Analyzing the topology of N-linked glycans by PNGase F accessibility assay

Summary

While N-glycans are synthesized in the lumens, some of them reach the cytosolic side of membranes through retro-translocation independent of endoplasmic-reticulum-associated degradation. Here, we present a protocol to measure the topology of N-glycans in a transmembrane protein, based on the principle that cytosolic but not luminal N-glycans are trimmed by PNGase F in the absence of detergent. We describe the procedures for this protocol consisting of microsome preparation from cells, PNGase F accessibility assay, and western blot analysis.

For complete details on the use and execution of this protocol, please refer to Wang et al.¹

Graphical abstract

Highlights

• •Protocol for identifying localization of N-glycans on intracellular organelle proteins
• •Steps for preparing microsomes and performing PNGase F accessibility assay
• •Assay to measure the deglycosylation of proteins in microsomes

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

While N-glycans are synthesized in the lumens, some of them reach the cytosolic side of membranes through retro-translocation independent of endoplasmic-reticulum-associated degradation. Here, we present a protocol to measure the topology of N-glycans in a transmembrane protein, based on the principle that cytosolic but not luminal N-glycans are trimmed by PNGase F in the absence of detergent. We describe the procedures for this protocol consisting of microsome preparation from cells, PNGase F accessibility assay, and western blot analysis.

Before you begin

Verify the subcellular localization of the protein of interest

This protocol will determine PNGase F accessibility on sealed microsomes produced through cell homogenization. Thus, the protein of interest should localize on intracellular organelles such as endoplasmic reticulum (ER) or Golgi. In contrast, this assay is not applicable to proteins located on plasma membranes, which form membranous sheets instead of microsomes after cell homogenization. For proteins located at both plasma membranes and intracellular organelles, plasma membranes should be removed from cell homogenates before the assay. The subcellular localization of a protein can be determined by approaches such as immunocytochemistry and cell surface chemical labeling, which will not be covered by this protocol.

Verify the detection of the deglycosylation reaction through Western blot analysis

Before conducting this protocol, perform a deglycosylation assay on the protein of interest denatured by SDS, reducing agents and high-temperature treatment following the recommended protocols from PNGase F vendors. This analysis ensures that all N-glycans of the protein are removed by PNGase F. Samples treated with or without PNGase F are subject to SDS-PAGE followed by Western blot analysis. The deglycosylation reaction should reduce the apparent molecular weight of the protein by ∼3 kDa per N-glycan on SDS-PAGE dependent on the nature of the polysaccharide attached to the protein.² Thus, optimizing the SDS-PAGE conditions such as selecting the right percentage of acrylamide may be required to see the reduction in the molecular weight. A well-controlled experiment should compare the deglycosylation patterns of samples with and without the target protein to exclude unspecific signals.

Prepare stock and working solutions

Timing: 1 h, plus sufficient time prior to experiment to cool down the solutions.

Prepare the solutions in materials and equipment section prior to the experiment.

Prepare cultured cells as starting material

Timing: A few days, depending on cell growth.

The protein to be studied can be either endogenously expressed or transiently expressed. Please refer to the step-by-step method details for the recommended amount of starting material. Routine cell culture techniques will not be covered in this protocol.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit anti-Myc tag (Dilution: 1/10,000)	Bethyl	A190-205A. RRID:AB_67391
Mouse anti-CREB3L1 (Dilution: 0.6 μg/mL)	Purified in-house (Denard et al., 20183)	Clone 10H1
Anti-GRP 94 Antibody (9G10) Alexa Fluor® 790 (Dilution: 1/300)	Santa Cruz Biotechnology	sc-32249 AF790. RRID:AB_627676
Horseradish-peroxidase-conjugated donkey anti-mouse	Jackson ImmunoResearch	715-035-150 RRID:AB_2340770
Horseradish-peroxidase-conjugated donkey anti-Rabbit	Jackson ImmunoResearch	111-035-003 RRID:AB_2313567
Chemicals, peptides, and recombinant proteins
Pierce Protease Inhibitor (Cocktail) Mini Tablets	Thermo Fisher	A32955
PNGase F	Promega	V4831
0.5 M Tris-HCl, pH 6.8	Bio-Rad	1610799
(PBS) Dulbecco’s Phosphate-Buffered Saline without calcium and magnesium	Corning	21-031
(NP-40) NP-40 Alternative 10% solution	Millipore	492018-50ML
Critical commercial assays
SuperSignal West Pico PLUS Chemiluminescent Substrate	Thermo Fisher	34580
Roche X-tremeGENE™ HP DNA Transfection Reagent	Sigma-Aldrich	6366546001
Experimental models: Cell lines
Human: SV589 cells	Laboratory of Brown & Goldstein	N/A
Recombinant DNA
pCDNA3.1-Hygro(+) (Empty vector)	Invitrogen	V87020
pCMV-TM4SF20-Myc	Chen et al., 20144	N/A
Software and algorithms
LI-COR Image Studio	LI-COR, Inc	https://www.licor.com/bio/image-studio/
Other
Plastic cell lifter	Corning	3008
0.22-μm sterile filter, syringe, or vacuum assembly
15-mL tubes and a centrifuge that holds such tubes and runs at 4°C.
2-mL Ultracentrifuge tubes and a high-speed ultracentrifuge that holds such tubes and runs at 4°C.
Reaction tubes. Example: PCR tubes or plates, with caps or sealings.
Thermocycler (recommended), heat block or incubator.
22-Guage needles	BD	305155
1-mL syringes	BD	309659
1-mL syringes	Air-Tite	ML1
Equipment for cell culture, SDS-PAGE and Western blot.

