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Jul 2019

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Photoactivable Cholesterol as a Tool to Study Interaction of Influenza Virus Hemagglutinin with Cholesterol
利用光活化胆固醇研究流感病毒血凝素与胆固醇相互作用   

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Abstract

Non-covalent binding of cholesterol to the transmembrane region of proteins affect their functionalities, but methods to prove such an interaction are rare. We describe our protocol to label the hemagglutinin (HA) of Influenza virus with a cholesterol derivative in living cells or with immunoprecipitated protein. We synthesized a “clickable” photocholesterol compound, which closely mimics authentic cholesterol. It contains a reactive diazirine group that can be activated by UV-illumination to form a covalent bond with amino acids in its vicinity. Incorporation of photocholesterol into HA is then visualized by “clicking” it to a fluorophore, which can be detected in an SDS-gel by fluorescence scanning. This method provides a convenient and practical way to demonstrate cholesterol-binding to other proteins and probably to identify the binding site.

Keywords: Influenza virus (流感病毒), Hemagglutinin (血凝素), Cholesterol (胆固醇), Lipid-interaction (脂类相互作用), Membrane (膜), Photocholesterol (Photocholesterol), Click-chemistry (点击化学)

Background

Non-covalent interactions of proteins with cholesterol are supposed to regulate trafficking and functionalities of many proteins (de Vries et al., 2015). However, due to the transient and rather weak nature of this interaction, cholesterol-binding proteins and the respective binding sites are notoriously difficult to identify. Highly sophisticated methods, such as NMR or crystallography require large amounts of purified proteins which need to be integrated into artificial lipid membranes and are thus accessible only to specialized labs. A simple procedure involves measuring the cholesterol content in purified proteins using commercial “kits” (e.g., Amplex red cholesterol assay kit, Molecular Probes). However, this method is insensitive and requires solubilization of proteins from membranes with detergent which often removes non-covalently bound lipids.

One improvement is the synthesis of clickable–photocholesterol compounds which can be covalently linked to a protein. They can be added to cells where they are rapidly integrated into membranes and thus can interact with proteins in their native environment. They contain a diazirine group at position 6 of the sterol ring, which disintegrates upon uv-illumination into molecular nitrogen plus a highly reactive carbene-group that forms a covalent bond with amino acid side chains in close vicinity. To detect cross-linked proteins, probes have a latent affinity handle, an alkyne group for chemical conjugation under physiological conditions to azide-reporters by copper-catalyzed azide-alkyne cycloaddition (“click chemistry”). Reporters are either present on “beads” to enrich and identify probe-interacting proteins by mass spectrometry or are fluorophores which allow their in-gel detection.

We synthesized a compound, 6,6'-Azi-25-ethinylcholesterol [termed improved photoclick-cholesterol, complete synthesis is described in Hu et al. (2019)], which is more similar to genuine cholesterol than a commercially available photoclick-cholesterol (Hex-5'-ynyl 3ß-hydroxy-6-diazirinyl-5α-cholan- 24-oate, Avanti Polar lipids, number 700147) since it contains (besides the alkyne group) no further alterations in cholesterol’s alkyl side chain (Figure 1). A previous study showed that 6-photocholesterol is a faithful mimetic of authentic cholesterol (Mintzer et al., 2002). Note, however, that some of the diazirine groups might be photoactivated to other reactive species that have a longer half time than the carbene-group which might unspecifically label proteins. Thus, photocrosslinking is a qualitative rather than a quantitative measure of the cholesterol affinity of a protein. Nevertheless, both cholesterol probes label only a few specific proteins out of all cellular membrane proteins (Thiele et al., 2000; Hulce et al., 2013), indicating that they are suitable tools for analyzing possible interactions between cholesterol and target proteins.



Figure 1. The structure of cholesterol and of two photoactivatable derivatives. A. Cholesterol. B. Improved Photoclick-cholesterol (6,6'-Azi-25-ethinyl-cholesterol) used in this protocol. C. Photoclick-cholesterol (Hex-5'-ynyl 3ß-hydroxy-6-diazirinyl-5α-cholan-24-oate) commercially available from Avanti lipids. The photolabile azide-group is highlighted blue and the alkyne-group in red in B.

Materials and Reagents

  1. Cell culture consumables
    1. 6-well/24-well tissue culture plates, flasks, pipettes (Sarstedt, Germany)
    2. Pipette tips (Sarstedt, Germany)
    3. Aluminum foil
    4. CHO-K1 (Chinese hamster ovary cells) (ATCC, catalog number: CCL-61)
    5. Plasmid (pCAGGS, Niwa et al., 1991)
    6. DMEM (PAN Biotech, catalog number: P04-04500)
    7. EDTA-Trypsin (PAN Biotech, catalog number: P10-023100)
    8. DPBS w/o calcium/magnesium (PAN Biotech, catalog number: P04-36500)
    9. Penicillin/Streptomycin (10,000 U/ml) (PAN Biotech, catalog number: P06-07100)
    10. FBS (fetal bovine serum) (PAN Biotech, catalog number: P30-3306)
    11. Opti-MEMTM I Reduced Serum Medium (Thermo Fisher, GibcoTM, catalog number: 31985070)
    12. TurboFect Transfection Reagent (Thermo Fisher, Thermo ScientificTM, catalog number: R0531)
    13. Growth medium (see Recipes)

  2. Immunoprecipitation
    1. Ethanol (Sigma, CAS number: 64-17-5, catalog number: 34852-1L-M)
    2. Tris-HCl (TRIS hydrochloride) (Carl Roth, CAS number: 1185-53-1, catalog number: 9090.3)
    3. EDTA (AppliChem, CAS number: 6381-92-6, catalog number: A1104)
    4. Sodium Pyrophosphate (Fisher Scientific, Alfa Aesar, CAS number: 7722-88-5, catalog number: A17546.30)
    5. Sodium Fluoride (Fisher Scientific, Alfa Aesar, CAS number: 7681-49-4, catalog number: A13019.30)
    6. Sodium Orthovanadate (Sigma, CAS number: 13721-39-6, catalog number: S6508-10G)
    7. Benzamidine Hydrochloride (AppliChem, CAS number: 1670-14-0, catalog number: A1380)
    8. PMSF (AppliChem, CAS number: 329-98-6, catalog number: A0999)
    9. NEM (N-Ethylmaleimide) (Sigma, CAS number: 128-53-0, catalog number: E3876-5G)
    10. cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Sigma, Roche, catalog number: 11873580001)
    11. NP-40 (Thermo Fisher, Thermo ScientificTM, catalog number: 85124)
    12. Protein-G-Sepharose 4 Fast Flow (Sigma, GE Healthcare, GE17-0618-01)
    13. Anti-HA2 antiserum (Hu et al., 2019)
    14. IP buffer (see Recipes)
    15. Cell lysis buffer (see Recipes)

