108 lines
No EOL
31 KiB
XML
108 lines
No EOL
31 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="NBK593868" /><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="/NBK593868/?report=printable" /><meta name="ncbi_app" content="bookshelf" />
|
||
<!-- Logger end -->
|
||
|
||
<title>Purification and analysis of free N-glycans in Saccharomyces cerevisiae - 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="Purification and analysis of free N-glycans in Saccharomyces cerevisiae" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/24" /><meta name="citation_author" content="Hiroto Hirayama" /><meta name="citation_author" content="Tadashi Suzuki" /><meta name="citation_pmid" content="37590614" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593868/" /><meta name="citation_keywords" content="free N-glycans" /><meta name="citation_keywords" content="misfolded glycoproteins" /><meta name="citation_keywords" content="peptide:N-glycanase" /><meta name="citation_keywords" content="endoplasmic reticulum-associated degradation" /><meta name="citation_keywords" content="yeast" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Purification and analysis of free N-glycans in Saccharomyces cerevisiae" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Hiroto Hirayama" /><meta name="DC.Contributor" content="Tadashi Suzuki" /><meta name="DC.Date" content="2022/03/24" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593868/" /><meta name="description" content="In eukaryotes, free N-glycans (fNGs) are liberated either from lipid donors or glycoproteins and are mainly accumulated in the cytosol. The fNGs are generated by two distinct pathways (Figure 1) (1). First, fNGs are generated from misfolded glycoproteins by the action of cytosolic peptide:N-glycanase (PNGase; Png1 in yeast) during endoplasmic reticulum-associated degradation (ERAD). Alternatively, mature forms of dolichol-linked oligosaccharides (Glc3Man9GlcNAc2) can be liberated in the luminal side of the endoplasmic reticulum (ER) by oligosaccharyltransferase (2, 3). The fNGs are then processed by glycosidases (e.g., α-glucosidase I/II and mannosidase I) in the ER and are eventually transported to the cytosol by an unidentified mechanism (4). In budding yeast, almost all cytosolic fNGs are generated from misfolded glycoproteins in PNGase-dependent manner (Figure 1) (5). The structural and quantitative analysis of fNGs, therefore, would serve as a powerful method for monitoring the overall processing of N-glycans on misfolded glycoproteins that undergo ERAD in yeast cells (5)." /><meta name="og:title" content="Purification and analysis of free N-glycans in Saccharomyces cerevisiae" /><meta name="og:type" content="book" /><meta name="og:description" content="In eukaryotes, free N-glycans (fNGs) are liberated either from lipid donors or glycoproteins and are mainly accumulated in the cytosol. The fNGs are generated by two distinct pathways (Figure 1) (1). First, fNGs are generated from misfolded glycoproteins by the action of cytosolic peptide:N-glycanase (PNGase; Png1 in yeast) during endoplasmic reticulum-associated degradation (ERAD). Alternatively, mature forms of dolichol-linked oligosaccharides (Glc3Man9GlcNAc2) can be liberated in the luminal side of the endoplasmic reticulum (ER) by oligosaccharyltransferase (2, 3). The fNGs are then processed by glycosidases (e.g., α-glucosidase I/II and mannosidase I) in the ER and are eventually transported to the cytosol by an unidentified mechanism (4). In budding yeast, almost all cytosolic fNGs are generated from misfolded glycoproteins in PNGase-dependent manner (Figure 1) (5). The structural and quantitative analysis of fNGs, therefore, would serve as a powerful method for monitoring the overall processing of N-glycans on misfolded glycoproteins that undergo ERAD in yeast cells (5)." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK593868/" /><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/g1-purificationanal/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK593868/" /><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="CE8CE4137D66C9E10000000000680051.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="_NBK593868_"><span class="title" itemprop="name">Purification and analysis of free <i>N</i>-glycans in <i>Saccharomyces cerevisiae</i></span></h1><div class="contrib half_rhythm"><span itemprop="author">Hiroto Hirayama</span>, D. Sci.