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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="_NBK593909_"><span class="title" itemprop="name">Endo-β-galactosidase digestion</span></h1><div class="contrib half_rhythm"><span itemprop="author">Michiko Fukuda, N</span>, Dr<div class="affiliation small">Sanford-Burnham-Prebys Medical Discovery Institute<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="gro.yrevocsidPBS@okihcim" class="oemail">gro.yrevocsidPBS@okihcim</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Tomoya Akama, O</span>, Dr<div class="affiliation small">Kansai Medical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.umk.atakarih@tamaka" class="oemail">pj.ca.umk.atakarih@tamaka</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">September 30, 2021</span>; Last Revision: <span itemprop="dateModified">March 22, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g32-endogalact.Introduction"><h2 id="_g32-endogalact_Introduction_">Introduction</h2><p>Endo-β-galactosidase has been widely used for structural characterization of glycoproteins and glycolipids (<a class="bk_pop" href="#g32-endogalact.REF.1">1</a>) containing <i>N</i>-acetyllactosaminoglycan [GlcNAcβ1-3Galβ1-4]<sub>n</sub> (n > 2) structure (Note 1). Method for analyzing the structure of each glycan varies. Three typical analyses are shown below.</p></div><div id="g32-endogalact.Protocol"><h2 id="_g32-endogalact_Protocol_">Protocol</h2><p>Endo-β-galactosidase is used to eliminate polylactosamines and GlcNAc-sulfated keratan sulfate structures from glycoproteins, glycolipids, and cell surfaces. It is also used to release and analyze nonreducing terminal glycans attached to core glycoconjugates by repeating LacNAc as it can cleave di-LacNAc structures present inside carbohydrate chains.</p><div id="g32-endogalact.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Endo-β-galactosidase from <i>Citrobacter freundii</i> (previously called as <i>Enterobacter freundii</i>). This enzyme had been available at Seikagaku Kogyo, but currently it is discontinued. Remaining stocks may be available from venders. Enzymes from other bacteria can be used for analysis, but the enzymatic activity must be confirmed before use.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Keratan sulfate (8618-KS (R&D systems, MN, USA); CSR-NaKS2(PNC)1 (Cosmo Bio, Tokyo, Japan); etc.)</p></dd><dt>3.</dt><dd><p class="no_top_margin">0.2 M sodium-acetate buffer, pH 5.8</p></dd><dt>4.</dt><dd><p class="no_top_margin">Sodium deoxytaurocholate (86339, Sigma-Aldrich, MO, USA)</p></dd><dt>5.</dt><dd><p class="no_top_margin">Phosphate buffered saline (PBS)</p></dd><dt>6.</dt><dd><p class="no_top_margin">Blood group antigen-specific antibodies (MA1-7675, Invitrogen, MA, USA)</p></dd><dt>7.</dt><dd><p class="no_top_margin">Galactose oxidase (G7907, Sigma-Aldrich)</p></dd></dl></div><div id="g32-endogalact.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">A 37°C incubator</p></dd><dt>2.</dt><dd><p class="no_top_margin">A glass column (1 × 130 cm) packed with Sephadex G-50 (superfine) and equilibrated with 0.2 M NaCl</p></dd><dt>3.</dt><dd><p class="no_top_margin">Fraction collector</p></dd><dt>4.</dt><dd><p class="no_top_margin">Thin-layer chromatography (TLC) plate (Baker HPTLC) and glass chromatography chamber</p></dd><dt>5.</dt><dd><p class="no_top_margin">Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) system (gel and power supply)</p></dd><dt>6.</dt><dd><p class="no_top_margin">Scintillation counter</p></dd></dl></div><div id="g32-endogalact.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Digestion of keratan sulfates by endo-β-galactosidase (<a class="bk_pop" href="#g32-endogalact.REF.2">2</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Dissolve keratan sulfate (3.0 mg) in 300 µL of 0.2 M sodium-acetate buffer, pH 5.8.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Dissolve endo-β-galactosidase (0.1 unit) with 100 µL of water, and add 20 µL (20 mUnits) to the keratan sulfate solution (<b>Note 2</b>).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Incubate at 37°C for 1 h or 48 h.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Apply the digest to Sephadex G-50 (superfine) column (1 × 130 cm). Collect 1.15 mL each/tube using a fraction collector and assay for hexose (<a class="figpopup" href="/books/NBK593909/figure/g32-endogalact.F1/?report=objectonly" target="object" rid-figpopup="figg32endogalactF1" rid-ob="figobg32endogalactF1">Figure 1</a>).</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Digestion of glycolipids by endo-β-galactosidase (<a class="bk_pop" href="#g32-endogalact.REF.3">3</a>–<a class="bk_pop" href="#g32-endogalact.REF.5">5</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Dissolve glycosphingolipid (100 µg) in 50 µL of 0.2 M sodium-acetate buffer, pH 5.8, containing 200 µg of sodium deoxytaurocholate.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Dissolve endo-β-galactosidase (0.1 Units) with 100 μL of water, and add 50 µL (5 mUnits) to the glycolipid solution (<b>Note 2</b>).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Incubate at 37°C for 20 h.