Protocol to differentiate glycosylphosphatidylinositol-anchored prion protein from pro-prion protein in cancer cells

Summary Defects of glycosylphosphatidylinositol (GPI)-anchor synthesis lead to the production of pro-proteins with altered functions. However, pro-protein-specific antibodies for functional analysis are lacking. Here, we present a protocol to differentiate GPI-anchored prion protein (PrP) from pro-PrP in cancer cells using a complementary approach applicable to other GPI-anchored proteins. We first describe steps for phosphatidylinositol-specific phospholipase C treatment and flow-cytometry-based detection. We then detail the carboxypeptidase Y (CPDY) assay including antibody immobilization, affinity purification, CPDY treatment, and western-blot-based detection. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).1


SUMMARY
Defects of glycosylphosphatidylinositol (GPI)-anchor synthesis lead to the production of pro-proteins with altered functions. However, pro-protein-specific antibodies for functional analysis are lacking. Here, we present a protocol to differentiate GPI-anchored prion protein (PrP) from pro-PrP in cancer cells using a complementary approach applicable to other GPI-anchored proteins. We first describe steps for phosphatidylinositol-specific phospholipase C treatment and flow-cytometry-based detection. We then detail the carboxypeptidase Y (CPDY) assay including antibody immobilization, affinity purification, CPDY treatment, and western-blot-based detection. For complete details on the use and execution of this protocol, please refer to Li et al. (2022). 1

BEFORE YOU BEGIN
CRITICAL: All materials used for cell culture need to be sterilized, and all procedures for handling cells need to be carried out in a Level II biosafety cabinet under standard aseptic techniques.
Note: This protocol has been successfully used for identifying the expression form of prion protein (PrP) in pancreatic ductal adenocarcinoma cells and melanoma cells. 1,2 Here, we describe the detailed steps using hepatocellular carcinoma (HCC) cells as an example. But we expect this will be applicable to other cell lines.

Cell preparation
Timing: 1 week 1. Passage the proliferative human pancreatic cancer cell line AsPC-1 and liver cancer cell line HLE with 0.25% Trypsin-Ethylene Diamine Tetraacetie Acid (EDTA) solution.
Note: Cells need to be split from one 10 cm Perti dish to three 6 cm Petri dish or from one 10 cm Perti dish to two 10 cm Petri dish after 2-3 days of culture. Recommended continuous passage times for cells is less than 10. i. Plate the cells in a 10 cm Petri dish at a density of 1 3 10 6 cells. 3. Culture the cells at 37 C with 5% CO 2 in a humidified chamber.

Reagent preparation
Prepare the specified buffers and solutions as described below.
Timing: 2-4 h 4. PI-PLC stock solution a. Dissolve PI-PLC to a final concentration of 1 unit/mL in the PI-PLC storage buffer (Table 2). Aliquot and store the stock solution at À20 C, protected from light. 5. CPDY stock solution a. Dissolve CPDY in 10 mM Tris-HCl (pH7.5) (the final concentration is 1 unit/mL). Aliquot and store the stock solution at -20 C, protected from light.
CRITICAL: CPDY causes skin and eye irritation. Inhalation may cause allergic or asthmatic symptoms or breathing difficulties. This should be prepared in a fume hood.

MATERIALS AND EQUIPMENT
Note: Add 13 completeä the proteinase inhibitor cocktail and 1mM phenylmethanesulfonyl fluoride (PMSF) before use.
CRITICAL: SDS causes respiratory tract irritation, it should be weighed in the fume hood. Wear personal protective equipment.
Note: Store at 4 C for up to 3 months.
1 U/mL PI-PLC: 5 U PI-PLC in 5 mL PI-PLC Storage buffer Note: Store at -20 C for up to 2 years.
Note: Store at -20 C for up to 3 months.
Note: Use freshly prepared solution, sodium cyanoborohydride is toxic. Wear gloves when handling.

