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...
The macrophage scavenger receptor CD36 plays an important role in binding and uptake of oxidized forms of low-density lipoprotein (LDL), foam cell formation, and lesion development during atherosclerosis. The structural basis of CD36-lipoprotein ligand recognition is an area of intense interest. In a companion article we reported the characterization of a structurally conserved family of oxidized choline glycerophospholipids (ox-PC CD36 ) that serve as novel high affinity ligands for cells stably transfected with CD36, mediating recognition of multiple oxidized forms of LDL (Podrez, E. A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P., Shan, L., Gugiu, B., Fox, P. L., Hoff, H. F., Salomon, R. G., and Hazen, S. L. (July 8, 2002) J. Biol. Chem. 277, 10.1074/ jbc.M203318200). Here we use macrophages from wildtype and CD36 null mice to demonstrate that CD36 is the major receptor on macrophages mediating recognition of oxPC CD36 species when presented (؉/؊ plasma) in pure form, within PC bilayers in small unilamellar vesicles, and within liposomes generated from lipid extracts of native LDL. We also show that oxPC CD36 promote CD36-dependent recognition when present at only a few molecules per particle, resulting in macrophage binding, uptake, metabolism, cholesterol accumulation, and foam cell formation. Finally, using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS), we demonstrate that oxPC CD36 are generated in vivo and are enriched in atherosclerotic lesions. Collectively, our data suggest that formation of this novel family of oxidized phospholipids participates in CD36-mediated recognition of oxidized lipoproteins and foam cell formation in vivo.CD36 is a multifunctional cellular receptor with broad ligand specificity (1, 2). It is expressed in a number of cells including microvascular endothelial cells, platelets, adipocytes, striated muscles, macrophages, and some vascular smooth muscle cells (1, 2). CD36 regulates cellular adhesion and angiogenesis, serving as a receptor for thrombospondin; it also serves as a scavenger receptor in macrophages, mediating uptake of apoptotic cells and modified lipoproteins, and participates in carbohydrate and lipid metabolism, modulating insulin resistance and long chain fatty acid transport (3-8). CD36 has recently been implicated in a variety of pathologic conditions, including atherosclerosis, diabetes, and cardiomyopathy. Perhaps the most compelling data on the role of CD36 in atherosclerosis are from studies of CD36-deficient mice, which show a 70 -80% reduction in aortic lesion size (9). In vitro experiments demonstrate that macrophages from CD36-deficient mice take up different forms of oxidized LDL 1 poorly and are resistant to foam cell formation, providing a mechanism for the atheroprotection observed in CD36 null mice (9, 10). Given the potential clinical significance of this receptor, it is important to know the nature of the ligand(s) in oxidized LDL (oxLDL) that are recognize...
Clearance by the retinal pigment epithelium (RPE) of shed photoreceptor outer segments (OSs), a tissue with one of the highest turnover rates in the body, is critical to the maintenance and normal function of the retina. We hypothesized that there is a potential role for photo-oxidation in OS uptake by RPE via scavenger receptor-mediated recognition of structurally defined lipid peroxidation products. We now demonstrate that specific structurally defined oxidized species derived from arachidonyl, linoleoyl, and docosahexanoyl phosphatidylcholine may serve as endogenous ligands on OSs for uptake by RPE via the scavenger receptor CD36. Mass spectrometry studies of retinal lipids recovered from dark-adapted rats following physiological light exposure demonstrate in vivo formation of specific oxidized phosphatidylcholine molecular species possessing a CD36 recognition motif, an oxidatively truncated sn-2 acyl group with a terminal ␥-hydroxy(or oxo)-␣,-unsaturated carbonyl. Cellular studies using RPE isolated from wild-type versus CD36 null mice suggest that CD36 plays a role in engulfment, but not initial binding, of OSs via these oxidized phospholipids. Parallel increases in OS protein-bound nitrotyrosine, a post-translational modification by nitric oxide (NO)-derived oxidants, were also observed, suggesting a possible role for light-induced generation of NO-derived oxidants in the initiation of OS lipid peroxidation. Collectively, these studies suggest that intense light exposure promotes "oxidative tagging" of photoreceptor outer segments with structurally defined choline glycerophospholipids that may serve as a physiological signal for CD36-mediated phagocytosis under oxidant stress conditions.
Alpha-enolase (ENO1), also known as 2-phospho-D-glycerate hydrolase, is a metalloenzyme that catalyzes the conversion of 2-phosphoglyceric acid to phosphoenolpyruvic acid in the glycolytic pathway. It is a multifunctional glycolytic enzyme involved in cellular stress, bacterial and fungal infections, autoantigen activities, the occurrence and metastasis of cancer, parasitic infections, and the growth, development and reproduction of organisms. This article mainly reviews the basic characteristics and biological functions of ENO1.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced, in part, from NADPH oxidase in response to host invasion and tissue injury. Defects in NADPH oxidase impair host defense; however, the role of ROS and RNS in the response to tissue injury is not known. We addressed this issue by subjecting leukocyte oxidase (Nox2)-deficient (Nox2 ؊/؊ ) mice to arterial injury. Femoral artery injury was associated with increased Nox2 expression, ROS͞RNS production, and oxidative protein and lipid modification in wild-type mice. In Nox2 ؊/؊ mice, RNS-mediated protein oxidation, as monitored by protein nitrotyrosine content, was significantly diminished. This was accompanied by reduced neointimal proliferation, as monitored by intimal thickness and intimal͞medial ratio, in Nox2 ؊/؊ compared to wild-type mice. In addition, Nox2 deficiency led to reduced cellular proliferation and leukocyte accumulation. These data indicate that Nox2-mediated oxidant production has a requisite role in the response to tissue injury.
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