Oxidized low-density lipoprotein-binding specificity of Asp-hemolysin from Aspergillus fumigatus.

Oxidized low-density lipoprotein (Ox-LDL) is known to be involved in the generation and progression of atherosclerosis. Ox-LDL has a number of potentially atherogenic effects on vascular cells, including the uncontrolled uptake by scavenger receptors. We have previously shown that Asp-hemolysin binds to Ox-LDL in a concentration-dependent manner. The present study was undertaken to clarify the binding specificity of Asp-hemolysin to Ox-LDL. We examined the binding specificity of Asp-hemolysin to Ox-LDL using several modified lipoproteins and scavenger receptor ligands. Asp-hemolysin bound to Ox-LDL with shorter LDL oxidation times. However, Asp-hemolysin did not bind to the acetylated LDL. The native high-density lipoprotein (n-HDL) and modified HDL (e.g., acetylated HDL, oxidized HDL) also had no Asp-hemolysin binding. Furthermore, inhibitors of the scavenger receptor binding, including maleylated BSA, polyinosinic acid, dextran sulfate and fucoidin, had no effect on the binding of Ox-LDL to Asp-hemolysin. Surface plasmon resonance studies revealed that Ox-LDL binds with high affinity (K(D)=0.63 microg/ml) to Asp-hemolysin. We concluded that Asp-hemolysin is a specific binding protein with a high affinity for Ox-LDL, and its binding specificity is distinct from any receptor for Ox-LDL. The present studies suggest that Asp-hemolysin may bind to Ox-LDL using a mechanism different from the scavenger receptors.     

Endothelial scavenger receptors

In the past few decades, cDNAs for endothelial scavenger receptors that bind to negatively charged molecules, particularly acetylated low density lipoproteins (Ac-LDL), have been cloned by expression cloning using modified LDL as ligands. A prototypic members of endothelial scavenger receptor family, namely, scavenger receptor class B type I (SR-BI) has been characterized as a high density lipoprotein (HDL) receptor. Another prototypic member, CD36, has been determined as a multiple ligand receptor because it binds to oxidized LDLs (Ox-LDL), trombospondin, erythrocytes infected with Plasmodium falciparum, long-chain fatty acids, and Gram-negative and Gram-positive bacteria. Lectin-like oxidized LDL receptor-1 (LOX-1) has been discovered as the principal receptor that mediates the action of Ox-LDL in the vascular walls. Recently, the structure of oxidized phospholipids, originally found in Ox-LDL, and its molecular mechanism of action on endothelial cells were determined. Further, the use of genetically manipulated rodent models and the recent forward genetic screening technique revealed the physiological and pathological functions of these endothelial scavenger receptors in innate immunity and infection. In this review, the structure and function of these multiligand scavenger receptors of endothelial cells have been described mainly in relation with lipid metabolism.     

Enhanced macrophage uptake of elastase-modified high-density lipoproteins

Incubation of human HDL (d = 1.063-1.21 g/ml) with monocyte-derived elastase causes selective proteolysis of apoA-II and apoA-I apolipoproteins. We have found that elastase-digested HDL (ED-HDL) bind to J774-A1 murine macrophages with enhanced affinity and are internalized and degraded at a rate threefold higher than that of native HDL. Unlike oxidized LDL and HDL and proteolytically modified LDL, the uptake of ED-HDL lipoproteins does not affect the cellular lipid biosynthesis nor modify the cell lipid content. The cell surface binding of (125)I-ED-HDL can be competed by native HDL but not by acetylated LDL, consistent with the idea that ED-HDL are recognized by the class B type I scavenger receptor. The liberation of elastase by lipid-engorging macrophages is regarded as an important event during atherogenesis. By enhancing the cellular uptake of HDL this process can lead to a local decrease of antiatherogenic HDL particles.     

Human platelets exclusively bind oxidized low density lipoprotein showing no specificity for acetylated low density lipoprotein.

The widely studied macrophage scavenger receptor system is known to bind both acetylated low density lipoprotein and oxidized low density lipoprotein. Although only the latter ligand has been shown to occur in vivo, acetylated low density lipoprotein is often used to evaluate the contribution of scavenger receptors to different (patho)physiologic processes, assuming that all existing subtypes of scavenger receptors recognise both lipoproteins. In the present work, we identify human platelets as the first natural cell type to bind oxidized low density lipoprotein without showing specificity for acetylated low density lipoprotein. Consequently, platelets possess exclusive receptor(s) for oxidized low density lipoprotein distinct from the 'classical' scavenger receptor AI/AII. From the data presented in this work, we conclude that the class B scavenger receptor CD36 (GPIV) is responsible for this exclusive oxidized low density lipoprotein binding.     

