We are grateful to Drs. Mike S. Brown and Joseph L. Goldstein for their agreement to publish this method according to the methods described in their publications. We appreciate Dr. Peter Espenshade for sharing the GFP-SCAP plasmid. This work was supported by NIH grants NS072838 and NS079701 to D.G., American Cancer Society Research Scholar Grant RSG-14-228-01-CSM to D.G., and OSUCCC Pelotonia Postdoctoral Fellowship to C.C. We also appreciate the support from the Ohio State Neuroscience Core (P30 NS038526) and OSUCCC Translational Therapeutic Program seed grant and start-up funds to D.G.

1. Detection of Endogenous SCAP Protein in Human Cells
<ol>
	<li>Cell Culture and Treatment
	<ol>
		<li>Seed ~ 1 &#215; 106 U87 cells in a 10 cm dish with Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and incubate the cells at 37 &#176;C and 5% CO2 for 24 hr before treatment.</li>
		<li>Wash cells once with phosphate-buffered saline (PBS), then switch cells to fresh DMEM medium with or without glucose (5 mM) for 12 hr.</li>
	</ol>
	</li>
	<li>Preparation of Cell Membrane Fractions and Nuclear Extracts
	<ol>
		<li>Wash cells once with PBS, scrape into 1 ml PBS, and centrifuge at 1,000 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend cells in an ice-cold buffer containing 10 mM HEPES-KOH (pH 7.6), 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium ethylenediaminetetraacetic acid (EDTA), 1 mM sodium ethylene glycol tetraacetic acid (EGTA), 250 mM sucrose, and a mixture of protease inhibitors (5 &#181;g/ml pepstatin A, 10 &#181;g/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and 25 &#181;g/ml N-acetyl-Leu-Leu-Norleu-al (ALLN)) for 30 min on ice.</li>
		<li>Pass cell extracts through a 22 G x 1 1/2 needle 30 times and centrifuge at 890 x g at 4 &#176;C for 5 min to isolate nuclei.</li>
		<li>Use the supernatant for the separation of membrane fractions (step 1.2.7). Resuspend the nuclear pellet in 0.1 ml of buffer C (20 mM HEPES/KOH pH 7.6, 0.42 M NaCl, 2.5% (v/v) glycerol, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA and a mixture of protease inhibitors (5 &#181;g/ml pepstatin A, 10 &#181;g/ml leupeptin, 0.5 mM PMSF, 1 mM DTT, and 25 &#181;g/ml ALLN)).</li>
		<li>Rotate the suspension at 4 &#176;C for 60 min and centrifuge at 20,000 x g in a microcentrifuge for 20 min at 4 &#176;C. The supernatant is designated as &#34;nuclear extracts&#34;.</li>
		<li>Heat the nuclear extracts at 100 &#176;C for 10 min with 5x loading buffer (312.5 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 12.5% &#946;-mercaptoethanol, and 0.05% bromophenol blue) before subjecting to SDS-PAGE.</li>
		<li>Centrifuge the supernatant from the original 890 x g spin at 20,000 x g for 20 min at 4 &#176;C. For subsequent western blot analysis, dissolve the pellet in 0.1 ml of SDS lysis buffer (10 mM Tris-HCl pH 6.8, 100 mM NaCl, 1% (v/v) sodium dodecyl sulfate (SDS), 1 mM sodium EDTA, and 1 mM sodium EGTA). This is designated &#34;membrane fraction&#34;. 
		NOTE: We use 20,000 x g for 20 min to pellet membranes. 100,000 x g for 10 - 20 min has been used in the papers from the Brown and Goldstein laboratory15.</li>
		<li>Incubate the membrane fraction at 37 &#176;C for 30 min and determine the protein concentration by Bradford protein assay. Add 1 &#181;l 100x bromophenol blue solution before subjecting the samples to SDS-PAGE.</li>
		<li>Load 25 - 50 &#181;g of total membrane proteins on a 10% SDS-PAGE gel39. Run the gel at 80 V for 15 min, then change to 130 V and run for another 115 min. Transfer at 140 mA for 130 min39.
		<ol>
			<li>For Western blotting analysis, use anti-SCAP antibody to detect the total SCAP protein. Use protein disulfide isomerase (PDI), an ER-resident protein40, as an internal control. Use anti-SREBP-1 antibody to detect the N-terminal band of SREBP-1. Use Lamin A as an internal control for nuclear extracts31.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Analysis of SCAP N-glycosylation in Human Cells
<ol>
	<li>Cell Culture, Transfection, and Treatment
	<ol>