  3. SDS-PAGE and Western blot
    1. PVDF blotting-membrane, 0.2 µm (VWR, Peqlab, catalog number: 732-3200)
    2. 1.5 ml microcentrifuge tubes (Sarstedt, Germany)
    3. 30% acrylamide/ bisacrylamide (37.5:1) (Carl Roth, catalog number: 3029.1)
    4. Tetramethylethylenediamine (TEMED) (Carl Roth, CAS number: 110-18-9, catalog number: 2367.3)
    5. Ammonium persulfate (APS) (Carl Roth, CAS number: 7727-54-0, catalog number: 9592.2)
    6. Sodium dodecyl sulfate (SDS) (Carl Roth, CAS number: 151-21-3, catalog number: 2326.4)
    7. Bromophenol bule (Sigma, CAS number: 115-39-9, catalog number: 1081220005)
    8. 1,4-Dithiothreitol (DTT) (Carl Roth, CAS number: 3483-12-3, catalog number: 6908.2)
    9. Glycerol (Carl Roth, CAS number: 56-81-5, catalog number: 3783.1)
    10. Skimmed milk powder (Carl Roth, CAS number: 68514-61-4, catalog number: T145.3)
    11. Tween 20 (Carl Roth, CAS number: 9005-64-5, catalog number: 9127.1)
    12. PierceTM ECL Plus Western Blotting Substrate (Thermo Fisher, catalog number: 32132)
    13. Stacking-gel solution (see Recipes)
    14. Separating-gel solution (see Recipes)
    15. 5x Non-reducing loading buffer (see Recipes)
    16. 4x Reducing loading buffer (see Recipes)
    17. Blocking buffer (see Recipes)

  4. Crosslinking and click chemistry
    1. Blacklight Blue (UV) Lamp (Sankyo Denki, power 8W, 3.5 A, 60V, wavelength 320-365 nm, Figure 3A)
    2. Improved Photoclick-cholesterol (Figure 1B, dissolved in ethanol, the tube is sealed and stored at -20 °C). The long synthesis is described in all details in Hu et al. (2019). But an intermediate alkyne-cholesterol can be purchased from Click Chemistry Tools (https://clickchemistrytools.com/product/alkyne-cholesterol/). With this compound it might be easier to synthesize the final product, the improved Photoclick-cholesterol.
    3. Picolyl-Azide-Sulfo-Cy3 (Jena Bioscience, catalog number: CLK-1178-1)
    4. CuAAC Biomolecule Reaction Buffer Kit (THPTA-based) (Jena Bioscience, catalog number: CLK-1178-1)

Equipment

  1. -20 °C freezer
  2. Pipette controller (Hirschmann Laborgeräte)
  3. Heracell 240i CO2 incubator (Heraeus)
  4. Tabletop centrifuge (Eppendorf, model: 5424R)
  5. Power pack p25 (Analytik Jena, Biometra P25T)
  6. Chemiluminescence Imaging, Fusion SL (PeqLab)
  7. PerfectBlueTM Semi-Dry Electro Blotting Systems (VWR, PeqLab, model: SedecTM M)
  8. Tube-rotator (Carl Roth, ThermoFisher, model: ATX1.1)
  9. Typhoon FLA 9400 scanner (GE Healthcare) (Figure 2)


    Figure 2. Typhoon FLA 9400 scanner. The gel from Part I Section D, which contains the cross-linked samples and washed with ddH2O, is inserted between two glass plates for scanning with a laser. For results, see Figures 4A and 4B.

Software

  1. Typhoon Scanner Control v5.0 Software (GE Healthcare)
  2. ImageQuant (GE Healthcare)
  3. ImageJ (Fiji)
  4. Microsoft Excel
  5. GraphPad Prism

Procedure

Part I: Photocholesterol labeling of HA in living cells

  1. HA expression in CHO cells by transfection
    1. Seed 0.9 x 106-1.1 x 106 CHO cells into one well of a 6-well plate one day before transfection such that they reach 70-90% confluency the next day.
    2. Dilute 3 µg plasmid (pCAGGS) encoding HA in 300 µl serum-free Opti-MEM and mix by pipetting.
    3. Briefly vortex TurboFect reagent and add 6 µl to the diluted DNA. Mix immediately by vortexing.
    4. Incubate 15-20 min at room temperature.
    5. During the incubation time, remove the old cell medium and replace by 1 ml serum-free DMEM.
    6. Add the DNA/TurboFect reagent mixture dropwise to the cell medium.
    7. Gently rock the plate to achieve an even distribution of the DNA/TurboFect complexes.
    8. Incubate at 37 °C in a CO2 incubator.

  2. Photocholesterol labeling of HA inside cells and crosslinking    
    1. At 6 h post transfection, replace the cell medium with 1 ml fresh serum-free DMEM to remove the transfection reagent.
    2. Add 5 μl photocholesterol (from a 5 mg/ml stock in ethanol, which is stored at -20 °C, final concentration: 50 μM) to each well and gently rock the plate to achieve an even distribution.
    3. Incubate at 37 °C in a CO2 incubator until the next step.
    4. At 24 h post transfection, replace the cell medium with 1 ml fresh serum-free DMEM to remove dead cells floating in the culture supernatant.
    5. Put the plate in the cold room (~8 °C, Figure 3B) or on ice as long as the “UV lamp/6-well plate/ice box” can be protected from light. Cold temperature slows down the mobility of lipids within the membrane and prevents conformational changes of proteins, and is thus better suited for subsequent crosslinking.
    6. Take off the lid from the plate and from the UV lamp since plastic will absorb UV-light (Figure 3C).
    7. Put the UV lamp directly on top of the 6-well plate, turn on the UV lamp (wavelength 320-365 nm, power 8 W, 3.5 A, 60 V) to expose cells to UV light for 10-20 min in the dark to activate the diazirine group to trigger crosslinking (Figure 3D). The cells are still attached to the 6-well culture plate and are covered by 1 ml serum-free DMEM at this stage. Wear protective googles during illumination.


      Figure 3. Crosslinking with photocholesterol. A. UV lamp (wavelength 320-365 nm, power 8 W, 3.5 A, 60 V). B. Cold room (Tm: ~8 °C). C. Preparation before UV illumination by taking off the lid from the tissue culture plate and from the UV lamp. D. UV illumination of the culture plates in the dark.

