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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="_NBK593885_"><span class="title" itemprop="name">Enzyme assay of cerebroside sulfotransferase</span></h1><div class="contrib half_rhythm"><span itemprop="author">Koichi Honke</span>, MD, Ph.D.<div class="affiliation small">School of Medicine,
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Kochi Univ.<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-ihcok@eknohk" class="oemail">pj.ca.u-ihcok@eknohk</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 19, 2021</span>; Last Revision: <span itemprop="dateModified">March 15, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g86-assaycersulfot.Introduction"><h2 id="_g86-assaycersulfot_Introduction_">Introduction</h2><p>There are two major sulfoglycolipids in the mammal: one being the sulfatide, which is a sphingolipid, and the other being the seminolipid, which is an ether glycerolipid. Sulfatide is a major lipid component of the myelin sheath and is synthesized in myelin-generating cells, oligodendrocytes in the central nervous system, and Schwann cells in the peripheral nervous system. Seminolipid is synthesized in spermatocytes and maintained in the subsequent germ cell stages.</p><p>The carbohydrate moiety of sulfatide and seminolipid has the same structure and is biosynthesized via sequential reactions catalyzed by common enzymes: ceramide galactosyltransferase (CGT, EC 2.4.1.45) and cerebroside sulfotransferase (CST, EC 2.8.2.11) (<a class="figpopup" href="/books/NBK593885/figure/g86-assaycersulfot.F1/?report=objectonly" target="object" rid-figpopup="figg86assaycersulfotF1" rid-ob="figobg86assaycersulfotF1">Figure 1</a>). Cerebroside is the trivial name of galactosylceramide (GalCer). CST is also known as sulfatide synthase. CST catalyzes the transfer of sulfonate group from the activated sulfate, 3’-phosphoadenosine 5’-phosphosulfate (PAPS), to the C3 position of the nonreducing terminal galactose of glycolipid oligosaccharides (<a class="figpopup" href="/books/NBK593885/figure/g86-assaycersulfot.F2/?report=objectonly" target="object" rid-figpopup="figg86assaycersulfotF2" rid-ob="figobg86assaycersulfotF2">Figure 2</a>).</p><p>CST was homogeneously purified from human renal cancer cells where CST is very highly expressed (<a class="bk_pop" href="#g86-assaycersulfot.REF.1">1</a>). The purified CST acts not only on GalCer but also on galactosylglycerolipids, lactosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide, indicating that CST recognizes β-galactoside at the nonreducing termini of sugar chains attached to a lipid moiety.</p><p>Subsequently, a cDNA clone encoding human CST was isolated from a cDNA library of human renal cancer cells, using degenerate oligonucleotides synthesized on the basis of amino acid sequence data of the purified enzyme (<a class="bk_pop" href="#g86-assaycersulfot.REF.2">2</a>), followed by cloning of human genomic DNA (<a class="bk_pop" href="#g86-assaycersulfot.REF.3">3</a>) and mouse cDNA and genomic DNA (<a class="bk_pop" href="#g86-assaycersulfot.REF.4">4</a>). CST is composed of 423 amino acids and has type II transmembrane topology and two PAPS recognizing motifs as other Golgi-resident sulfotransferases do. Two <i>N</i>-glycan oligosaccharide chains are attached to CST, and the C-terminal one is essential for its enzymatic activity. CST is expressed in a tissue-specific manner and is highly expressed in the brain, the kidney, the testis, and the alimentary system. This tissue-specific expression of the CST gene is regulated by the alternative usage of multiple promoters (<a class="bk_pop" href="#g86-assaycersulfot.REF.4">4</a>). CST gene is assigned to human chromosome 22q12 and mouse chromosome 11 (<a class="bk_pop" href="#g86-assaycersulfot.REF.3">3</a>).</p><p>CST is the sole sulfotransferase responsible for the biosynthesis of sulfatide (<a class="bk_pop" href="#g86-assaycersulfot.REF.5">5</a>), which is rich in the myelin sheath, and seminolipid, which is unique in spermatogenic cells. <i>Cst</i>-null mice generated by gene targeting manifest some neurological disorders due to myelin dysfunction and an arrest of spermatogenesis, indicating that CST and its products or sulfoglycolipids are essential for organisms (<a class="bk_pop" href="#g86-assaycersulfot.REF.5">5</a>). CST is the first member identified in the βGal 3-<i>O</i>-sulfotransferase family. The other three members, Gal3ST-2-4, have been identified in the expressed sequence tag database using homology to the <i>CST</i> gene. All the Gal3ST-2-4 actually showed sulfotransferase activity <i>in vitro</i> but act on glycoproteins (<a class="bk_pop" href="#g86-assaycersulfot.REF.6">6</a>).