Materials and equipment

• •Reagents for cell culture, SDS-PAGE and Western blot.
• •Ultrapure water, typically 18.2 megohm water.
• •10% SDS (4 g sodium dodecyl sulfate in 40 mL Ultrapure water)

Refer to Safety Datasheet for proper handling of sodium dodecyl sulfate.

Buffer T

Reagent	Final concentration	Amount
0.5 M Tris-HCl pH 6.8	50 mM	50 mL
KCl	25 mM	0.932 g
MgCl2 · 6H2O	5 mM	0.508 g
Sucrose	250 mM	42.79 g
Ultrapure water	N/A	Fill to total volume
Protease Inhibitor tablet	1×	Add before use (see note below)
Total	N/A	500 mL

Note: After solubilization, filter the buffer through membranes with a pore size of 0.22 μm to reduce microorganism contamination. Aliquot and store the buffer at −20°C. Protease Inhibitor tablets are added before use: 1 mini tablet is added to 10 mL Buffer T. Since this buffer contains sucrose, to reduce microorganism contamination, it should be kept in 4°C for no more than a week after thawing. Formula adopted from Castle, 2003.⁵

2× Sample Buffer

Reagent	2× concentration
Tris-HCl pH 6.8	20 mM
SDS	4%
Glycerol	12%
Bromophenol blue	0.01%
DTT	0.2M
Make with Ultrapure water

Note: This is an example recipe of Sample buffer adapted from Laemlli., 1970.⁶ The solution can be stored at −20°C for several months.

Step-by-step method details

Microsome preparation from cells

Timing: 3 h

Timing: for Step 7: 1–2 min per sample

The following protocol is for producing microsomes from monolayer SV589 cells.These cells are seeded at 4 × 10⁵ cells per 6-cm dish and cultured for 2 days before harvesting. TM4SF20, the protein of interest, was transiently expressed via plasmid transfection. Alternatively, 90% confluent cells grown in a 15-cm dish can also be harvested to produce the same result as stated in Step 4.

Optional: Transfection of expression vectors for the protein of interest: For TM4SF20, we use SV589 cells seeded in 6-cm dishes as above for transfection. One day after seeding, for each dish, 0.1 μg pCMV-TM4SF20-Myc expression vector with 1.9 μg empty vector is transfected with 3 μL Roche X-tremeGENE HP DNA Transfection Reagent following manufacturer’s instruction (PDF). This approach yields at least 50% transfection efficiency. We harvest the cells 16 h post transfection.

When cells expressing the protein of interest reach the density indicated above, they are harvested and broken by mechanical force. The microsome fraction is collected by ultracentrifugation of the post-nuclear supernatant.

• 1.Thaw Buffer T and add the protease inhibitor tablet to 1×. Keep Buffer T and PBS at 4°C or on ice. Prepare the collection tubes (15-mL tube) for harvest of the cells.
• 2.At room temperature (22°C), aspirate the media from cultured cells and rinse the cells with ice-cold PBS in the culture dishes.
• 3.Aspirate the PBS and add 1.5 mL (per 6-cm dish) or 10 mL (per 15-cm dish) PBS to the dishes.
• 4.
  Scrape the cells from the dishes in PBS with plastic cell lifters.

  • a.
    For each microsome sample, combine the cell suspension from three 6-cm dishes or one 15-cm dish into one 15-mL collection tube.
  • b.
    After this step, the collection tubes are kept on ice throughout the experiment.
• 5.Centrifuge the cell suspension at 1,000 × g for 5 min to pellet the cells. Aspirate the supernatant.

