Identification of two antagonists of the scavenger receptor CD36 using a high-throughput screening model
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CD36
Scavenger Receptor
Foam cell
Sf9
IC50
Abstract Several lines of evidence suggest that clearance of oxidized LDL (oxLDL) immune complexes by macrophage IgG Fc receptors (FcγRs) plays a role in atherogenesis. OxLDL may also be cleared directly by FcγRs, as shown for murine FcγRII-B2. In humans, the homologous FcγR is FcγRIIA (CD32), which is abundantly expressed on monocytes and macrophages and shares 60% sequence identity with murine FcγRII-B2. As murine FcγRII-B2 and human FcγRIIA also share similar IgG ligand-binding properties, the purpose of this study was to test the hypothesis that human CD32 is a receptor for oxLDL. For these studies we used transfected Chinese hamster ovary (CHO) cells, monocytes, and cell lines that functionally express either of two FcγRIIA subtypes (R131 or H131) and assayed binding or degradation of several preparations of oxLDL. The integrity of all oxLDL preparations was checked by studying their ability to react with CHO cells expressing human type I scavenger receptors and by other characteristics of lipoprotein oxidation. Although we showed that each preparation of oxLDL could recognize class A or class B scavenger receptors, we did not detect any differences in the binding or degradation of any type of oxLDL preparation among control versus CHO cell transfectants. Using monocytes that express FcγRIIA and CD36, we showed that the binding of oxLDL was inhibited by antibodies to CD36, but not by FcγRIIA antibodies. Thus, the data do not support the hypothesis that human FcγRIIA is by itself a receptor for oxLDL. We conclude that human CD32 can mediate uptake of lipoprotein immune complexes, but does not mediate uptake of oxLDL in the absence of anti-oxLDL antibodies. OxLDL may interact with human mononuclear phagocytes directly via other types of receptors, such as class A and class B scavenger receptors or CD68.
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CD36
Scavenger Receptor
Foam cell
Sf9
IC50
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Scavenger Receptor
Scavenger
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Upon activation, human peripheral blood monocytes and U937 cells oxidized low density lipoprotein (LDL), converting it to a cytotoxin.The oxidized LDL loses its ability to interact specifically with the native LDL (apoB/E) receptor and becomes a ligand for the scavenger receptors and two other receptors, FqRII (CD32) and CD36.We performed a series of studies to evaluate the potential contribution of each of these receptors to the process of monocyte-mediated LDL oxidation.To assess the participation of the apoB/E recep tor, we tested the ability of activated human monocytes to oxidize LDL after u p and down-regulation of apoB/E recep tors.Neither up-regulation nor down-regulation of the apoB/E receptor significantly modified the level of LDL lipid oxidation.Acetylated LDL, a ligand for scavenger receptors, was also oxidized by the activated monocytes.Methylated LDL, a chemically modified LDL that is not recognized by the apoB/E or scavenger receptors, was oxidized as we1i.mThus, LDL does not need to interact with either the apoB/E receptor or scavenger receptors in order to undergo lipid oxidation.Additionally, monoclonal antibodies to CD36 and CD32 were used to block these two receptors that recognize oxidized LDL.Although both antibodies interfered with oxidized LDL binding to these receptors, neither treatment interfered with LDL lipid oxidation mediated by activated human monocytes.Our results suggest that interaction with these receptors is not a requirement for LDL lipid oxidation by activated human monocytes.-Cathcart,M.