</p></div><div id="g86-assaycersulfot.Protocol"><h2 id="_g86-assaycersulfot_Protocol_">Protocol</h2><p>A convenient assay method for CST activity was developed using anion-exchange chromatography (<a class="bk_pop" href="#g86-assaycersulfot.REF.7">7</a>). The reaction mixture contains the donor substrate, [<sup>35</sup>S]PAPS, the acceptor substrate GalCer, and enzyme protein in a reaction buffer supplemented with some cofactors. After incubation, the reaction product is isolated on a DEAE-Sephadex A-25 column and assayed for radioactivity using a liquid scintillation counter. The values are corrected for a blank value, which is obtained using a reaction mixture devoid of the acceptor.</p><p>Since the acidity of PAPS is stronger than sulfatide, PAPS is retained in the DEAE-Sephadex A-25 resin after elution with 90 mM of ammonium acetate in methanol. This method using anion-exchange chromatography applies to the enzyme assay for ganglioside synthases (<a class="bk_pop" href="#g86-assaycersulfot.REF.7">7</a>).</p><div id="g86-assaycersulfot.Materials"><h3>Materials</h3><p>1. 250 mM sodium cacodylate, pH 6.4 (Na cacodylate from FUJIFILM Wako Pure Chemical Corp., Osaka, Japan)</p><p>2. 0.5 mM GalCer in 5% TRX-100 (GalCer from Sigma, Missouri)</p><p>3. 3’-phospoadenosine-5’-phosphosulfate (PAPS from Sigma)</p><p>4. 1 mM [<sup>35</sup>S]PAPS (NEN-PerkinElmer, Massachusetts)</p><p>5. 0.1 M MnCl<sub>2</sub> (MnCl<sub>2</sub> from FUJIFILM Wako Pure Chemical Corp.)</p><p>6. 10% polyoxyethylene (23) lauryl ether (Lubrol PX from FUJIFILM Wako Pure Chemical Corp.)</p><p>7. 12.5 mM Dithiothreitol (DTT from FUJIFILM Wako Pure Chemical Corp.)</p><p>8. 0.25 M NaF (NaF from FUJIFILM Wako Pure Chemical Corp.)</p><p>9. 0.1 M ATP (ATP from FUJIFILM Wako Pure Chemical Corp.)</p><p>10. 0.5 M NaCl (NaCl from FUJIFILM Wako Pure Chemical Corp.)</p><p>11. DEAE-Sephadex A-25 (GE Healthcare Life Sciences, Illinois)</p><p>12. Chloroform (FUJIFILM Wako Pure Chemical Corp.)</p><p>13. Methanol (FUJIFILM Wako Pure Chemical Corp.)</p><p>14. Scintillation cocktail (PerkinElmer)</p></div><div id="g86-assaycersulfot.Instruments"><h3>Instruments</h3><p>1. Water bath</p><p>2. Mini glass columns</p><p>3. Accupenser (dispenser)</p><p>4. Liquid scintillation counter</p></div><div id="g86-assaycersulfot.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Preparation of the reaction mixture</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare the reaction mixture in a total volume of 50 μL containing 25 mM of Na cacodylate (pH 6.4), 50 μM GalCer in 0.5% TRX-100, 10 mM MnCl<sub>2</sub>, 1% Lubrol PX, 0.25 mM of DTT , 5 mM NaF, 2 mM ATP, 50 mM NaCl, 40 μM [<sup>35</sup>S]PAPS (ca 100 dpm/pmol (<i>see</i>
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<b>Note 1</b>))</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Enzyme reaction</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Add 20 μL of enzyme source.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Incubate at 37°C for 1 h.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Terminate the reaction with 1 mL of chloroform/methanol/water (30:60:8 v/v).</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Isolation of the reaction product (sulfatide) from PAPS</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Add the whole reaction product onto a mini column packed with 1 mL of DEAE-Sephadex A-25 resin.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Wash with 3 mL of chloroform/methanol/water (30:60:8 v/v).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Wash with 6 mL of methanol.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Elute with 5 mL of 90 mM of ammonium acetate in methanol.</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">Measurement of radioactivity</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Eluate is transferred into a scintillation vial.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Put the scintillation cocktail into the eluate.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Count the radioactivity with a liquid scintillation counter.</p></dd><dt>d.</dt><dd><p class="no_top_margin">The values are corrected for a blank value, which is obtained using a reaction mixture devoid of GalCer (<i>see</i>
