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.     

Dissociation of the high density lipoprotein and low density lipoprotein binding activities of murine scavenger receptor class B type I (mSR-BI) using retrovirus library-based activity dissection

The murine class B, type I scavenger receptor (mSR-BI) is a receptor for both high density lipoprotein (HDL) and low density lipoprotein (LDL) and mediates selective, rather than endocytic, uptake of lipoprotein lipid. We have developed a "retrovirus library-based activity dissection" method to generate mSR-BI mutants in which some, but not all, of the activities of this multifunctional protein have been disrupted. This method employs three techniques: 1) efficient in vitro cDNA mutagenesis (here error-prone PCR was used), 2) efficient retroviral delivery and high expression of single mutant cDNAs into individual cells, and 3) isolation of infected cells expressing the desired mutant phenotype using high sensitivity positive/negative screening by two-color fluorescence-activated cell sorting. A set of mutants, all having arginine substitutions at two common sites (positions 402 or 401 and position 418), were isolated and characterized. Mutation at either site alone did not generate as strong a mutant phenotype (loss of DiI uptake from DiI-HDL) as did the double mutations. "Activity-dissected" double mutants were as effective as wild-type mSR-BI in functioning as LDL receptors, mediating high affinity LDL binding and uptake of metabolically active cholesterol from LDL, but they lost most of their corresponding HDL receptor activity. Thus, these mutants provide support for the proposal that the interaction of SR-BI with HDL differs from that with LDL. Examination of the in vivo function of such mutants may provide insights into the differential roles of the LDL and HDL receptor activities of SR-BI in normal lipoprotein metabolism and in SR-BI's ability to protect against atherosclerosis.     

High density lipoprotein metabolism in low density lipoprotein receptor-deficient mice

The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein (¹²⁵I) and in the cholesteryl ester (CE) moiety ([³H]). The metabolism of ¹²⁵I-/[³H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([³H]). In contrast, in LDLR^−/− mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR^−/− mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR^−/− mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.     

Binding and cross-linking studies show that scavenger receptor BI interacts with multiple sites in apolipoprotein A-I and identify the class A amphipathic alpha-helix as a recognition motif

Scavenger receptor, class B, type I (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester without the uptake and degradation of the particle. In transfected cells SR-BI recognizes HDL, low density lipoprotein (LDL) and modified LDL, protein-free lipid vesicles containing anionic phospholipids, and recombinant lipoproteins containing apolipoprotein (apo) A-I, apoA-II, apoE, or apoCIII. The molecular basis for the recognition of such diverse ligands by SR-BI is unknown. We have used direct binding analysis and chemical cross-linking to examine the interaction of murine (m) SR-BI with apoA-I, the major protein of HDL. The results show that apoA-I in apoA-I/palmitoyl-oleoylphosphatidylcholine discs, HDL(3), or in a lipid-free state binds to mSR-BI with high affinity (K(d) congruent with 5-8 microgram/ml). ApoA-I in each of these forms was efficiently cross-linked to cell surface mSR-BI, indicating that direct protein-protein contacts are the predominant feature that drives the interaction between HDL and mSR-BI. When complexed with dimyristoylphosphatidylcholine, the N-terminal and C-terminal CNBr fragments of apoA-I each bound to SR-BI in a saturable, high affinity manner, and each cross-linked efficiently to mSR-BI. Thus, mSR-BI recognizes multiple sites in apoA-I. A model class A amphipathic alpha-helix, 37pA, also showed high affinity binding and cross-linking to mSR-BI. These studies identify the amphipathic alpha-helix as a recognition motif for SR-BI and lead to the hypothesis that mSR-BI interacts with HDL via the amphipathic alpha-helical repeat units of apoA-I. This hypothesis explains the interaction of SR-BI with a wide variety of apolipoproteins via a specific secondary structure, the class A amphipathic alpha-helix, that is a common structural motif in the apolipoproteins of HDL, as well as LDL.     

In vivo cholesteryl ester selective uptake of mildly and standardly oxidized LDL occurs by both parenchymal and nonparenchymal mouse hepatic cells but SR-BI is only responsible for standardly oxidized LDL selective uptake by nonparenchymal cells

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.     