  3. Immunoprecipitation of HA (buffers should be cold and procedures are performed at 4 °C)
    1. After exposure to UV light cells are still attached to the 6-well culture plate and are covered in DMEM. Put the 6-well plate on ice and detach the cells by scraping without changing medium.
    2. Transfer cells to 1.5 ml microcentrifuge tubes and centrifuge at 1,200 x g for 5 min at 4 °C.
    3. Wash the cell pellet once with 1 ml PBS and resuspend the cells in 500 μl cell lysis buffer.
    4. Lyse the cells for 30 min at 4 °C and centrifuge at maximal speed (13,500 rpm/17,000 x g ) for 15 min at 4 °C to remove insoluble material.
    5. Transfer the supernatant to a new 1.5 ml microcentrifuge tube without disturbing the pellet.
    6. Remove 50 μl from the lysate for analysis of protein expression by Western blot. Add 12 µl 5x non-reducing loading buffer to this 50 μl lysate, incubate at 95 °C for 5 min and store at -20 °C.
    7. Add anti-HA2 antiserum (1:1,000) to the remaining 450 μl lysate and incubate at 4 °C with agitation overnight (12-16 h) in the cold room.
    8. Prepare working solution of protein-G-sepharose beads according to the manufacturer’s instruction.
      a. The stock of protein-G-sepharose is stored in ethanol. Thus, centrifuge the stock solution at 12,000 x g for 30 s to remove the ethanol.
      b. Wash the protein-G-sepharose beads once with PBS by adding the same volume of PBS to the beads and centrifuge at 12,000 x g for 30 s.
      c. Resuspend the protein-G-sepharose beads in PBS by adding the same volume of PBS (1:1 dilution).
    9. Add 50 μl of the protein-G-sepharose beads to each sample and incubate at 4 °C for another 4 h in the cold room with agitation.
    10. Centrifuge at 12,000 x g for 30 s to pellet the HA/antibody/protein-G-sepharose complex.
    11. Remove the supernatant and wash the complex twice with 1 ml IP-buffer and then twice with 1 ml PBS to exclude the possibility that chemicals from the IP-buffer affect the click chemistry reaction in the next step (described in Part I Section D). For the same reason, remove the liquid as completely as possible after the last washing with PBS.
    12. Proceed to the click chemistry reaction (described in Part I Section D) as soon as possible. Storing the HA/antibody/protein-G-sepharose complex on ice for 1 h is fine, but storing for longer time (e.g., overnight at -20 °C) is not recommended.

  4. (Cu(I))-catalyzed Azide-Alkyne Click chemistry reactions (CuAAC) according to the manufacturer’s instruction
    1. Prepare stock solutions in ddH2O: 250 mM THPTA, 100 mM CuSO4, 1 M Na-Ascorbate. The efficiency of the reaction is higher when all the reagents are freshly prepared.
    2. Prepare stock solution of Picolyl-Azide-Sulfo-Cy3 (2 mM, dissolved in H2O, red color).
    3. Prepare CuSO4:THPTA-premix freshly (molar ratio of 1:5) for each experiment. 6 µl (2 µl CuSO4 + 4 µl THPTA) mixture is used here for each reaction. (The ratio of CuSO4:THPTA is recommended as 1:5 in the manufacturer’s instruction, but could be changed.)
    4. Resuspend HA/antibody/protein-G-sepharose complex in 164 µl of H2O.
    5. Add 10 µl of 2 mM Picolyl-Azide-Sulfo-Cy3 to the mixture and mix by pipetting.
      Note: The amount of Azide-Cy3 added here is adjusted based on the amount used in labeling of immunoprecipitated HA. The same final concentration of photocholesterol (50 µM) is used for HA labeling. The molar ratio of alkyne:azide in click chemistry reaction is 1:4, which could be adjusted to get a better result. A higher ratio would give a stronger fluorescence signal. A ratio of 1:10 is recommended in the manufacturer’s instruction.
    6. Add 6 µl CuSO4:THPTA-premix to the mixture and mix by pipetting.
    7. Add 20 µl Na-Ascorbate to the mixture, such that the final volume of each reaction is 200 µl. Mix by pipetting.
    8. Incubate samples at room temperature for 1 h with gentle mixing (avoid vortexing) every 10-15 min. Protect samples from light by wrapping the tubes in aluminum foil.
    9. Wash samples with 1 ml PBS (at least 4 times) until no red color from Azide-Cy3 is seen in the supernatant. However, extensive washing of the beads is not recommended since the amount of beads becomes slightly reduced after each washing step.
    10. Resuspend samples in 40 µl 1x reducing loading buffer (diluted from 4x reducing loading buffer in ddH2O) and incubate at 95 °C for 5 min.
    11. Run SDS-PAGE.
    12. Wash the gel with ddH2O for at least 1 h with periodic changes (e.g., 10 min each). Sometimes overnight (> 12 h) washing is required to reduce the background signal from uncoupled Picolyl-Azide-Sulfo-Cy3.
    13. Scan the gel with a biomolecular imager (Typhoon FLA 9400 scanner, Excitation = 550 nm, Emission = 560 nm for Cy3).

    Note: The order of Steps D4-D7 should not be changed.


Part II: Photocholesterol labeling of immunoprecipitated HA


Transfect CHO cells to express HA as described in Section A of Part I. 24 h after transfection, immunoprecipitate HA with anti-HA2 antiserum as described in Section C of Part I, and then proceed to crosslinking.
Crosslinking between immunoprecipitated HA and photocholesterol:

  1. Resuspend the washed HA/antibody/protein-A-sepharose complex in 100 µl PBS.
  2. Add 0.5 µl photocholesterol (from a 5 mg/ml stock dissolved in ethanol, final concentration: 50 µM), and mix by pipetting. Transfer the solution into one well of a 24-well plate such that a thin liquid layer covering the bottom of the well is formed.
  3. Expose samples to UV light (see Figure 3).
  4. Transfer the labeled protein to a microcentrifuge tube and wash the mixture twice with 1 ml IP buffer and then twice with 1 ml PBS.
  5. Proceed to click chemistry reaction as described in Section D of Part I.

Part III: Western blot to determine the expression level of HA

  1. Prepare gel for SDS-PAGE. The concentration of acrylamide in the stacking gel is 4%, while that in the separation gel is 12%.
  2. Run SDS-PAGE at constant voltage. Use low voltage (80-100 V) while proteins are in the stacking gel and high voltage (160 V) for the separation gel.
  3. Transfer the proteins from the SDS-gel onto a polyvinylidene difluoride (PVDF) membrane at 200-220 mA for 1 h using a semi-dry transfer system.
    Note: The time could be adjusted based on the molecular weight of the protein under study. Larger proteins require longer transfer time.
  4. Incubate the membrane with blocking solution for 30 min at room temperature.
  5. Incubate the membrane with primary antibody (anti-HA2 antiserum, diluted 1:1,000 in blocking solution) for 2 h at room temperature or overnight (12-16 h) at 4 °C.
  6. Wash the membrane 4 times (5 min each) with PBST (see Recipes) on an orbital shaker.
  7. Incubate the membrane with secondary antibody coupled to horseradish peroxidase (1:5,000 dilution in blocking solution) for 1 h at room temperature.
  8. Repeat Step 6 in Part III.
  9. Apply the ECL plus reagent to the membrane according to manufacturer’s instruction, which reacts with horseradish peroxidase to give the chemiluminescent signals.
  10. Detect the protein with a Fusion SL camera system.

Data analysis

  1. For quantification of band intensities, Western blot and the fluorogram were analyzed with ImageJ (Fiji) software. The ratios of photo-cross-linking (fluorogram) to protein expression (Western blot) were calculated for a HA-mutant lacking the cholesterol binding site, and results were normalized to values of HA wild type (HA-wt, set to 100%).
  2. For detailed instruction of using ImageJ please go to https://imagej.net/ImageJ. Here we briefly describe the process of quantification (see Figure 4C). 1) Make sure that the bands are horizontally aligned. 2) Subtract the background signal from cells transfected with empty vector. 3) Select the bands [the left panel in Figure 4C(b) and second panel in Figure 4C(c)]. 4) Plot the band, which gives histograms indicating the intensity of each bands [the middle panel in Figure 4C(b) and the third panel in Figure 4C(c)]. 5) Draw lines to seal each histogram. 6) Select the “magic wand” button and click inside each histogram, which will give a value indicating the intensity of the analyzed band in a new “Result” window [the last panel in Figure 4C(b) and Figure 4C(c)]. Values are exported as csv. file and analyzed by Microsoft Excel (see Table 1). Cholesterol incorporation into the HA-mutant lacking the cholesterol-binding site is reduced to 34% relative to HA-wt.