Nitrosylated high density lipoprotein is recognized by a scavenger receptor in rat liver

In order to assess the presence of specific recognition sites for high density lipoprotein (HDL) in vivo, HDL was nitrosylated with tetranitromethane and the decay and liver uptake were compared with that of native HDL. The association of intravenously injected nitrosylated HDL (TNM-HDL) with liver was greatly increased as compared to native HDL. Using a cold cell isolation method, it became evident that the liver endothelial cells were responsible for the increased uptake of the modified HDL. The involvement of the endothelial cells in the uptake of TNM-HDL from the circulation could also be demonstrated morphologically by using the fluorescent dye dioctadecyl-tetramethyl-indocarbocyanine perchlorate (Dil) to label HDL. In vitro competition studies with isolated liver endothelial cells indicated that unlabeled modified HDL and acetylated LDL displaced iodine-labeled TNM-HDL, while no competition was seen with LDL and a slight displacement was seen with unlabeled native HDL. Nonlipoprotein competitors of the scavenger receptor such as fucoidin and polyinosinic acid blocked the interaction of TNM-HDL with the liver endothelial cells. Also the degradation of TNM-HDL was blocked by low concentrations of chloroquine. It can be concluded that a scavenger receptor on liver endothelial cells is involved in the clearance of tetranitromethane-modified HDL, which excludes the possibility of using TNM-HDL in vivo to assess the non-receptor-dependent uptake of HDL. The use of nitrosylated HDL in vitro as a low affinity control is limited to cell types that do not possess scavenger receptors, because cell types with scavenger receptors will recognize and internalize TNM-HDL by a high affinity scavenger pathway.     

CD36 does not play a direct role in HDL or LDL metabolism

CD36 and scavenger receptor class B, type I (SR-BI) are both class B scavenger receptors that recognize a broad variety of ligands, including oxidized low density lipoprotein (oxLDL), HDL, anionic phospholipids, and apoptotic cells. In this study we investigated the role of mouse CD36 (mCD36) as a physiological lipoprotein receptor. We compared the association of various lipoprotein particles with mCD36 and mSR-BI expressed in COS cells by adenovirus-mediated gene transfer. mCD36 bound human oxLDL and mouse HDL with high affinity. Human LDL bound poorly to mCD36, indicating that mCD36 is unlikely to play a significant role in LDL metabolism. The ability of mCD36 to mediate the selective uptake of cholesteryl esters (CE) from receptor-bound HDL was assessed. In comparison with mSR-BI, mCD36 inefficiently mediated the selective uptake of CE. Hepatic overexpression of mCD36 in C57BL/6 mice by adenovirus-mediated gene transfer did not result in significant alterations in plasma LDL and HDL levels. We conclude that mCD36, while able to bind HDL with high affinity, does not contribute significantly to HDL or LDL metabolism.     

Metabolism of modified LDL and foam cell formation in murine macrophage-like RAW 264 cells.

The uptake of modified low density lipoprotein (LDL) by arterial macrophages is a key event in the atherogenesis. We studied 1) the uptake and degradation of modified LDL, 2) LDL recognition by specific receptors, and 3) the foam cell formation with murine macrophage-like RAW 264 cells in vitro. The cells took up and degraded effectively 125I-labeled acetylated LDL (Ac-LDL) and aggregated LDL (Aggr-LDL). Also oxidized LDL (Ox-LDL) was taken up but it was degraded poorly. The degradation of 125I-Ac-LDL was efficiently competed by both unlabeled Ac-LDL and Ox-LDL, whereas the degradation of 125I-Ox-LDL was partially competed by unlabeled Ox-LDL and Aggr-LDL but not at all by unlabeled Ac-LDL. The incubation with increasing concentrations of Ac-LDL, Aggr-LDL or Ox-LDL resulted in marked foam cell formation in the RAW 264 cells. Ox-LDL was cytotoxic at 500 to 1000 microg/ml concentrations. The results show that RAW 264 cells have at least two classes of receptors for modified lipoproteins: one that recognizes both Ox-LDL and Ac-LDL, and is similar to the scavenger receptors, and another that recognizes Ox-LDL but not Ac-LDL. RAW 264 cells are a convenient model cell line for examining the metabolism of modified lipoproteins, not only that of Ac-LDL but also that of Ox-LDL and Aggr-LDL, and cellular accumulation of lipids derived from modified LDL.     

