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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="_NBK594001_"><span class="title" itemprop="name">Expression and binding assay of endoplasmic reticulum-Golgi intermediate compartment protein 53 (ERGIC-53, LMAN1)</span></h1><div class="contrib half_rhythm"><span itemprop="author">Kazuo Yamamoto</span>, Ph.D.<div class="affiliation small">Grad School of Frontier Sciences, The University of Tokyo<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.oykot-u.k.ude@otomamay" class="oemail">pj.ca.oykot-u.k.ude@otomamay</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">September 29, 2021</span>; Last Revision: <span itemprop="dateModified">March 21, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g105-bindingERGIC53.Introduction"><h2 id="_g105-bindingERGIC53_Introduction_">Introduction</h2><p>ERGIC-53 (ER-Golgi intermediate compartment, 53 kDa) is one of the cargo receptors, which transport glycoproteins in intracellular secretory pathway. This belongs to a new class of type I transmembrane sorting receptor of 53 kDa comprising L-type lectin domain, stalk domain, transmembrane domain, and short cytoplasmic domain. ERGIC-53 associates with MCFD2 (Multiple Coagulation Factor Deficiency 2) protein in calcium-dependent manner, and its interaction is essential for expressing carbohydrate-binding activity of lectin domain (<a class="bk_pop" href="#g105-bindingERGIC53.REF.1">1</a>). MCFD2 protein contains two EF-hand motifs near the C-terminus. The defect of the gene encoding either ERGIC-53 (LMAN1) or MCFD2 causes autosomal recessive bleeding disorder and combined deficiency of coagulation factors V and VIII (F5F8D) (<a class="bk_pop" href="#g105-bindingERGIC53.REF.2">2</a>,<a class="bk_pop" href="#g105-bindingERGIC53.REF.3">3</a>). ERGIC-53/MCFD2 preferentially binds to M8B high mannose-type glycan and transports glycoproteins from the endoplasmic reticulum to the Golgi.</p></div><div id="g105-bindingERGIC53.Protocol"><h2 id="_g105-bindingERGIC53_Protocol_">Protocol</h2><p>The following protocol describes the method to obtain recombinant proteins as a probe for biochemical analysis. Because sugar-binding activity of intracellular lectin is not so strong, tetramerization of the lectin is useful for enhancing the binding. To analyze the functional properties of intracellular lectins, usual expression in mammalian cells is used.</p><div id="g105-bindingERGIC53.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Solubilization buffer: 50 mM of Tris-HCl, pH 8.0, containing 6 M guanidine, 1 mM of DTT, and 0.1 mM of ethylenediaminetetraacetic acid (EDTA)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Refolding buffer: 100 mM Tris-HCl, pH 7.5, containing 0.4 M L-arginine, 5 mM of reduced glutathione, 0.5 mM of oxidized glutathione, and 0.5 mM of phenylmethanesulfonyl fluoride</p></dd><dt>3.</dt><dd><p class="no_top_margin">Dialysis buffer: 20 mM of Tris-HCl, pH 7.5, containing 25 mM of NaCl and 0.1 mM of EDTA</p></dd><dt>4.</dt><dd><p class="no_top_margin">Isopropyl β-thiogalactopyranoside</p></dd><dt>5.</dt><dd><p class="no_top_margin">Biotin ligase, BirA</p></dd><dt>6.</dt><dd><p class="no_top_margin">R-phycoerythrin (PE)-labeled streptavidin</p></dd><dt>7.</dt><dd><p class="no_top_margin">Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA)</p></dd><dt>8.</dt><dd><p class="no_top_margin">HBS: 20 mM of HEPES-NaOH, pH 7.4, containing 150 mM of NaCl and 1 mM of EDTA</p></dd><dt>9.</dt><dd><p class="no_top_margin">Propidium iodide (PI)</p></dd></dl></div><div id="g105-bindingERGIC53.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">FACS Calibur (BD Biosciences, San Jose, CA)</p></dd><dt>2.</dt><dd><p class="no_top_margin">CellQuest software (BD Biosciences)</p></dd></dl></div><div id="g105-bindingERGIC53.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Expression and preparation of soluble ERGIC-53 tetramer</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Construct plasmid encoding a soluble lectin domain of ERGIC-53 with an enzymatic biotinylation sequence (<a class="bk_pop" href="#g105-bindingERGIC53.REF.4">4</a>) (<b>Note 1</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Transform <i>E. coli</i> cell with plasmid and induce expression by adding isopropyl β-thiogalactopyranoside.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Recover expressed lectin domain as soluble proteins or inclusion bodies.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Solubilize recovered inclusion bodies in solubilization buffer, diluted with refolding buffer to a protein concentration of 6 mM, and refolded <i>in vitro</i> by dialysis against dialysis buffer at 4°C for 24 h.</p></dd><dt>e.</dt><dd><p class="no_top_margin">After removal of insoluble material by centrifugation, apply the soluble fraction to anion-exchange chromatography and gel chromatography.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add biotin ligase BirA for biotinylation of soluble ERGIC-53 lectin domain.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Mix soluble ERGIC-53 lectin domain with PE-labeled streptavidin to make PE-labeled soluble ERGIC-53 tetramer (<b>Note 2</b>).</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Binding assay of soluble ERGIC-53 tetramer to cells by flow cytometry</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Harvest cultured mammalian cells and suspend in HBS at a concentration of 2 × 10<sup>7</sup> cells/mL (<b>Note 3</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add PE-labeled soluble ERGIC-53 tetramer at a concentration of 10–100 μg/mL to 10 μL of the cell suspension in a 96-well plate (<b>Note 4</b>).