    Figure 4. Image processing. A-B. Increasing the voltage from 600 (A) to 800 (B) of the scanner increases the contrast. The molecular weight is shown on the left. The next three lanes (L1, L2 and L3) are samples from cells transfected with empty vector (mock), plasmid expressing a HA mutant lacking the cholesterol binding site (HA-mutant), and plasmid expressing the wild-type HA (HA-wt), respectively. The scan can be exported as a tif-file and evaluated with ImageJ. C. Images processed by ImageJ (Fiji). (a) The working panel of ImageJ (Fiji). (b-c) Quantification of the band intensity of HA-mutant and HA-wt from fluorescence scanner (b) and Western blot (c). The density of bands in the yellow boxes is determined and the area under the curves is calculated for each peak. The results obtained from cells transfected with empty vector (mock) is subtracted and the relative incorporation of photo-cholesterol into the HA-mutant relative to HA-wt is calculated (Table 1).

    Table 1. Data processing to compare photocholesterol incorporation into HA-mutant with HA-wt (an example)

Notes

  1. Since the cholesterol concentration in membranes is very high (up to 50% in the plasma membrane) and since both lipids and proteins are mobile within the membrane, every transmembrane region is likely to have transient contact with cholesterol. Thus, in principle every transmembrane protein can be crosslinked to photocholesterol, especially if it is strongly expressed. In order to exclude such an “unspecific” interaction, it is advisable to include a mutant in which the supposed cholesterol-binding site is mutated. In the case of HA labeling, a mutant with exchange of the site was reduced to 58%, relative to wt HA and normalized for the expression level of both proteins.
  2. Since HA with mutations in the cholesterol-binding site are transported to the plasma membrane, but are no longer present in cholesterol-enriched domains (de Vries et al., 2015), one can argue that its reduced labeling inside cells is due to the fact that less photocholesterol is present in their immediate neighborhood. Therefore, we also labeled immunoprecipitated HA, which, however, also revealed a reduced incorporation of photocholesterol (38% relative to wt HA).

Trouble shooting:

  1. Efficient cross-linking requires large amount of the target protein. Therefore, the corresponding gene should be cloned into a vector with a strong promoter to achieve a high expression level.
  2. The amount of photocholesterol used for labeling can be varied. We tested 20 µM, 50 µM and 100 µM (final concentration) for labeling with immunoprecipitated HA. Higher concentration gives stronger fluorescence signal in the gel. In the final protocol, we used the same final concentration of 50 µM for labeling HA both in living cells (5 µl photocholesterol in 1 ml DMEM) and with immunoprecipitated protein (0.5 µl photocholesterol in 0.1 ml PBS).
  3. If no signal is seen in the target protein after labeling of living cells, one could still try labeling of the immunoprecipitated protein since higher concentration of photcholesterol can be achieved and the compound does not compete with authentic cholesterol present in the membrane.
  4. If the analyzed protein is unstable, the step of washing with PBS (Step D9 in Part I) can be omitted. 4x reducing loading buffer can be directly added to the samples, which are boiled and subjected to SDS-PAGE. In this case, overnight washing of the gel with ddH2O is necessary to wash away background signal from Picolyl-Azide-Sulfo-Cy3.
  5. If a positive result cannot be repeated, consider to use a new CuAAC kit.

Recipes

  1. Growth medium (stored at 4 °C)
    DMEM
    10% FCS
    100 U/L Penicillin/Streptomycin
  2. IP buffer (filtered and stored at 4 °C for up to 1 year)
    500 mM Tris-HCl (pH 7.4)
    20 mM EDTA
    30 mM sodium pyrophosphate decahydrate
    10 mM sodium fluoride
    1 mM sodium orthovanadate
    2 mM benzamidine
    1 mM PMSF (phenylmethylsulfonyl fluoride)
    1 mM NEM
    1x protease inhibitor cocktail (freshly added for each experiment)
    Note: Some protease inhibitor could be omitted depending on the studied protein.
  3. Lysis buffer (freshly prepared)
    1% NP40 in IP buffer
  4. Stacking-gel solution (freshly prepared)
    4% (w/v) acrylamide/bisacrylamide
    0.1% SDS
    125 mM Tris-HCl (pH 6.8)
    0.075% (w/v) APS
    0.15% (v/v) TEMED
  5. Separating-gel solution (freshly prepared)
    12% (w/v) acrylamide/bisacrylamide stock solution
    0.1% (w/v) SDS
    375 mM Tris-HCl (pH 8.8)
    0.05% (w/v) APS
    0.1% (v/v) TEMED
  6. 5x non-reducing loading buffer (stored at -20 °C)
    1 M Tris-HCl
    10% (w/v) SDS
    50% (v/v) glycerin
    0.1 % (w/v) bromophenol blue
    pH 6.8
  7. 4x reducing loading buffer (stored at -20 °C)
    1 ml 5x non-reducing buffer
    250 µl 2 M (20x) DTT
  8. PBST (stored at room temperature)
    PBS with 0.1% Tween-20
  9. Blocking solution (freshly prepared)
    3% skimmed milk in PBST

Acknowledgments

This work was supported by the German Research Foundation (SFB 740, TP C3) and by the Human Frontiers Science Program (awarded to M.V.). B.H. is the recipient of a Ph.D. fellowship from the China Scholarship Council (CSC). C.T. was funded by the German Research Foundation under Germany’s Excellence Strategy (EXC2151-390873048). M.R.G is recipient of a Ph.D. fellowship from the DAAD. This protocol was adapted and modified from Hu et al. (2019).

Competing interests

B.H. and co-authors declare no conflicts of interest.

References

  1. de Vries, M., Herrmann, A. and Veit, M. (2015). A cholesterol consensus motif is required for efficient intracellular transport and raft association of a group 2 HA from influenza virus. Biochem J 465(2): 305-314.
  2. Hu, B., Höfer, C. T., Thiele, C. and Veit, M. (2019). Cholesterol Binding to the Transmembrane Region of a Group 2 Hemagglutinin (HA) of Influenza Virus Is Essential for Virus Replication, Affecting both Virus Assembly and HA Fusion Activity. J Virol 93(15): e00555-00519.
  3. Hulce, J. J., Cognetta, A. B., Niphakis, M. J., Tully, S. E. and Cravatt, B. F. (2013). Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells. Nat Methods 10(3): 259-264.
  4. Mintzer, E. A., Waarts, B. L., Wilschut, J. and Bittman, R. (2002). Behavior of a photoactivatable analog of cholesterol, 6-photocholesterol, in model membranes. FEBS Lett 510(3): 181-184.
  5. Niwa, H., Yamamura, K. and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108(2): 193-199.
  6. Thiele, C., Hannah, M. J., Fahrenholz, F. and Huttner, W. B. (2000). Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles. Nat Cell Biol 2(1): 42-49.

