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Method Article

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

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DOI:

10.3791/54709

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November 8th, 2016

 ,  ,  ,  ,  ,  , 
1Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine

In This Article

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

1. Detection of Endogenous SCAP Protein in Human Cells

  1. Cell Culture and Treatment
    1. Seed ~ 1 × 106 U87 cells in a 10 cm dish with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and incubate the cells at 37 °C and 5% CO2 for 24 hr before treatment.
    2. Wash cells once with phosphate-buffered saline (PBS), then switch cells to fresh DMEM medium with or without glucose (5 mM) for 12 hr.
  2. Preparation of Cell Membrane Fractions and Nuclear Extracts
    1. Wash cells once with PBS, scrape into 1 ml PBS, and centrifuge at 1,000 x g for 5 min at 4 °C.
    2. 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 µg/ml pepstatin A, 10 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and 25 µg/ml N-acetyl-Leu-Leu-Norleu-al (ALLN)) for 30 min on ice.
    3. Pass cell extracts through a 22 G x 1 1/2 needle 30 times and centrifuge at 890 x g at 4 °C for 5 min to isolate nuclei.
    4. 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 µg/ml pepstatin A, 10 µg/ml leupeptin, 0.5 mM PMSF, 1 mM DTT, and 25 µg/ml ALLN)).
    5. Rotate the suspension at 4 °C for 60 min and centrifuge at 20,000 x g in a microcentrifuge for 20 min at 4 °C. The supernatant is designated as "nuclear extracts".
    6. Heat the nuclear extracts at 100 °C for 10 min with 5x loading buffer (312.5 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 12.5% β-mercaptoethanol, and 0.05% bromophenol blue) before subjecting to SDS-PAGE.
    7. Centrifuge the supernatant from the original 890 x g spin at 20,000 x g for 20 min at 4 °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 "membrane fraction".
      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.
    8. Incubate the membrane fraction at 37 °C for 30 min and determine the protein concentration by Bradford protein assay. Add 1 µl 100x bromophenol blue solution before subjecting the samples to SDS-PAGE.
    9. Load 25 - 50 µg 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.
      1. 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.

2. Analysis of SCAP N-glycosylation in Human Cells

  1. Cell Culture, Transfection, and Treatment
    1. For membrane analysis, seed ~1 × 106 HEK293T cells in a 10 cm dish with DMEM supplemented with 5% FBS and incubate the cells at 37°C and 5% CO2 for 24 hr before transfection.
    2. Dilute 4 - 8 µg GFP-SCAP plasmid (a gift from Dr. Peter Espenshade)22 in 1 ml reduced serum medium and mix gently.
    3. Add 8 - 16 µl DNA transfection reagent by pipetting directly into the 1 ml reduced serum medium containing plasmids and mix gently.
    4. Incubate the transfection reagent/plasmid complex for 25 min at room temperature.
    5. Add transfection complex to the cells with fresh medium and continue to culture for 24 hr at 5% CO2 and 37 °C.
    6. 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 µg/ml), an N-glycosylation inhibitor.
  2. Prepare cell membrane pellets as described in steps 1.2.1, 1.2.2, 1.2.3, and 1.2.7.
  3. Detection of SCAP N-glycosylation
    1. Trypsin Proteolysis
      1. Resuspend the membrane pellets from cells overexpressing GFP-SCAP in 114 µl of buffer containing 10 mM HEPES·KOH (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, and 100 mM NaCl.
      2. Incubate 57 µl aliquots of membrane proteins in the absence or presence of 1 µl of trypsin (1 µg/µl), in a total volume of 58 µl, for 30 min at 30 °C.
      3. Stop reactions by the addition of 2 µl (400 units) of soybean trypsin inhibitor.
    2. Deglycosylation by Peptide-N-Glycosidase F (PNGase F)
      1. For subsequent treatment with PNGase F, add 10 µl of solution containing 3.5% (wt/vol) SDS and 7% (vol/vol) 2-mercaptoethanol to each sample.
      2. After heating at 100 °C for 10 min, sequentially add 7 µl of 0.5 M sodium phosphate (pH 7.5), 7 µl of 10% (v/v) Nonidet P-40 with 12× protease inhibitors, and 2 µl (0.5 U/µl) of PNGase F.
      3. After incubation at 37 °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% β-mercaptoethanol, and 0.05% bromophenol blue). Heat the mixtures at 100 °C for 10 min and subject to SDS-PAGE. Load 25-50 µg 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.
      4. For the Western blot, use 10 µg/ml anti-SCAP (9D5) antibody to detect N-glycosylation and total SCAP protein.

3. Detection of SCAP Trafficking from ER to the Golgi by Using GFP-SCAP in Human Cells

  1. Seed 2.5 × 105 U87 cells in a 60 mm dish with DMEM supplemented with 5% FBS and incubate the cells at 37 °C and 5% CO2 for 24 hr before transfection.
  2. Dilute 4 µg GFP-SCAP plasmid in 0.5 ml reduced serum medium and mix.
  3. Add 8 µl DNA transfection reagent by pipetting directly into 0.5 ml reduced serum medium containing plasmids and mix gently.
  4. Incubate the transfection reagent/plasmid complex for 25 min at room temperature.
  5. Add transfection complex to the cells with fresh medium and continue to culture for 24 hr at 5% CO2 and 37 °C.
  6. Reseed 5 - 10 × 104 cells into a 6-well plate containing a glass cover slip (22 mm × 22 mm) and continue to culture at 37 °C and 5% CO2 for 24 hr.
  7. 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 µg/ml).
  8. Wash cells once with PBS and fix in 4% formaldehyde for 10 min.
  9. Wash cells three times with PBS, invert the coverslips, mount on slides together with antifade reagent, and seal the coverslips using nail polish.
  10. Check the GFP-SCAP trafficking using a 63X oil immersion objective in a laser scanning confocal microscope41.

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Results


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 "membrane fraction". The nucleus form (N-terminal) of SREBP-1 is detected in the "nuclear extracts". 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 stimulatio...

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Discussion

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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
X-treme GENE HP DNA Transfection ReagentRoche6366236001
Opti-MEMI mediumLife Technologies31985-070
trypsin SigmaT6567
soybean trypsin inhibitorSigmaT9777
PNGase F SigmaP7367
Anti-SCAP (9D5) antibody  Santa Cruzsc-13553
GFP antibody Roche11814460001
SREBP-1 antibody (IgG-2A4)BD Pharmingen557036
PDI Antibody (H-17)Santa Cruzsc-30932
Lamin A Antibody (H-102)Santa Cruzsc-20680
SCAP antibody (a.a 450-500)Bethyl Laboratories, Inc.A303-554A
Dulbecco’s modified Eagle’s medium (DMEM) without glucose, pyruvate and glutamine  Cellgro17-207-CVAdd 1 mM Pyravate and 4 mM Glutamine before use
22 G x 1 1/2 needleBD305156
pepstatin ASigmaP5318
leupeptinSigmaL2884
PMSFSigmaP7626
DTTSigma43819
ALLNSigmaA6185
Nitrocellulose membraneGERPN3032D
EDTA Solution 0.5 M pH 8.5 100 mlVWR82023-102
EGTA 0.5 M sterile (pH 8.0) 50 mlFisher Scentific50255956
HyClone FBSThermo scientificSH3007103
glycerolSigmaG5516
β-mercaptoethanol SigmaM3148
bromophenol blueSigmaB8026
prolong gold antifade reagent with dapiLife TechnologiesP36935
L-glutamine (200 mM)Life Technologies25030081
sodium pyruvateLife Technologies11360-070

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