		<li>For membrane analysis, seed ~1 &#215; 106 HEK293T cells in a 10 cm dish with DMEM supplemented with 5% FBS and incubate the cells at 37&#176;C and 5% CO2 for 24 hr before transfection.</li>
		<li>Dilute 4 - 8 &#181;g GFP-SCAP plasmid (a gift from Dr. Peter Espenshade)22 in 1 ml reduced serum medium and mix gently.</li>
		<li>Add 8 - 16 &#181;l DNA transfection reagent by pipetting directly into the 1 ml reduced serum medium containing plasmids and mix gently.</li>
		<li>Incubate the transfection reagent/plasmid complex for 25 min at room temperature.</li>
		<li>Add transfection complex to the cells with fresh medium and continue to culture for 24 hr at 5% CO2 and 37 &#176;C.</li>
		<li>Wash once with PBS and treat the cells for 12 hr with or without glucose (5 mM) in the presence or absence of tunicamycin (1 &#181;g/ml), an N-glycosylation inhibitor.</li>
	</ol>
	</li>
	<li>Prepare cell membrane pellets as described in steps 1.2.1, 1.2.2, 1.2.3, and 1.2.7.</li>
	<li>Detection of SCAP N-glycosylation
	<ol>
		<li>Trypsin Proteolysis
		<ol>
			<li>Resuspend the membrane pellets from cells overexpressing GFP-SCAP in 114 &#181;l of buffer containing 10 mM HEPES&#183;KOH (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, and 100 mM NaCl.</li>
			<li>Incubate 57 &#181;l aliquots of membrane proteins in the absence or presence of 1 &#181;l of trypsin (1 &#181;g/&#181;l), in a total volume of 58 &#181;l, for 30 min at 30 &#176;C.</li>
			<li>Stop reactions by the addition of 2 &#181;l (400 units) of soybean trypsin inhibitor.</li>
		</ol>
		</li>
		<li>Deglycosylation by Peptide-N-Glycosidase F (PNGase F)
		<ol>
			<li>For subsequent treatment with PNGase F, add 10 &#181;l of solution containing 3.5% (wt/vol) SDS and 7% (vol/vol) 2-mercaptoethanol to each sample.</li>
			<li>After heating at 100 &#176;C for 10 min, sequentially add 7 &#181;l of 0.5 M sodium phosphate (pH 7.5), 7 &#181;l of 10% (v/v) Nonidet P-40 with 12&#215; protease inhibitors, and 2 &#181;l (0.5 U/&#181;l) of PNGase F.</li>
			<li>After incubation at 37 &#176;C for 3 hr, stop reactions by the addition of 5x loading buffer (312.5 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 12.5% &#946;-mercaptoethanol, and 0.05% bromophenol blue). Heat the mixtures at 100 &#176;C for 10 min and subject to SDS-PAGE. Load 25-50 &#181;g of total membrane proteins on a 10% SDS-PAGE gel39. Run the gel at 80 V for 15 min, then change to 130 V for another 115 min. Transfer at 140 mA for 130 min.</li>
			<li>For the Western blot, use 10 &#181;g/ml anti-SCAP (9D5) antibody to detect N-glycosylation and total SCAP protein.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Detection of SCAP Trafficking from ER to the Golgi by Using GFP-SCAP in Human Cells
<ol>
	<li>Seed 2.5 &#215; 105 U87 cells in a 60 mm dish with DMEM supplemented with 5% FBS and incubate the cells at 37 &#176;C and 5% CO2 for 24 hr before transfection.</li>
	<li>Dilute 4 &#181;g GFP-SCAP plasmid in 0.5 ml reduced serum medium and mix.</li>
	<li>Add 8 &#181;l DNA transfection reagent by pipetting directly into 0.5 ml reduced serum medium containing plasmids and mix gently.</li>
	<li>Incubate the transfection reagent/plasmid complex for 25 min at room temperature.</li>
	<li>Add transfection complex to the cells with fresh medium and continue to culture for 24 hr at 5% CO2 and 37 &#176;C.</li>
	<li>Reseed 5 - 10 &#215; 104 cells into a 6-well plate containing a glass cover slip (22 mm &#215; 22 mm) and continue to culture at 37 &#176;C and 5% CO2 for 24 hr.</li>
	<li>Wash once with PBS and treat the cells for 12 hr with or without glucose (5 mM) in the presence or absence of tunicamycin (1 &#181;g/ml).</li>
	<li>Wash cells once with PBS and fix in 4% formaldehyde for 10 min.</li>
	<li>Wash cells three times with PBS, invert the coverslips, mount on slides together with antifade reagent, and seal the coverslips using nail polish.</li>
	<li>Check the GFP-SCAP trafficking using a 63X oil immersion objective in a laser scanning confocal microscope41.</li>
</ol>

