Profiling the Lipid Raft Proteome from Human MEC1 Chronic Lymphocytic Leukemia Cells

Munther Alomari

doi:10.4172/jpb.s7-005

Abstract

Lipid rafts are specialized micro-domains located in the outer plasma membrane of cells and play important roles in various cellular functions, including cell signalling, secretory and endocytic pathways. Cell surface profiling, in particular the lipid raft proteome, has attracted interest in oncology due to the potential use of raft proteins as novel targets for diagnostics and therapeutics. Three different methods have been used to identify the lipid raft proteome from the human chronic lymphocytic leukemia (CLL) cell line MEC1. Firstly, lipid raft proteins were enriched and identified using sucrose gradient ultracentrifugation and 2D liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). To confirm protein localization to the lipid raft, proteomes were compared before and after cholesterol depletion by methyl-β-cyclodextrin (MβCD) using isobaric tags for relative and absolute quantitation (iTRAQ)-labeling coupled to 2D LC-MS/MS. Lipid raft proteins were also identified by immuno-precipitation of crosslinked CD20, a tetraspanin protein that translocates to lipid raft following treatment with the therapeutic antibody, rituximab. In total, 643 proteins were found in lipid rafts of CLL cells (580 following sucrose gradient ultracentrifugation, 181 depleted by MβCD and 199 isolated by immunoprecipitation) and 64 proteins were identified by all 3 methods). These data represents the first comprehensive profile of the lipid raft proteome in CLL cells and includes 30 proteins with no previous known association to the lipid raft. These proteins may represent novel diagnostic and therapeutic targets for CLL.

Highlights

Lipid rafts (LR) were first characterized as functional membrane micro-domains about two decades ago [1]
Externalization of phosphatidylserine on cells during apoptosis was quantified by Annexin V-PE binding, while the loss of cell membrane integrity was demonstrated by binding of 7-amino-actinomycin D (7-AAD) to DNA (Annexin V-PE apoptosis detection kit 1, BD Biosciences, San Diego, CA)
No significant increase in apoptosis or necrosis was observed when cells were incubated with rituximab (10 μg/mL, 24 h; Figure 1), as expected

Summary

Introduction

Lipid rafts (LR) were first characterized as functional membrane micro-domains about two decades ago [1] Their existence was controversial for many years, investigations of the structure and function of LRs are at the forefront of cell biology and bio-membrane research [2,3]. Proteins are recruited to LRs in response to various stimuli, e.g., radiation [5], hormones [6] and therapeutic antibodies [7]. This recruitment is primarily mediated by acylation or farnesylation of a GPI (glycosylphosphatidylinositol) anchor [8]. In B-lymphocytes, LRs may be concentrated in microvilli-rich regions [11]

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