Glucosidase Inhibition to Study Calnexin-assisted Glycoprotein Folding in Cells

Materials and Reagents

1. Pipette tips (any brand)
2. 1.5 ml microcentrifuge tubes (any brand)
3. 6-well cell culture plates (CytoOne, catalog number: CC7682-7506)
   
4. 100 mm cell culture dishes (Corning, catalog number: 430167)
5. HEK293 cells (ATCC, catalog number: CRL-1573)
6. Dulbecco's Modified Eagle’s medium (Lerner Research Institute Cell Culture Core, catalog number: 11-500)
7. Fetal bovine serum (FBS) (Corning, catalog number: 35-011-CV)
8. Plasmid pcDNA3.1-corin-V5
   Note: This plasmid was generated by inserting a cDNA encoding human corin into a pcDNA3.1/V5 vector (Thermo Fisher, catalog number: K480001), as published previously (Knappe et al., 2003).
9. Plasmid pcDNA3.1-prothrombin-V5
   Note: This plasmid was generated by inserting a cDNA encoding human prothrombin into a pcDNA3.1/V5 vector (Thermo Fisher, catalog number: K480001), as published previously (Wang et al., 2018).
10. OPTI-MEM (Thermo Fisher, catalog number: 11058021)
11. Fugene transfection reagent (Promega, catalog number: E2311)
12. Phosphate buffered saline (PBS), 10x (Affymetrix, catalog number: 75889)
13. Trypsin-EDTA solution (Corning, catalog number: 25053CI)
14. Geneticin (Fisher Scientific, catalog number: BP6731)
15. Glycine (Research Products International (RPI), catalog number: G36050)
16. Tris-base (Fisher Scientific, catalog number: 502-13-709)
17. Sodium chloride (RPI, catalog number: S23020)
18. Nonidet P-40 (Affymetrix, catalog number: 19628)
19. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340)
20. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166500)
21. SDS-PAGE protein sample buffer (2x) (Bio-Rad, catalog number: 1610737)
22. β-mercaptoethanol (Fisher Scientific, catalog number: BP176-100)
23. Pre-stained protein ladder (Thermo Fisher, catalog number: 26616)
24. Tris-Glycine gel (4-20%) (Thermo Fisher, catalog number: XP04200BOX)
25. Methanol (any brand, ACS grade)
26. Polyvinylidene difluoride membrane for Western blotting (Millipore, catalog number: IPVH00010)
27. Tris-buffered saline and Tween 20 (TBST), 20x (Santa Cruz Biotechnology, catalog number: sc-362311)
28. Non-fat milk (Bio-Rad, catalog number: 1706404)
29. Horseradish peroxidase (HRP) conjugated anti-V5 antibody (Thermo Fisher, catalog number: R96125)
30. 1-deoxynojirimycin (DNJ) (Alfa Aesar, catalog number: J62602)
31. Cell lysis buffer (see Recipes)
32. SDS-PAGE buffer (see Recipes)
33. Western blot transfer buffer (see Recipes)
34. Western blot blocking buffer (see Recipes)
    

Equipment

1. Pipetman P20 (Gilson, catalog number: F144801)
2. Pipetman P200 (Gilson, catalog number: F123601)
3. Pipetman P1000 (Gilson, catalog number: F123602)
4. Humidified cell culture incubator (any brand)
5. Microcentrifuge (Thermo Fisher, catalog number: 75002446)
6. Benchtop rocker (any brand)
7. Heating block up to 100 °C (any brand)
8. Mini gel apparatus (Thermo Fisher, catalog number: A25977)
9. Gel transfer apparatus (Bio-Rad, catalog number: 1660827EDU)
10. Power Supply (Bio-Rad, catalog number: 1645052)
11. Ice bucket (any brand)
    

Procedure

1. Generation of HEK293 cells stably expressing recombinant glycoproteins (corin or prothrombin)
   1. Seed HEK293 cells in a 6-well plate (3 x 10⁵ cells per well) with Dulbecco’s Modified Eagle’s medium (DMEM, 2 ml per well) containing 10% FBS.
   2. Culture the cells at 37 °C in a humidified incubator to 70-80% of confluency (24 h).
   3. Transfect HEK293 cells with the plasmid (2 μg, expressing human corin or prothrombin containing a C-terminal V5 tag) using OPTI-MEM medium (85 μl) and Fugene transfection reagent (6 μl), according to the manufacturer’s protocol.
   4. Culture the cells at 37 °C for 2 days.
   5. Remove the medium and wash the cells once with PBS (5 ml per well).
   6. Treat the cells with trypsin-EDTA solution (1 ml) at 37 °C for 1-2 min. Check the cells under a microscope to ensure that > 70% of cells are detached. (HEK293 cells attached to the culture plate are flat, whereas the detached cells are round.)
   7. Tap the culture plate gently so that > 90% of cells are detached. Stop the trypsin activity by adding fresh DMEM with 10% FBS (1 ml per well).
   8. Transfer the transfected HEK293 cells into 100 mm dishes (2.2 x 10⁶ cells per dish) with 10 ml of DMEM containing 10% FBS and geneticin (G418, 400 μg/ml), an antibiotic commonly used for the selection of stably transfected mammalian cells. The concentration of G418 was based on the published information and the laboratory’s previous experience. 
   9. Culture the transfected cells and replace the culture medium with fresh medium every other day, for 2 weeks. No need to passage cells during this time.
   10. Maintain the remaining G418 resistant cells for the following experiments.
       