Note: Cell pellet is usually visible at the bottom of the tubes.

• 6.Add 600 μL Buffer T to the cell pellet.
• 7.
  Break the cells in Buffer T by passing through a 22-Guage needle for 25 times.

  • a.
    Assemble the 22-Guage needle with 1 mL syringe.
  • b.
    In the first cycle, draw the cell pellet together with the buffer and blow to disperse the suspension. Repeat the draw-blow cycle for 25 times.

CRITICAL: Now that the cells are broken, it is critical to keep the samples on ice or at 4°C to minimize degradation and undesired reactions.

Note: Sharp needles. Use proper personal protective equipment.

• 8.Centrifuge the homogenate at 1,000 × g for 5 min at 4°C to pellet the heavy membrane fractions, mostly the nucleus.

Note: The supernatant should be clear without white particles. Troubleshooting 1

• 9.Transfer 540 μL of the supernatant (post-nuclear supernatant) to a 2-mL ultracentrifuge tube.
• 10.Ultracentrifuge the post-nuclear supernatant at 100,000 × g for 30 min at 4°C to pellet the microsomes. Carefully remove the supernatant.

Note: Microsome pellets are usually a visible thin layer. Troubleshooting 2

• 11.Resuspend the microsome pellet with 100 μL Buffer T by pipetting with 200 μL tips.

Note: Volume can be adjusted as desired. For one 15-cm dish of cells, 150–200 μL Buffer T is usually used.

Note: Usually, 10‒20 times of pipetting are needed to disperse visible membrane particles in the buffer. The resulting suspension should be partially opaque without visible membrane particles. Troubleshooting 3

• 12.Move the resuspended microsomes to another tube (1.5 mL Eppendorf tube) for easier handling.

Note: Ultracentrifuge tubes are usually round-bottom and without caps, making it at risk of spilling or rolling on ice.

Pause point: Resuspended microsomes can be left on ice for a few hours, although we recommend immediately advancing to the next steps of experiment. We have not tested if it could be stored longer or flash frozen.

PNGase F accessibility assay

Timing: 1.5 h (for steps 13 to 19)

After obtaining the microsomes, this section will describe the setup of the PNGase F accessibility assay.

• 13.Dilute the PNGase F by 1:4 with buffer T for easier handling. The amount of PNGase F can be calculated from the Assay assembly chart below.
• 14.For each microsome sample, assemble four reaction conditions in reaction tubes on ice.

Assay assembly

Reaction tube #	1	2	3	4
Resuspended microsomes	12 μL	12 μL	12 μL	12 μL
PNGase F (1:4 diluted)		1 μL	1 μL	1 μL
10% NP-40			1 μL	1 μL
10% SDS				1 μL
Buffer T	8 μL	7 μL	6 μL	5 μL
Total = 20 μL

Note: It is convenient to perform these reactions in strip tubes or plates with thermocyclers as used for polymerase chain reaction (PCR).

• 15.Incubate the reactions at 30°C for 45 min, then cool down to 4°C or on ice for ∼5 min.
• 16.
  Preheat the sample buffer for denaturation.

  • a.
    Dispense 20 μL of 2× Sample Buffer in a tube/well per reaction in the previous step.
  • b.
    When the reaction in Step 15 is about to finish, preheat the sample buffer to 95°C.

Note: Step 16 was implemented to prevent undesired PNGase F activity after the addition of Sample Buffer.

Note: Refer to Safety Datasheet of DTT and perform the relevant steps in a well-ventilated area.

• 17.When the sample buffer reaches 95°C, quickly transfer the finished reaction from step 15 to corresponding well containing the 2× Sample Buffer. Briefly pipette to mix the sample with Sample Buffer.
• 18.Cap or seal the tubes or wells and incubate the samples at 95°C for 5 min, then at 80°C for 5 min. If using a thermocycler, set the cap temperature to 105°C to avoid condensation.

Note: This step denatures the sample for SDS-PAGE and ensures inactivation of PNGase F. The heat inactivation condition of PNGase F was reported by New England Biolabs to be “75°C for 10 min”.⁷

• 19.Cool the samples on ice.

Note: Steps 16‒19 can be set as a thermocycler program as below.

Temperature	Time
95°C	Pause or keep at this temperature for Steps 16–17. Jump to next step when starting Step 18.
95°C	5 min
80°C	5 min
4°C	Forever

Pause point: Now the samples can be frozen at −20°C for a few weeks, depending on the stability of the proteins of interest.

Western blot analysis

Timing: 1–2 days

• 20.
  Analyze the samples with SDS-PAGE followed by Western blot.

