Identification of Lipid Raft Associated Proteins from Human Lens Epithelial Cells

Abstract

Abstract: : Purpose: To identify the presence, the subcellular localization and potential function of lipid rafts in human lens epithelial cells. This study provides new information on lipid raft constituents from the entire cell. Methods: Lipid raft fractions were prepared from the human lens epithelial cell line, FHL124. Two triton insoluble floating fractions (TIFF) were collected; a top and a bottom band at the 20–28% and 28–38 % sucrose interfaces, respectively. Cholesterol and phospholipids were analyzed by matrix–assisted laser desorption/ionization mass spectrometry (MALDI–MS) using 2,5 dihydroxybenzoic acid as the matrix. Proteins from both TIFF were fractionated by SDS–PAGE then proteolyzed in situ prior to liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Results: The TIFF were enriched in cholesterol and sphingomyelins. After separation of protiens by SDS–PAGE and colloidal blue staining, ∼25 bands were visible. However, by LC–ESI–MS/MS, greater than 1000 proteins were identified from each of the two fractions; ∼65% of the proteins were present in both TIFF. Proteins found in both TIFF must be common to more than one type of lipid raft. The other ∼ 35% of identified proteins are unique to either the top or bottom TIFF. The lipid raft marker proteins, caveolin–1, flotillin–1 and flotillin–2, were identified in the top and bottom TIFF by western blotting and LC–ESI–MS/MS. Other constituents, including raftlin, a protein necessary for the integrity of lipid rafts, were identified by LC–ESI–MS/MS. Proteins from both the top and bottom TIFF fall into 5 major classes, signal transduction (e.g., src), membrane trafficking (e.g., rab GTPases), ATP binding proteins (e.g., ATP binding cassette proteins), cytoskeletal proteins (e.g., actin), and adhesion proteins (e.g., integrins). The bottom TIFF was enriched in mitochondrial raft proteins. Conclusions: The exquisite sensitivity of in situ proteolysis, coupled with LC–ESI–MS/MS allows the identification of proteins associated with lipid rafts from human lens epithelial cells. This technique will be extremely useful to characterize changes in lipid raft constituents of lens epithelial cells that distinguish normal aging from pathological states, and may provide insight into interventions that delay or prevent cataract formation.

Keywords: cell membrane/membrane specializations • proteomics • protein purification and characterization