Negatively cooperative binding of high-density lipoprotein to the HDL receptor SR-BI

Scavenger receptor class B, type I (SR-BI), is a high-density lipoprotein (HDL) receptor, which also binds low-density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a coreceptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high-affinity binding sites (one site model). We have reinvestigated the ligand concentration dependence of (125)I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [(3)H]CE from [(3)H]CE-HDL using an expanded range of ligand concentrations (<1 μg of protein/mL, lower than previously reported). Scatchard and nonlinear least-squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects ("lattice model"). Similar results were observed for LDL. Application of the "infinite dilution" dissociation rate method established that the binding of (125)I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport, and cell signaling.     

The role of high-density lipoprotein cholesterol in the prevention and possible treatment of cardiovascular diseases

Despite significant progress in the management of atherosclerosis and its resultant complications, cardiovascular disease remains the principal cause of death in the world. The National Cholesterol Education Project Adult Treatment Panel III (NCEP ATP III) recognizes low levels of high-density lipoprotein cholesterol (HDL) as a risk factor for coronary heart disease (CHD) and high levels of HDL as a risk-reducing factor; however, the elevation of HDL as a specific therapeutic target for the prevention and treatment of CHD has yet to be accepted on the same level as low-density lipoprotein (LDL)-reducing therapies. Current HDL elevators including nicotinic acid, fibric acid derivatives, peroxisome proliferator activated receptor (PPAR) agonists and statins also affect other lipid constituents which make interpretation of the clinical trials of these drugs difficult in teasing out the independent effect of HDL elevation. Ample laboratory investigation suggests that HDL elevation would reduce atherosclerotic burden through multiple independent mechanisms. In this review, we explore HDL biology, its potential mechanisms in the treatment of atherosclerotic disease, and promising new drugs with HDL-raising activity.     

Differential roles of cysteine residues in the cellular trafficking, dimerization, and function of the high-density lipoprotein receptor, SR-BI

The scavenger receptor, class B, type I (SR-BI) binds high-density lipoprotein (HDL) and mediates selective delivery of cholesteryl esters (CEs) to the liver and steroidogenic cells of the adrenal glands and gonads. Although it is clear that the large extracellular domain (ECD) of SR-BI binds HDL, the role of ECD in the selective HDL-CE transport remains poorly understood. In this study, we used a combination of mutational and chemical approaches to systematically evaluate the contribution of cysteine residues, especially six cysteine residues of ECD, in SR-BI-mediated selective HDL-CE uptake, intracellular trafficking, and SR-BI dimerization. Pretreatment of SR-BI-overexpressing COS-7 cells with a disulfide (S-S) bond reducing agent, β-mercaptoethanol (100 mM) or dithiothreitol (DTT) (10 mM), modestly but significantly impaired SR-BI-mediated selective HDL-CE uptake. Treatment of SR-BI-overexpressing COS-7 cells with the optimal doses of membrane permeant alkyl methanethiosulfonate (MTS) reagents, positively charged MTSEA or neutral MMTS, that specifically react with the free sulfhydryl group of cysteine reduced the rate of SR-BI-mediated selective HDL-CE uptake, indicating that certain intracellular free cysteine residues may also be critically involved in the selective cholesterol transport process. In contrast, use of membrane impermeant MTS reagent, positively charged MTSET and negatively charged MTSES, showed no such effect. Next, the importance of eight cysteine residues in SR-BI expression, cell surface expression, dimer formation, and selective HDL-derived CE transport was evaluated. These cysteine residues were replaced either singly or in pairs with serine, and the mutant SR-BIs were expressed in either COS-7 or CHO cells. Four mutations, C280S, C321S, C323S, and C334S, of the ECD, either singly or in various pair combinations, resulted in significant decreases in SR-BI (HDL) binding activity, selective CE uptake, and trafficking to the cell surface. Surprisingly, we found that mutation of the two remaining cysteine residues, C251 and C384 of the ECD, had no effect on either SR-BI expression or function. Other cysteine mutations and substitutions were also without effect. Western blot data indicated that single and double mutations at C280, C321, C323, and C334 residues strongly favor dimer formation. However, they are rendered nonfunctional presumably because of mutation-induced formation of aberrant disulfide linkages resulting in inhibition of optimal HDL binding and, thus, selective HDL-CE uptake. These results provide novel insights into the functional role of four cysteine residues, C280, C321, C323, and C334, of the SR-BI ECD in SR-BI expression and trafficking to the cell surface, its dimerization, and associated selective CE transport function.     