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Apply the digest to the TLC plate, and develop the TLC plate in a solvent comprising chloroform/methanol/water (2:1:0.1, v/v). The TLC plate is sprayed with orcinol solution and baked in an oven for 5 min or until a purple color develops (<a class="figpopup" href="/books/NBK593909/figure/g32-endogalact.F2/?report=objectonly" target="object" rid-figpopup="figg32endogalactF2" rid-ob="figobg32endogalactF2">Figure 2</a>).</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Cell surface modification of endo-β-galactosidase (<a class="bk_pop" href="#g32-endogalact.REF.6">6</a>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Wash human red blood cells with PBS by centrifugation. Suspend red cells with an equal volume of PBS.</p></dd><dt>b.</dt><dd><p class="no_top_margin">To the cells (100 µL), add 20 µL of endo-β-galactosidase (20 mUnits) dissolved in PBS and incubate at 37°C for 30 min (<b>Note 2</b>).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Determine levels of carbohydrate antigens by hemagglutination using specific antibody for each blood group. Alternatively, galactose and/or <i>N</i>-acetylgalactosamine residues of cell surface glycoconjugates are labeled by galactose oxidase/NaB[<sup>3</sup>H]<sub>4</sub> followed by endo-β-galactosidase treatment. Released oligosaccharides are then analyzed by Sephadex G-50 chromatography (<a class="figpopup" href="/books/NBK593909/figure/g32-endogalact.F3/?report=objectonly" target="object" rid-figpopup="figg32endogalactF3" rid-ob="figobg32endogalactF3">Figure 3</a>), glycoproteins are analyzed by SDS–polyacrylamide gel electrophoresis (<a class="figpopup" href="/books/NBK593909/figure/g32-endogalact.F4/?report=objectonly" target="object" rid-figpopup="figg32endogalactF4" rid-ob="figobg32endogalactF4">Figure 4</a>), and glycolipids are analyzed by TLC (<a class="figpopup" href="/books/NBK593909/figure/g32-endogalact.F5/?report=objectonly" target="object" rid-figpopup="figg32endogalactF5" rid-ob="figobg32endogalactF5">Figure 5</a>).</p></dd></dl></dd></dl></div><div id="g32-endogalact.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Endo-β-galactosidase hydrolyzes linear polylactosaminoglycans but not branched polylactosamines (<a class="bk_pop" href="#g32-endogalact.REF.1">1</a>). Any modification of galactose residue hinders the access of this enzyme.</p></dd><dt>2.</dt><dd><p class="no_top_margin">The amount of enzyme required for complete digestion of the substrate depends on the structure, purity, and amount of the substrate.</p></dd></dl></div></div><div id="g32-endogalact.References"><h2 id="_g32-endogalact_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g32-endogalact.REF.1">Fukuda MN. CRC Handbook of endoglycosidases and glycoaminases. Boca Raton (FL): CRC press; 1992. Chapter 4, Endo-β-galactosidases; p. 55-103.</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g32-endogalact.REF.2">Fukuda MN, Matsumura G. Endo-beta-galactosidase of Escherichia freundii. Purification and endoglycosidic action on keratansulfates, oligosaccharides, and blood group active glycoprotein. <span><span class="ref-journal">J Biol Chem. </span>1976 Oct 25;<span class="ref-vol">251</span>(20):6218–25.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/135762" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 135762</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g32-endogalact.REF.3">Fukuda MN, Dell A, Oates JE, Wu P, Klock JC, Fukuda M. Structures of glycosphingolipids isolated from human granulocytes. The presence of a series of linear poly-N-acetyllactosaminylceramide and its significance in glycolipids of whole blood cells. <span><span class="ref-journal">J Biol Chem. </span>1985 Jan 25;<span class="ref-vol">260</span>(2):1067–82.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/3881421" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3881421</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g32-endogalact.REF.4">Fukuda MN, Bothner B, Lloyd KO, Rettig WJ, Tiller PR, Dell A. Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence. <span><span class="ref-journal">J Biol Chem. </span>1986 Apr 15;<span class="ref-vol">261</span>(11):5145–53.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/3514610" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3514610</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g32-endogalact.REF.5">Fukuda MN, Watanabe K, Hakomori S. Release of oligosaccharides from various glycosphingolipids by endo-beta-galactosidase. J Biol Chem. 1978 Oct 10;253(19):6814-9. PMID: 99449. [<a href="https://pubmed.ncbi.nlm.nih.gov/99449" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 99449</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g32-endogalact.REF.6">Fukuda MN, Fukuda M, Hakomori S. Cell surface modification by endo-beta-galactosidase. Change of blood group activities and release of oligosaccharides from glycoproteins and glycosphingolipids of human erythrocytes. J Biol Chem. 1979 Jun 25;254(12):5458-65. PMID: 87393. [<a href="https://pubmed.ncbi.nlm.nih.gov/87393" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 87393</span></a>]</div></dd></dl></div><h2 id="NBK593909_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g32-endogalact.