<div class="affiliation small">RIKEN<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.nekir@amayarih-otorih" class="oemail">pj.nekir@amayarih-otorih</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Tadashi Suzuki</span>, D. Sci.<div class="affiliation small">RIKEN<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.nekir@mg_ikuzust" class="oemail">pj.nekir@mg_ikuzust</a></div></div></div><p class="small">Created: <span itemprop="datePublished">September 15, 2021</span>; Last Revision: <span itemprop="dateModified">March 24, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g1-purificationanal.Introduction"><h2 id="_g1-purificationanal_Introduction_">Introduction</h2><p>In eukaryotes, free <i>N</i>-glycans (fNGs) are liberated either from lipid donors or glycoproteins and are mainly accumulated in the cytosol. The fNGs are generated by two distinct pathways (<a class="figpopup" href="/books/NBK593868/figure/g1-purificationanal.F1/?report=objectonly" target="object" rid-figpopup="figg1purificationanalF1" rid-ob="figobg1purificationanalF1">Figure 1</a>) (<a class="bk_pop" href="#g1-purificationanal.REF.1">1</a>). First, fNGs are generated from misfolded glycoproteins by the action of cytosolic peptide:<i>N</i>-glycanase (PNGase; Png1 in yeast) during endoplasmic reticulum-associated degradation (ERAD). Alternatively, mature forms of dolichol-linked oligosaccharides (Glc3Man9GlcNAc2) can be liberated in the luminal side of the endoplasmic reticulum (ER) by oligosaccharyltransferase (<a class="bk_pop" href="#g1-purificationanal.REF.2">2</a>, <a class="bk_pop" href="#g1-purificationanal.REF.3">3</a>). The fNGs are then processed by glycosidases (e.g., α-glucosidase I/II and mannosidase I) in the ER and are eventually transported to the cytosol by an unidentified mechanism (<a class="bk_pop" href="#g1-purificationanal.REF.4">4</a>). In budding yeast, almost all cytosolic fNGs are generated from misfolded glycoproteins in PNGase-dependent manner (<a class="figpopup" href="/books/NBK593868/figure/g1-purificationanal.F1/?report=objectonly" target="object" rid-figpopup="figg1purificationanalF1" rid-ob="figobg1purificationanalF1">Figure 1</a>) (<a class="bk_pop" href="#g1-purificationanal.REF.5">5</a>). The structural and quantitative analysis of fNGs, therefore, would serve as a powerful method for monitoring the overall processing of <i>N</i>-glycans on misfolded glycoproteins that undergo ERAD in yeast cells (<a class="bk_pop" href="#g1-purificationanal.REF.5">5</a>).</p></div><div id="g1-purificationanal.Protocol"><h2 id="_g1-purificationanal_Protocol_">Protocol</h2><p>In this chapter, detailed protocols for isolation and structural analysis of neutral fNGs in yeast cells are described.</p><div id="g1-purificationanal.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Yeast extract peptone dextrose (YPD) broth (2% peptone, 1% yeast extract, and 2% glucose, Fisher Scientific (Pittsburgh, PA), catalog number: DF0428-17-5)</p></dd><dt>2.</dt><dd><p class="no_top_margin">20 mM of Tris-HCl (pH 8.0)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Ethanol (Wako (Tokyo, Japan), catalog number: 057-00456)</p></dd><dt>4.</dt><dd><p class="no_top_margin">Acetonitrile (high-performance liquid chromatography [HPLC] grade, Wako) catalog number: 015-08633)</p></dd><dt>5.</dt><dd><p class="no_top_margin">AG1-X2 for anion-exchange chromatography (200–400 mesh; acetate form; Bio-Rad (Hercules, CA), catalog number: 1401253)</p></dd><dt>6.</dt><dd><p class="no_top_margin">AG50-X8 for cation-exchange chromatography (200–400 mesh; H<sup>+</sup> form; Bio-Rad, catalog number: 1421451)</p></dd><dt>7.</dt><dd><p class="no_top_margin">InertSep graphite carbon (GC) column for solid phase extraction (150 mg/3 mL; GL-Science (Tokyo, Japan) catalog number: 5010-68000)</p></dd><dt>8.</dt><dd><p class="no_top_margin">2-aminopyridine (PA) (special grade for fluorescence labeling; Wako, catalog number: 011-14181)</p></dd><dt>9.</dt><dd><p class="no_top_margin">PA-labeling solution (dissolve 522 mg of 2-aminopyridine in 200 µL of acetate)</p></dd><dt>10.</dt><dd><p class="no_top_margin">Dimethylamine-borane (Wako, catalog number: 026-08402)</p></dd><dt>11.