Specific, saturable, and high affinity binding of 125I-low density lipoprotein to glass beads.

¹²⁵I-Low density lipoprotein (¹²⁵I-LDL)¹ binds tightly to glass beads at physiologic pH and ionic strength. This binding shows saturability, high affinity (half maximal binding achieved at 10–15 μg protein/ml), and specificity (unlabeled LDL but not HDL or albumin competes with ¹²⁵I-LDL for binding to the glass beads). In contrast to the binding of ¹²⁵I-LDL to the physiologic LDL receptor on the surface of human fibroblasts and lymphocytes, the binding of ¹²⁵I-LDL to bind to inert substances such as glass must be considered in the interpretation of studies in which ¹²⁵I-LDL binding to membrane receptors is measured. The data emphasize the importance of correlating observed ¹²⁵I-LDL binding with a physiologic action of the lipoprotein.     

Recognition of lipoproteins by scavenger receptor class A members

Scavenger receptor class A (SR-A) proteins are type II transmembrane glycoproteins that form homotrimers on the cell surface. This family has five known members (SCARA1 to 5, or SR-A1 to A5) that recognize a variety of ligands and are involved in multiple biological pathways. Previous reports have shown that some SR-A family members can bind modified low-density lipoproteins (LDLs); however, the mechanisms of the interactions between the SR-A members and these lipoproteins are not fully understood. Here, we systematically characterize the recognition of SR-A receptors with lipoproteins and report that SCARA1 (SR-A1, CD204), MARCO (SCARA2), and SCARA5 recognize acetylated or oxidized LDL and very-low-density lipoprotein in a Ca²⁺-dependent manner through their C-terminal scavenger receptor cysteine-rich (SRCR) domains. These interactions occur specifically between the SRCR domains and the modified apolipoprotein B component of the lipoproteins, suggesting that they might share a similar mechanism for lipoprotein recognition. Meanwhile, SCARA4, a SR-A member with a carbohydrate recognition domain instead of the SRCR domain at the C terminus, shows low affinity for modified LDL and very-low-density lipoprotein but binds in a Ca²⁺-independent manner. SCARA3, which does not have a globular domain at the C terminus, was found to have no detectable binding with these lipoproteins. Taken together, these results provide mechanistic insights into the interactions between SR-A family members and lipoproteins that may help us understand the roles of SR-A receptors in lipid transport and related diseases such as atherosclerosis.     

Class B scavenger receptors CD36 and SR-BI are receptors for hypochlorite-modified low density lipoprotein

The presence of HOCl-modified epitopes inside and outside monocytes/macrophages and the presence of HOCl-modified apolipoprotein B in atherosclerotic lesions has initiated the present study to identify scavenger receptors that bind and internalize HOCl-low density lipoprotein (LDL). The uptake of HOCl-LDL by THP-1 macrophages was not saturable and led to cholesterol/cholesteryl ester accumulation. HOCl-LDL is not aggregated in culture medium, as measured by dynamic light scattering experiments, but internalization of HOCl-LDL could be inhibited in part by cytochalasin D, a microfilament disrupting agent. This indicates that HOCl-LDL is partially internalized by a pathway resembling phagocytosis-like internalization (in part by fluid-phase endocytosis) as measured with [14C]sucrose uptake. In contrast to uptake studies, binding of HOCl-LDL to THP-1 cells at 4 degrees C was specific and saturable, indicating that binding proteins and/or receptors are involved. Competition studies on THP-1 macrophages showed that HOCl-LDL does not compete for the uptake of acetylated LDL (a ligand to scavenger receptor class A) but strongly inhibits the uptake of copper-oxidized LDL (a ligand to CD36 and SR-BI). The binding specificity of HOCl-LDL to class B scavenger receptors could be demonstrated by Chinese hamster ovary cells overexpressing CD36 and SR-BI and specific blocking antibodies. The lipid moiety isolated from the HOCl-LDL particle did not compete for cell association of labeled HOCl-LDL to CD36 or SR-BI, suggesting that the protein moiety of HOCl-LDL is responsible for receptor recognition. Experiments with Chinese hamster ovary cells overexpressing scavenger receptor class A, type I, confirmed that LDL modified at physiologically relevant HOCl concentrations is not recognized by this receptor.     