Cubilin, a high-density lipoprotein receptor

The metabolism of HDL particles is a complex biological process involving various regulating factors in plasma and different cellular receptors. In addition to the well-established scavenger receptor BI-mediated selective HDL-cholesteryl ester uptake in liver and steroidogenic tissues, evidence has been provided that HDL also undergoes holoparticle endocytosis in different tissues. Recently, a novel receptor expressed in various absorptive epithelia was disclosed as a high affinity receptor for endocytosis of HDL and lipid-poor apolipoprotein AI. This receptor, designated cubilin, may play an important role in the renal clearance of filterable apolipoprotein AI/HDL and in the maternal-fetal transport of cholesterol.     

Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein

Cultures of human skin fibroblasts were labeled to high cholesterol specific activity with [3H]cholesterol and incubated briefly (1-3 min) with normal human plasma. The plasma was fractionated by two-dimensional agarose-polyacrylamide gel electrophoresis and the early appearance of cholesterol label among plasma lipoproteins determined. A major part of the label at 1-min incubation was in a pre-beta-migrating apo A-I lipoprotein fraction with a molecular weight of ca. 70,000. Label was enriched about 30-fold in this fraction relative to its content of apo A-I (1-2% of total apo A-I). The proportion of label in this lipoprotein was strongly correlated with its concentration in plasma. Further incubation (2 min) in the presence of unlabeled cells demonstrated transfer of label from this fraction to a higher molecular weight pre-beta apo A-I species, to low-density lipoprotein, and to the alpha-migrating apo A-I that made up the bulk (96%) of total apo A-I in plasma. The data suggest that a significant part of cell-derived cholesterol is transferred specifically to a pre-beta-migrating lipoprotein A-I species as part of a cholesterol transport transfer sequence in plasma.     

Scavenger receptor BI (SR-BI) clustered on microvillar extensions suggests that this plasma membrane domain is a way station for cholesterol trafficking between cells and high-density lipoprotein

Receptor-mediated trafficking of cholesterol between lipoproteins and cells is a fundamental biological process at the organismal and cellular levels. In contrast to the well-studied pathway of LDL receptor-mediated endocytosis, little is known about the trafficking of high-density lipoprotein (HDL) cholesterol by the HDL receptor, scavenger receptor BI (SR-BI). SR-BI mediates HDL cholesteryl ester uptake in a process in which HDL lipids are selectively transferred to the cell membrane without the uptake and degradation of the HDL particle. We report here the cell surface locale where the trafficking of HDL cholesterol occurs. Fluorescence confocal microscopy showed SR-BI in patches and small extensions of the cell surface that were distinct from sites of caveolin-1 expression. Electron microscopy showed SR-BI in patches or clusters primarily on microvillar extensions of the plasma membrane. The organization of SR-BI in this manner suggests that this microvillar domain is a way station for cholesterol trafficking between HDL and cells. The types of phospholipids in this domain are unknown, but SR-BI is not strongly associated with classical membrane rafts rich in detergent-resistant saturated phospholipids. We speculate that SR-BI is in a more fluid membrane domain that will favor rapid cholesterol flux between the membrane and HDL.     

Acute changes in high-density lipoprotein cholesterol with exercise of different intensities

The purpose of this study was to investigate the acute effects of exercise on plasma high-density lipoprotein cholesterol (HDL-C) and to determine whether the magnitude of this response would be affected by the intensity of the exercise. Twelve men (19-41 yr) ran an equivalent distance (9-12 km) on a treadmill on two separate occasions. On one occasion the exercise was performed at a speed that elicited 60% of the subject's maximal O2 uptake (VO2max), and on the other occasion exercise was performed at a speed that elicited 90% of VO2max. Changes in total cholesterol, triglycerides (TG), HDL-C, HDL apoprotein A (HDL-A), HDL saturation, lactate (LA), and free fatty acids (FFA) were measured during the course of each run, and all values were corrected for changes in plasma volume as indicated by hematocrit. There were significant increases (P less than 0.01) in HDL-C, HDL-A, and HDL saturation with exercise at both intensities, but greater increases in HDL-C (25 vs. 14%) and HDL-A (18 vs. 8%) were observed with the higher intensity exercise. Plasma FFA and TG did not differ between conditions, but LA concentrations rose significantly during the high-intensity exercise. These results indicate that increases in HDL components can occur with a relatively moderate exercise session and that the magnitude of these increases are directly related to the exercise intensity.     