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="g32-endogalact.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593909/bin/g32-endogalact-Image001.jpg" alt="Figure 1: . Sephadex G-50 chromatography of bovine corneal keratan sulfate digested by C." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Sephadex G-50 chromatography of bovine corneal keratan sulfate digested by <i>C. freundii</i> endo-β-galactosidase (<a class="bk_pop" href="#g32-endogalact.REF.2">2</a>). Chromatograms were monitored by anthrone color reaction for hexose. The smallest oligosaccharide peak IX contains GlcNAcβ1-3Gal and SO<sub>4</sub>-6GlcNAcβ1-3Gal. Peak VIII contains sulfated tetrasaccharides.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g32-endogalact.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593909/bin/g32-endogalact-Image002.jpg" alt="Figure 2: . Thin-layer chromatography (TLC) of glycolipids digested by endo-β-galactosidase (4)." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Thin-layer chromatography (TLC) of glycolipids digested by endo-β-galactosidase (<a class="bk_pop" href="#g32-endogalact.REF.4">4</a>). Each purified glycolipid was digested by endo-β-galactosidase and was separated into organic phases and water in chloroform/methanol/water (2:1:0.1, v/v). Left panel shows the organic phase of the products of Ga4a (lane 1), Ga4b (lane 2), and Ga6 (lane 3), which identified glucosylceramide as the product. Right panel shows the TLC of the oligosaccharide products separated into water phase, the structure of which was identified as monosialylated tetrasaccharide (1’ and 2’) and disialylated pentasaccharide (lane 3’) (<a class="bk_pop" href="#g32-endogalact.REF.4">4</a>). NE1, glucosylceramide; NE2, lactosylceramide; NE3, globotriaosylceramide; TC, taurodeoxycholate (detergent).</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g32-endogalact.F3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593909/bin/g32-endogalact-Image003.jpg" alt="Figure 3: . Gel filtration analysis of oligosaccharides released by endo-β-galactosidase from cell surface labeled human erythrocytes (6)." /></div><h3><span class="label">Figure 3: </span></h3><div class="caption"><p>Gel filtration analysis of oligosaccharides released by endo-β-galactosidase from cell surface labeled human erythrocytes (<a class="bk_pop" href="#g32-endogalact.REF.6">6</a>). Human erythrocytes were labeled using galactose oxidase/NaB[<sup>3</sup>H]<sub>4</sub> treatment. Erythrocytes were then treated with endo-β-galactosidase and released oligosaccharides were applied to Sephadex G-50 column. Radioactivity in each fraction was counted by a scintillation counter. Open circle: released radioactivity after endo-β-galactosidase treatment; closed circle, released radioactivity by control treatment.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g32-endogalact.F4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593909/bin/g32-endogalact-Image004.jpg" alt="Figure 4: . Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of surface labeled human red blood cells treated with or without endo-β-galactosidase (6)." /></div><h3><span class="label">Figure 4: </span></h3><div class="caption"><p>Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of surface labeled human red blood cells treated with or without endo-β-galactosidase (<a class="bk_pop" href="#g32-endogalact.REF.6">6</a>). Lanes 1 and 2 are Coomassie blue staining of the gel showing erythrocyte membrane proteins. Bands 3 (anion transporter) and 4.5 (glucose transporter) are heterogeneously glycosylated by polylactosamines, giving broad bands. Lanes 3 and 4 are fluorogram of cell surface labeled erythrocyte membranes after treatment without (lane 3) or with endo-β-galactosidase (lane 4). Note that radioactivity associated with bands 3 and 4.5 disappeared after endo-β-galactosidase digestion.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g32-endogalact.F5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593909/bin/g32-endogalact-Image005.jpg" alt="Figure 5: . Fluorogram of thin-layer chromatography (TLC) analysis of cell surface labeled erythrocyte glycolipids (6)." /></div><h3><span class="label">Figure 5: </span></h3><div class="caption"><p>Fluorogram of thin-layer chromatography (TLC) analysis of cell surface labeled erythrocyte glycolipids (<a class="bk_pop" href="#g32-endogalact.REF.6">6</a>). Glycolipids were extracted from the membranes of cell surface labeled human erythrocytes treated with (lane 2) or without (lane 1) endo-β-galactosidases. Radioactive glycolipids were separated using TLC and visualized using fluorography. Note that endo-β-galactosidase removed labels at H2, H3, and H4 regions, which are made of polylactosaminyl structure, whereas short lacto-series glycolipids (H1) or globo-series (Glo) structure were not susceptible to endo-β-galactosidase.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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