5 polysaccharides are partially N-deacetylated with hydrazine and subjected to deaminative cleavage with nitrous acid at pH 3.9. The resulting mixture of oligosaccharides was fractionated by gel filtration chromatography. The decasaccharide fraction was used as a substrate for GlcNAcT-II. The digestion of a tetradecasaccharide fraction with β-D-glucuronidase yielded tridecasaccharides with nonreducing terminal GlcNAc residues, which are suitable substrates for HS-GlcAT-II.</p><p>4. To determine the molecular size of polymerized HS chains, a Superdex 200 10/300 GL column should be used instead of a Superdex Peptide HR 10/30 column.</p><p>5. Unit is defined as follows: one unit of the enzyme is the amount required for the eliminative cleavage of typical CS or HS, yielding ultraviolet-absorbing materials corresponding to 1 μmol Δ<sup>4,5</sup>-hexuronate residues/min at 37°C, pH 7.0, as calculated with a value of 5,500 (<a class="bk_pop" href="#g78-gaggths.REF.7">7</a>) for the molar extinction coefficient of Δ<sup>4,5</sup>-hexuronated oligosaccharides.</p></div></div><div id="g78-gaggths.References"><h2 id="_g78-gaggths_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g78-gaggths.REF.1">Nadanaka S, Zhou S, Kagiyama S, Shoji N, Sugahara K, Sugihara K, Asano M, Kitagawa H. EXTL2, a member of the EXT family of tumor suppressors, controls glycosaminoglycan biosynthesis in a xylose kinase-dependent manner. <span><span class="ref-journal">J Biol Chem. </span>2013 Mar 29;<span class="ref-vol">288</span>(13):9321–33.</span> [<a href="/pmc/articles/PMC3611003/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3611003</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23395820" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23395820</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g78-gaggths.REF.2">Kim BT, Kitagawa H, Tanaka J, Tamura J, Sugahara K. In vitro heparan sulfate polymerization: crucial roles of core protein moieties of primer substrates in addition to the EXT1-EXT2 interaction. <span><span class="ref-journal">J Biol Chem. </span>2003 Oct 24;<span class="ref-vol">278</span>(43):41618–23.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12907685" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12907685</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g78-gaggths.REF.3">Kitagawa H, Tsuchida K, Ujikawa M, Sugahara K. Detection and characterization of UDP-GalNAc: chondroitin N-acetylgalactosaminyltransferase in bovine serum using a simple assay method. <span><span class="ref-journal">J Biochem. </span>1995 May;<span class="ref-vol">117</span>(5):1083–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8586623" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8586623</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g78-gaggths.REF.4">Kim BT, Kitagawa H, Tamura J, Saito T, Kusche-Gullberg M, Lindahl U, Sugahara K. Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode alpha 1,4- N-acetylglucosaminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2001 Jun 19;<span class="ref-vol">98</span>(13):7176–81.</span> [<a href="/pmc/articles/PMC34642/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC34642</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11390981" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11390981</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g78-gaggths.REF.5">Busse M, Feta A, Presto J, Wilen M, Gronning M, Kjellen L, Kusche-Gullberg M. Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation. <span><span class="ref-journal">J Biol Chem. </span>2007 Nov 9;<span class="ref-vol">282</span>(45):32802–10.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17761672" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17761672</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g78-gaggths.REF.6">Lidholt K, Weinke JL, Kiser CS, Lugemwa FN, Bame KJ, Cheifetz S, Massague J, Lindahl U, Esko JD. A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>1992 Mar 15;<span class="ref-vol">89</span>(6):2267–71.</span> [<a href="/pmc/articles/PMC48638/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC48638</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/1532254" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1532254</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g78-gaggths.REF.7">Hovingh P, Linker A. The disaccharide repeating-units of heparan sulfate. <span><span class="ref-journal">Carbohydr Res. </span>1974 Oct;<span class="ref-vol">37</span>(1):181–92.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/4279141" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 4279141</span></a>] [<a href="http://dx.crossref.org/10.1016/s0008-6215(00)87073-1" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="g78-gaggths.REF.8">Nadanaka S, Kitagawa H. Heparan sulphate biosynthesis and disease. <span><span class="ref-journal">J Biochem. </span>2008 Jul;<span class="ref-vol">144</span>(1):7–14.