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

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 Ox-LDL with high affinity. This binding was very effectively inhibited (ϳ50%) 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.

Oxidized Phospholipids as Endogenous Pattern Recognition Ligands in Innate Immunity

“…In studies with copper oxLDL, at least half of the macrophage binding activity was observed to remain in the protein moieties of lipoprotein following solvent extraction, suggesting that protein-oxPL adducts may also serve as ligands for macrophage recognition and phagocytosis via CD36 (34). This has also been observed in model studies employing protein-lipid and peptide-lipid adducts.…”

“…For example, studies from the collaborative research groups of Steinberg and Witztum (35) first reported that receptors for oxLDL on elicited mouse peritoneal macrophages recognize both the oxidized lipid component and the modified protein moieties of oxLDL. In an extension of those studies, these investigators later identified the mouse scavenger receptor CD36 responsible for mediating binding to copper oxLDL via oxPC (34). In competition studies ϳ50% of the CD36 binding activity was shown to reside within the lipid (organic solvent)-extractable fraction (34).…”

“…In an extension of those studies, these investigators later identified the mouse scavenger receptor CD36 responsible for mediating binding to copper oxLDL via oxPC (34). In competition studies ϳ50% of the CD36 binding activity was shown to reside within the lipid (organic solvent)-extractable fraction (34). Concurrently, Podrez et al (33) reported results from studies employing LDL exposed to the myeloperoxidase-H 2 O 2 -nitrite system of monocytes, a more physiologically relevant oxidized form of LDL.…”

“…Over the past several years, much effort has focused on our understanding of the chemical and structural nature of oxPL ligands recognized by macrophage pattern recognition receptors. There is general consensus that a significant portion of ligand binding activity on oxidized lipoproteins and senescent or apoptotic cells resides within extractable lipids (33)(34)(35)(36)(37). For example, studies from the collaborative research groups of Steinberg and Witztum (35) first reported that receptors for oxLDL on elicited mouse peritoneal macrophages recognize both the oxidized lipid component and the modified protein moieties of oxLDL.…”

“…2 The abbreviations used are: oxLDL, oxidized low density lipoprotein; oxPC, oxidized phosphatidylcholine; oxPL, oxidized phospholipid(s); oxPC CD36 , oxidized PC species possessing an sn-2 acyl group that incorporates a terminal ␥-hydroxy(or oxo)-␣,␤-unsaturated carbonyl; oxPS, oxidized phosphatidylserine; MS, mass spectrometric/spectrometry; PLA 2 , phospholipase A 2 . (34,38,39). Alternative members of the innate immune system such as the acute-phase reactant C-reactive protein (38) and antibodies to protein-oxPC adducts have been shown to bind to both oxLDL and apoptotic cells through recognition of oxPC as a common ligand (5,38).…”