Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Summary

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Abstract

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.

Protocol

1. Introduction

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

Discussion

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 spe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Annexin V-conjugated magnetic beads and MACS micro and minicolumns	Miltenyi Biotec
Lipids (of highest purity)	Avanti Polar Lipid, Inc