</dt><dd><p class="no_top_margin">Reducing reagent (dissolve 20 mg of dimethylamine-borane in 100 µL of acetate)</p></dd><dt>12.</dt><dd><p class="no_top_margin">MonoSpin Hydrophilic Interaction Liquid Chromatography (HILIC) Columns (NH<sub>2</sub>, S-Type; GL-Science, catalog number: 5010-21710)</p></dd><dt>13.</dt><dd><p class="no_top_margin">NH2P-50 4E column (4.6 × 250 mm; Shodex (Tokyo, Japan), catalog number: F7630001)</p></dd><dt>14.</dt><dd><p class="no_top_margin">Mobile phase A for HPLC (93% acetonitrile in 0.3% acetate [pH adjusted to 7.0 with ammonia])</p></dd><dt>15.</dt><dd><p class="no_top_margin">Mobile phase B for HPLC (20% acetonitrile in 0.3% acetate [pH adjusted to 7.0 with ammonia])</p></dd><dt>16.</dt><dd><p class="no_top_margin">PA-labeled glycans (TaKaRa (Kyoto, Japan))</p></dd><dt>17.</dt><dd><p class="no_top_margin">PA-Glucose Oligomer (DP=3–15; TaKaRa, catalog number: 4108)</p></dd></dl></div><div id="g1-purificationanal.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Speed Vac concentrator (EYELA (Tokyo, Japan))</p></dd><dt>2.</dt><dd><p class="no_top_margin">HPLC system (LaChrom Elite; Hitachi High-Tech (Tokyo, Japan))</p></dd></dl></div><div id="g1-purificationanal.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Extraction of free <i>N</i>-glycans from yeast cells</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Grow overnight culture (5 mL scale).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Dilute saturated cells to OD<sub>600</sub> = 0.5 with YPD and grow to OD<sub>600</sub> = 2.0 (3–4 h) at 30°C (50 mL scale).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Harvest the cells and resuspend 100 OD<sub>600</sub> unit cells in 500 µL of 20 mM of Tris-HCl (pH 8.0).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Add 500 µL of ethanol.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Mix and centrifuge 20,000 ×<i>g</i> for 5 min at 4°C (<b>Note 1</b>).</p></dd><dt>f.</dt><dd><p class="no_top_margin">Transfer the supernatant to a new tube and lyophilize by Speed Vac or an equivalent.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Desalting and purification of free <i>N</i>-glycans</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Resuspend lyophilized sample in 500 µL of distilled water (DW).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Apply samples onto AG1-X2 and AG50-X8 (resin volume, 500 μL each (<b>Note 2</b>)), wash the column with 3 mL of water, and collect flow through as a desalted fraction.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Activate InertSep GC column by adding 3 mL of 100% acetonitrile.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Equilibrate the GC column by 6 mL of DW.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Apply the desalted sample onto the equilibrated GC column.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Wash the column with 3 mL of water (×2 times).</p></dd><dt>g.</dt><dd><p class="no_top_margin">Elute purified free <i>N</i>-glycans with 3 mL of 25% acetonitrile.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Evaporate samples to dryness by Speed Vac or an equivalent.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Pyridyl amino labeling (PA-labeling) of free <i>N</i>-glycans</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Dissolve the dried-up samples in 20 µL of 2-aminopyridine labeling solution.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Incubate the sample at 80°C for 1 h.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 20 µL of reducing reagent to the sample.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Incubate the sample at 80°C for 1 h.</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">Removal of excessive 2-aminopyridine from the sample by MonoSpin HILIC Columns</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Load 500 µL of DW onto a spin column and centrifuge 5,000 ×<i>g</i> for 1 min.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Discard the flow through.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Load 700 µL of 100% acetonitrile onto the spin column for equilibration and centrifuge 5,000 ×<i>g</i> for 1 min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Discard the flow through.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add 500 µL of 100% acetonitrile to the sample, and mix it well.