简介

[摘要 ] 胆固醇与蛋白质跨膜区的非共价结合会影响其功能,但很少有证明这种相互作用的方法。我们描述了我们的协议,以活细胞中的胆固醇衍生物或免疫沉淀蛋白标记流感病毒的血凝素(HA)。我们合成了一种“可点击的” 光胆固醇化合物,该化合物紧密模拟真实的胆固醇。它包含一个反应性重氮基团,该基团可以通过紫外线照射激活,并与其附近的氨基酸形成共价键。然后将光胆固醇掺入HA中,通过将其“点击”到荧光团上进行可视化,可以通过荧光扫描在SDS凝胶中检测到。 该方法提供了一种方便实用的方法来证明胆固醇与其他蛋白质的结合,并可能确定结合位点。

[背景 ] 蛋白质与胆固醇的非共价相互作用被认为可以调节许多蛋白质的运输和功能(de Vries 等,2015)。然而,由于这种相互作用的短暂且相当弱的性质,众所周知,胆固醇结合蛋白和各自的结合位点很难鉴定。精细的制作方法,如NMR或晶体需要大量需要被整合到人造脂质膜,并因此只能访问特殊纯化的蛋白质的我捷思实验室。一个简单的程序包括使用商业的“试剂盒”测量纯化的蛋白质中的胆固醇含量(例如,的Amplex 红胆固醇测定试剂盒,分子探针)。但是,这种方法不灵敏,需要用去污剂将蛋白溶解在膜上,去污剂通常会去除非共价结合的脂质。

一个改进是合成的clickable- photocholesterol 化合物,其可以是共价链接编到的蛋白质。可以将它们添加到细胞中,在那里它们迅速整合到膜中,因此可以在其天然环境中与蛋白质相互作用。它们包含在甾醇环,6位置的二吖丙因基团,其崩解时UV -illumination成分子氮加上一个高反应性碳烯基,其形成一个共价键与氨基酸密切附近侧链。为了检测交联的蛋白质,探针具有潜在的亲和力,一个炔基可在生理条件下通过铜催化的叠氮化物- 炔烃环加成反应与叠氮化物-报告物化学偶联(“点击化学”)。报道者要么出现在“珠子”上以通过质谱富集和鉴定与探针相互作用的蛋白质,要么是允许其凝胶内检测的荧光团。

我们合成了化合物,6,6 ' -Azi-25-ethinylcholesterol [ 称为改进photoclick -胆固醇,全合成是d 在旁切胡等人。(2019 )] ,其更类似于真正的胆固醇比市售photoclick -胆固醇(十六进制-5'-炔基3SS羟基-6- diazirinyl-5α- 胆- 24 -酸酯,Avanti极性脂质,编号700147) ,因为它含有(除了ALK ÿ NE组)中胆固醇没有进一步的改动' 1-8烷基侧链(图1)。先前的研究表明6-光胆固醇是真实胆固醇的忠实模拟物(Mintzer 等,2002)。但是请注意,某些重氮基可能被光活化为其他反应性物种,该物种的半衰期比可能非特异性标记蛋白质的碳烯基更长。因此,光交联是蛋白质胆固醇亲和力的定性而非定量度量。尽管如此,两种胆固醇探针仅标记所有细胞膜蛋白中的少数特定蛋白(Thiele 等,2000;Hulce 等,2013),表明它们是分析胆固醇与靶蛋白之间可能相互作用的合适工具。



D:\ Reformatting \ 2020-1-6 \ 1902917--1281 BodanHu 794678 \ Figs jpg \图1.jpg

图1.胆固醇和两种可光活化衍生物的结构。A.胆固醇。B.改进Photoclick -胆固醇(6,6 ' -Azi-25乙炔基胆固醇)在此协议中使用。C. 可购自Avanti脂质的Photoclick- 胆固醇(Hex-5'-炔基3ß-羟基-6-二氮杂酰基-5α-胆素-24-酸酯)。光不稳定的叠氮化物基团以B突出显示为蓝色,炔烃基团以红色突出显示。

关键字:流感病毒, 血凝素, 胆固醇, 脂类相互作用, 膜, Photocholesterol, 点击化学

材料和试剂


 


细胞培养耗材
6孔/ 24孔组织培养板,烧瓶,移液器(德国萨尔斯特)
移液器吸头(德国萨尔斯特)
铝箔
CHO-K1(中国卵巢细胞)(ATCC ,目录号:CCL-61)
质粒(pCAGGS ,Niwa 等,1991 )
DMEM(PAN Biotech,目录号:P04-04500)
EDTA-胰蛋白酶(PAN Biotech,目录号:P10-023100)
不含钙/镁的DPBS(PAN Biotech,目录号:P04-36500)
青霉素/链霉素(10,000 U / ml )(PAN Biotech,目录号:P06-07100)
FBS(胎牛血清)(PAN Biotech,目录号:P30-3306)
Opti-MEM TM I降低血清培养基(Thermo Fisher,Gibco TM ,目录号:31985070)
TurboFect 转染试剂(热费舍尔,热科学TM ,目录号:R0531)
生长培养基(请参见食谱)
 


免疫沉淀
乙醇(Sigma,CAS号:64-17-5 ,目录号:34852-1L-M)
Tris-HCl(TRIS盐酸盐)(Carl Roth,CAS号:1185-53-1,目录号:9090.3)
EDTA(AppliChem,CAS号:6381-92-6,目录号:A1104)
焦磷酸钠(Fisher Scientific,Alfa Aesar ,CAS号:7722-88-5,目录号:A17546.30)
氟化钠(Fisher Scientific,Alfa Aesar ,CAS号:7681-49-4,目录号:A13019.30)
原钒酸钠(Sigma ,CAS号:13721-39-6,目录号:S6508-10G)
盐酸苄am(AppliChem,CAS号:1670-14-0,目录号:A1380)
PMSF(AppliChem,CAS号:329-98-6,目录号:A0999)
NEM(N-乙基马来酰亚胺)(Sigma,CAS号:128-53-0,目录号:E3876-5G)
cOmplete TM ,不含EDTA的蛋白酶抑制剂混合物(Sigma,Roche,目录号:11873580001)
NP - 40(Thermo Fisher,Thermo Scientific TM ,目录号:85124)
Protein-G- Sepharose 4 Fast Flow(Sigma,GE Healthcare,GE17-0618-01)
抗HA 2 抗血清(Hu 等,2019)
IP缓冲区(请参阅食谱)
细胞裂解缓冲液(请参阅食谱)
 