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18367479" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18367479</span></a>] [<a href="http://dx.crossref.org/10.1093/jb/mvn040" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="g78-gaggths.REF.9">Yamada S. Specific functions of Exostosin-like 3 (EXTL3) gene products. <span><span class="ref-journal">Cell Mol Biol Lett. </span>2020;<span class="ref-vol">25</span>:39.</span> [<a href="/pmc/articles/PMC7441721/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC7441721</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/32843889" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 32843889</span></a>] [<a href="http://dx.crossref.org/10.1186/s11658-020-00231-y" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594024_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g78-gaggths.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g78-gaggths.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK594024/bin/g78-gaggths-Image001.jpg" alt="Figure 1: . The five members of the exostosin (EXT) gene family." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>The five members of the exostosin (EXT) gene family. (A) Schematic structures of the proteins of five EXT gene family are shown. Highly conserved regions, exostosin family domain (pfam03016) and glycosyl transferase family 64 domain (pfam09258), are indicated by yellow-green and green boxes, respectively. White and yellow bars indicate DXD motifs, which are involved in the enzyme activity and nucleotide diphosphate sugar binding capacity of the glycosyltransferases. DXD motif registered as a region involved in “substrate binding” in UniProt database is indicated by yellow. a.a., amino acid; TM, transmembrane domain. Additionally, glycosyltransferase activities detected in EXT members are summarized. Although EXTs are type II integral transmembrane proteins, the stem region connected to the luminal C-terminal catalytic domain of EXTs can be cleaved by proteases in the lumen to release active soluble enzymes. Therefore, the activities of secreted EXTs in serum and biological fluids can be measured. (B) The biosynthesis of heparan sulfate (HS) in mammals. Mammalian HS is biosynthesized by EXT1/EXT2 polymerase complex. Although three EXTLs possess <i>in vitro</i> glycosyltransferase activities in relation to HS biosynthesis, <i>in vivo</i> significance in the biosynthesis of HS remains unclear (<a class="bk_pop" href="#g78-gaggths.REF.8">8</a>, <a class="bk_pop" href="#g78-gaggths.REF.9">9</a>).</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g78-gaggths.F2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202%3A%20.%20Measurement%20of%20GlcNAcT-I%20activity.&p=BOOKS&id=594024_g78-gaggths-Image002.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK594024/bin/g78-gaggths-Image002.jpg" alt="Figure 2: . Measurement of GlcNAcT-I activity." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Measurement of GlcNAcT-I activity. GlcNAcT-I-reaction products were subjected to high-performance liquid chromatography analysis using a Nova-Pak C18 column. The separated fractions (2 mL each) were measured for radioactivity. White arrow, UDP-[<sup>3</sup>H]GlcNAc; black arrow, [<sup>3</sup>H]GlcNAcα1-4GlcAβ1-3Galβ1-<i>O</i>-C<sub>2</sub>H<sub>4</sub>NH-benzyloxycarbonyl.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g78-gaggths.F3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK594024/bin/g78-gaggths-Image003.jpg" alt="Figure 3: . GlcNAcT-II and HS-GlcAT-II assays using a syringe column." /></div><h3><span class="label">Figure 3: </span></h3><div class="caption"><p>GlcNAcT-II and HS-GlcAT-II assays using a syringe column.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g78-gaggths.F4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204%3A%20.%20Analysis%20of%20polymerized%20heparan%20sulfate%20(HS)%20chains.&p=BOOKS&id=594024_g78-gaggths-Image004.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK594024/bin/g78-gaggths-Image004.jpg" alt="Figure 4: . Analysis of polymerized heparan sulfate (HS) chains." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4: </span></h3><div class="caption"><p>Analysis of polymerized heparan sulfate (HS) chains. (A) The polymerization reaction products were analyzed by gel filtration on a Superdex peptide column. (B) Molecular size of HS chains was determined by gel filtration chromatography on a Superdex 200 column. White arrowheads indicate the elution positions of commercial dextrans of known molecular weights.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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