REAGENT or RESOURCE SOURCE IDENTIFIER
Milli-Q water purification system Millipore Cat#C85358 Sonicator with tip probe Fisher Scientific Cat#FB505 Water bath (at 37 C) Shanghai Jinghong Technical Cell culture dishes, 6 cm, round NEST Cat#705001 Cell culture dishes, 10 cm, round NEST Cat#704002

OPEN ACCESS
Note: Store at À20 C for up to 3 months.
Note: Store at room temperature for up to 3 months.

STEP-BY-STEP METHOD DETAILS
We describe two assays that are performed independently. For cell surface PI-PLC treatment assay, please refer to steps 1-15. For CPDY treatment assay, please refer to steps 16-48.

Cell surface PI-PLC treatment assay Collection of cells
Timing: 30 min; day 1 This procedure details how to prepare and collect cells.
1. Collect cells. a. Seeding 5 3 10 5 cells in a 6 cm Petri dish 12-24 h before experiment. Once cell number reaches 1 3 10 6 , aspirate off the cell culture medium and discard. b. Wash the three 6 cm Petri dishes once with 2 mL PBS for each Petri dish and discard the PBS. c. Add 1 mL of 0.05% trypsin-EDTA into each Petri dish for 2 min at 37 C in a humidified chamber. d. Add 3 mL of cell culture medium into each Petri dish to stop the cell digestion and transfer the cells with a pipette into a 15 mL centrifuge tube. e. Centrifuge the tubes at 300 3 g for 3 min at room temperature. 2. Discard the supernatant, resuspend the cells of three 15 mL centrifuge tube with 1 mL ice-cold PBS, and transfer the cells to three 1.5 mL microcentrifuge tubes.

PI-PLC treatment assay
Timing: 1 h; day 1 The following steps detail how to treat cells with PI-PLC.
3. Wash the cells of three microcentrifuge tubes twice with 1 mL ice-cold PBS by centrifuging at 300 3 g at 4 C for 3 min. 4. Add 200 mL (0.1 U/mL) PI-PLC into one of the three microcentrifuge tubes and add 200 mL of PBS into the two remaining microcentrifuge tubes, label the tubes for PI-PLC treatment or no PI-PLC treatment, and resuspend the cells with gentle pipetting (essential) Note: PI-PLC enzymatic activity is not affected by the presence of small amount of divalent metal ions. 1 U PI-PLC can be diluted with ice-cold PBS. Diluted PI-PLC can be stored at -20 C.
5. Incubate the microcentrifuge tubes at 37 C for 1 h and gently flick the tubes every 10 min (essential) 6. Centrifuge the tubes at 300 3 g for 3 min and discard the supernatant. 7. Wash the cells once with 1 mL of PBS and centrifuge the tubes at 300 3 g for 3 min.
Note: From this step on, a common problem frequently encountered is the loss of cells, be careful to handle the cells. This procedure details how to stain cells with indicated antibody for flow cytometry and/or immunofluorescence staining analysis.
8. Discard the supernatant and add antibodies to the tubes.     (Figure 2A).
Note: To ensure the amount of intact resins in all columns is equal, the 200 mL pipette tip that is used to transfer the beads is cut off with scissors at 5 millimeter from the tip ( Figure 2B). Shake the beads solution well each time and transfer the equilibrated resins into the columns.
18. Centrifuge the columns at 1,000 3 g for 1 min at room temperature and discard the flowthrough. 19. Wash the beads twice with 300 mL binding buffer (Table 7), centrifuge at 1,000 3 g for 1 min, and discard the flow-through. 20. Add 100 mg of 8B4 antibody and adjust the volume to 300 mL with binding buffer (Table 7) (essential) Note: Make sure that the antibody solution does not contain reducing reagent as it will greatly affect the efficiency of antibody coupling.
21. Add 4.5 mL of 5 M sodium cyanoborohydride solution into the antibody-beads column (essential). 22. Rotate the column for 120 min at room temperature. 23. Centrifuge the column at 1,000 3 g for 1 min at room temperature and save the flow-through to check the efficacy of antibody conjugating.
Note: Using bicinchonininc acid (BCA) protein assay kit to determine antibody concentration in the flow-through to check the efficiency of antibody coupling before step 24.
24. Wash the beads twice with 300 mL of binding buffer (Table 7) each time, centrifuge and discard the flow-through. 25. Wash the beads once with 300 mL of tris buffer (Table 8), centrifuge and discard the flowthrough. 26. Add 300 mL of tris buffer (Table 8) to the column and 4.5 mL of the sodium cyanoborohydride solution in a fume hood, rotate the column for 15 min at room temperature. 27. Centrifuge the column at 1,000 3 g for 1 min at room temperature and discard the flow-through. 28. Wash the antibody-bead mixture six times with 200 mL of washing buffer each time, centrifuge at 1,000 3 g for 1 min and discard the flow-through.  29. If the column is not used right away, wash the beads twice with 300 mL of binding buffer (Table 7), centrifuge at 1,000 3 g for 1 min and discard the flow-through. 30. Store the resin in binding buffer (Table 7) containing 0.1% Antibacterial and preservative agents (ProClean 300) at 4 C.