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<li>Guo, D., Bell, E. H., Mischel, P., Chakravarti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+SREBP-1-driven+lipid+metabolism+to+treat+cancer%5BTitle%5D)+AND+%22Curr+Pharm+Des%22%5BJournal%5D)">Targeting SREBP-1-driven lipid metabolism to treat cancer.</a> Curr Pharm Des. 20, 2619-2626 (2014).</li>
<li>Bell, E. H., Guo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomarkers+for+malignant+gliomas%5BTitle%5D)+AND+%22Malignant+Gliomas%2C+Radiation+Medicine+Rounds%22%5BJournal%5D)">Biomarkers for malignant gliomas.</a> Malignant Gliomas, Radiation Medicine Rounds. 3, 339-357 (2012).</li>
<li>Geng, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+SOAT1+suppresses+glioblastoma+growth+via+blocking+SREBP-1-mediated+lipogenesis%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Inhibition of SOAT1 suppresses glioblastoma growth via blocking SREBP-1-mediated lipogenesis.</a> Clin Cancer Res. , (2016).</li>
<li>Ru, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Feedback+Loop+Regulation+of+SCAP%2FSREBP-1+by+miR-29+Modulates+EGFR+Signaling-Driven+Glioblastoma+Growth%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Feedback Loop Regulation of SCAP/SREBP-1 by miR-29 Modulates EGFR Signaling-Driven Glioblastoma Growth.</a> Cell Rep. , (2016).</li>
<li>Green, M. R., Sambrook, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGreen%2C+MR+%22Molecular+Cloning%3A+A+Laboratory+Manual+%28Fourth+Edition%29%22">Molecular Cloning: A Laboratory Manual (Fourth Edition)</a>. , (2012).</li>
<li>Uehara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((S-nitrosylated+protein-disulphide+isomerase+links+protein+misfolding+to+neurodegeneration%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration.</a> Nature. 441, 513-517 (2006).</li>
<li>Miyashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Confocal+microscopy+for+intracellular+co-localization+of+proteins%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Confocal microscopy for intracellular co-localization of proteins.</a> Methods Mol Biol. 1278, 515-526 (2015).</li>
<li>Cao, J., Guo, S., Arai, K., Lo, E. H., Ning, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+extracellular+signaling+utilizing+a+glycoproteomic+approach%3A+lectin+blot+surveys%2C+a+first+and+important+step%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Studying extracellular signaling utilizing a glycoproteomic approach: lectin blot surveys, a first and important step.</a> Methods Mol Biol. 1013, 227-233 (2013).</li>
</ol>

The authors have nothing to disclose.

