Antibodies and reagents
The antibodies and reagents we used in this study were: Hoechst 33342, trihydrochloride, trihydrate, Alexa Fluor 488 goat anti-mouse IgG (H+L) (Invitrogen, Carlsbad, CA, used 1:100), anti-Myc tag antibody (clone 9E10, used 1:1000 for western blotting, 1:100 for immunofluorescence), stabilized goat anti-mouse IgG (H+L), peroxidase conjugated (Pierce, Rockford, IL, used 1:1000), and anti-HA tag antibody (clone 16B12, Covance, Princeton, NJ, used 1:1000).
Cell culture and transfection
Human embryonic kidney (HEK293) cells and human cervix epitheloid carcinoma (HeLa) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 08458-74, Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% FBS (Cat. 171012, Lot. 9B0148, Nichirei Biosciences Inc., Tokyo, Japan), in 5% CO2 at 37°C. Transient transfection was performed using transfection reagent PEI Max (1 µg/mL, Polysciences, Inc., Warrington, PA). DNA, PEI Max, and serum-free DMEM were mixed in a ratio of 1 µg: 2 µL: 25 µL. The mixture was incubated at room temperature for 15 min and then added to the cells. The cells were cultured in 5% CO2 at 37°C for 1 or 2 overnights.
To construct plasmids for the expression of Laforin, pcDNA-LDH+His/Myc was provided by Dr. Yamakawa (RIKEN Brain Science Institute, Saitama, Japan). Myc-tagged Laforin was subcloned into pQCXIP (Clontech Laboratories, Inc, Mountain View, CA) or pGEX6P1 (GE Healthcare, Piscataway, NJ) for expression in mammalian or bacterial cells respectively. Point mutations of Laforin were introduced using a QuikChange kit (Agilent, Santa Clara, CA) or a KOD-Plus-Mutagenesis kit (Toyobo Co., Ltd., Osaka, Japan). Plasmids for the expression of Malin (pcDNA3-HA-Malin) and NSF (pcDNA3-Myc-NSF) were provided by Profs. Carlos Roma-Mateo (Instituto de Biomedicina de Valencia, CSIC and Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Valencia, Spain) and Yasuko Iwakiri (Yale University, CT), respectively. All the plasmids used in this study were verified by DNA sequencing.
Biotin switch assay
HEK293 cells were transfected with pQCXIP-Laforin-myc or Laforin mutants. Cells were lysed in lysis buffer (50 mM NaCl, 250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, 0.1% (v/v) Triton X-100, 0.1 mM neocuproine). After sonication, the lysates were clarified by centrifugation at 4°C, 11,700 × g for 10 min. The supernatants were collected and their protein concentrations were determined using Bio-Rad Protein Assay Dye Reagent Concentrate (Cat. 500-0006, Bio-Rad, Hercules, CA) and diluted to 0.4 mg of protein solution into 1 mL of HEN buffer (250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, 0.1 mM neocuproine). Then 200 µM CysNO (100 µM NaNO2, 100 µM cysteine, 0.5 mM HCl) was added and allowed to react at room temperature for 20 min. After incubation, 4 mL of blocking buffer (HEN buffer + 20 mM methyl methane thiosulfonate (MMTS, Lot. BCBH0692V, Sigma-Aldrich, St. Louis, MO), 2.5% (v/v) SDS) was added at 50°C for 20 min with gentle vortex every 4 min to block free thiol groups. After removing excess MMTS by acetone precipitation, the pellets were re-suspended in 100 µL HENS buffer (HEN buffer + 1% (v/v) SDS) and supplemented with 2 µL of ascorbate solution (50 mM (+)-sodium L-ascorbate (Sigma-Aldrich) in HEN buffer) and 33 µL of biotinylating reagent (4 mM EZ-Link HPDP-biotin (Prod. 21341, Lot. OG188581, Thermo Fisher Scientific, Inc.) in N,N-dimethylformamide) to reduce nitrosothiols to thiols and biotinylate. After removing excess biotin by acetone precipitation, the pellets were resuspended in 225 µL HENS buffer and 10 µL samples were saved for western blot. The remaining suspensions were mixed with 450 µL neutralization buffer (100 mM NaCl, 20 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, 0.5% (v/v) Triton X-100) and the biotinylated proteins were isolated with 20 µL NeutrAvidin UltraLink Resin (Prod. 53150, Lot. LI149138, Thermo Fisher Scientific, Inc.) at 4°C overnight with constant rotation. After washing 3 times with wash buffer (neutralization buffer + 600 mM NaCl), the beads were resuspended in 20 µL neutralization buffer. Finally, 3×SDS-sample buffer was added and boiled at 100°C for 5 min for SDS-PAGE.
HEK293 cells were transfected with pQCXIP-Laforin-myc or Laforin mutants. Cells were lysed in lysis buffer (50 mM Hepes-KOH (pH 7.5), 150 mM KCl, 0.1% (v/v) Triton X-100, 1 mM PMSF, 0.1% beta-mercaptoethanol). After incubation for 5 min on ice, the lysate was clarified by centrifugation at 4°C, 11,700 × g for 10 min. The supernatants were divided into half, and CysNO, at a final concentration of 300 µM, was added to one of the samples for S-nitrosylation in a dark at room temperature for 20 min. After incubation, the samples were incubated with amylose resin (Cat. E8021S, Lot. 319-42; New England Biolabs Inc., Ipswitch, MA) at 4°C for 1 h. Amylose is a glucan that partially shares its structure with glycogen. After washing 3 times with lysis buffer, the sample volume was adjusted to 20 µL. Finally, 3×SDS-sample buffer was added and boiled at 100°C for 5 min for SDS-PAGE.