Making cell lysates
Timing: 1 h; day 1 This procedure details how to prepare cell lysate for affinity purification.
31. 24 h before making cell lysate, seed 5 3 10 6 cells in a 10 cm Petri dish. Discard cell culture medium and wash the cells gently three times with ice-cold PBS by pipetting along the edge of the Petri dish. Discard the PBS after each wash. 32. Add 500 mL ice-cold cell lysis buffer (Table 3) to the cells, scrape the cells and incubate the celllysate at 4 C for 5 min.
Note: Cool down the lysis buffer and PBS at 4 C. Cool down the table-top centrifuge to 4 C before step 31. Add protease and phosphatase inhibitors to cell lysis buffer just before cell lysis.
33. Transfer the lysate to a 1.5 mL tube and sonicate the cell lysate with an ultrasonic cell pulverizer (Scientz-11D, Ningbo Scientz Biotechnology CO.LTD, China) for 1 min at 120 Watts (Ultrasonic 5 s/ interval 5 s).
Note: Adjust the ultrasonic condition to reduce the generation of foam and splashes. Sonicate 2 minutes at 60 Watts to produce homogenous cell lysates. The ultrasonic horn must be inserted into the sample before starting; the probe should be centered and not attached to the tube; the ultrasonic time should not exceed 5 s each time, and the interval time should be greater than or equal to the ultrasonic time.
34. Place the tube on ice for 20 min and then centrifuge the tube at 13,000 3 g for 10 min at 4 C to get rid of non-dissolved cell debris.
Note: Cell collection, sonication, and the long-term incubations are performed at 4 C.
Note: To decrease nonspecific binding, the cell lysates can be pre-cleaned with 80 mL agarose beads. Incubate the agarose beads with the cell lysate on a rotator for 30 min to 1 h at 4 C. Spin down the beads at 1,000 3 g for 1 min and collect the supernatant.

Affinity purification of PrP
Timing: 12 h; day 1

OPEN ACCESS
This procedure details the purification of PrP in cells.
35. Transfer the supernatant to a spin column containing the aminolinked antibody (volume: about 500 mL) for affinity purification. 36. Incubate the column on a rotator overnight at 4 C (at least 12 h). 37. Centrifuge the column at 1,000 3 g for 1 min at 4 C and discard the flow-through. 38. Wash the beads six times with 300 mL lysis buffer (Table 3) by turning the column upside down 10 times, and centrifuge the column at 1,000 3 g for 1 min at 4 C. Discard flow-though.
Note: We do recommend washing the beads with lysis buffer (Table 3) but not with PBS or other buffers during step 38.
39. Add 10 mL of neutralization buffer (Table 9) to the collection tube, add 20 mL of elution buffer (Table 10) in the column and incubate for 5 s, centrifuge the column at 1,000 3 g for 1 min at 4 C (essential) 40. Add 100 mL of elution buffer (Table 10) to the column and incubate for 15 min at 4 C. The column does not need to be mixed (essential) 41. Centrifuge the column at 1,000 3 g for 1 min at 4 C to collect the eluent.
Note: In case the eluent is not enough for carboxypeptidase treatment, repeat affinity purification 2-3 times to collect more eluent.
Note: In case the amount of antibody available for PrP purification is 10-75 mg, the affinity purification step shall be repeated 2-3 times to get enough purified PrP for CPDY treatment (Please see following).