In this study, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using GFP-labeling and confocal microscopy. The method is specifically used to analyze membrane protein and is an important tool to investigate SCAP N-glycosylation and trafficking.
Compared with the method using various lectins to recognize different N-glycan chains and identify N-glycosylated proteins42, our method described here uses PNGase F to remove protein-linked N-glycan and detects the migration shift to identify the glycosylation of SCAP protein fragment a.a 540-707. The limitation of the lectin method is dependent upon the identification of the appropriate type of lectin to recognize the specific N-glycan. Our PNGase F cleavage method provides a simple procedure to analyze SCAP N-glycosylation. Furthermore, the method can also be applied to analyze SCAP N-glycosylation and protein level in human patients or animal tissues after homogenization2. Our study provides a new tool to analyze the signature of lipid metabolism in cancer and metabolic diseases.
In this study, the plasmid of GFP-labeled SCAP used for analysis of N-glycosylation and trafficking in human cells is hamster-origin. SCAP antibody (IgG-9D5) that is raised against the amino acids 540-707 fragment of SCAP protein can only detect the protein and N-glycosylation of the hamster SCAP, such as in the Chinese hamster ovarian (CHO) cell line15. The antibody against amino acids 450 - 500 of human SCAP is able to detect the endogenous SCAP protein in human cells, as shown in Figure 1. To detect SCAP N-glycosylation in human cells or tissues, we need to develop a specific antibody against the amino acids 540 - 707 fragment of human SCAP. In addition, identification of a specific lectin to recognize SCAP-bound N-glycan is an alternative way to analyze human SCAP N-glycosylation42. Moreover, defining the composition and structure of each N-glycan chain binding in SCAP (N263, N590 and N641) will facilitate our understanding of the underlying mechanism of SCAP trafficking from the ER to the Golgi.
Furthermore, to obtain the best results for the detection of SCAP N-glycosylation, we adjusted some reaction parameters according to our experimental conditions, which are modified from the methods described in the publications from the Brown and Goldstein Laboratory15. To successfully detect SCAP protein and its N-glycosylation, the quality of the membrane fractions and nuclear extracts are very important. We directly determined the protein concentration after the membrane fractions incubated at 37 &#176;C, avoiding putting the sample back on ice. The method described here can be widely applied to different fields of research, including cancer, cardiovascular disease, diabetes, and obesity.

Deregulation of lipid metabolism is a common characteristic of cancer and metabolic diseases1-6. In these processes, sterol regulatory element-binding proteins (SREBPs), a family of transcription factors, play a critical role in controlling the expression of genes important for the uptake and synthesis of cholesterol, fatty acids, and phospholipids7-10.
SREBPs, including SREBP-1a, SREBP-1c, and SREBP-2, are synthesized as inactive precursors that bind to the endoplasmic reticulum (ER) membrane by virtue of two transmembrane domains7. The N-terminus of SREBPs contains a DNA-binding domain for the transactivation of target genes11. The C-terminus of SREBP precursors binds to SREBP cleavage-activating protein (SCAP)12,13, a polytopic membrane protein that plays a pivotal role in the regulation of SREBP stability and activation14-16.
The implementation of transcriptional function requires the translocation of the SCAP/SREBP complex from the ER to the Golgi, where two proteases sequentially cleave SREBP and release its N-terminal fragment, which then enters into the nucleus for the transactivation of lipogenic genes7. In these processes, the levels of sterols in the ER membrane control the exit of the SCAP/SREBP complex from the ER7,17. Under high sterol conditions, sterol binds to SCAP or ER-resident insulin-induced gene protein-1 (Insig-1) or -2 (Insig-2), enhancing the association of SCAP with Insigs that retain the SCAP/SREBP complex in the ER18-20. When sterol levels decrease, SCAP dissociates with Insigs. This leads to a SCAP conformational change, which allows SCAP interaction with common coat proteins (COP)II complex. The complex mediates the incorporation of the SCAP/SREBP complex into the budding vesicles and directs its transport from the ER to the Golgi21,22. Upon translocation to the Golgi, SREBPs are sequentially cleaved by Site-1 and Site-2 proteases, leading to the release of the N-terminus7,23-29.
SCAP protein carries three N-linked oligosaccharides at asparagine (N) positions N263, N590, and N64115. We recently revealed that glucose-mediated N-glycosylation of SCAP in these sites is a prerequisite condition for SCAP/SREBP trafficking from the ER to the Golgi under low sterol conditions30-32. Loss of SCAP glycosylation via mutation of all three asparagine to glutamine (NNN to QQQ) disables the trafficking of the SCAP/SREBP complex and results in the instability of SCAP protein and reduction of SREBP activation31. Our recent data also demonstrate that SREBP-1 is highly activated in glioblastoma and regulated by SCAP N-glycosylation4,31,33,34. Targeting SCAP/SREBP-1 signaling is emerging as a new strategy to treat malignancies and metabolic syndromes1,3,35-38. Therefore, it is important to develop an effective method to analyze SCAP protein and N-glycosylation levels and monitor its trafficking in human cells and patient tissues.
The luminal region of SCAP protein (amino acids 540-707) in the ER contains two N-glycosylation sites (N590 and N641) that are protected from proteolysis when intact membranes are treated with trypsin15. This luminal fragment has a molecular weight of ~ 30 kDa that is small enough to allow the resolution of individual glycosylated variants of SCAP by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)15,31. Here, we provide a method to detect N-glycosylation of SCAP and the total protein in human cells. This protocol is derived from the method described in the publications from the Brown and Goldstein laboratory15 and our recent publication31. The protocol can be used in the study of SCAP protein from mammalian cells.