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<b>Note 2</b>).</p></dd></dl></dd></dl></div><div id="g86-assaycersulfot.Notes"><h3>Notes</h3><p>1. Since the half time of <sup>35</sup>S is relatively short (87.5 d), specific activity of [<sup>35</sup>S]PAPS changes day by day. Therefore, the radioactivity of donor substrate must be checked every time when used.</p><p>2. Enzyme activity is calculated by the following formula:</p><p>Enzyme activity (pmol/h/mL) = (sample − blank [dpm])/specific activity of [<sup>35</sup>S]PAPS (dpm/pmol) × 50.</p></div></div><div id="g86-assaycersulfot.References"><h2 id="_g86-assaycersulfot_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g86-assaycersulfot.REF.1">Honke K, Yamane M, Ishii A, Kobayashi T, Makita A. Purification and characterization of 3'-phosphoadenosine-5'-phosphosulfate:GalCer sulfotransferase from human renal cancer cells. <span><span class="ref-journal">J Biochem (Tokyo). </span>1996 Mar;<span class="ref-vol">119</span>(3):421–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8830034" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8830034</span></a>] [<a href="http://dx.crossref.org/10.1093/oxfordjournals.jbchem.a021258" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g86-assaycersulfot.REF.2">Honke K, Tsuda M, Hirahara Y, Ishii A, Makita A, Wada Y. Molecular cloning and expression of cDNA encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase. <span><span class="ref-journal">J Biol Chem. </span>1997 Feb 21;<span class="ref-vol">272</span>(8):4864–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9030544" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9030544</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.272.8.4864" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g86-assaycersulfot.REF.3">Tsuda M, Egashira M, Niikawa N, Wada Y, Honke K. Cancer-associated alternative usage of multiple promoters of human GalCer sulfotransferase gene. <span><span class="ref-journal">Eur J Biochem. </span>2000 May;<span class="ref-vol">267</span>(9):2672–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10785389" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10785389</span></a>] [<a href="http://dx.crossref.org/10.1046/j.1432-1327.2000.01281.x" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g86-assaycersulfot.REF.4">Hirahara Y, Tsuda M, Wada Y, Honke K. cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase. Eur J Biochem. 2000 Apr;267(7):1909-17. doi: 10.1046/j.1432-1327.2000.01139.x.PMID: 10727929. [<a href="https://pubmed.ncbi.nlm.nih.gov/10727929" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10727929</span></a>] [<a href="http://dx.crossref.org/10.1046/j.1432-1327.2000.01139.x.PMID" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g86-assaycersulfot.REF.5">Honke K, Hirahara Y, Dupree J, Suzuki K, Popko B, Fukushima K, Fukushima J, Nagasawa T, Yoshida N, Wada Y, Taniguchi N. Paranodal junction formation and spermatogenesis require sulfoglycolipids. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2002 Apr 2;<span class="ref-vol">99</span>(7):4227–32.</span> [<a href="/pmc/articles/PMC123630/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC123630</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11917099" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11917099</span></a>] [<a href="http://dx.crossref.org/10.1073/pnas.032068299" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g86-assaycersulfot.REF.6">Honke K, Taniguchi N. Sulfotransferases and sulfated oligosaccharides. <span><span class="ref-journal">Med Res Rev. </span>2002 Nov;<span class="ref-vol">22</span>(6):637–54.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12369092" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12369092</span></a>] [<a href="http://dx.crossref.org/10.1002/med.10020" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g86-assaycersulfot.REF.7">Kawano M, Honke K, Tachi M, Gasa S, Makita A. An assay method for ganglioside synthase using anion-exchange chromatography. Anal Biochem 19891Oct;182(1):9-15. doi: 10.1016/0003-2697(89)90709-4. PMID: 2513740. [<a href="https://pubmed.ncbi.nlm.nih.gov/2513740" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2513740</span></a>] [<a href="http://dx.crossref.org/10.1016/0003-2697(89)90709-4" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593885_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g86-assaycersulfot.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="g86-assaycersulfot.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593885/bin/g86-assaycersulfot-Image001.jpg" alt="Figure 1: . Biosynthetic pathway of sulfatide and seminolipid." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Biosynthetic pathway of sulfatide and seminolipid.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g86-assaycersulfot.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593885/bin/g86-assaycersulfot-Image002.jpg" alt="Figure 2: . Reaction mediated by cerebroside sulfotransferase (CST)." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Reaction mediated by cerebroside sulfotransferase (CST).</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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