       
       
2. Verification of recombinant protein expression in stably transfected HEK293 cells
   1. Seed stably transfected HEK293 cells in a 6-well plate (3 x 10⁵ cells in 2 ml DMEM per well) and culture the cells at 37 °C to 90% of confluency (36 h).
   2. Gently remove the medium by suction and wash the cells once with PBS (pre-chilled on ice, 1 ml per well).
   3. Detach the cells by flushing with 1 ml of pre-chilled PBS using a pipet until all cells are detached.
   4. Transfer the cells to a 1.5 ml microcentrifuge tube.
   5. Spin down the cells with a microcentrifuge (900 x g at 4 °C for 5 min) and discard the supernatant.
   6. Lyse the pelleted cells in a cell lysis buffer (50 μl) at 4 °C for 30 min (tap the tube every 5 min to ensure the cells were completely lysed).
   7. Centrifuge the lysis mixture at 16,200 x g, 4 °C, for 10 min.
   8. Collect the supernatant (cell lysate, 50 μl) and discard the insoluble cellular debris.
   9. Denature the cell lysate with SDS-PAGE sample buffer (2x, 50 μl) containing 5% β-mercaptoethanol at 95 °C for 5 min and cool down the protein sample to room temperature.
   10. Confirm the expression of recombinant glycoproteins by SDS-PAGE and Western blotting with the conditions indicated in the table below.
       
       
       
3. Inhibition of α-glucosidases I and II by DNJ
   1. Seed HEK293 cells stably expressing recombinant proteins (corin or prothrombin) in a 6-well plate or a 100 mm dish, depending on the further analysis, with DMEM containing 10% FBS and G418 (400 μg/ml) (optional but recommended).
   2. Culture the cells to 70-80% of confluency.
   3. Replace the medium with fresh DMEM containing 10% FBS, G418 (400 μg/ml) (optional), and DNJ (2 mM) to block the activity of α-glucosidases I and II.
   4. Culture the cells at 37 °C for 24-48 h.
   5. Analyze the glycoproteins of interest in the cells and the conditioned medium, depending on research purposes (see Notes 4-6 below).

Notes

1. This protocol was used for studying the calnexin-assisted folding and extracellular expression of recombinant corin (a cell surface protein) and prothrombin (a secreted protein) (Wang et al., 2018).
2. The expression plasmids used in transfection (in Step A2) and the verification (in Procedure B) should be modified based on glycoproteins of interest.
3. The time of DNJ inhibition (in Steps C3-C4) can be adjusted depending on specific cell types and glycoproteins of interest. This may require pilot experiments.
4. In general, DNJ inhibition blocks the calnexin- and/or calrecticulin-assisted glycoprotein folding, resulting in protein retention by ER chaperones such as binding immunoglobulin protein (BiP), which can be further verified by co-immunoprecipitation with ER proteins and/or co-immune staining with ER markers (Wang et al., 2018).
5. For glycoproteins that are expressed on the cell surface, e.g., corin, DNJ inhibition is expected to impair intracellular trafficking and reduce cell surface expression, which can be verified by cell surface protein labeling and/or immune staining (Wang et al., 2018).
6. For glycoproteins that are secreted, e.g., prothrombin, DNJ inhibition is expected to reduce their extracellular secretion, which can be verified by Western blotting and/or ELISA (Wang et al., 2018).
   

Data analysis

Depending on proteins of interest and the experimental design, enhanced ER chaperone binding, reduced cell surface expression, and reduced protein secretion can be analyzed by methods described in Notes 4-6. Western blotting can be analyzed qualitatively with visual inspection and/or quantitatively with densitometry. Immune staining can be analyzed with ImageJ, a Java-based image processing program (the National Institutes of Health) (Wang et al., 2018). As an example, DNJ treatment reduced corin expression on the surface of HEK293 cells, which was analyzed by Western blotting (Figure 3) (Wang et al., 2018).


Figure 3. Western blotting analysis of corin protein. Transfected HEK293 cells expressing human corin were cultured in the absence (-) or presence (+) of DNJ (2 mM). After 48 h, the cells were lysed and analyzed by Western blotting using an anti-V5 antibody. In the cells treated with DNJ, corin expression on the cell surface, but not inside the cell, was decreased. In previous studies with trypsin digestion, cell surface protein labeling, and glycosidase endo H digestion, the top and lower bands were shown to represent cell surface and intracellular corin proteins, respectively (Chen et al., 2015; Wang et al., 2018).

Recipes

1. Cell lysis buffer
   50 mM Tris-base (pH 8.0)
   150 mM sodium chloride
   1% Nonidet P-40 (vol/vol)
   1% protease inhibitor cocktail (vol/vol)
2. SDS-PAGE buffer
   25 mM Tris-base
   250 mM glycine
   3.5 mM SDS
3. Western blot transfer buffer
   25 mM Tris-base
   190 mM glycine
   20% methanol (vol/vol)
4. Western blot blocking buffer
   5% non-fat milk in TBST (w/w)
   

Acknowledgments

This work was supported in part by grants from the NIH (HL126697, HD093727, and 1S10RR031537-01).

Competing interests

The authors have no competing interests to declare.

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