<table><tbody><tr><td>X-treme GENE HP DNA Transfection Reagent</td><td>Roche</td><td>6366236001</td><td></td></tr><tr><td>Opti-MEMI medium</td><td>Life Technologies</td><td>31985-070</td><td></td></tr><tr><td>trypsin&amp;nbsp;</td><td>Sigma</td><td>T6567</td><td></td></tr><tr><td>soybean trypsin inhibitor</td><td>Sigma</td><td>T9777</td><td></td></tr><tr><td>PNGase F&amp;nbsp;</td><td>Sigma</td><td>P7367</td><td></td></tr><tr><td>Anti-SCAP (9D5) antibody&amp;nbsp;&amp;nbsp;</td><td>Santa Cruz</td><td>sc-13553</td><td></td></tr><tr><td>GFP antibody&amp;nbsp;</td><td>Roche</td><td>11814460001</td><td></td></tr><tr><td>SREBP-1 antibody (IgG-2A4)</td><td>BD Pharmingen</td><td>557036</td><td></td></tr><tr><td>PDI Antibody (H-17)</td><td>Santa Cruz</td><td>sc-30932</td><td></td></tr><tr><td>Lamin A Antibody (H-102)</td><td>Santa Cruz</td><td>sc-20680</td><td></td></tr><tr><td>SCAP antibody (a.a 450-500)</td><td>Bethyl Laboratories, Inc.</td><td>A303-554A</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM) without glucose, pyruvate and glutamine&amp;nbsp;&amp;nbsp;</td><td>Cellgro</td><td>17-207-CV</td><td>Add 1 mM Pyravate and 4 mM Glutamine before use</td></tr><tr><td>22 G x 1 1/2 needle</td><td>BD</td><td>305156</td><td></td></tr><tr><td>pepstatin A</td><td>Sigma</td><td>P5318</td><td></td></tr><tr><td>leupeptin</td><td>Sigma</td><td>L2884</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>43819</td><td></td></tr><tr><td>ALLN</td><td>Sigma</td><td>A6185</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>GE</td><td>RPN3032D</td><td></td></tr><tr><td>EDTA Solution 0.5 M pH 8.5 100 ml</td><td>VWR</td><td>82023-102</td><td></td></tr><tr><td>EGTA 0.5 M sterile (pH 8.0) 50 ml</td><td>Fisher Scentific</td><td>50255956</td><td></td></tr><tr><td>HyClone FBS</td><td>Thermo scientific</td><td>SH3007103</td><td></td></tr><tr><td>glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>bromophenol blue</td><td>Sigma</td><td>B8026</td><td></td></tr><tr><td>prolong gold antifade reagent with dapi</td><td>Life Technologies</td><td>P36935</td><td></td></tr><tr><td>L-glutamine (200 mM)</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>sodium pyruvate</td><td>Life Technologies</td><td>11360-070</td><td></td></tr></tbody></table>

analysis of scap n-glycosylation and trafficking in human cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Figure 1 shows the detection of endogenous SCAP protein and SREBP-1 nuclear form in human glioblastoma U87 cells in response to glucose stimulation by using western blot. The membrane protein SCAP is detected in the &#34;membrane fraction&#34;. The nucleus form (N-terminal) of SREBP-1 is detected in the &#34;nuclear extracts&#34;. The level of SCAP protein (PDI serves as an internal control of ER membrane proteins) and SREBP-1 nuclear form are markedly enhanced by glucose stimulation. These results demonstrate that the protocol is able to detect the endogenous SCAP protein in human cells.
 