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Allow it to stand at 25°C for 30 min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Wash the cells twice with HBS.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Suspend in 200 μL of HBS containing 1 μg/mL PI.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Measure the fluorescence intensity of PE-labeled ERGIC-53 tetramer at 575 nm by flow cytometry.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Other biochemical analysis of ERGIC-53 in mammalian cells</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Construct plasmid encoding ERGIC-53 with FLAG-tag at N-terminus (<b>Note 5</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Introduce plasmid into mammalian cells by lipofectamine 2000 according to manufacturer’s protocol.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Culture transformed cells at 37°C for 24–48 h.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Precipitate or stain ERGIC-53 using anti-FLAG antibody under appropriate conditions.</p></dd></dl></dd></dl></div><div id="g105-bindingERGIC53.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Enzymatic biotinylation sequence is as follows: GGGLNDIFEAQKIEWHE (<a class="bk_pop" href="#g105-bindingERGIC53.REF.4">4</a>). Side chain of lysine is enzymatically biotinylated.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Partial purification of ERGIC-53 from cell lysates had been reported using mannose-immobilized beads (<a class="bk_pop" href="#g105-bindingERGIC53.REF.5">5</a>). However, several mannose-binding lectins associated with quality control of glycoproteins are also present in the ER and Golgi apparatus. The interaction between these lectins and mannose is quite weak; thus, it is difficult to purify these proteins using conventional affinity chromatography methods.</p></dd><dt>3.</dt><dd><p class="no_top_margin">To modify cell surface glycans, cultured cells are treated with castanospermine, deoxynojirimycin, deoxymannojirimycin, kifunensine, or swainsonine at concentrations of 1 mM, 1 mM, 2 μg/mL, or 10 μg/mL, respectively. Several lectin-resistant Chinese hamster ovary cell variants, such as Lec1, Lec2, or Lec8 with different <i>N</i>-glycan expressions are also available.</p></dd><dt>4.</dt><dd><p class="no_top_margin">Calcium ions are required for sugar-binding of ERGIC-53, and EDTA inhibits its activity. Usually, 1 mM of calcium chloride is added to the buffer.</p></dd><dt>5.</dt><dd><p class="no_top_margin">Because cytoplasmic tail of ERGIC-53 contains anterograde and retrograde transport signals in cells, the modification of C-terminus with tagged sequence may cause abnormal localization of the proteins.</p></dd></dl></div></div><div id="g105-bindingERGIC53.References"><h2 id="_g105-bindingERGIC53_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g105-bindingERGIC53.REF.1">Kawasaki N, Ichikawa Y, Matsuo I, Totani K, Matsumoto N, Ito Y, Yamamoto K. The sugar-binding ability of ERGIC-53 is enhanced by its interaction with MCFD2. <span><span class="ref-journal">Blood. </span>2008 Feb 15;<span class="ref-vol">111</span>(4):1972–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18056485" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18056485</span></a>] [<a href="http://dx.crossref.org/10.1182/blood-2007-06-097022" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g105-bindingERGIC53.REF.2">Nichols WC, Seligsohn U, Zivelin A, Terry VH, Wheatley MA, Moussalli MJ, Hauri HP, Ciavarella N, Kaufman RJ, Ginsburg D. Mutations in the ER-Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII. <span><span class="ref-journal">Cell. </span>1998 Apr 3;<span class="ref-vol">93</span>(1):61–70.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9546392" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9546392</span></a>] [<a href="http://dx.crossref.org/10.1016/s0092-8674(00)81146-0" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g105-bindingERGIC53.REF.3">Zhang B, Cunningham MA, Nichols WC, Bernat JA, Seligsohn U, Pipe SW, McVey JH, Schulte-Overberg U, de Bosch NB, Ruiz-Saez A, White GC, Tuddenham EG, Kaufman RJ, Ginsburg D. Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex. <span><span class="ref-journal">Nat Genet. </span>2003 Jun;<span class="ref-vol">34</span>(2):220–5.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12717434" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12717434</span></a>] [<a href="http://dx.crossref.org/10.1038/ng1153" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g105-bindingERGIC53.REF.4">Yamamoto K., Kawasaki N. Detection of weak-binding sugar activity using membrane-based carbohydrates. <span><span class="ref-journal">Methods Enzymol. </span>2010;2010;<span class="ref-vol">478</span>:233–40.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20816483" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20816483</span></a>] [<a href="http://dx.crossref.org/10.1016/S0076-6879(10)78011-3" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g105-bindingERGIC53.REF.5">Pimpaneau V, Midoux P, Monsigny M, Roche AC. Characterization and isolation of an intracellular D-mannose-specific receptor from human promyelocytic HL60 cells. <span><span class="ref-journal">Carbohydr Res. </span>1991 Jun 25;<span class="ref-vol">213</span>:95–108.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1933956" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1933956</span></a>] [<a href="http://dx.crossref.org/10.1016/s0008-6215(00)90601-3" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594001_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g105-bindingERGIC53.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div id="bk_toc_contnr"></div></div></div>
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