Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

Summary

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

Abstract

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

Introduction

The quaternary structure of a protein plays a crucial role in many cellular processes. Signaling pathways, gene expression, and enzyme activation/deactivation all rely on the proper assembly of protein complexes ^1-4. This process also known as homo- or hetero-oligomerization is due to irreversible covalent or reversible electrostatic and hydrophobic protein-protein interactions. Oligomerization not only diversifies the different cellular processes without increasing the genome size, but also provides a strategy for proteins to build stable complexes that are more resistant towards denaturation and degradation ⁵. Defects in oligomerization have an impact on the function of proteins and can lead to the development of diseases. For example, the enzyme phenylalanine hydroxylase forms a tetrameric complex. Some mutations within the protein complex can weaken the tetramer formation and lead to the disease phenylketonuria ⁶.

The human MxA protein is an interferon (IFN)-induced antiviral effector protein exerting a broad antiviral activity against various RNA as well as DNA viruses ⁷. It belongs to the superfamily of dynamin-like large GTPases and has the capacity to form large oligomeric structures in vitro ⁸. Oligomerization has been suggested to protect MxA from rapid degradation ^9,10. Despite intense efforts by many research groups, the molecular mechanism of action remains largely elusive and the role of the oligomerization state of MxA for its antiviral function is under debate ^9,11,12. In this regard, Gao and coworkers proposed a model where MxA exerts its antiviral activity by interacting with viral nucleoproteins in form of large ring-like oligomeric structures ¹¹. However, more recently, we demonstrated that MxA dimers exhibit antiviral activity and interact with the nucleoprotein of influenza A virus ¹². Based on the crystal structure of MxA, Gao and coworkers identified several amino acid residues in the interface regions that are critical for its oligomerization in vitro and its antiviral function ^11,13. Therefore, in order to elucidate which oligomeric state of MxA exerts antiviral activity, we sought to establish a simple protocol to rapidly determine the oligmeric state of MxA interface mutants expressed in human cells as well as endogenous MxA expressed after IFNα stimulation.

Although there are many techniques that are commonly used to investigate the interaction between proteins such as the split-Green Fluorescent Protein (split-GFP) complementation assay ¹⁴, surface plasmon resonance ¹⁵ and Förster resonance energy transfer (FRET) ¹⁶, they do not provide information of the exact stoichiometry of an oligomeric protein complex. For investigation of this particular aspect, techniques such as multi-angle light scattering (MALS) ¹⁷ and analytical ultracentrifugation ¹⁸ are very useful. Usually, the proteins analyzed using these methods are purified proteins. Oligomerization processes may also depend on other cellular factors. If these factors are unknown, the analysis is more difficult. Additionally, some proteins are difficult to express in E. coli and to purify. Therefore, these methods are not the optimal choice to analyze protein oligomerization in the cellular environment. In addition, these techniques require expensive instruments which are not readily available.

Non-denaturing polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography or chemical crosslinking followed by conventional Sodium dodecyl sulfate (SDS)-PAGE are useful tools for the characterization of the formation of oligomers from cell lysates ^2,19,20. These methods do not require specialized equipment and can be easily performed in a standard laboratory. We initially evaluated various chemical cross-linking protocols that invariantly led to non-specific aggregation and precipitation of MxA. Therefore, we next tested non-denaturing PAGE protocols. As non-denaturing PAGE excludes the use of SDS, the migration of proteins depends on their native charge. Blue-native PAGE uses coomassie brilliant blue G250 to load proteins with an overall negative charge, similar to SDS, but does not denature the protein ²¹. Unfortunately, coomassie brilliant blue precipitates in the presence of high salts and divalent cations (e.g. Mg²⁺) that are often included in lysis buffers. Depending on the buffers used, it may be difficult to analyze the sample without further optimization of steps that could have an effect on the oligomeric protein complex.

Here we present a simple protocol based on a previously published method ²² to determine oligomerization of human MxA protein derived from cellular lysates using non-denaturing PAGE.

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Protocol

NOTE: This protocol is based on the previously published non-denaturing PAGE protocol ¹². In that study, the oligomeric state of the MxA protein was assessed using either Vero cells overexpressing MxA or IFN-α-stimulated A549 cells expressing endogenous MxA. The protocol described below can be used to analyze the oligomeric state of any protein in addition to MxA. However, further optimization may be required.

1. Preparation of Cell Lysate for Non-denaturing PAGE

NOTE: To analyze the oligomeric state of the human MxA protein in either Vero or A549 cells, 1.0 x 10⁶ cells were harvested. Depending on the cell type or the abundance of the protein to analyze, the cell number should be adjusted. It is also important to protect the lysis buffer from light exposure, as soon as the light-sensitive iodoacetamide is added.