Figure 2 shows the analysis of SCAP N-glycosylation and total GFP-SCAP protein levels in response to glucose stimulation and tunicamycin treatment in HEK293T cells expressing GFP-labeled hamster SCAP. Figure 2A shows the thematic structure of SCAP protein across the ER membrane carrying three N-glycosylation sites. The fragment of SCAP containing a.a 540 - 707, located in the 6th luminal loop in the ER, is trypsin-resistant15 and is used for analysis of N-glycosylation on the sites N590 and N641. As shown in Figure 2B (upper panel), after trypsin treatment, the higher weight bands (containing a.a 540 - 707) indicate SCAP protein containing one or two N-linked oligosaccharides (N590 and N641) in the presence of glucose and absence of PNGase or tunicamycin (detected by IgG-9D5 antibody). In the presence of PNGase F, N-linked oligosaccharides were removed from the SCAP protein, causing the fragment (a.a 540 - 707) to move faster in SDS-PAGE. Consistently, blocking the N-glycosylation initiation process by tunicamycin completely abolished SCAP N-glycosylation, indicated by the appearance of a lower band of SCAP fragment (Figure 2B, upper panel). Moreover, total GFP-SCAP protein (detected by an antibody against GFP) was reduced in the absence of glucose or via tunicamycin treatment (Figure 2B, lower panel). These results demonstrate that the protocol is suitable for the analysis of SCAP N-glycosylation.
 
Figure 3 shows that GFP-SCAP trafficking from the ER to the Golgi in response to glucose stimulation was suppressed by tunicamycin treatment in U87 cells. As shown in Figure 3A, confocal microscopy images show that GFP-SCAP protein resides in the ER in the absence of glucose, as shown by its co-localization with PDI, an ER-resident protein (red). In contrast, in the presence of glucose, GFP-SCAP moves to the Golgi, shown by the co-localization with the Golgi protein marker Giantin (red) (Figure 3B). Moreover, tunicamycin treatment suppressed glucose-induced trafficking of GFP-SCAP from the ER to the Golgi (Figure 3A and B).
<img alt="Figure 1" src="/files/ftp_upload/54709/54709fig1.jpg" /> 
Figure 1. Detection of Endogenous SCAP Protein and SREBP-1 Nuclear Form in U87 Cells. Western blot analysis of membrane and nuclear extracts from human primary glioblastoma U87 cells cultured in serum-free media with or without glucose (5 mM) for 12 hr. Anti-SCAP antibody against human SCAP a.a 450 - 500 was used to detect the endogenous SCAP in human cells. The active form (N-terminal) of SREBP-1 is detected in the &#34;nuclear extracts&#34; using antibody against the a.a 301 - 407 fragment of human SREBP-1. This figure has been modified with permission from 31. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54709/54709fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54709/54709fig2.jpg" /> 
Figure 2. Western Blot Analysis of SCAP N-glycosylation and GFP-SCAP Protein Levels. A) Schematic diagram showing the SCAP structure spanning cross the ER membrane. There are three asparagine (N) residues in SCAP, all of which reside in the ER lumen and are modified by N-linked oligosaccharides. The fragment of SCAP containing amino acids 540-707 is resistant to trypsin digestion. It contains two N-linked oligosaccharide modification sties, N590 and N641, which can be detected by IgG-9D5 antibody against the a.a 540 - 707 of hamster SCAP by western blot. B) Western blot analysis of SCAP N-glycosylation (upper: with trypsin treatment) or GFP-SCAP protein levels (lower: no trypsin treatment) from HEK293T cells transiently transfected with GFP-SCAP for 24 hr and then cultured in serum-free media with or without glucose (5 mM) in the presence or absence of tunicamycin (1 &#181;g/ml), a N-glycosylation inhibitor, for 12 hr. The numbers on the left side of the blot indicate the number of N-glycosylation sites on SCAP protein (N590 and N641 sites) (upper, detected by IgG-9D5 antibody). Lower panel for GFP-SCAP was detected by an antibody against GFP protein. This figure has been modified with permission from 31. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54709/54709fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54709/54709fig3.jpg" /> 
Figure 3. Detection of SCAP Trafficking from the ER to the Golgi. A, B) Confocal microscopy images show that GFP-SCAP resides in the ER or moves to the Golgi in the absence or presence of glucose (5 mM) with/without tunicamycin (1 &#181;g/ml) treatment in U87 cells for 12 hr. PDI shows ER staining (red) (A). Giantin, the Golgi marker (red) (B). DAPI (blue) stains the cell nucleus. The scale bars represent 10 &#181;m. Figure 3A is new data and has not been published before. Figure 3B has been modified with permission from 31. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54709/54709fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of SCAP N-glycosylation and Trafficking in Human Cells at JoVE.com