SDS-PAGE和W 酯印迹
PVDF 印迹膜,0.2 µm(VWR,Peqlab ,目录号:732-3200)
1.5 ml 微量离心管(德国萨尔斯特)
30%丙烯酰胺/ bisacrylami 德(37.5:1)(卡尔罗斯,目录号:3029.1)
四甲基乙二胺(TEMED)(Carl Roth,CAS号:110-18-9,目录号:2367.3)
过硫酸铵(APS)(Carl Roth,CAS号:7727-54-0,目录号:9592.2)
十二烷基硫酸钠(SDS)(Carl Roth,CAS号:151-21-3,目录号:2326.4)
溴酚蓝(Sigma,CAS号:115-39-9,目录号:1081220005)
1,4-二硫苏糖醇(DTT)(Carl Roth,CAS号:3483-12-3,目录号:6908.2)
甘油(Carl Roth,CAS号:56-81-5,目录号:3783.1)
脱脂奶粉(Carl Roth,CAS号:68514-61-4,目录号:T145.3)
Twee n 20(Carl Roth,CAS号:9005-64-5,目录号:9127.1)
Pierce TM ECL Plus Western印迹底物(Thermo Fisher,目录号:32132)
堆积凝胶解决方案(请参阅食谱)
分离凝胶溶液(请参见配方)
5x非还原装载缓冲区(请参阅食谱)
4x减少加载缓冲区(请参阅食谱)
阻塞缓冲区(请参见食谱)
 


交联和点击化学
黑光蓝(UV)灯(三共电气,功率8W,3.5 A,60V,波长320 - 365纳米,图3A)
改进的Photoclick- 胆固醇(图1B,溶于乙醇,将管密封并保存在-20 °C )。在Hu 等人的文章中详细描述了长合成。(2019)。但是可以从Click Chemistry Tools(https://clickchemistrytools.com/product/alkyne-cholesterol/)购买中间体炔烃。使用这种化合物,可能更容易合成最终产品,即改良的Photoclick- 胆固醇。
Picolyl-Azide-Sulfo-Cy3(Jena Bioscience,目录号:CLK-1178-1)
CuAAC Biomole cule反应缓冲液试剂盒(基于THPTA )(Jena Bioscience,目录号:CLK-1178-1)
 


设备


 


-20°C冷冻室
移液器对照奥勒(赫斯曼Laborgeräte )
Heracell 240i CO 2 培养箱(Heraeus)
台式离心机(Eppendorf,型号:5424R)
电源组P25(Analytik的耶拿,Biometra公司P25T)
化学发光成像,Fusion SL(PeqLab )
蓝色恐惧TM 半干电印迹系统(VWR,PEQLAB ,米Odel等:Sedec TM M)
管转子(Carl Roth,ThermoFisher ,型号:ATX1.1)
台风FLA 9 4 00扫描仪(GE Healthcare)(图2)
 


 


D:\ Reformatting \ 2020-1-6 \ 1902917--1281 BodanHu 794678 \ Figs jpg \图2.jpg


图2. 台风FLA 9 4 00扫描仪。将来自第I部分D部分的凝胶(其中包含交联的样品并用ddH 2 O 洗涤)插入两个玻璃板之间,以进行激光扫描。为结果,参见图4 阿和4 乙。


 


软件


 


台风扫描仪控制v 5 。0软件(GE ħ ealthcare)
ImageQuant (GE ħ ealthcare)
图片J(斐济)
微软Excel
GraphPad棱镜
 


程序


 


第一部分:活细胞中HA的光胆固醇标记


 


通过转染在CHO细胞中表达HA
见d 0.9 ×10 6 -1.1 X 10个6 CHO细胞进入一个孔的6孔培养板的转染的前一天,例如使得它们达到70-90%汇合第二天。
稀UTE 3微克质粒(pCAGGS中)在300编码HAμ 升无血清的Opti -MEM和通过移液混合。
简言之涡流TurboFect 试剂,并添加6 μ 升至稀释的DNA。立即涡旋混合。
在室温下孵育15-20分钟。
在温育期间,移出旧的细胞培养基并替换为1 ml无血清的DMEM。
将DNA / TurboFect 试剂混合物滴加到细胞培养基中。
轻轻摇动板,以使DNA / TurboFect 复合物均匀分布。
在CO 2 培养箱中于37°C 孵育。
 


 


 


细胞内HA的光胆固醇标记和交联
转染后6 h ,用1 ml 新鲜的无血清DMEM 代替细胞培养基以去除转染试剂。
加入5 μ 升pH值otocholesterol (从5毫克/米升乙醇股票,其被存储在-20 ℃下,网络连接最终浓度:50 μM )到每个孔中,轻轻地摇动平板,以实现均匀的分布。
在CO 2 培养箱中于37°C孵育,直至下一步。
转染后24小时,用1 ml 新鲜的无血清DMEM 代替细胞培养基,以除去漂浮在培养上清液中的死细胞。
只要可以保护“ UV灯/ 6孔板/冰盒”免受光照,就可以将板放在冷藏室(〜8°C,图3B)中或冰上。低温会减慢脂质在膜内的迁移,并阻止蛋白质的构象变化,因此更适合于后续的交联。
因为塑料会吸收紫外线,所以请从盘子和紫外线放大器上取下盖子(图3C)。
将紫外线灯直接放在6孔板的顶部,打开紫外线灯(波长320-365 nm,功率8 W,3.5 A,60 V),使细胞在黑暗中暴露于紫外线下10-20分钟激活重氮基团以触发交联(图3D)。所述细胞仍附着到6孔培养板中,用1 m的覆盖升在此阶段无血清DMEM。在照明过程中戴防护用的Google。
 


D:\ Reformatting \ 2020-1-6 \ 1902917--1281 BodanHu 794678 \ Figs jpg \图3.jpg


图3.与光胆固醇的交联。A.紫外灯(波长320-365 nm,功率8 W,3.5 A,60 V)。B.冷藏室(Tm:〜8°C)。C.从组织培养板和紫外线灯上取下盖子,在紫外线照射前进行准备。D.在黑暗中用紫外线照射培养板。


HA的免疫沉淀(缓冲液应该是冷的,并且程序应在4 °C下进行)
暴露于紫外线后,细胞仍附着在6孔培养板上,并被DMEM覆盖。将6 孔板放在冰上,通过刮擦分离细胞,而无需更换培养基。
将细胞转移到1.5 ml 微量离心管中,并在4 °C下以1200 x g 离心5分钟。
洗涤细胞沉淀一次用1M 升PBS重悬在500细胞μ 升细胞裂解缓冲液。
在4°C下将细胞裂解30分钟,然后在4°C下以最大速度(13,500 rpm / 17,000 x g )离心15分钟,以去除不溶物。
将上清液转移到新的1.5 ml 微量离心管中,而不会干扰沉淀。
除去50 μ 升从裂解物用于通过蛋白表达的分析W¯¯ 西部时代印迹。添加12μ 升5×非还原加样缓冲液,这50个μ 升溶胞产物,孵育95℃5分钟,然后储存在-20℃。
抗HA添加2 抗血清(1:1000)到剩余的450 μ 升溶胞产物并孵育在4 ℃下搅拌过夜(12-16 在冷室小时)。
P repare的蛋白质G-工作液琼脂糖凝胶根据制造商的说明书珠。
蛋白质-G- 琼脂糖浆的库存储存在乙醇中。因此,将原液以12,000 xg离心30秒钟以除去乙醇。
W¯¯ 灰蛋白-G- 琼脂糖珠一次,用PBS通过添加PBS的相同体积的珠和离心机以12,000 ×g离心30秒。
ř esuspend 蛋白质-G- 本身pharose 珠在PBS中通过添加PBS的相同体积(1:1倍稀释)。
加入50 μ 升的蛋白G-的琼脂糖珠到每个样品中并孵育在4℃下对另一个4 h中的冷室中搅拌。
以12,000 xg离心30 s沉淀HA /抗体/蛋白质-G- 琼脂糖复合物。
除去上清液和洗复杂两次用1 米升用IP缓冲液,然后两次1 米升PBS,以排除从化学品IP-缓冲器影响在下一步骤中,点击化学反应(在第I部分中描述的可能性科d )。出于同样的原因,在最后一次用PBS洗涤后,应尽可能完全除去液体。
尽快进行点击化学反应(在第I 部分D 部分中描述)。将HA /抗体/蛋白质-G- 琼脂糖复合物在冰上保存1 小时是可以的,但不建议保存更长的时间(例如,在-20 °C 过夜)。
 