HEK293 cells transfected with pQCXIP-Laforin-myc and pCDNA3-HA-Malin were treated with 100 µM CysNO in 5% CO2 at 37°C for 2 h. Cells were lysed in lysis buffer (50 mM NaCl, 250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, 0.1%(v/v) Triton X-100, 0.1 mM neocuproine, 1 mM PMSF). After incubation for 5 min on ice, the lysates were clarified by centrifugation at 4°C, 11,700 × g for 10 min. For assay, the supernatants were incubated with Anti-c-Myc Agarose Affinity Gel antibody produced in rabbit (Sigma-Aldrich; Cat. A7470, Lot. 093M4823) at 4°C for 2 h. After washing 3 times with lysis buffer, the sample volume was adjusted to 20 µL. Finally, 3×SDS-sample buffer was added and boiled at 100°C for 5 min for SDS-PAGE.
The western blot samples were fractionated by SDS-PAGE using a suitable composition of polyacrylamide gel and transferred to a PVDF membrane (Merck Millipore Ltd., Darmstadt, Germany). The membrane was blocked with 4% skim milk in phosphate-buffered saline (PBS) with 1% Tween 20 (PBST) for 1 h and then incubated for 1 h with primary antibodies diluted in 4% skim milk in PBST. After incubation, the membrane was washed 3 or 4 times with PBST for 5–10 min each wash and incubated for 30 min with secondary antibodies conjugated to horseradish peroxidase (HRP). After the membrane was again washed 3 or 4 times with PBST for 5–10 min each wash, it was reacted with AmershamTM ECLTM Western Blotting Detection Reagents (GE Healthcare) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.) and target proteins were detected using LAS-4000 (GE Healthcare). Quantification of the western blots was performed using Image J software (https://imagej.nih.gov/ij/).
Protein purification and phosphatase assay
E. coli BL21 (DE3) transformants harboring different glutathione S-transferase (GST)-tagged Laforins were grown in 3–5 mL LB/ampicillin at 37°C overnight. The bacterial culture was transferred to new 500 mL LB/ampicillin and grown at 37°C until the absorbance at 660 nm reached about 0.8. Isopropyl-β-D-thiogalactoside (IPTG) was then added to a final concentration of 0.1 mm, and the cultures were maintained overnight at 25°C. Cells were harvested and resuspended in 20 mL sonication buffer (50 mM HEPES-NaOH (pH 7.0), 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 2 mM DTT, 2 mM PMSF). Cells were disrupted by sonication and clarified by centrifugation at 4°C, 2,500 × g for 15 min. The supernatants were incubated with 20–30 µL of Glutathione SepharoseTM 4B (Code No. 17-0756-01, Lot. 301195; GE Healthcare). After washing 3 times with sonication buffer, the glutathione beads were resuspended in 100–200 µL of sonication buffer. Protein concentration was determined using Bio-Rad Protein Assay Dye Reagent Concentrate (Cat. 500-0006, Bio-Rad), SDS-PAGE, and Coomassie Brilliant Blue (CBB) staining using CBB Stain One (Lot. L1K9496, Nacalai Tesque, Inc.). Samples were stored at 4°C. In vitro phosphatase assay was performed using 1.5 µg of the recombinant proteins on glutathione beads. The beads with the proteins were washed once with phosphatase buffer (0.2 M Tris-HCl (pH 6.8), 1 mM DTT) and suspended in 100 µL of phosphatase buffer. Then 100 mM CysNO was added to a final concentration of 200 µM and reacted in the dark at room temperature for 20 min. After washing once with phosphatase buffer and resuspending in 100 µL of phosphatase buffer, 1 M para-nitrophenylphosphate (pNPP, Lot. MOT4381, Nacalai Tesque, Inc.) was added to a final concentration of 150 mM for the phosphatase reaction at 37°C for 5 min. 90 µL of the reaction supernatants were taken and transferred to 96 well plates. The reactions were then stopped by adding 10 µL of 5 M NaOH and the absorbance was measured at 405 nm.
Lafora body assay
HeLa cells on coverslips were transfected with pQCXIP-Laforin-myc. After 14 h of transfection, the coverslips were transferred to fresh culture medium in the presence or absence of 100 µM CysNO or GSNO (50 µM NaNO2, 50 µM reduced glutathione, 0.5 mM HCl) and incubated in 5% CO2 at 37°C for 2 h. LB formation in the cells was observed by immunofluorescence microscopy, as described below. Approximately 40–60 cells expressing wild-type Laforin, and the numbers of those cells with LB, were counted in each experiment.
Immunofluorescence microscopy and image data analysis
HeLa cells on coverslips were fixed with 10% Formalin in PBS for 15 min, and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were blocked with 4% bovine serum albumin (BSA) in PBS for 15 min, and then incubated for 15 min with primary antibodies diluted in 4% BSA in PBS. The cells were washed 3 times with PBS, and incubated for 15 min with secondary antibodies conjugated to Alexa fluorophores (Invitrogen). After washing, the coverslips were mounted on microscope slides (Matsunami Glass Ind., Ltd., Osaka, Japan) and the cells were imaged using a microscope (AXIO OBSERVER A1 system, LD A-Plan 32x/0.40 Ph1, Carl Zeiss, Oberkochen, Germany) equipped CCD camera (1392×1040 pixels, Nippon Roper, Tokyo, Japan) or FV1000 confocal microscope system (Olympus, Tokyo, Japan). Image data were processed and quantified using Image J software.