Name: pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells
Uploaded: 2010-04-27T17:10:00
Description: Watch this Scientific Journal Video about Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells at JoVE.com

Wij danken Zehavit Frenkel en Sandra Tolchinsky voor technische bijstand. Onderzoek in verband met dit werk wordt ondersteund door subsidies van de Israel Science Foundation (1229-1207) en de Duits-Israëlische Project Samenwerking (DIP-DFG).

Het volgende protocol is bedoeld voor de analyse van suiker ketens van in totaal glycoproteïnen of van een specifiek glycoproteïne van belang. De procedure is in principe hetzelfde voor beide gevallen met een paar kleine aanpassingen, zoals vermeld in het gehele protocol. <ol><li> Voor metabole de etikettering van N-gekoppelde glycanen moet men een subconfluent cultuur van zoogdiercellen &#39;s nachts gekweekt in een 90 mm schaal voor elk monster gebruik (monsters kunnen vertegenwoordigen verschillende tijdstippen...

<ol>
	<li>Parodi, A. J., Behrens, N. H., Leloir, L. F., Carminatti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+polyprenol-bound+saccharides+as+intermediates+in+glycoprotein+synthesis+in+liver.">The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver.</a> Proc Natl Acad Sci U S A. 69 (11), 3268-3272 (1972).</li><li>Avezov, E., Frenkel, Z., Ehrlich, M., Herscovics, A., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+(ER)+mannosidase+I+is+compartmentalized+and+required+for+N-glycan+trimming+to+Man5-6GlcNAc2+in+glycoprotein+ER-associated+degradation.">Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation.</a> Mol Biol Cell. 19 (1), 216-225 (2008).</li><li>Frenkel, Z., Gregory, W., Kornfeld, S., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-associated+degradation+of+mammalian+glycoproteins+involves+sugar+chain+trimming+to+Man6-5GlcNAc2.">Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.</a> J Biol Chem. 278 (36), 34119-34124 (2003).</li><li>Hosokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+endoplasmic+reticulum+(ER)+degradation+of+misfolded+Null+Hong+Kong+alpha1-antitrypsin+by+human+ER+mannosidase+I.">Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.</a> J Biol Chem. 278 (28), 26287-26294 (2003).</li><li>Jakob, C. A., Burda, P., Roth, J., Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+misfolded+endoplasmic+reticulum+glycoproteins+in+Saccharomyces+cerevisiae+is+determined+by+a+specific+oligosaccharide+structure.">Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.</a> J Cell Biol. 142 (5), 1223-1233 (1998).</li><li>Tolchinsky, S., Yuk, M. H., Ayalon, M., Lodish, H. F., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+versus+secreted+forms+of+human+asialoglycoprotein+receptor+subunits.+Role+of+a+juxtamembrane+pentapeptide.">Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide.</a> J Biol Chem. 271 (24), 14496-14503 (1996).</li></ol>

De pulse-chase analyse van suikers in levende cellen met HPLC-scheiding biedt een methode voor het bestuderen van de dynamiek van oligosaccharide structurele veranderingen gedurende de gehele levensduur van een glycoproteïne. Er zijn steeds meer aanwijzingen dat dergelijke wijzigingen zijn betrokken bij de productie van de signalen voor ER vouwing, kwaliteitscontrole en-handel systemen 2-5. De methode kan worden toegepast, niet alleen voor een specifiek glycoproteïne van belang, maar ook voor het analyseren...