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.     

Endocytosis is not required for the selective lipid uptake mediated by murine SR-BI

The scavenger receptor class B, type I (SR-BI) mediates the cellular selective uptake of cholesteryl esters and other lipids from high-density lipoproteins (HDL) and low-density lipoproteins (LDL). This process, unlike classical receptor-mediated endocytosis, does not result in lipoprotein degradation. Instead, the lipid depleted particles are released into the medium. Here we show that selective lipid uptake mediated by murine SR-BI can be uncoupled from the endocytosis of HDL or LDL particles. We found that blocking selective lipid uptake by incubating cells with the small chemical inhibitors BLT-1 or BLT-4 did not affect endocytosis of HDL. Similarly, blocking endocytosis by hyperosmotic sucrose or K+ depletion did not prevent selective lipid uptake from HDL or LDL. These findings suggest that mSR-BI-mediated selective uptake occurs at the cell surface upon the association of lipoproteins with mSR-BI and does not require endocytosis of HDL or LDL particles.     

Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I,but not apoB,in human apoB transgenic mice

Scavenger receptor BI (SR-BI) is a multi-ligand lipoprotein receptor that mediates selective lipid uptake from HDL, and plays a central role in hepatic HDL metabolism. In this report, we investigated the extent to which SR-BI selective lipid uptake contributes to LDL metabolism. As has been reported for human LDL, mouse SR-BI expressed in transfected cells mediated selective lipid uptake from mouse LDL. However, LDL-cholesteryl oleoyl ester (CE) transfer relative to LDL-CE bound to the cell surface (fractional transfer) was approximately 18-fold lower compared with HDL-CE. Adenoviral vector-mediated SR-BI overexpression in livers of human apoB transgenic mice ( approximately 10-fold increased expression) reduced plasma HDL-cholesterol (HDL-C) and apolipoprotein (apo)A-I concentrations to nearly undetectable levels 3 days after adenovirus infusion. Increased hepatic SR-BI expression resulted in only a modest depletion in LDL-C that was restricted to large LDL particles, and no change in steady-state concentrations of human apoB. Kinetic studies showed a 19% increase in the clearance rate of LDL-CE in mice with increased SR-BI expression, but no change in LDL apolipoprotein clearance. Quantification of hepatic uptake of LDL-CE and LDL-apolipoprotein showed selective uptake of LDL-CE in livers of human apo B transgenic mice. However, such uptake was not significantly increased in mice over-expressing SR-BI. We conclude that SR-BI-mediated selective uptake from LDL plays a minor role in LDL metabolism in vivo.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

Opposite effect of caveolin-1 in the metabolism of high-density and low-density lipoproteins

Receptors of the scavenger class B family were reported to be localized in caveolae, the cell surface microdomains rich in free cholesterol and glycosphyngolipids, which are characterized by the presence of caveolin-1. Parenchymal hepatic and hepatoma HepG2 cells express very low levels of caveolin-1. In the present study, stable transformants of HepG2 cells expressing caveolin-1 were generated to address the effect of caveolin-1 on receptor activity. Compared to normal cells, these cells show higher (125)I-bovine serum albumin (BSA) uptake and cholesterol efflux, two indicators of functional caveolae. By immunoprecipitation, cell fractionation and confocal analyses, we found that caveolin-1 is well colocalized with the cluster of differentiation-36 (CD36) and the low-density lipoprotein (LDL) receptor (LDLr) but to a lesser extent with the scavenger receptor class B type I (SR-BI) in HepG2 cells expressing caveolin-1. However, caveolin-1 expression favors the dimerization of SR-BI. Two clones of cells expressing caveolin-1 were investigated for their lipoprotein metabolism activity. Compared to normal cells, these cells show a 71-144% increase in (125)I-LDL degradation. The analysis of the cholesteryl esters (CE)-selective uptake (CE association minus protein association) revealed that the expression of caveolin-1 in HepG2 cells decreases by 59%-73% LDL-CE selective uptake and increases high-density lipoprotein (HDL)-CE selective uptake by 44%-66%. We conclude that the expression of caveolin-1 in HepG2 cells moves the balance of LDL degradation/CE selective uptake towards degradation and favors HDL-CE selective uptake. Thus, in the normal hepatic parenchymal situation where caveolin-1 is poorly expressed, LDL-CE selective uptake is the preferred pathway.     