1. Seed 0.3 x 10⁶ A549 or Vero cells per well into 6 well-dishes. Keep the cells in 2 ml growth medium per well (see Table 1). Incubate cells overnight in a cell culture incubator (37 °C, 5% CO₂).
2. Harvest the cells by washing with 1 ml of phosphate buffered saline (PBS) and detach by adding 0.5 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) 1x solution for approximately 5 min at room temperature.
3. As soon as the cells detach from the dish, add 0.5 ml growth medium and carefully mix by pipetting up and down.
4. Transfer the cells of each well into one 2 ml tube and pellet them using a table top centrifuge (5,000 x g, 4 °C, 5 min).
5. Carefully remove the supernatant by pipetting without disturbing the cell pellet.
6. Wash the cells with 1 ml ice-cold PBS by carefully pipetting the cell suspension up and down.
7. Pellet cells in a table top centrifuge (5,000 x g, 4 °C, 5 min).
8. Carefully remove the supernatant by pipetting without detaching the cell pellet.
9. Resuspend cells in 200 μl ice-cold lysis buffer (see Table 1) by pipetting up and down and put on ice.
10. Immediately, protect lysate from light by covering the tubes using tin foil and incubate for 30 min on ice.
    NOTE: After incubation for 30 min on ice, it is no longer essential to protect the lysate from light exposure, since the protection of the free thiol groups is irreversible.
11. Remove cell debris by centrifugation in a pre-chilled table top centrifuge (13,000 x g, 4 °C, 20 min).
12. Equilibrate dialysis columns in dialysis buffer (Table 1) in the cold room at 4 °C for 20 min during the centrifugation step. Use a column with a molecular weight cut off of 10,000.
    1. Attach the columns to a float buoy and put them into a beaker filled with dialysis buffer. To ensure gentle stirring, use a magnetic stirrer. Do not touch the membrane.
       NOTE: Dialysis columns can be purchased or prepared from 1.5 ml tubes according to the protocol described by Fiala and coworkers ¹⁹.
13. Remove the columns from the dialysis buffer and the float buoy. Transfer the cleared lysates into the prepared dialysis column by pipetting without touching the membrane. Attach the columns to a float buoy and put them back into the beaker filled with dialysis buffer.
14. Dialyze the lysate in a beaker containing ice-cold dialysis buffer (Table 1) for at least 4 hr (or preferably overnight) at 4 °C while carefully stirring using a magnetic stirrer. Use at least 100 ml dialysis buffer for a 200 µl lysate.
15. Transfer the dialysed sample into a 1.5 ml tube. Remove precipitates by centrifugation in a table top centrifuge (13,000 x g, 4 °C, 20 min). To prevent the dissociation of the oligomeric protein complexes continue with the protocol (section 2) immediately after dialysis. Do not freeze the prepared lysates.

2. Electrophoresis

NOTE: Electrophoresis was performed as described before with some modifications ²². In the protocol described below, pre-cast gradient gels were used (4-15% gradient). Alternatively, the gels can be prepared in the laboratory. It is very important to exclude any denaturing agent such as SDS to prevent the dissociation of the oligomeric protein complexes. Time of electrophoresis was optimized for the different oligomeric states of the human MxA protein. However, it can vary for other proteins, depending on the size of the oligomeric complex as well as the range of separation that is supposed to be achieved to analyze the complex. Therefore, the optimal time of electrophoresis should be determined empirically. For optimal resolution of the oligomers to be analyzed the current should not exceed 25 mA.

1. Assemble the non-denaturing PAGE gel in the gel chamber. Fill the inner and outer chamber with pre-chilled running buffer (Table 1).
2. Pre-run the gel with pre-chilled running buffer at 25 mA per gel for 15 min in the cold room at 4 °C.
3. Mix 15 µl of the above prepared lysates with 5 µl of 4x sample buffer (Table 1). Do not boil the sample.
4. Load 15 µl of sample and a native protein standard of choice on the gel. Run the gel at 25 mA for 4 hr in the cold room at 4 °C.
   NOTE: For semi-quantitative analyses, a protein quantification protocol (e.g. a Bradford protein assay ²³) can be performed in order to ensure loading of equal amounts of total protein per lane.

3. Western Blot

NOTE: Described below is the protocol of a wet western blot system. Any blotting membrane can be used. Activate polyvinylidene fluoride (PVDF) membranes in 100% methanol before equilibration in blotting buffer. The Semi-dry western blot technique can be used alternatively, but has to be optimized for large oligomeric complexes.

