An Assay System for Plate-based Detection of Endogenous Peptide:N-glycanase/NGLY1 Activity Using A Fluorescence-based Probe

Materials and reagents

Biological materials

1. Rat brain tissues (a rat outbred strain, Sprague-Dawley)

2. Cell lines (e.g., HeLa and HEK293 cells)

3. Fibroblast derived from healthy subjects and NGLY1-deficiency patients (available from Coriell.org)

Reagents

1. 5-Carboxyfluorescein- and dabcyl-labeled glycosylated cyclo-heptapeptide (fGCP, Mw: 2924.9) (GlyTech, Inc.)

2. Sucrose (FUJIFILM Wako Chemicals, catalog number: 196-00015)

3. EDTA (FUJIFILM Wako Chemicals, catalog number: 345-01865)

4. Trizma base (Sigma-Aldrich, catalog number: T1503)

5. NP-40 (IGEPAL^® CA-630) (MPBIO, catalog number: 198596)

6. Hydrochloric acid (HCl) (FUJIFILM Wako Chemicals, catalog number: 080-01066)

7. cOmplete EDTA-free protease inhibitor cocktail (Merck-Millipore, catalog number: 11836170001)

8. Pefabloc SC (Merck-Millipore, catalog number: 11429868001)

9. Rabeprazole sodium salt (Tokyo Chemical Industry Co. Ltd., catalog number: R0115)

10. Dithiothreitol (DTT) (FUJIFILM Wako Chemicals, catalog number: M02712)

11. Powermasher II (Nippi-Inc., catalog number: 891-300)

12. Biomasher II (1.5 mL tube) (Nippi-Inc., catalog number: 320-103)

Solutions

1. 10× NGLY1 buffer (see Recipes)

2. Lysis buffer for animal tissues (see Recipes)

3. Lysis buffer for cultured cells (see Recipes)

4. 1 mM fGCP stock solution (see Recipes)

5. 100 μM fGCP working solution (see Recipes)

Recipes

1. 10× NGLY1 buffer

Store at room temperature. This buffer remains stable at room temperature for at least one year.

2. Lysis buffer for animal tissues

40 μL of lysis buffer is used for one reaction. The reagent should be prepared immediately before use.

3. Lysis buffer for cultured cells

40 μL of the buffer is used for each reaction. The reagent should be prepared immediately before use.

4. 1 mM fGCP stock solution

Store at -20 °C. This stock remains stable at -20 °C for at least 1–2 years.

5. 100 μM fGCP working solution

10 μL of the solution is used for each reaction.

Laboratory supplies

1. 96-well black polystyrene microplate (clear flat bottom) (Corning, catalog number: CLS3603)

Equipment

1. Refrigerated microcentrifuge

2. Sonicator (TOMY, model: UR-21P)

3. Varioskan LUX multimode microplate reader (Thermo, model: VL0000D0)

Procedure

A. Preparation of cell lysate from cultured cells

1. Collect cultured cells (5 × 10⁶ cells) into a tube and wash them with PBS (see Note 1).

2. Resuspend the cells in 50 μL of lysis buffer for cultured cells.

3. Incubate the suspension on ice for 10 min to disrupt the cells.

4. Clarify the samples by centrifugation at 20,000× g for 5 min at 4 °C.

5. Transfer the supernatant to a new tube and use as the enzyme source (see Notes 2 and 3).

B. Preparation of cell lysate from rodent tissues

1. Transfer tissues of interest (e.g., 25–50 mg of the brain) to 1.5 mL Biomasher II tube (see Note 4).

2. Resuspend the tissue into 250 μL of NGLY1 buffer.

3. Lyse the tissue four times by homogenizing for 20 s followed by a cooling period of 20 s on ice using Powermasher II.

4. Clarify the samples by centrifugation at 20,000× g for 5 min at 4 °C.

5. Transfer the supernatant to a new tube and use as the enzyme source (see Note 3).

6. Calculate protein concentration in the cell lysate by protein assay (e.g., BCA assay) (see Note 5).

C. Real-time measurement of PNGase/NGLY1 activity

1. Transfer 40 μL of the enzyme source to a 96-well black polystyrene microplate (see Note 6).

2. Add 10 μL of 100 μM fGCP solution to the well loaded with the enzyme source and mix the solution by pipetting.

3. Immediately set the plate on the Varioskan LUX multimode microplate reader (see Note 7).

4. Measure fluorescence intensity every 15 min with incubation at 25 °C for 6–12 h (λ excitation: 353 nm; λ emission: 450 nm) (Figure 2A and C, Figure 3A and C) (see Notes 8 and 9).