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Load the PA-labeled sample onto the equilibrated spin column and centrifuge 5,000 ×<i>g</i> for 1 min (<b>Note 3</b>).</p></dd><dt>g.</dt><dd><p class="no_top_margin">Discard the flow through.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Load 700 µL of 90% acetonitrile onto the spin column and centrifuge 5,000 ×<i>g</i> for 1 min.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Discard the flow through.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Elute the PA-labeled fNGs by loading 200 µL of DW and centrifugation (5,000 ×<i>g</i> for 1 min).</p></dd></dl></dd><dt>5.</dt><dd><p class="no_top_margin">Analysis of fNGs by size-fractionation HPLC</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Analyze aliquots (10 μL) of the fNGs solutions by HPLC at 25°C on NH2P-50 4E column by the following elution condition:<br />flow rate: 0.8 mL/min; <br />column temperature: 25°C,<br />detection of the fluorescence: λ<sub>ex</sub>, 310 nm and λ<sub>em</sub>, 380 nm;<br />gradient profile (expressed as the percentage of solvent B): 0–5 min, isocratic 3%; 5–8 min, 3%–33%; and 8–40 min, 33%–71%.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Assign the separated fNGs and calculate the amount of each glycan (<a class="figpopup" href="/books/NBK593868/figure/g1-purificationanal.F2/?report=objectonly" target="object" rid-figpopup="figg1purificationanalF2" rid-ob="figobg1purificationanalF2">Figure 2</a>) (<b>Notes 4–6</b>).</p></dd></dl></dd></dl></div><div id="g1-purificationanal.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Cells will burst with this treatment, and fNGs can be recovered in the supernatant, whereas proteins will be precipitated.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Prepare AG1-X2 and AG50-X8 resin as 50% slurry.</p></dd><dt>3.</dt><dd><p class="no_top_margin">The insoluble precipitate that appears by adding 100% acetonitrile in Step 4e can also be loaded onto the spin column.</p></dd><dt>4.</dt><dd><p class="no_top_margin">Each peak is assigned by comparing their elution position with those of authentic standards, which are available from TaKaRa.</p></dd><dt>5.</dt><dd><p class="no_top_margin">The number of each fNGs is calculated based on the peak area with PA-Glc<sub>6</sub> (2 pmol) in 1 µL of the PA-glucose oligomer (TaKaRa) as a reference.</p></dd><dt>6.</dt><dd><p class="no_top_margin">Approximately 500 pmol of PA-labeled fNGs (Hex5-12HexNAc2 forms of glycans) will be isolated from 100 OD<sub>600</sub> units of yeast cells.</p></dd></dl></div></div><div id="g1-purificationanal.References"><h2 id="_g1-purificationanal_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g1-purificationanal.REF.1">Hirayama H, Hosomi A, Suzuki T. Physiological and molecular functions of the cytosolic peptide:N-glycanase. <span><span class="ref-journal">Semin Cell Dev Biol. </span>2015;<span class="ref-vol">41</span>:110–20.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/25475175" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25475175</span></a>] [<a href="http://dx.crossref.org/10.1016/j.semcdb.2014.11.009" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g1-purificationanal.REF.2">Harada Y, Buser R, Ngwa EM, Hirayama H, Aebi M, Suzuki T. Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation. <span><span class="ref-journal">J Biol Chem. </span>2013;<span class="ref-vol">288</span>(45)</span> [<a href="/pmc/articles/PMC3820902/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3820902</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/24062310" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24062310</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M113.486985" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g1-purificationanal.REF.3">Yamasaki T, Kohda D. Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase. <span><span class="ref-journal">J Biol Chem. </span>2020;<span class="ref-vol">295</span>(47):16072–85.</span> [<a href="/pmc/articles/PMC7681024/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC7681024</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/32938717" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 32938717</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.RA120.015013" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g1-purificationanal.REF.4">Harada Y, Hirayama H, Suzuki T. Generation and degradation of free asparagine-linked glycans. <span><span class="ref-journal">Cell Mol Life Sci. </span>2015;<span class="ref-vol">72</span>(13):2509–33.