1. Disassemble the gel and carefully transfer it into SDS buffer (Table 1).
2. Incubate for 10 min at room temperature while gently shaking.
3. Prepare 2 sponges, 4 cellulose filter paper sheets and a blotting membrane per gel. Soak them in blotting buffer (Table 1).
4. Assemble the sandwich as follows (bottom to top): 1 sponge, 2 cellulose filter paper sheets, membrane, gel, 2 cellulose filter paper sheets, 1 sponge.
5. Put the sandwich into the blotting tank. Make sure that the membrane faces the plus pole while the gel faces the minus pole.
6. Fill the blotting tank with pre-chilled blotting buffer.
7. Blot at 90 mA overnight at 4 °C for best protein transfer results.
8. Disassemble the sandwich and visualize the protein standard by incubating the membrane in Ponceau S solution for 5 min at room temperature.
9. Destain the membrane by carefully washing off the Ponceau S with deionized water until you can clearly see the bands of the protein standard.
10. Mark the bands of the protein standard using a pen.
    NOTE: Residual Ponceau S can interfere with the immunostaining. To avoid this, the membrane can be destained further by incubation in 0.1 M NaOH for 1 min and subsequent washing with deionized water.
11. Block the membrane with Blocking buffer (see Table 1) for at least 1 hr at room temperature or overnight at 4 °C.
12. Visualize protein(s) of interest by immunostaining using antibodies directed against the protein to be analyzed.
    NOTE: The human MxA protein was visualized using the rabbit polyclonal antibody specific for human Mx1 diluted 1:1,000 in Blocking buffer (Table 1). The antibody solution was incubated overnight at 4 °C. Alternatively, the monoclonal anti-MxA antibody (clone 143) can be used (data not shown) ²⁴.

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Results

Using non-denaturing PAGE, we analyzed the oligomeric state of the human wild type MxA, the dimeric interface mutants MxA(R640A) and MxA(L617D) as well as the monomeric interface mutant MxA(M527D) from cell lysates ¹². Cells were lysed in a buffer containing 1% octylphenoxypolyethoxyethanol (NP-40) and iodoacetamide to ensure protein solubilization and protection of free thiol groups (see Figure 1). As described before, salt and small metabolites were removed by dialysis ¹⁹. Protein...

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Discussion

Here we describe a simple method that allows the rapid determination of the oligomeric state of proteins expressed in mammalian cells by non-denaturing PAGE followed by Western blot analysis. The major advantage of this approach is that the oligomeric state of a given protein can be determined from whole cell lysates without prior protein purification. This may be important for proteins that oligomerize or exert their function in association with auxiliary factors. In addition, the proteins are still in their native stat...

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Materials

Name	Company	Catalog Number	Comments
Slide-A-Lyzer MINI Dialysis Units, 10K MWCO, 0.5 ml	Thermo Fisher Scientific	69570	Pre-equilibrate in dialysis buffer (if Glycerol removal is desired) Can be self-made according to Fiala et al. 2011
4–15% Mini-PROTEAN TGX Precast Protein Gels, 10-well,	Bio-Rad	456-1083	Pre-run in running buffer to adjust buffer system
cOmplete, Mini, EDTA-free	Roche	11836170001	use 1 tablet per 50 ml
PBS, pH 7.4  bottle a 500 ml Gibco	Thermo Fisher Scientific	14190-094
Ponceau S solution	Sigma-Aldrich	P7170	TOXIC wear gloves and protect eyes
NativeMark Unstained Protein Standard  50 µl	Invitrogen	P/N 57030	load 5 µl/well
A549 cells	ATCC	ATCC CCL185	Grow in growth medium (see Table 1)
Vero cells	ATCC	ATCC CCL81	Grow in growth medium (see Table 1)
anti-Mx1 antibody	Novus Biologicals	H00004599_D01P	Use at a 1:1,000 dilution
ECL Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (from donkey)	GE-Healthcare	NA934V	Use at a 1:10,000 dilution
0.5% Trypsin-EDTA (1x)        Life Technologies	Thermo Fisher	15400-054
Iodoacetamide   5 g	Sigma-Aldrich	I-6125	stock  100 mM
Bromphenolblue	Sigma-Aldrich	B0126-25G
DMEM +4.5g/l Gluc,+L-Glut,+Pyruvate life technologies	Thermo Fisher Scientific	41966-029
Pen  Strep 100 x     100ml               life technologies	Thermo Fisher Scientific	15140 - 130
Glutamax 100x Stock, 100 ml     life technologies	Thermo Fisher Scientific	350500-038
Fetal Bovine Serum, Dialyzed , US Origin 500 ml Gibco Lot:42G9552K	Thermo Fisher Scientific	10270-106
Cellulose filter paper	Bio-Rad	1703965
PVDF blotting  membrane	GE-Healthcare	10600022
Tris(hydroxymethyl)aminomethane	Biosolve	0020092391BS
sodium fluoride (NaF)	Sigma Aldrich	S-7920
NP-40	Calbiochem	492015
cOmplete, Mini, EDTA-free	Roche	11836170001
Tween 20	Calbiochem	6555204
CHAPS 10% solution	Amresco	N907
DL-Dithiothreitol (DTT)	Sigma Aldrich	43819
Glycine	Biosolve	0007132391BS
sodium orthovanadate (Na3VO4)	Sigma Aldrich	450243
Glycerol	Sigma Aldrich	G7757
β-Glycerophospate	Sigma Aldrich	G9422
Milk powder	Migros/Switzerland
Methanol	Millipore	1.06009
sodium cloride (NaCl)	Sigma Aldrich	71380
magnesium chloride (MgCl2)	Amresco	288
Sodium dodecyl sulphate (SDS)	Sigma Aldrich	L4509
sodium hydroxide (NaOH)	Sigma Aldrich	S-8045