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<td>Dialyzed Foetal Calf Serum</td>
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<td>04-011-1A</td>
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<td>Dulbecco's Modi&#xFB01;ed Eagle's Medium</td>
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<td>41965039</td>
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<td>Roche</td>
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<td>N-octylglucoside</td>
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<td>O3757</td>
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<td>P2714</td>
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<td>Rabbit polyclonal anti-H2 carboxy-terminal 6</td>
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<td>Buffer A: 1% Triton X-100, 0.5% w/v sodium deoxycholate, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Buffer B: 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Buffer C: N-glycosidase F reaction buffer: 200mM Na3PO4, pH 8.0, 4mM EDTA, 1.5% N-octylglucoside</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>NIH 3T3 stably expressing H2a 6</td>
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<td>Buffer E: 0.5% w/v SDS, 1% v/v 2-Mercaptoethanol, Protease inhibitor cocktail 2% v/v in PBS </td>
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<td>2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M3148</td>
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pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

Bevestiging van de Glc<sub&gt; 3</sub&gt; Man<sub&gt; 9</sub&gt; GlcNAc<sub&gt; 2</sub&gt; Voorloper oligosaccharide op beginnende polypeptiden in de ER is een gebruikelijke aanpassing voor de secretorische eiwitten. Hoewel deze wijziging was betrokken bij verschillende biologische processen, zijn bijkomende aspecten van zijn functie in opkomst, met de recente bewijs van haar rol in de productie van signalen voor glycoproteïne kwaliteitscontrole en mensenhandel. Zo worden verschijnselen in verband met N-gekoppelde glycanen en hun verwerking intensief onderzocht. Methoden die onlangs zijn ontwikkeld voor proteoomanalyse zijn sterk verbeterd de karakterisering van glycoproteïne N-gekoppelde glycanen. Toch hebben ze geen inzicht in de dynamiek van de suiker betrokken keten verwerking. Voor deze, de etikettering en de pulse-chase analyse protocollen die worden gebruikt die zijn meestal complexe en geven een zeer lage opbrengsten. We beschrijven hier een eenvoudige methode voor de isolatie en analyse van metabolisch gelabeld N-gekoppelde Oligosacchariden. Het protocol is gebaseerd op de etikettering van cellen met [2 -<sup&gt; 3</sup&gt; H] mannose, denaturerende lysis en enzymatische afgifte van de Oligosacchariden van ofwel een specifiek immunogeprecipiteerd eiwit van belang of uit de algemene glycoproteïne zwembad door opeenvolgende behandelingen met endo H en N-glycosidase F, gevolgd door moleculaire filtratie (Amicon). In deze methode wordt de geïsoleerde oligosaccharides dienen als input voor HPLC-analyse, die discriminatie tussen de verschillende glycaan structuren kunnen op basis van het aantal monosaccharide-eenheden die ze bevatten, met een resolutie van een monosaccharide. Met behulp van deze methode waren we in staat om hoge mannose N-gekoppelde oligosaccharide profielen van totale cel glycoproteïnen studie na pulse-chase in normale omstandigheden en onder proteasoomremming. Deze profielen werden vergeleken met die verkregen uit een immunogeprecipiteerd ER-geassocieerde degradatie (ERAD) substraat. Onze resultaten suggereren dat de meeste NIH 3T3-cellulaire glycoproteïnen zijn relatief stabiel is en dat de meeste van hun oligosacchariden zijn bijgesneden tot Man<sub&gt; 9-8</sub&gt; GlcNAc<sub&gt; 2</sub&gt;. In tegenstelling, zijn instabiel ERAD substraten bijgesneden tot Man<sub&gt; 6-5</sub&gt; GlcNAc<sub&gt; 2</sub&gt; En glycoproteïnen met deze soorten zich ophopen bij de remming van de proteasomale degradatie.

Watch this Scientific Journal Video about Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells at JoVE.com

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

We beschrijven een methode voor de analyse van de verandering van N-gekoppelde glycanen door het vroege leven van glycoproteïnen na hun biosynthese in zoogdiercellen. Dit wordt bereikt door pulse-chase analyse van metabolisch gelabeld glycanen, enzymatische vrijlating uit glycoproteïnen en onderzoek door HPLC.

Pulse-chase analyse van N-gebonden suikerketens van glycoproteïnen in zoogdiercellen

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

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