CPDY treatment assay
Timing: 12 h; day 2 This procedure details how to perform the CPDY treatment.
42. Prepare four 1.5 mL microcentrifuge tubes. Label the tubes as following: CPDY treatment 0, 30, 60 and 120 min. Add 20 mL sample from step 41 to each tube. 43. Add 0.5 U CPDY to each tube and incubate for indicated periods at 37 C(essential).
Note: It is better to prepare fresh CPDY or use the aliquoted CPDY stored at À20 C, and the amount of CPDY can be adjusted according to the amount of eluent.
44. Add 7 mL 43 SDS Laemmli buffer (Table 11) to each tube once the reaction is done and heat the tube at 100 C for 10 min, then place the tube on ice for at least 5 min. 45. Analyze 10 mL of sample by western blot (Figure 3). a. Prepare a 10% resolving polyacrylamide gels with 5% stacking gel. b. Install the gel in the gel running cassette (BioRad, 1658001) and fill in with 13 running buffer. load the samples and the molecular weight marker. Gel running conditions: set the Voltage at 80 V and run for 30 min. Once the proteins enter the resolving gel, re-set the voltage to 120 V and run the gel for 1 h. c. Transfer separated proteins to 0.45 mm nitrocellulose (NC) membrane (Merck Millipore, USA) as following: i. Soak NC membranes, sponges, and thickened filter paper in transfer buffer for few seconds.
ii. Remove the gel from the running apparatus and prepare the transfer sandwich by building the following layers in the transfer holder: sponge -thickened filter paper -gel -NC membrane -thickened filter paper-sponge. iii. Roll a 25 mL glass centrifuge tube over the NC membrane to get rid of air bubbles trapped between gel and the NC membrane. d. Lock the transfer holder and place it into a transfer apparatus. Fill the transfer apparatus with transfer buffer. Conditions of transfer: set current at 0.12 mA (A piece of polyacrylamide gel), transfer for 1 h 30 min. Proteins will migrate from the cathode (-) to the anode (+). e. After transfer, block the NC membrane with 3% bovine serum albumin (BSA) in TBST at room temperature for 2 h. After blocking, discard the block solution. f. NC membrane is then incubated with 4H2 (final concentration is 1 ng/mL) on a rocker at 4 C overnight. After primary antibody reaction, discard the solution. g. Wash the NC membrane three times with TBST for 10 min each time at room temperature and discard TBST. h. Add HRP-conjugated secondary antibody (goat-anti-mouse, final concentration is 0.2 ng/mL) and incubate for 1 h at room temperature. After secondary antibody reaction, discard the solution. i. Wash the NC membrane three times with TBST for 10 min each time at room temperature and discard the TBST. j. Detect chemiluminescence reaction with ECL substrate using a chemiluminescence imager (Tanon 4800).

Used resin storage
Timing: 1 h; day 2

OPEN ACCESS
This procedure details how to store the used spin column.
Note: To save time and expense, antibody conjugated resin can be re-used, the used spin column is stored in a 4 C refrigerator.
46. Add 200 mL of binding buffer (Table 7) to the column, centrifuge at 1,000 3 g for 1 min and discard the flow-through. 47. Repeat this step once. 48. Add 200 mL of binding buffer (Table 7) to the column. For long-term storage, add 0.1% Antibacterial and preservative agents (ProClean 300).
Note: The used spin column can be re-used for about 3 times. Seal the lid with sterile parafilm.