Low-density lipoprotein
Monocyte
PCSK9
Lipoprotein(a)
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Syrian hamsters of the APA strain (APA hamsters) have recently been demonstrated to develop atheromatous lesions in the aortic arches under the diabetic condition induced by a single injection of streptozotocin (SZ). Various lipoprotein receptors are reported to play important roles in atherogenesis mainly in vitro, while there are few reports on the relative expressions of these receptors in vivo. In this study, we therefore examined messenger RNA (mRNA) expressions of several lipoprotein receptors on the aortic arches of diabetic APA hamsters at 6, 14 and 26 weeks after the injection (WAI) of SZ. In semi-quantitative RT-PCR, scavenger receptor (SR)-AI, macrosialin (MS)/CD68, and receptor for advanced glycation end-products (RAGE) mRNAs showed significant increases at 6 WAI of SZ, and SR-AI and CD36 mRNA obviously increased until 26 WAI, as compared with the control. Low-density lipoprotein receptor mRNA showed a significant decrease at 14 and 26 WAI, and SR-BI mRNA significantly decreased at 6 and 14 WAI, as compared with the control. Very low-density lipoprotein receptor mRNA was at the same level as the control. By means of in situ hybridization, SR-AI, MS/CD68 and RAGE mRNA were detected in the foam cells of the fatty streaks at 6 WAI, which suggested that SR-AI, MS/CD68 and RAGE play crucial roles in the formation of the fatty streaks, the initial lesions of atherogenesis in diabetic APA hamsters. SR-AI and CD36 were also believed to be related to the progression of atherogenesis in this model.
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RAGE
CD68
Apolipoprotein E
Foam cell
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The binding and uptake of oxidatively modified low density lipoprotein (OxLDL) by mouse peritoneal macrophages occurs, in part, via the well characterized acetyl LDL receptor. However, several lines of evidence indicate that as much as 30-70% of the uptake can occur via a distinct receptor that recognizes OxLDL with a higher affinity than it recognizes acetyl LDL. We describe the partial purification and characterization of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that specifically binds OxLDL. This receptor is shown to be distinct from the acetyl LDL receptor as well as from two other macrophage proteins that also bind OxLDL--the Fc gamma RII receptor and CD36. We suggest that this OxLDL-binding membrane protein participates in uptake of OxLDL by murine macrophages and also represents a receptor responsible for macrophage binding and phagocytosis of oxidatively damaged cells.
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Scavenger Receptor
Cell surface receptor
LRP1B
Foam cell
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Type I and type II scavenger receptors, which have been implicated in the development of atherosclerosis and other macrophage-associated functions, differ only by the presence in the type I receptor of an extracellular cysteine-rich C-terminal domain. Stable Chinese hamster ovary (CHO) cell transfectants expressing high levels of either the type I or type II bovine scavenger receptors have been generated. Type I and type II receptors in these cells mediated high-affinity saturable endocytosis of both 125I-labeled acetylated low density lipoprotein (LDL) and 125I-labeled oxidized LDL with the distinctive broad ligand specificity characteristic of scavenger receptors. After incubation for 2 days with acetylated LDL, the transfected cells accumulated oil red O-staining lipid droplets reminiscent of those in macrophage foam cells, whereas untransfected CHO cells did not. Thus, macrophage-specific gene products other than the scavenger receptor are not required for modified-LDL-induced intracellular lipid accumulation. In transfected cells, acetylated LDL efficiently competed for both its own endocytosis and that of oxidized LDL. In contrast, oxidized LDL competed effectively for its own endocytosis but only poorly for that of acetylated LDL. This nonreciprocal cross competition suggests that these ligands may bind to nonidentical but interacting sites on a single receptor. Results were similar for transfectants expressing either type I or type II scavenger receptors. Therefore, the nonreciprocal cross competition previously reported for cultured peritoneal macrophages may not be the result of differences between the type I and type II receptors. The nonreciprocal cross competition seen in the transfected CHO cells differs from that previously observed with cultured macrophages.