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/25772500" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25772500</span></a>] [<a href="http://dx.crossref.org/10.1007/s00018-015-1881-7" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g1-purificationanal.REF.5">Hirayama H, Seino J, Kitajima T, Jigami Y, Suzuki T. Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae. <span><span class="ref-journal">J Biol Chem. </span>2010;<span class="ref-vol">285</span>(16):12390–404.</span> [<a href="/pmc/articles/PMC2852977/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2852977</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20150426" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20150426</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M109.082081" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g1-purificationanal.REF.6">Hossain TJ, Hirayama H, Harada Y, Suzuki T. Lack of the evidence for the enzymatic catabolism of Man1GlcNAc2 in Saccharomyces cerevisiae. <span><span class="ref-journal">Biosci Biotechnol Biochem. </span>2016;<span class="ref-vol">80</span>(1):152–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/26264652" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 26264652</span></a>] [<a href="http://dx.crossref.org/10.1080/09168451.2015.1072464" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593868_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g1-purificationanal.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="g1-purificationanal.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593868/bin/g1-purificationanal-Image001.jpg" alt="Figure 1: . Formation/catabolic pathway of free N-glycans (fNGs) in yeast (1)." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Formation/catabolic pathway of free <i>N</i>-glycans (fNGs) in yeast (<a class="bk_pop" href="#g1-purificationanal.REF.1">1</a>). On the luminal side of the ER, a few amounts (~3.5%) of fNGs are generated by oligosaccharyltransferase (OST). After sequential trimming of the liberated fNGs by ER-resident hydrolases (glucosidase I, glucosidase II, and mannosidase I), M8-form fNGs is retrotranslocated to the cytosol by an unidentified mechanism (<a class="bk_pop" href="#g1-purificationanal.REF.2">2</a>-<a class="bk_pop" href="#g1-purificationanal.REF.4">4</a>). Conversely, most fNGs (~96.5%) are generated by cytosolic peptide:<i>N</i>-glycanase (Png1) cleaves <i>N</i>-glycans from misfolded glycoproteins, which are recognized by the ERAD and retrotranslocated from the ER to the cytosol (<a class="bk_pop" href="#g1-purificationanal.REF.1">1</a>,<a class="bk_pop" href="#g1-purificationanal.REF.5">5</a>). fNGs in the cytosol are further trimmed by a sole cytosolic α-mannosidase (Ams1), giving rise to M1 form fNGs (<a class="bk_pop" href="#g1-purificationanal.REF.6">6</a>).</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g1-purificationanal.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593868/bin/g1-purificationanal-Image002.jpg" alt="Figure 2: . The elution profile of yeast fNGs by size-fractionation high-performance liquid chromatography (HPLC) (5)." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>The elution profile of yeast fNGs by size-fractionation high-performance liquid chromatography (HPLC) (<a class="bk_pop" href="#g1-purificationanal.REF.5">5</a>). Hex5–Hex12 represents Hex5HexNAc2–Hex12HexNAc2. The arrowheads indicate the elution position of PA-isomaltooligosaccharides (PA-glucose oligomer) for elution standard. *, non-specific peak.</p></div></div></div></div><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: NBK593868</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/37590614" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">37590614</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/glycoproteins/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/glycopodv2/g2-synthesisglyco/" 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=NBK593868&ncbi_domain=glycopodv2&ncbi_report=printable&ncbi_type=fulltext&ncbi_objectid=&ncbi_pcid=/NBK593868/?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> |