Institute of Molecular Medicine and Genetics, Georgia Health Sciences University
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Bieberich, E. Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction. J. Vis. Exp. (50), e2657, doi:10.3791/2657 (2011).
The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid.
We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.
The lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS) technique was developed in our laboratory to isolate ceramide-associated protein complexes 1-3. Originally, the lipid vesicles were made of ceramide and phosphatidylserine, which allowed for MACS using magnetic particle-conjugated Annexin V (highly affine to phosphatidylserine) to isolate the vesicles and their associated proteins. We have used the LIMACS technique for the in vitro reconstitution of a ceramide-associated polarity complex and the isolation of ceramide-binding proteins from cell lysates 3. LIMACS can be modified using other interaction partners for the isolation of the vesicles (e.g., glycolipid-specifc lectins or lipid antibodies).
2. Experimental Procedures
Preparation of Lipid Vesicles and aPKCBinding Assays
In vitro lipid-protein polarity complex
LIMACS purification of PKCζ-EGFP and the ceramide binding domain C20ζ-EGFP
A detergent-free lysate of MDCK cells expressing full length PKCζ C-terminally linked to green fluorescent protein (FLζ-EGFP) or a ceramide binding domain in the C-terminus of PKCζ (C20ζ-EGFP) was incubated with phosphatidylserine/ceramide vesicles as described in Experimental Procedures. After elution of the MACS column, protein was analyzed using immunoblotting and antibodies against PKCζ and EGFP for detection of the eluted protein 2.
Figure 1. LIMACS of EGFP-labeled PKCζ and its C-terminal fragment C20ζ using phosphatidylserine/ceramide vesicles.
Detergent-free lysates of MDCK cells expressing EGFP (as a non-binding control), full length PKCζ-EGFP, or the ceramide binding, C-terminal fragment C20ζ-EGFP were incubated with phosphatidylserine/ceramide vesicles as described in the Experimental Procedures section. After using LIMACS, protein was eluted with SDS sample buffer and analyzed by SDS-PAGE and immunoblotting. The left panel shows that EGFP did not bind to the vesicles retained with the Annexin V-linked magnetic beads. The middle and right panel shows that full length PKCζ-EGFP and C20ζ-EGFP were retained due to binding to ceramide.
To test the specific interaction between a lipid and its binding protein is hampered by embedding of lipids in the cell membrane. The cell membrane consists of a mixture of several lipids and proteins and it is organized in lipid microdomains or rafts. Therefore, the co-purification of microdomains and proteins cannot clearly distinguish if a protein directly binds to a lipid or is only enriched in a microdomain structure. Other methods using defined lipids coated on surfaces such as lipid ELISAs or Plasmon resonance spectroscopy can detect the interaction of a specific lipid with its binding protein.. However, to determine this interaction in a physiologically relevant membrane environment, the surface (e.g., for Plasmon resonance spectroscopy) has to be coated with lipid vesicles or liposomes.
We developed a novel lipid vesicle binding assay as an alternative to these previous methods. The novelty comes from incorporating an anchor lipid (phosphatidylserine) into the vesicles, which allows for the isolation of lipid vesicles with Annexin V-conjugated magnetic beads. Therefore, we termed this assay lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (LIMACS). LIMACS can be performed for endogenous proteins, proteins expressed in cells, or recombinant proteins reconstituted in a lipid protein complex 1-3.
The protein bound to the lipid vesicles can then be recovered by simply taking the MACS column from the magnetic stand and eluting it with an appropriate buffer. This can be an enzyme compatible buffer to measure enzyme activity in the eluate or SDS sample buffer to perform protein analysis using SDS-PAGE and immunoblotting. If using SDS sample buffer the MACS column does not have to be removed from the magnetic stand, which allows for retention of the magnetic beads on the stand.
There are precautions to be taken when using LIMACS. Evidently, LIMACS cannot be used with a detergent lysate because this will destroy the lipid vesicles. Also, it has to be tested if the binding protein of the target lipid also binds to phosphatidylserine because this lipid is used as an anchor for binding of the vesicles to Annexin V-conjugated magnetic beads. . In some cases, phosphatidylserine may impair binding to the target protein. Therefore, negative controls and controls with various amounts of phosphatidylserine have to be included into the assay. Thesecontrols can be easily performed by using lipid vesicles containing only phosphatidylserine or a mixture of phosphatidylserine with non-binding lipids. Including negative controls is also required for cell lysates that contain a significant amount of endogenous phosphatidylserine. To avoid contamination with endogenous lipid membranes or vesicles it is recommended to clear the cell lysate by including an ultracentrifugation step at 100,000xg for 1 h. It is evident that lipid binding proteins in the supernatant can only be cytosolic, which is a limitation of the LIMACS procedure. There is the possibility to extend LIMACS to other anchor lipids such as GM1 and cholera toxin B subunit conjugated with magnetic beads.
We are currently exploring the use of lipid-specific antibodies for LIMACS, which would make the use of anchor lipids for preparation of the vesicles unnecessary. One of the beneficial aspects of LIMACS is the isolation of the lipid protein complex which allows for a variety of protein analytical methods that are not implementable.. For example, we have used LIMACS to isolate a reconstituted polarity protein complex of Par6/Cdc42 associated with ceramide-bound aPKC. Therefore, LIMACS is a powerful novel method for the analysis and isolation of lipid-associated protein complexes.
No conflicts of interest declared.
This work was supported by the NIH grants R01NS046835 and R01AG034389, and the March of Dimes grant 6FY08-322. A special thank you is devoted to Mrs. Eleanor Brown (Miltenyi Biotec, Auburn, CA) who helped tremendously with her insight into the MACS technology. Miltenyi has generously provided the material used for the demonstration of the experiments at no cost. I am also grateful to Dr. Guanghu Wang (Medical College of Georgia/Georgia Health Sciences University, Augusta, GA) who generated the PKCζ expressing cell lines. Support by the Institute of Molecular Medicine at the Medical College of Georgia/Georgia Health Sciences University (under directorship of Dr. Lin Mei) is also acknowledged.
|Annexin V-conjugated magnetic beads and MACS micro and minicolumns||Miltenyi Biotec|
|Lipids (of highest purity)||Avanti Polar Lipid, Inc|