EXPECTED OUTCOMES
The PrP in HLE cell lines is pro-PrP. (1) the PrP on the cell surface of HLE cell lines is resistant to PI-PLC treatment ( Figure 1). (2) whereas GPI-anchored proteins are resistant to carboxypeptidase Y treatment, 6 the PrP purified from HLE cell lines is more sensitive to CPDY treatment in a time-dependent manner than PrP from AsPC-1 ( Figure 3). After treatment with the enzyme for 120 min, the amount of purified PrP from HLE cells is reduced by more than 80% ( Figure 3).

LIMITATIONS
Although most GPI-anchored proteins can be cleaved by PI-PLC on cell surface, there are GPIanchored proteins resistant to PI-PLC digestion due to the presence of an additional acyl chain, which can't be cut by PGAP1. Two approaches can be adopted. First, treating the purified protein with carboxypeptidases which cleave proteins from the carboxyl-terminus if it is not protected by GPI-anchor. This approach requires the target protein to be purified. However, purification of a protein could be a daunting project by itself in the absence of a good amount of high-quality antibody.
An alternate approach is to check the expression level of PGAP1 and to over-express PGAP1, then perform PI-PLC treatment to check the effect on the target protein.

TROUBLESHOOTING Problem 1
Double peaks appear in the PI-PLC treated or not treated cells (PI-PLC assay-Date Collection, step 15).

Potential solution
Increase the amount of primary antibody and/or lengthen the incubation time of primary antibody. Shake the antibody-cell mixture every 10 min. Make sure all the steps were performed at 4 C.

Problem 2
No peak shift under PI-PLC treatment in positive control group (PI-PLC assay-Date Collection, step 15).

Potential solution
PI-PLC is not working, use freshly made PI-PLC solution. Antibody may lose efficacy during storage or re-usage, try a new batch of antibody. In some cases, the protein epitope may be lost causing the monoclonal antibody not recognizing the target, use another monoclonal antibody targeting another epitope. Increase the incubation time of PI-PLC treatment for an additional 30 min, and flick the tubes five times with fingers for every 10 min. Reduce cells by one-third to increase the efficiency of PI-PLC cleavage.

Potential solution
Centrifuge at 660 3 g for 3 min, and reduce washing from 6 times to 3 times. Vortex the tubes instead of pipetting up-and-down.

Problem 4
The amount of antibody used for affinity purification is relatively big and the cost is high (Antibody immobilization, step 20).

Potential solution
The amount of antibody can be reduced from 100 mg to 10-75 mg. In this case, purification must be repeated as stated in the second note of step 41.

Problem 5
Antibody is not linked to the resin (Antibody immobilization, step 23).

Potential solution
Dialyze the antibody extensively against 13 PBS to get rid of glycine during antibody purification process. Buffers should not contain reducing agents (for example: DL-dithiothreitol (DTT) or b-mercaptoethanol) as they can affect the integrity of the antibody.

Problem 6
Target protein is not purified from sample (CPDY treatment assays-affinity purification steps, step 41).

Potential solution
Re-make antibody-resin, use freshly made sodium cyanoborohydride solution.
Use a different batch of antibody. The expression level of a bait protein is too low, repeat steps 35-36 with cell lysates as much as possible or alternatively, incubate a large volume of cell lysate with the antibody conjugated resins in a 10-15 mL tube overnight at 4 C. After this step, transfer the cell lysate-resins back to the spin column at step 35, centrifuge at 1,000 3 g for 1 min to collect the resins. Repeat the steps as necessary to collect all the resins before step 37.

Potential solution
Use a new column. When centrifuging columns, the collection column should not contain excess liquid to reduce back pressure.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Chaoyang Li (chaoyangli@gzhmu.edu.cn).

Materials availability
All reagents generated in this study are available from the lead contacts upon completing a Materials Transfer Agreement.

Data and code availability
This study did not generate and analyze any original datasets/codes.