  • a.
    To control for unspecific bands, include a microsome sample without the protein of interest, such as that from cells transfected with an empty vector or those in which the gene encoding the protein of interest is knocked out.
  • b.
    In addition to blotting for the protein of interest, proteins known to contain N-linked glycans in the lumen should be analyzed as quality controls to indicate whether the microsomes are sealed during PNGase F treatment. We have used an ER luminal protein GRP94 and a membrane protein CREB3L1 for this purpose.

Expected outcomes

The identification of a cytosolic N-glycan in TM4SF20 raises the necessity to develop an assay capable of determining the location of N-glycans relative to membranes. We thus developed the PNGase F accessibility assay, the principle of which is indicated in Figure 1. In Tube 2, PNGase F was added to the microsomes in the absence of any detergents. If there were N-linked glycans on the cytosolic side of membranes that are accessible to the enzyme, they should be removed following this treatment. In Tube 3, NP-40 was added into the reaction to dissolve membranes without unfolding glycosylated proteins. N-linked glycans that were removed in Tube 3 but not 2 should reside in the lumen. In Tube 4, SDS was added further into the reaction to unfold glycosylated proteins. Glycans removed only under this condition were those inaccessible to PNGase F without denature of the proteins. Their location relative to membranes cannot be determined by this assay.¹

Figure 1.

Schematic illustration of the PNGase F accessibility assay

The N-linked glycosylation sites and glycans are highlighted in yellow and red, respectively. Reused from Wang et al.¹

SDS is not used alone because PNGase F activity is inhibited by SDS in the absence but not presence of NP-40.⁷

Figure 2A shows the deglycosylation patterns of two proteins, CREB3L1 and GRP94, which contain only luminal N-glycans. As expected, both proteins were deglycosylated by PNGase F when membranes were dissolved by NP-40 (Tube 3). These results suggest that microsomes remained intact in the assay.

Figure 2.

Expected outcome of the assay

(A‒C) Panels A and C: PNGase F accessibility assay results for CREB3L1, GRP94 and TM4SF20(C). Proteins were separated by 8% (for CREB3L1 and GRP94) or 13% SDS-PAGE (for TM4SF20), and detected by Western blot with anti-CREB3L1, anti-GRP94, and anti-Myc as indicated. Panel B: Topology for TM4SF20(C), an isoform of TM4SF20 with a cytosolic N-linked glycan. The dashed circle highlighted in green contains the unknown structure by which the retrotranslocated TM4SF20 traverses membranes, and glycosylated N148 the localization of which remains undetermined in this study as it is inaccessible to PNGase F without unfolding of the protein by SDS. Reused from Wang et al.¹

The deglycosylation pattern is different for TM4SF20(C), an isoform of TM4SF20 with a cytosolic N-glycan (Figure 2B). Each of the three deglycosylation conditions reduces the apparent molecular weight of TM4SF20(C) (Figure 2C), as the three N-linked glycans locate at different environments: N163 at the cytosolic side, which was deglycosylated in Tube 2; N132 at the luminal side, which was deglycosylated in Tube 3 but not 2; and N148 that is inaccessible to PNGase F, which was only deglycosylated in Tube 4 when the protein was unfolded by SDS.¹

Limitations

This assay depends on the sealed microsomal membranes to protect luminal N-glycans from deglycosylation catalyzed by PNGase F. Thus, it can only determine the topology of glycoproteins that reside on membranes that form sealed microsomes after cell homogenization, such as ER and Golgi. In addition, the localization of inaccessible glycans such as N148 in TM4SF20(C) that are digested only in the presence of SDS (Tube 4) cannot be determined through this assay.

Troubleshooting

Problem 1

Nuclear or large membrane particles are still present in the supernatant after centrifuge. (Related to Step 8)

Potential solution

Gently tap the tube and repeat centrifugation for another 5 min.

Problem 2

No visible microsome pellets after ultracentrifuge. (Related to Step 10)

Potential solution

When cell count or membrane content is low, it could be difficult to visualize the microsome pellet in the ultracentrifuge tube. Under this circumstance, we recommend starting with more cells for easier handling.

Problem 3

Cannot disperse the microsome pellet with pipetting (Related to Step 11).

Potential solution

Dispersion of the pellet relies on the small diameter of tip opening. For pipette tips, this can be achieved by pushing the tip against the tube bottom to create a small opening when pipetting. Alternatively, gel loading tips or 0.1-mL microsyringes may be used to solve the problem.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jin Ye (jin.ye@utsouthwestern.edu).

Materials availability

This protocol does not use any uncommercialized reagent.

Data and code availability

This protocol does not present new results. Original data can be obtained from the referred to article.¹