Figure 2. Real-time measurement of NGLY1 activity in cultured cell lines and evaluation of the endogenous NGLY1 activity in patients’ fibroblast using fGCP assay [20]. A. Analysis of endogenous NGLY1 activity in HeLa and HEK293 cells. NGLY1-KO cells show no endogenous NGLY1 activity in both cell lines. Each cell extract prepared from 5 × 10⁶ cells was incubated with fGCP for 12 h at 25 °C. The fluorescence intensity of fGCP was measured every 15 min. B. Two parameters, initial slope (0–90 min) and maximum fluorescence intensity of endogenous NGLY1 activity were measured in HeLa and HEK293 cells. C. Real-time measurement of endogenous NGLY1 activity (measured every 15 min) in fibroblasts. The fluorescence intensity of each sample was measured by incubating fGCP with crude cell extract prepared from 5 × 10⁶ cells for 8 h at 25 °C. D. Quantitative analysis of endogenous NGLY1 activity in fibroblasts. The horizontal line represents the means of biological triplicates for control- or patient-derived samples. Error bars are mean ± S.D. from biological triplicates. For statistical analysis, a Student’s t-test was applied. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 3. Real-time measurement of NGLY1 activity in rat tissues: liver and brain [20]. A. Real-time measurement of endogenous Ngly1 activity (measured every 15 min) in rat liver of various ages. The fluorescence intensity of each sample was measured by incubating fGCP with 116 μg of protein extract from the liver for 12 h at 25 °C. B. Quantitative analysis of (A). C. Real-time measurement of endogenous Ngly1 activity (measured every 15 min) in rat brain. Fluorescence intensity of each sample was measured by incubating fGCP with 116 μg of protein extract from the brain for 12 h at 25 °C. D. Quantitative analysis of C. Error bars are means ± S.D. (n = 3 rats in each age). For statistical analysis, a Student’s t-test was applied. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001, respectively.

Data analysis

1. For calculating the fluorescence intensity in each sample, the blank value should be subtracted from all other sample values.

2. Fluorescence intensity should be normalized by cell number or protein concentration.

3. Reaction curves can be evaluated using the initial slope (slope of 0–90 min) and the maximum intensity of the reaction (Figure 2B and D, Figure 3B and D) (see Notes 10 and 11).

Validation of protocol

This protocol has been used and validated in the following research article:

• Hirayama et al. [20]. Development of a fluorescence and quencher-based FRET assay for detection of endogenous peptide: N-glycanase/NGLY1 activity. J Biol Chem 300(4): 107121 (Figures 4–6).]

General notes and troubleshooting

General notes

1. The collected cells can be flash frozen in liquid nitrogen and stored at -80 °C until use.

2. For one assay, 1–5 × 10⁶ cells were required to prepare the crude extract.

3. Cells or tissue lysates should be prepared immediately before the assay, as NGLY1 activity is drastically decreased in frozen crude lysates.

4. The preserved frozen tissues (stored at -80 °C) are also used as a source of the enzyme in this assay.

5. Lysates containing 50–100 μg of proteins are required for one assay.

6. Prepare a blank well containing 40 μL of the same buffer as samples (lysis buffer for animal tissues or cultured cells) and 10 μL of 100 μM fGCP working solution.

7. Keep the lid of the assay plate closed to prevent evaporation of the solution.

8. For the evaluation of endogenous NGLY1 activity, it is preferable to perform assays of the lysates prepared from NGLY1-KO cells or lysates treated with 100 μM of zVAD-fmk, a potent NGLY1-inhibitor [21], as a negative control.

9. Replicate experiments (biological or technical) should be carried out to ensure accurate interpretation of the results through statistical analysis.

10. It is possible to determine the initial slope by measuring two time points (0 and 90 min) to compare the relative enzyme activity among the samples. However, real-time measuring of the reaction for 8–12 h is preferable to obtain more information on the enzyme properties (e.g., background level, maximum activity, and time to reach plateau)

11. To calculate the specific activity of NGLY1 (expressed as pmol of deglycosylated fGCP per minute normalized by protein concentration; pmol/min/mg protein), create a standard curve of the concentration of fGCP treated with PNGase F and the fluorescence intensity. Then, calculate the pmol of the product (i.e., deglycosylated substrate) from the standard curve. Alternatively, the 5FAM-GCP assay [18], which can measure enzyme activity through the separation of the substrate and product using HPLC, can also be used.

Troubleshooting

Problem 1: Fluorescence signal is low throughout the experiment.

Possible cause: Number of cells/total tissues for the preparation of cell lysates is too low.

Solution(s): Prepare more cells/tissues.

Acknowledgments

Research reported in this publication was supported by the T-CiRA Program (NGLY1 Deficiency Project), the RIKEN Pioneering Project (“Glyco-lipidologue Initiative”), Japan Agency for Medical Research and Development Core Research for Evolutional Science and Technology (AMED-CREST) Grant JP24gm14100003h0005 (to T.S.), and by KAKENHI Grant Number JP22K06155 (to H.H.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS). The image of cells in the Graphical overview was provided by DBCLS Togo Picture Gallery (2016 DBCLS TogoTV; https://togotv.dbcls.jp/pics.html). We thank Enago (https://www.enago.jp) for the English language review. This protocol was adapted and modified from Hirayama et al. [20].

Competing interests

The authors declare that they have no competing interests.

Ethical considerations

Care procedures and experiments of a rat outbred strain, Sprague–Dawley, conformed to the association for assessment and accreditation of laboratory animal care guidelines. All experiments using animals were approved by the experimental animal care and use committee of our organization.

References

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