Characterization of low density and high density lipoprotein receptors in the rat corpus luteum and regulation by gonadotropin

Freshly prepared plasma membranes from rat corpora lutea were examined for the presence of low density lipoprotein (LDL) and high density lipoprotein (HDL) receptors by determining the specific binding of 125I-LDL and 125I-HDL. These membranes have two types of binding site for 125I-LDL, one with high affinity (Kd = 7.7 micrograms of LDL protein/ml), the other with low affinity (Kd = 213 micrograms of LDL protein/ml) and one type of binding site for 125I-HDL with Kd = 17.8 micrograms of HDL protein/ml. LDL receptor is sensitive to pronase and trypsin; HDL receptor, however, is resistant. The binding reaction was further characterized with respect to effect of time and temperature of incubation, requirement of divalent metal ion, influence of ionic strength, and binding specificity. In vivo pretreatment of rats with human choriogonadotropin (hCG) resulted in induction of both LDL and HDL receptors in a dose- and time-dependent manner when compared with saline-injected controls. The induction of lipoprotein receptors by hCG treatment is target organ-specific since the increase was seen only in the ovarian tissue. Membranes prepared from liver, kidney, and heart did not show an increase in lipoprotein receptors after hCG injection. An examination of the equilibrium dissociation constants for 125I-LDL and 125I-HDL binding after hCG administration revealed that the increase in binding activity was due to an increase in the number of binding sites rather than to a change in the binding affinity. In conclusion, rat corpus luteum possesses specific receptors for both LDL and HDL and these receptors are regulated by gonadotropins.     

The scavenger cell pathway for lipoprotein degradation: Specificity of the binding site that mediates the uptake of negatively-charged LDL by macrophages

Macrophages isolated from a variety of organs in several animal species exhibit high affinity binding sites that recognize chemically modified proteins. One of these binding sites recognizes human plasma low density lipoprotein (LDL) in which the positive charges on the epsilon-amino groups of lysine have been removed or neutralized by chemical modification, thus giving the protein an enhanced negative charge. Effective treatments include reaction of LDL with organic acid anhydrides (acetylation or maleylation) and reaction with aldehydes, such as treatment with malondialdehyde. After the negatively-charged LDL binds to the surface receptor sites, it is rapidly internalized by the macrophages by endocytosis and hydrolyzed in lysosomes. The liberated cholesterol is reesterified in the cytoplasm, producing massive cholesteryl ester deposition. The binding site for negatively-charged LDL has been demonstrated so far only on macrophages and other scavenger cells. It is not expressed in cultured fibroblasts, smooth muscle cells, lymphocytes, or adrenal cells. In addition to its affinity for acetylated LDL and malondialdehyde-treated LDL, the macrophage site binds a variety of polyanions. It exhibits a particularly high affinity for certain sulfated polysaccharides (dextran sulfate and fucoidin), certain polynucleotides (polyinosinic acid and polyguanylic acid), polyvinyl sulfate, and maleylated albumin. It is possible that the site that binds negatively-charged LDL may be responsible for the massive accumulation of cholesteryl esters that occurs in vivo in macrophages and other scavenger cells in patients with high levels of circulating plasma LDL.     

Uptake and fate of class B scavenger receptor ligands in HepG2 cells.

Class B scavenger receptors (SR-Bs) interact with native, acetylated and oxidized low-density lipoprotein (LDL, AcLDL and OxLDL), high-density lipoprotein (HDL3) and maleylated BSA (M-BSA). The aim of this study was to analyze the catabolism of CD36- and LIMPII-analogous-1 (CLA-1), the human orthologue for the scavenger receptor class B type I (SR-BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate-affinity binding sites for all the SR-B ligands tested with dissociation constants ranging from 20 to 30 microg.mL-1. Competition binding studies at 4 degrees C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I-LDL and 125I-HDL3 and vice versa, and that only M-BSA and LDL may have distinct binding sites. Degradation/association ratios for SR-B ligands show that LDL is very efficiently degraded, while M-BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6-4.7-fold CE-selective uptake ([3H]CE association/125I-protein association) was measured. M-BSA efficiently competed for the CE-selective uptake of LDL-, OxLDL-, AcLDL- and HDL3-CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes, whereas HDL3-CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR-B ligands clearly suggests that CLA-1 and/or CD36 are involved at least partially in this process in HepG2 cells.     

Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.     