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CD36
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Engagement of the receptor for advanced glycation end products (RAGE) by its signal transduction ligands evokes inflammatory cell infiltration and activation of the vessel wall. However, soluble RAGE (sRAGE), the truncated form spanning the extracellular binding domain of RAGE, has potent anti-inflammatory properties by acting as a decoy for RAGE ligands. We now show that sRAGE binds with high affinity to atherogenic low-density lipoprotein (LDL) modified by hypochlorous acid (HOCl), the major oxidant generated by the myeloperoxidase-H2O2-chloride system of phagocytes activated during inflammation. We further demonstrate that sRAGE can be coprecipitated with HOCl-LDL from spiked serum. To determine the functional significance of sRAGE binding to HOCl-LDL, cell association studies with macrophages were performed. sRAGE effectively inhibited cellular uptake of HOCl-LDL and subsequent lipid accumulation. Using Chinese hamster ovary cells overexpressing class B scavenger receptor CD36 or SR-BI, two preferential scavenger receptors for HOCl-LDL, we demonstrate that sRAGE only interferes with CD36-mediated uptake of HOCl-LDL. The present findings indicate that sRAGE acts as a sink for HOCl-LDL, which is abundantly present in human atherosclerotic lesions. We propose that sRAGE represents a physiological antagonist that interferes with scavenger receptor-mediated cholesterol accumulation and foam cell formation of macrophages.
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CD36
Hypochlorous acid
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Foam cell
Low-density lipoprotein
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Interaction of advanced glycation end products (AGE) with AGE-receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE-receptors identified so far are RAGE (receptor for AGE), 80 K-H, OST-48, galectin-3, and SR-A (macrophage scavenger receptor type I and II). Since SR-A belongs to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to the class B scavenger receptor family (SR-B) can recognize AGE-proteins as a ligand. This was tested in the present study at the cellular level using CHO (Chinese hamster ovary) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-BSA (bovine serum albumin) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36 but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized LDL (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE-proteins. Because CD36 is one of the major Ox-LDL receptors and is upregulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE-proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.
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CD36
RAGE
Foam cell
Advanced glycation end-product
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Both CD36 and SR-B1 are glycosylated integral membrane proteins belonging to the class B scavenger receptor family and are known to play roles in cardiovascular and atherosclerotic disease. CD36 serves as the receptor for oxidized low density lipoprotein (oxLDL) and facilitates cholesterol accumulation in macrophages and development of atherosclerotic plaques. SR-B1 (scavenger receptor class B type 1) is the primary receptor for high density lipoprotein (HDL), which removes cholesterol from the peripheral atherosclerotic plaques and delivers it to the liver for excretion. Current structural information about class B scavenger receptors is limited to only purified extracellular regions, transmembrane segments, or transient transfection studies in cultured cells, and remains a roadblock to studying the isolated functions of these lipid and cholesterol transporters. Recently, we have successfully expressed and purified full-length human SR-B1 and CD36 using an Sf9 insect cell/baculoviral infection system, which allows for the expression of glycosylated proteins. Purified full-length receptors are a valuable addition to our scientific toolbox and allow us to ask novel questions about the structure-function relationships of scavenger receptors. To verify function within Sf9 cell membranes, we expressed human full-length constructs in plated Sf9 cells and observed significantly increased binding of DiI-HDL and DiI-oxLDL, as well as increased DiI-lipid uptake when comparing SR-B1 and CD36 to empty vector infected cells, respectively. When purified and solubilized in detergent micelles, both receptors remain stable over time as shown by Prometheus thermal shift assays. We have also demonstrated that purified proteins are able to bind to their native ligands (HDL to SR-B1 and oxLDL to CD36) with high affinity by microscale thermophoresis (MST). However, as SR-B1 and CD36 are both scavenger receptors, they are able to bind to a variety of lipoprotein ligands and we have demonstrated these interactions with varying binding affinities by MST. Glycosylation of both CD36 and SR-B1 has been shown to be important for cell surface expression, but the role of glycosylation in ligand binding has remained uncharacterized. We are the first to demonstrate that the glycosylation status of CD36 and SR-B1 does not alter binding affinities or protein stability. These purified receptors and functional assays provide promise for understanding the ways in which class B scavenger receptors perform their critical functions and lay the groundwork for future structure-function studies.
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Sf9
Foam cell
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