(Cu(I))催化的叠氮化物- 炔烃点击化学反应(CuAAC )根据制造商的说明
制备储备溶液在DDH 2 ○:250毫THPTA,100mM的硫酸铜4 , 1M的钠抗坏血酸。当所有试剂都是新鲜制备时,反应效率更高。
制备Picolyl-Azide-Sulfo-Cy3(2 mM,溶于H 2 O,红色)的储备溶液。
为每个实验新鲜准备CuSO 4 :THPTA- 预混物(摩尔比为1:5)。6 μ 升(2 μ升的CuSO 4 + 4μ 升THPTA)混合物在这里用于各反应。(CuSO 4 :THPTA 的比例在制造商的说明中建议为1:5,但可以更改。)
重悬HA /抗体/蛋白G ^ - 琼脂糖在164μ复杂升H的2 O.
向混合物中加入10 µl 2 mM的Picolyl-Azide-Sulfo-Cy3,然后通过移液混合。
注意:此处添加的叠氮基-Cy3的量是根据标记免疫沉淀的HA的量进行调整的。相同的终浓度光胆固醇(50 µM)用于HA标记。在点击化学反应中炔烃:叠氮化物的摩尔比为1:4,可以对其进行调节以获得更好的结果。比率越高,荧光信号越强。在制造商的说明中建议比例为1:10。


向混合物中加入6 µl CuSO 4 :THPTA- 预混物,然后通过移液混合。
向混合物中添加20 µl 抗坏血酸钠,以使每个反应的最终体积为200 µl 。通过移液混合。
每隔10-15分钟将样品在室温下温育1小时,然后缓慢混合(避免涡旋)。将试管包裹在铝箔中以保护样品免受光照。
洗液样品用1 米升PBS(至少4倍),直到没有红色从叠氮基的Cy3的被认为是在上清液中。但是,不建议对珠子进行大量清洗,因为在每个清洗步骤之后,珠子的数量会略有减少。
将样品重悬于40 µl 1x还原负载缓冲液(从ddH 2 O中的4x还原负载缓冲液中稀释)中,并在95°C下孵育5分钟。
运行SDS-PAGE。
定期更换(例如每次10分钟),并用ddH 2 O 洗涤凝胶至少1 h 。有时需要整夜(> 12 小时)洗涤,以减少未偶联的Picolyl-Azide-Sulfo-Cy3产生的背景信号。
用生物分子成像仪(Typhoon FLA 9400扫描仪,对于Cy3 ,激发= 550 nm,发射= 560 nm )扫描凝胶。
注意:不应更改步骤s D4 - D7 的顺序。


 


第二部分:免疫沉淀HA的光胆固醇标记


 


转染CHO细胞以表达HA,如P art I的A 部分所述。转染后24小时,用HA HA的抗HA 2 抗血清免疫沉淀的HA,如P art I的C 部分所述,然后进行交联。


免疫沉淀HA和光胆固醇之间的交联:


将洗涤过的HA /抗体/蛋白-A- 琼脂糖复合物重悬于100 µl PBS中。
加入0.5 µl光胆固醇(从溶于乙醇的5 mg / ml 储备液中,最终浓度:50 µM),并通过移液混合。将溶液转移到24孔板的一个孔中,从而形成覆盖孔底部的薄液体层。  
将样品暴露在紫外线下(见图3)。
所述标记的蛋白质转移到微量离心管中,并用1洗两次混合物米升IP缓冲液1,然后两次米升PBS。
进行到点击化学反应如上述节的d P 技术I.
第三部分:Western blot检测HA的表达水平


 


准备用于SDS-PAGE的凝胶。堆积凝胶中丙烯酰胺的浓度为4%,而分离凝胶中丙烯酰胺的浓度为12%。
在恒定电压下运行SDS-PAGE。当蛋白质在堆叠凝胶中时,请使用低电压(80-100 V),在分离凝胶中请使用高电压(160 V)。
使用半干转移系统将蛋白从SDS凝胶转移到200-220 mA的聚偏二氟乙烯(PVDF)膜上1小时。
注意:时间可以根据所研究蛋白质的分子量进行调整。较大的蛋白质需要更长的转移时间。


在室温下,将膜与封闭溶液孵育30 分钟。
将膜与一抗(抗HA 2 抗血清,在封闭溶液中以1:1,000稀释)在室温下孵育2小时,或在4 °C过夜(12-16 小时)。
在定轨摇床上用PBST (请参阅R ecipes)将膜清洗4次(每次5分钟)。
将膜与偶联辣根过氧化物酶的二抗(在封闭溶液中以1:5,000的稀释度)在室温下孵育1小时。
重复第三部分中的步骤6 。
根据制造商的应用ECL Plus试剂至膜“ S代码,其与辣根过氧化物酶反应以得到化学发光信号。
使用Fusion SL摄影机系统检测蛋白质。
 


数据分析


 


为了定量分析结合强度,使用Image J(Fiji)软件分析了W 酯印迹和荧光图。计算缺少胆固醇结合位点的HA突变体的光交联(荧光图)与蛋白质表达的比率(W 酯印迹),并将结果标准化为HA野生型的值(HA - wt ,设置为100 %)。
有关使用Image J的详细说明,请访问https://imagej.net/ImageJ。在这里,我们简要描述量化过程(参见图4C)。1)确保频段水平对齐。2)从空载体转染的细胞中减去背景信号。3)选择频带[ 在图4C(b)中左侧面板和第二面板在图4C(c)中] 。4)绘制带,绘制直方图,指示每个带的强度[ 图4C(b)中的中间面板和图4C(c)中的第三面板] 。5)画线以密封每个直方图。6)选择“魔术棒”按钮,然后在每个直方图内部单击,这将在新的“结果”窗口[ 图4C(b)和图4C(c)中的最后一个面板中给出一个值,指示分析带的强度。)] 。值导出为csv。文件并由Microsoft Excel分析(请参见表1)。相对于HA-wt,将胆固醇掺入缺乏胆固醇结合位点的HA突变体中减少到34%。
 