Oxidized low-density lipoprotein increases cultured human endothelial cell tissue factor activity and reduces protein C activation

Increasing evidence suggests that the formation of oxidized low-density lipoprotein (Ox-LDL) in vivo is associated with the development of atherosclerotic vascular disease. We investigated the effects of Ox-LDL on two vascular endothelial cell coagulant properties, tissue factor expression, and protein C activation. The Ox-LDL increased human arterial and venous endothelial cell tissue factor activity, with 100 micrograms/ml of Ox-LDL increasing factor activity fourfold. Native LDL modified by incubation with cultured human arterial and venous endothelial cells also induced endothelial cell tissue factor activity. This modification was blocked by coincubation with the antioxidants, probucol or ascorbic acid. It was determined, based on inhibition by known scavenger receptor antagonists (fucoidin, dextran sulfate), that binding of Ox-LDL via the acetyl LDL (scavenger) receptor was partially responsible for the increase in tissue factor expression. Whereas endothelial cell tissue factor expression was increased by incubation with Ox-LDL, protein C activation was reduced approximately 80% by incubating cultured endothelial cells with Ox-LDL. The effect of Ox-LDL on protein C activation was not inhibited by antagonists to the scavenger receptor. These data indicating that an atherogenic lipoprotein can regulate key vascular coagulant activities provide an additional link between vascular disease and thrombosis.     

FEEL-1 and FEEL-2 are endocytic receptors for advanced glycation end products

Advanced glycation end products (AGEs) are nonenzymatically glycosylated proteins, which accumulate in vascular tissues in aging and diabetes. Receptors for AGEs include scavenger receptors, which recognize acetylated low density lipoproteins (Ac-LDL) such as scavenger receptor class AI/AII (SR-A), cell surface glycoprotein CD36, scavenger receptor class B type I (SR-BI), and lectin-like oxidized low density lipoprotein receptor-1. The broad ligand repertoire of these receptors as well as the diversity of the receptors for AGEs have prompted us to examine whether AGEs are also recognized by the novel scavenger receptors, which we have recently isolated from a cDNA library prepared from human umbilical vein endothelial cells, such as the scavenger receptor expressed by endothelial cells-I (SREC-I); the fasciclin EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor-1 (FEEL-1); and its paralogous protein, FEEL-2. At 4 degrees C, (125)I-AGE-bovine serum albumin (BSA) exhibited high affinity specific binding to Chinese hamster ovary (CHO) cells overexpressing FEEL-1 (CHO-FEEL-1) and FEEL-2 (CHO-FEEL-2) with K(d) of 2.55 and 1.68 microg/ml, respectively, but not to CHO cells expressing SREC (CHO-SREC) and parent CHO cells. At 37 degrees C, (125)I-AGE-BSA was taken up and degraded by CHO-FEEL-1 and CHO-FEEL-2 cells but not by CHO-SREC and parent CHO cells. Thus, the ability to bind Ac-LDL is not necessarily a prerequisite to bind AGEs. The (125)I-AGE-BSA binding to CHO-FEEL-1 and CHO-FEEL-2 cells was effectively inhibited by Ac-LDL and polyanionic SR-A inhibitors such as fucoidan, polyinosinic acids, and dextran sulfate but not by native LDL, oxidized LDL, or HDL. FEEL-1, which is expressed by the liver and vascular tissues, may recognize AGEs, thereby contributing to the development of diabetic vascular complications and atherosclerosis.     

Scavenger receptor class B type I-mediated reverse cholesterol transport is inhibited by advanced glycation end products

Cellular interactions of advanced glycation end products (AGE) are mediated by AGE receptors. We demonstrated previously that class A scavenger receptor types I and II (SR-A) and CD36, a member of class B scavenger receptor family, serve as the AGE receptors. In this study, we investigated whether scavenger receptor class B type I (SR-BI), another receptor belonging to class B scavenger receptor family, was also an AGE receptor. We used Chinese hamster ovary (CHO) cells overexpressed hamster SR-BI (CHO-SR-BI cells). (125)I-AGE-bovine serum albumin (AGE-BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-SR-BI cells. (125)I-AGE-BSA exhibited saturable binding to CHO-SR-BI cells (K(d) = 8.3 microg/ml). Endocytic uptake of (125)I-AGE-BSA by CHO-SR-BI cells was completely inhibited by oxidized low density lipoprotein (LDL) and acetylated LDL, whereas LDL exerted only a weak inhibitory effect (<20%). Cross-competition experiments showed that AGE-BSA had no effect on HDL binding to these cells and vice versa. Interestingly, however, SR-BI-mediated selective uptake of HDL-CE was completely inhibited by AGE-BSA in a dose-dependent manner (IC(50) <10 microg/ml). Furthermore, AGE-BSA partially inhibited (by <30%) the selective uptake of HDL-CE in human hepatocarcinoma HepG2 cells (IC(50) <30 microg/ml). In addition, [(3)H]cholesterol efflux from CHO-SR-BI cells to HDL was significantly inhibited by AGE-BSA in a dose-dependent manner (IC(50) <30 microg/ml). Our results indicate that AGE proteins, as ligands for SR-BI, effectively inhibit both SR-BI-mediated selective uptake of HDL-CE and cholesterol efflux from peripheral cells to HDL, suggesting that AGE proteins might modulate SR-BI-mediated cholesterol metabolism in vivo.     