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

analysis-of-scap-n-glycosylation-and-trafficking-in-human-cells

Nanyang Technological University

Research

JoVE Journal

Biology

8.7K Views.  The Ohio State University Comprehensive Cancer Center and College of Medicine.  We beschrijven een aangepaste werkwijze voor membraanfractie isolatie uit humane cellen en monstervoorbereiding voor de detectie van SCAP N -glycosylation en totaal eiwit door Western blot. We verdere invoering van een GFP-labeling methode om SCAP mensenhandel te monitoren met behulp van confocale microscopie. Dit protocol kan in normale biologische laboratoria. 

Video: Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

Chemistry

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Biochemistry

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies &#8212; small, monomeric, high-affinity protein binders &#8212; that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters&#39; sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

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

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

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

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological matrices is often necessary due to their low stoichiometry in comparison to non-modified analytes. Most enrichment workflows of PTMs on peptides in bottom-up proteomics workflows, where proteins are enzymatically digested before the resulting peptides are analyzed, only enrich one type of modification. It is the entire complement of PTMs, however, that leads to biological functions, and enrichment of a single type of PTM may miss such crosstalk of PTMs. PTM crosstalk has been observed between protein glycosylation and phosphorylation, the two most common PTMs in human proteins and also the two most studied PTMs using mass spectrometry workflows. Using the simultaneous enrichment strategy described herein, both PTMs are enriched from post-mortem human pancreatic tissue, a complex biological matrix. Dual-functional Ti(IV)-immobilized metal affinity chromatography is used to separate various forms of glycosylation and phosphorylation simultaneously in multiple fractions in a convenient spin tip-based method, allowing downstream analyses of potential PTM crosstalk interactions. This enrichment workflow for glyco- and phosphopeptides can be applied to various sample types to achieve deep profiling of multiple PTMs and identify potential target molecules for future studies.

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological ...

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Neuroscience

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Investigating ATG9A Localization in Cells

Imaging ATG9A, a Multi-Spanning Membrane Protein

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive pathological conditions. A set of proteins, called ATG proteins, comprise the core autophagy machinery and work together in a defined hierarchy. Studies in recent years have improved our knowledge of the autophagy pathway. Most recently, it has been proposed that ATG9A vesicles are at the heart of autophagy, as they control the rapid de novo synthesis of an organelle called the phagophore. The study of ATG9A has proven challenging, since ATG9A is a transmembrane protein, and it is present in different membrane compartments. As such, understanding its trafficking is an important element for understanding autophagy. Here, detailed methods are presented that can be used to study ATG9A and, in particular, its localization using immunofluorescence techniques, which can be assessed and quantified. The pitfalls of transient overexpression are also addressed. The correct characterization of ATG9A function and the standardization of techniques to analyze its trafficking are crucial to further characterize the events governing autophagy initiation.

Author Spotlight: Imaging ATG9A, a Multi-Spanning Membrane Protein  

This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot.

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

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