The role of human and mouse hepatic scavenger receptor class B type I (SR-BI) in the selective uptake of low-density lipoprotein-cholesteryl esters

Low-density lipoprotein (LDL)-cholesteryl ester (CE) selective uptake has been demonstrated in nonhepatic cells overexpressing the scavenger receptor class B type I (SR-BI). The role of hepatic SR-BI toward LDL, the main carrier of plasma CE in humans, remains unclear. The aim of this study was to determine if SR-BI, expressed at its normal level, is implicated in LDL-CE selective uptake in human HepG2 hepatoma cells and mouse hepatic cells, to quantify its contribution and to determine if LDL-CE selective uptake is likely to occur in the presence of human HDL. First, antibody blocking experiments were conducted on normal HepG2 cells. SR-BI/BII antiserum inhibited (125)I-LDL and (125)I-HDL(3) binding (10 microg of protein/mL) by 45% (p < 0.05) and CE selective uptake by more than 85% (p < 0.01) for both ligands. Second, HepG2 cells were stably transfected with a eukaryotic vector expressing a 400-bp human SR-BI antisense cDNA fragment. Clone 17 (C17) has a 70% (p < 0.01) reduction in SR-BI expression. In this clone, (3)H-CE-LDL and (3)H-CE-HDL(3) association (10 microg of protein/mL) was 54 +/- 6% and 45 +/- 7% of control values, respectively, while (125)I-LDL and (125)I-HDL(3) protein association was 71 +/- 3% and 58 +/- 5% of controls, resulting in 46% and 55% (p < 0.01) decreases in LDL- and HDL(3)-CE selective uptake. Normalizing CE selective uptake for SR-BI expression reveals that SR-BI is responsible for 68% and 74% of LDL- and HDL(3)-CE selective uptake, respectively. Thus, both approaches show that, in HepG2 cells, SR-BI is responsible for 68-85% of CE selective uptake. Other pathways for selective uptake in HepG2 cells do not require CD36, as shown by anti-CD36 antibody blocking experiments, or class A scavenger receptors, as shown by the lack of competition by poly(inosinic acid). However, CD36 is a functional oxidized LDL receptor on HepG2 cells, as shown by antibody blocking experiments. Similar results for CE selective uptake were obtained with primary cultures of hepatic cells from normal (+/+), heterozygous (-/+), and homozygous (-/-) SR-BI knockout mice. Flow cytometry experiments show that SR-BI accounts for 75% of DiI-LDL uptake, the LDL receptor for 14%, and other pathways for 11%. CE selective uptake from LDL and HDL(3) is likely to occur in the liver, since unlabeled HDL (total and apoE-free HDL(3)) and LDL, when added in physiological proportions, only partially competed for LDL- and HDL(3)-CE selective uptake. In this setting, human hepatic SR-BI may be a crucial molecule in the turnover of both LDL- and HDL(3)-cholesterol.     

Identification of the N-linked glycosylation sites on the high density lipoprotein (HDL) receptor SR-BI and assessment of their effects on HDL binding and selective lipid uptake

The murine class B, type I scavenger receptor mSR-BI, a high density lipoprotein (HDL) receptor that mediates selective uptake of HDL lipids, contains 11 potential N-linked glycosylation sites and unknown numbers of both endoglycosidase H-sensitive and -resistant oligosaccharides. We have examined the consequences of mutating each of these sites (Asn --> Gln or Thr --> Ala) on post-translational processing of mSR-BI, cell surface expression, and HDL binding and lipid transport activities. All 11 sites were glycosylated; however, disruption of only two (Asn-108 and Asn-173) substantially altered expression and function. There was very little detectable post-translational processing of these two mutants to endoglycosidase H resistance and very low cell surface expression, suggesting that oligosaccharide modification at these sites apparently plays an important role in endoplasmic reticulum folding and/or intracellular transport. Strikingly, although the low levels of the 108 and 173 mutants that were expressed on the cell surface exhibited a marked reduction in their ability to transfer lipids from HDL to cells, they nevertheless bound nearly normal amounts of HDL. Indeed, the affinity of (125)I-HDL binding to the 173 mutant was similar to that of the wild-type receptor. Thus, N-linked glycosylation can influence both the intracellular transport and lipid-transporter activity of SR-BI. The ability to uncouple the HDL binding and lipid transport activities of mSR-BI by in vitro mutagenesis should provide a powerful tool for further analysis of the mechanism of SR-BI-mediated selective lipid uptake.     

Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor

The murine scavenger receptor class B, type I (mSR-BI) is a receptor for high density lipoprotein (HDL), low density lipoprotein (LDL), and acetylated LDL (AcLDL). It mediates selective uptake of lipoprotein lipid and stimulates efflux of [(3)H]cholesterol to lipoproteins. SR-BI-mediated [(3)H]cholesterol efflux was proposed to be independent of ligand binding. In this study, using anti-mSR-BI antibody KKB-1 and two mSR-BI mutants with altered ligand binding properties, we demonstrated that SR-BI-mediated [(3)H]cholesterol efflux to lipoproteins was correlated with ligand binding and lipid uptake activities of the receptor. The KKB-1 antibody, which blocked lipoprotein binding without substantially altering the cholesterol oxidase-accessible cellular [(3)H]cholesterol, also blocked [(3)H]cholesterol efflux to HDL and LDL. One of the SR-BI mutants, which has a double substitution of arginines for glutamines at positions 402 and 418 (Q402R/Q418R), exhibited a high level of LDL binding and lipid uptake from LDL, but lost most of the corresponding HDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to LDL, but not to HDL. Another mutant, M158R, with an arginine in place of methionine at position 158, exhibited reduced HDL and LDL receptor activities, but apparently normal AcLDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to AcLDL, but not to HDL or LDL. These results suggest that SR-BI-stimulated [(3)H]cholesterol efflux to lipoproteins critically depends on ligand binding to this receptor and raise the possibility that the mechanisms of selective lipid uptake and [(3)H]cholesterol efflux may be intimately related.     

ApoA-II modulates the association of HDL with class B scavenger receptors SR-BI and CD36

The class B scavenger receptors SR-BI and CD36 exhibit a broad ligand binding specificity. SR-BI is well characterized as a HDL receptor that mediates selective cholesteryl ester uptake from HDL. CD36, a receptor for oxidized LDL, also binds HDL and mediates selective cholesteryl ester uptake, although much less efficiently than SR-BI. Apolipoprotein A-II (apoA-II), the second most abundant HDL protein, is considered to be proatherogenic, but the underlying mechanisms are unclear. We previously showed that apoA-II modulates SR-BI-dependent binding and selective uptake of cholesteryl ester from reconstituted HDL. To investigate the effect of apoA-II in naturally occurring HDL on these processes, we compared HDL without apoA-II (from apoA-II null mice) with HDLs containing differing amounts of apoA-II (from C57BL/6 mice and transgenic mice expressing a mouse apoA-II transgene). The level of apoA-II in HDL was inversely correlated with HDL binding and selective cholesteryl ester uptake by both scavenger receptors, particularly CD36. Interestingly, for HDL lacking apoA-II, the efficiency with which CD36 mediated selective uptake reached a level similar to that of SR-BI. These results demonstrate that apoA-II exerts a marked effect on HDL binding and selective lipid uptake by the class B scavenger receptors and establishes a potentially important relationship between apoA-II and CD36.     

Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain

Scavenger receptor BI (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without degradation of the HDL particle. In addition, SR-BI stimulates the bi-directional flux of free cholesterol (FC) between cells and lipoproteins, an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL. SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase. In contrast, CD36, a closely related class B scavenger receptor, has none of these activities despite binding HDL with high affinity. In the present study, analyses of chimeric SR-BI/CD36 receptors and domain-deleted SR-BI have been used to test the various domains of SR-BI for functional activities related to HDL CE selective uptake, bi-directional FC flux, and the alteration of membrane cholesterol mass and distribution. The results show that each of these activities localizes to the extracellular domain of SR-BI. The N-terminal cytoplasmic tail and transmembrane domains appear to play no role in these activities other than targeting the receptor to the plasma membrane. The C-terminal tail of SR-BI is dispensable for activity as well for targeting to the plasma membrane. Thus, multiple distinct functional activities are localized to the SR-BI extracellular domain.     