Highly purified scavenger receptor class B,type I reconstituted into phosphatidylcholine/cholesterol liposomes mediates high affinity high density lipoprotein binding and selective lipid uptake

The murine class B, type I scavenger receptor mSR-BI is a high and low density lipoprotein (HDL and LDL) receptor that mediates selective uptake of cholesteryl esters. Here we describe a reconstituted phospholipid/cholesterol liposome assay of the binding and selective uptake activities of SR-BI derived from detergent-solubilized cells. The assay, employing lysates from epitope-tagged receptor (mSR-BI-t1)-expressing mammalian and insect cells, recapitulated many features of SR-BI activity in intact cells, including high affinity and saturable (125)I-HDL binding, selective lipid uptake from [(3)H]cholesteryl ether-labeled HDL, and poor inhibition of HDL receptor activity by LDL. The novel properties of a mutated receptor (Q402R/Q418R, normal LDL binding but loss of most HDL binding) were reproduced in the assay, as was the ability of the SR-BI homologue CD36 to bind HDL but not mediate efficient lipid uptake. In this assay, essentially homogeneously pure mSR-BI-t1, prepared by single-step immunoaffinity chromatography, mediated high affinity HDL binding and efficient selective lipid uptake from HDL. Thus, SR-BI-mediated HDL binding and selective lipid uptake are intrinsic properties of the receptor that do not require the intervention of other proteins or specific cellular structures or compartments.     

Molecular cloning of a novel scavenger receptor for oxidized low density lipoprotein, SR-PSOX, on macrophages

Receptor-mediated endocytosis of oxidized low density lipoprotein (OxLDL) by macrophages has been implicated in foam cell transformation in the process of atherogenesis. Although several scavenger receptor molecules, including class A scavenger receptors and CD36, have been identified as OxLDL receptors on macrophages, additional molecules on macrophages may also be involved in the recognition of OxLDL. From a cDNA library of phorbol 12-myristate 13-acetate-stimulated THP-1 cells, we isolated a cDNA encoding a novel protein designated SR-PSOX (scavenger receptor that binds phosphatidylserine and oxidized lipoprotein), which acts as a receptor for OxLDL. SR-PSOX was a type I membrane protein consisting of 254 amino acids, expression of which was shown on human and murine macrophages with a molecular mass of 30 kDa. SR-PSOX could specifically bind with high affinity, internalize, and degrade OxLDL. The recognition of OxLDL was blocked by polyinosinic acid and dextran sulfate but not by acetylated low density lipoprotein. Taken together, SR-PSOX is a novel class of molecule belonging to the scavenger receptor family, which may play important roles in pathophysiology including atherogenesis.     

Oxidized low density lipoprotein inhibits the hemolytic activity of Asp-hemolysin from Aspergillus fumigatus

We have examined the effect of chemically modified human low density lipoproteins (LDLs) , acetylated LDL and oxidized LDL, on the hemolytic activity of Asp-hemolysin. Oxidized LDL, but not acetylated LDL, inhibited the hemolytic activity of this toxin. The inhibitory effects of oxidized LDL increased with the time of Cu²⁺-induced LDL oxidation. Similar inhibition was observed in the filtrate which was separated from the incubation mixture of Asp-hemolysin with oxidized LDL (for 2 h of oxidation) following ultrafiltration through a membrane with a molecular mass cutoff of 100 000. However, at longer LDL oxidation times, the inhibition by the filtrates was less than the control mixture without ultrafiltration. We suggest that the inhibition by oxidized LDL was due to the binding of oxidized LDL to Asp-hemolysin at shorter LDL oxidation times .