The Binding of Oxidized Low Density Lipoprotein to Mouse CD36 Is Mediated in Part by Oxidized Phospholipids That Are Associated with Both the Lipid and Protein Moieties of the Lipoprotein

Agnès Boullier,Kristin L Gillotte,Sohvi Hörkkö,Simone R Green,Peter Friedman,Edward A Dennis,Joseph L Witztum,Daniel Steinberg,Oswald Quehenberger

doi:10.1074/jbc.275.13.9163

Abstract

There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact OxLDL with high affinity. This binding was very effectively inhibited ( approximately 50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.

Highlights

The oxidative modification of low density lipoprotein (LDL)1 and the subsequent uptake of oxidized LDL (OxLDL) by macrophages, leading to foam cell formation, is an important pathway in atherogenesis [1, 2]
The studies reported below show that CD36, which is transiently expressed in COS-7 cells, exhibits ligand binding characteristics similar to those reported for intact macrophages, i.e. it can directly bind either apoB or the reconstituted lipids derived from OxLDL
To determine whether similar epitopes are responsible for the binding of apoB and lipids from OxLDL, we examined in separate experiments the binding of 125I-apoB derived from OxLDL and dihexadecylooxacarbocyanine perchlorate (DiO)-labeled microemulsions of lipids isolated from OxLDL to CD36-transfected cells in the presence and absence of EO6

Summary

Introduction

The oxidative modification of low density lipoprotein (LDL)1 and the subsequent uptake of oxidized LDL (OxLDL) by macrophages, leading to foam cell formation, is an important pathway in atherogenesis [1, 2]. Products derived by oxidation of pure phospholipids (e.g. 1-palmitoyl 2-arachidonoyl phosphatidylcholine) have been shown to inhibit the binding of intact OxLDL [11] and that of isolated apoB and microemulsions of OxLDL lipids [10, 11].

Results

Conclusion