D:\ Reformatting \ 2020-1-6 \ 1902917--1281 BodanHu 794678 \ Figs jpg \图4.jpg


图4.图像处理。A- B。将扫描仪的电压从600(A)增加到800(B)会增加对比度。分子量显示在左侧。接下来的三个泳道(L1,L2和L3)是用空载体转染的细胞(模拟),表达缺少胆固醇结合位点的HA突变体的质粒(HA突变体)和表达野生型HA的质粒(HA- wt )。扫描可被导出为TIF - 文件,并用图像评价J. C. 通过图像处理的图像Ĵ(斐济)。(a)图像J(斐济)的工作面板。(BC)HA-突变体和HA的条带强度进行定量- 重量从荧光扫描仪(b)和w ^ 西部时代印迹(C)。确定黄色框中的条带密度,并为每个峰计算曲线下的面积。减去从用空载体(模拟物)转染的细胞获得的结果,并计算光胆固醇相对于HA突变体相对于HA- wt 的相对掺入(表1)。






表1 。数据处理以比较光胆固醇与HA- wt 掺入HA突变体中(一个例子)


 


 


带强度


-嘲笑


突变体/ wt (%)


 


 


 


嘲笑


4,596.9


 


62.2%


荧光性


HA-MUT 蚂蚁


30,246.0


25649。1个


 


重量


45,830.8


41,233.9


 


嘲笑


1,378.3


 


183.0%


免疫印迹


HA突变体


48,514.1


47,135.8


 


HA- 重量


27,134.8


25,756.5


Fluro / 白平衡


 


 


 


34.0%


 


笔记


 


由于膜中的胆固醇浓度非常高(质膜中高达50%),并且由于脂质和蛋白质都在膜内移动,因此每个跨膜区域都可能与胆固醇短暂接触。因此,原则上每个跨膜蛋白都可以与光胆固醇交联,特别是如果它被强烈表达的话。为了排除这种“非特异性”相互作用,建议包括一个假定的胆固醇结合位点发生突变的突变体。在HA标记的情况下,相对于wt HA,具有位点交换的突变体降低至58%,并针对两种蛋白质的表达水平进行了标准化。
由于具有胆固醇结合位点突变的HA被转运至质膜,但不再存在于富含胆固醇的域中(de Vries 等人,2015年),因此可以认为其在细胞内的标记减少是由于事实上,他们附近的光胆固醇较少。因此,我们还标记了免疫沉淀的HA,但是,这也表明光胆固醇的掺入减少了(相对于wt HA,减少了38%)。
 


牛逼卢布拍摄:


有效的交联需要大量的目标蛋白。因此,应将相应的基因克隆到具有强启动子的载体中,以实现高表达水平。
用于标记的光胆固醇的量可以变化。我们测试了20 μ 男,50 μM 和100 μM(最终浓度),用于与免疫沉淀HA标签。浓度越高,凝胶中的荧光信号越强。在最后的协议中,我们使用相同的终浓度50 μM用于标记HA两者在活细胞(5 μ 升photocholesterol 在1M 升DMEM)并用免疫沉淀蛋白(0.5 μ 升photocholesterol 在0.1M 升PBS) 。
如果在标记活细胞后在目标蛋白中没有看到信号,则仍可以尝试标记免疫沉淀的蛋白,因为可以实现更高浓度的酚胆固醇,并且该化合物不会与膜中存在的真实胆固醇竞争。
如果分析的蛋白质不稳定,则可以省略用PBS洗涤的步骤(第I部分中的S tep D 9)。可以将4 x 还原负载缓冲液直接添加到样品中,煮沸并进行SDS- PAGE。在这种情况下,必须用ddH 2 O 过夜清洗凝胶,以洗去Picolyl-Azide-Sulfo-Cy3的背景信号。
如果不能重复得出阳性结果,请考虑使用新的CuAAC 试剂盒。
 


菜谱


 


生长培养基(储存在4°C)
记忆体


10%FCS


100 U / L青霉素/链霉素


IP缓冲区(在4°C下过滤并存储长达1年)
500 mM Tris-HCl(pH 7.4)


EDTA 20毫米


30 mM焦磷酸钠十水合物


10 mM氟化钠


1 mM原钒酸钠


2 mM苄am


1 mM PMSF(苯甲基磺酰氟)


1 mM NEM


1x蛋白酶抑制剂混合物(每个实验新添加)


注意:根据所研究的蛋白质,可以省略某些蛋白酶抑制剂。


裂解缓冲液(新鲜配制)
IP缓冲区中的1%NP40


堆积凝胶溶液(新鲜配制)
4%(w / v)丙烯酰胺/ 双丙烯酰胺


0.1%SDS


125 mM Tris - HCl(pH 6.8)


0.075%(w / v)APS


0.15%(v / v)适时


分离凝胶溶液(新鲜配制)
12%(w / v)丙烯酰胺/ 双丙烯酰胺储备溶液


0.1%(w / v)的SDS


375 mM Tris - HCl(pH 8.8)


0.05%(w / v)APS


0.1%(v / v)TEMED


5个非还原负载缓冲器(存储在-20 °C下)
1 M Tris - HCl


10%(w / v)SDS


50%(v / v)甘油


0.1%(w / v)溴酚蓝             


pH值6.8


4倍减少负载缓冲液(存储在-20 °C时)
1 m l 5x非还原缓冲器


250 µl 2 M(20x)DTT


PBST(室温保存)
含0.1%Tween-20的PBS


封闭液(新鲜配制)
PBST中的3%脱脂牛奶


 


致谢


 


这项工作得到了德国研究基金会(SFB 740,TP C3)和人类前沿科学计划(授予MV)的支持。BH是博士学位的获得者。中国国家奖学金委员会(CSC)的研究金。CT由德国研究基金会根据德国卓越战略(EXC2151-390873048)资助。MRG获得博士学位。d 。来自DAAD的研究金。该协议改编自Hu 等人。(2019 )。


 


利益争夺


 


BH和合著者声明没有利益冲突。


 


参考文献


 


de Vries,M.,Herrmann,A.和Veit ,M.(2015年)。胆固醇共有基序是有效的细胞内运输和流感病毒第2组HA的筏筏结合所必需的。生物化学Ĵ 465(2):305-314。
Hu,B.,Höfer ,CT,Thiele,C.和Veit ,M.(2019)。胆固醇与流感病毒第2组血凝素(HA)的跨膜区结合对于病毒复制至关重要,既影响病毒装配又影响HA融合活性。J Virol 93(15):e00555-00519。
Hulce ,JJ,Cognetta ,AB,Niphakis ,MJ,Tully,SE和Cravatt ,BF(2013)。哺乳动物细胞中与胆固醇相互作用的蛋白质的全蛋白质组定位。Nat Methods 10(3):259-264。
Mintzer,EA,Waarts,BL,Wilschut,J。和Bittman,R。(2002)。在模型膜中胆固醇可光活化类似物6-光胆固醇的行为。FEBS Lett 510(3):181-184。
Niwa ,H.,Yammura,K。和Miyazaki,J。(1991)。具有新型真核载体的高效表达转染子的高效选择。基因108(2):193-199。
Thiele,C.,Hannah,MJ,Fahrenholz,F.和Huttner,WB(2000)。胆固醇与突触素结合,是突触小泡的生物发生所必需的。Nat Cell Biol 2(1):42-49。
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hu, B., Gadalla, M. R., Thiele, C. and Veit, M. (2020). Photoactivable Cholesterol as a Tool to Study Interaction of Influenza Virus Hemagglutinin with Cholesterol. Bio-protocol 10(4): e3523. DOI: 10.21769/BioProtoc.3523.
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