Scavenger receptor class B, type I is expressed in porcine brain capillary endothelial cells and contributes to selective uptake of HDL-associated vitamin E

It is clearly established that an efficient supply to the brain of alpha-tocopherol (alphaTocH), the most biologically active member of the vitamin E family, is of the utmost importance for proper neurological functioning. Although the mechanism of uptake of alphaTocH into cells constituting the blood-brain barrier (BBB) is obscure, we previously demonstrated that high-density lipoprotein (HDL) plays a major role in the supply of alphaTocH to porcine brain capillary endothelial cells (pBCECs). Here we studied whether a porcine analogue of human and rodent scavenger receptor class B, type I mediates selective (without concomitant lipoprotein particle internalization) uptake of HDL-associated alphaTocH in a similar manner to that described for HDL-associated cholesteryl esters (CEs). In agreement with this hypothesis we observed that a major proportion of alphaTocH uptake by pBCECs occurred by selective uptake, exceeding HDL3 holoparticle uptake by up to 13-fold. The observation that selective uptake of HDL-associated CE exceeded HDL3 holoparticle up to fourfold suggested that a porcine analogue of SR-BI (pSR-BI) may be involved in lipid uptake at the BBB. In line with the observation of selective lipid uptake, RT-PCR and northern and western blot analyses revealed the presence of pSR-BI in cells constituting the BBB. Adenovirus-mediated overexpression of the human analogue of SR-BI (hSR-BI) in pBCECs resulted in a fourfold increase in selective HDL-associated alphaTocH uptake. In accordance with the proposed function of SR-BI, selective HDL-CE uptake was increased sixfold in Chinese hamster ovary cells stably transfected with murine SR-BI (mSR-BI). Most importantly stable mSR-BI overexpression mediated a twofold increase in HDL-associated [14C]alphaTocH selective uptake in comparison with control cells. In line with tracer experiments, mass transfer studies with unlabelled lipoproteins revealed that mSR-BI overexpression resulted in a twofold increase in endogenous HDL3-associated alphaTocH uptake. The results of this study indicate that SR-BI promotes the uptake of HDL-associated alphaTocH into cells constituting the BBB and plays an important role during the supply of the CNS with this indispensable micronutrient.     

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.     

Evidence for a Trypanosoma brucei lipoprotein scavenger receptor

African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids.     

Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake

Serum amyloid A is an acute phase protein that is carried in the plasma largely as an apolipoprotein of high density lipoprotein (HDL). In this study we investigated whether SAA is a ligand for the HDL receptor, scavenger receptor class B type I (SR-BI), and how SAA may influence SR-BI-mediated HDL binding and selective cholesteryl ester uptake. Studies using Chinese hamster ovary cells expressing SR-BI showed that (125)I-labeled SAA, both in lipid-free form and in reconstituted HDL particles, functions as a high affinity ligand for SR-BI. SAA also bound with high affinity to the hepatocyte cell line, HepG2. Alexa-labeled SAA was shown by fluorescence confocal microscopy to be internalized by cells in a SR-BI-dependent manner. To assess how SAA association with HDL influences HDL interaction with SR-BI, SAA-containing HDL was isolated from mice overexpressing SAA through adenoviral gene transfer. SAA presence on HDL had little effect on HDL binding to SR-BI but decreased (30-50%) selective cholesteryl ester uptake. Lipid-free SAA, unlike lipid-free apoA-I, was an effective inhibitor of both SR-BI-dependent binding and selective cholesteryl ester uptake of HDL. We have concluded that SR-BI plays a key role in SAA metabolism through its ability to interact with and internalize SAA and, further, that SAA influences HDL cholesterol metabolism through its inhibitory effects on SR-BI-mediated selective lipid uptake.