Identification of a Novel Family of Oxidized Phospholipids That Serve as Ligands for the Macrophage Scavenger Receptor CD36

Abstract

The macrophage scavenger receptor CD36 plays an important role in the uptake of oxidized forms of low density lipoprotein (LDL) and contributes to lesion development in murine models of atherosclerosis. However, the structural basis of CD36 lipoprotein ligand recognition is unknown. We now identify a novel class of oxidized phospholipids that serve as high affinity ligands for CD36 and mediate recognition of oxidized forms of LDL by CD36 on macrophages. Small unilamellar vesicles of homogeneous phosphatidylcholine (PC) molecular species were oxidized by the myeloperoxidase (MPO)-H 2 O 2 -NO 2 ؊ system, and products were separated by sequential LC/ESI/MS/MS. In parallel, fractions were tested for their ability to bind to CD36. Four major structurally related phospholipids with CD36 binding activity were identified from oxidized 1-palmitoyl-2-arachidonyl-PC, and four corresponding structural analogs with CD36 binding activity were identified from oxidized 1-palmitoyl-2-linoleoyl-PC. Each was then synthetically prepared, its structure confirmed by multinuclear NMR and high resolution mass spectrometry, and shown to possess identical CD36 binding activity and LC/ESI/MS/MS characteristics in both native and derivatized forms. Based upon the structures of the active compounds identified, and structure-function studies with a variety of synthetic analogs, we conclude that the structural characteristics required for high affinity binding of oxidized PC species to CD36 are a phospholipid with an sn-2 acyl group that incorporates a terminal ␥-hydroxy(or oxo)-␣,␤-unsaturated carbonyl (oxPC CD36 ). LC/ESI/MS/MS studies demonstrate that oxPC CD36 are formed during LDL oxidation by multiple distinct pathways. Formation of this novel class of oxidized PC species contributes to CD36-mediated recognition of LDL oxidized by MPO and other biologically relevant mechanisms. The present results offer structural insights into the molecular patterns recognized by the scavenger receptor CD36 and provide a platform for the development of potential therapeutic inhibitory agents.CD36 is a heavily glycosylated, single chain, integral plasma membrane protein that belongs to an evolutionarily conserved family of proteins that serve as scavenger and lipid receptors (1, 2). It is expressed on the surface of adipocytes, microvascular endothelial cells, macrophages, platelets, and specialized epithelial cells (1, 2). CD36 functions in vivo in scavenger recognition of oxidized lipoproteins and senescent or apoptotic cells, fatty acid transport, cell-matrix interactions, and antiangiogenic actions (3-5). Its deficiency in humans has been correlated with alterations in myocardial fatty acid uptake, hypertrophic cardiac myopathy, and insulin resistance (6 -8). Recent studies (3, 4, 9 -11) have focused attention on CD36 as a participant in the atherosclerotic process because of its ability to recognize oxidized forms of LDL (oxLDL).1 CD36 mediates lipid accumulation and macrophage foam cell formation in vitro and in vivo (3,12,13). It is heavily expre...

CD36: Implications in cardiovascular disease

“…In terms of modified LDL, the exact nature of the CD36 ligand has been recently defined as a truncated phosphatidylcholine species, with a specific sn-2 esterified γ-hydroxy (or oxo) -α,β-unsaturated carbonyl containing fatty acid (Podrez, et al, 2002a,Podrez, et al, 2002b. These modifications can occur as a result of the action of reactive oxygen or nitrogen species.…”

Localization of oxidized phosphatidylcholine in nonalcoholic fatty liver disease: Impact on disease progression

“…[12][13][14] Cellular uptake of oxLDL is mediated by the binding of oxidized phosphatidylcholine (oxPC) molecules, the principal oxidized phospholipid present in oxLDL, to scavenger receptors. 15,16 Recently, a specific monoclonal antibody against oxPC was developed by one of us. 17,18 Using this antibody, we have investigated the influence of oxidative stress on various diseases.…”

Oxidized Phospholipids as Endogenous Pattern Recognition Ligands in Innate Immunity

“…The structures of oxPC CD36 identified were established using a combination of cell binding studies and multiple distinct chromatographic and MS methods in conjunction with (i) results of numerous derivatization strategies to ascertain functional groups on isolated products, (ii) inference of structures of products that appeared plausible based upon MS results and known mechanisms of lipid oxidation and fragmentation, and (iii) de novo synthesis of each oxidized lipid. The identities of the oxidation products and synthetic standards were confirmed by demonstration that synthetic species recapitulate all biological, chemical, MS, and chromatographic characteristics of the lipids isolated from oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-PC, oxidized 1-palmitoyl-2-lineoyl-sn-glycero-3-PC, and oxidized 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-PC (19,20,22,40,41). Liquid chromatographic/electrospray ionization/tandem MS analyses have demonstrated the generation of these species in lipoproteins oxidized by multiple distinct pathways in vitro (40), in atherosclerotic lesions in vivo (41), in retina following light exposure in dark-adapted rodents (20), and in cerebral tissues following ischemia-reperfusion injury (42).…”

“…Although initially defined using PC molecular species, as outlined below, subsequent studies have shown that all classes of phospholipids examined thus far (PC, phosphatidylethanolamine, PS, and phosphatidic acid) harboring the high affinity motif for CD36 promote CD36-mediated recognition and phagocytosis (19,20,22,40,41). The structures of oxPC CD36 identified were established using a combination of cell binding studies and multiple distinct chromatographic and MS methods in conjunction with (i) results of numerous derivatization strategies to ascertain functional groups on isolated products, (ii) inference of structures of products that appeared plausible based upon MS results and known mechanisms of lipid oxidation and fragmentation, and (iii) de novo synthesis of each oxidized lipid.…”

“…Podrez et al (40,41) reported the first systematic study aimed at identifying the structures of specific oxidized lipids that serve as ligands for the scavenger receptor CD36. Structure/function analyses with multiple synthetic species defined critical structural elements of endogenous ligands for CD36 that promote high affinity binding to the scavenger receptor: a phospholipid with a truncated sn-2 acyl group that incorporates a terminal ␥-hydroxy(or oxo)-␣,␤-unsaturated carbonyl (oxPC CD36 ) (40,41). The structures of the oxidized lipid ligands of CD36 are illustrated in Fig.…”