Lecithin-cholesterol acyltransferase: role in lipoprotein metabolism, reverse cholesterol transport and atherosclerosis

In the past several years significant advances have been made in our understanding of lecithin-cholesterol acyltransferase (LCAT) function. LCAT beneficially alters the plasma concentrations of apolipoprotein B-containing lipoproteins, as well as HDL. In addition, its proposed role in facilitating reverse cholesterol transport and modulating atherosclerosis has been demonstrated in vivo. Analysis of LCAT transgenic animals has established the importance of evaluating HDL function, as well as HDL plasma levels, to predict atherogenic risk.     

Linkage of the apo CIII microsatellite with isolated low high-density lipoprotein cholesterol

To evaluate whether a highly polymorphic mic-rosatellite region within intron 3 of the apolipoprotein (apo) CIII gene is linked to the isolated low HDL-C phenotype, we studied eight unrelated probands (mean HDL-C=10 ±5mg/dl) and 157 biological family members. After PCR amplification of genomic DNA and denaturing polyacrylamide gel electrophoresis, 26 alleles were identified in this microsatellite including 9 alleles heretofore unreported. Quantitative sib-pair linkage analysis demonstrated strong evidence of linkage between the isolated low HDL-C phenotype and the apo CIII microsatellite region (P=0.007). The microsatellite was also linked to apo AI (P=0.001), the primary apolipoprotein of HDL-C. Therefore, this highly polymorphic microsatellite region is a potentially important marker in the genetic evaluation of the isolated low HDL-C phenotype. Received: 25 October 1996 / Accepted: 29 October 1997     

Immunochromatographic membrane strip assay system for a single-class plasma lipoprotein cholesterol, exemplified by high-density lipoprotein cholesterol measurement

In assessing risk factors of coronary heart disease, a membrane immunochromatographic system that minimizes requirements of instrument and reagent handling was investigated by utilizing high-density lipoprotein (HDL) cholesterol (HDL-C) as model analyte. The system is composed of four functional membrane strip pads connected in sequence as follows (from the bottom): immunoseparation based on the biotin-streptavidin reaction; catalytic conversion of cholesterol to hydrogen peroxide; production of a colorimetric signal; and induction of a continuous wicking of medium. For immunochromatography, a monoclonal antibody, specific to apolipoprotein B100 that is present on the surfaces of low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL), with a high binding constant (5 x 10(10) L/mol), was raised and chemically conjugated to streptavidin. The conjugate was first reacted with lipoprotein particles, and this mixture was absorbed by the capillary action into the biotin pad of the system. After being transferred by medium, immunocapture of LDL and VLDL particles onto the biotin pad took place, and in situ generation of a colorimetric signal in proportion to HDL-C occurred consecutively. The capture was selective as well as effective (minimum 88% of LDL and VLDL in clinical concentration ranges), and the detection limit of the HDL-C was far lower than 20 mg per 100 mL. The same concept may also be applicable to LDL cholesterol measurement provided suitable antibodies specific to HDL and VLDL are available.     

Apolipoprotein M--a novel player in high-density lipoprotein metabolism and atherosclerosis

PURPOSE OF REVIEW: Apolipoprotein M is a recently described apolipoprotein predominantly associated with high-density lipoprotein, but also found in chylomicrons, very low-density lipoproteins, and low-density lipoprotein. The purpose is to review recent information on the unusual structural properties of apolipoprotein M and its possible role in formation of pre-beta high-density lipoprotein and reverse cholesterol metabolism. RECENT FINDINGS: Apolipoprotein M is a lipocalin having a coffee filter-like structure with a hydrophobic ligand-binding pocket. Mature apolipoprotein M retains its signal peptide, which serves as a hydrophobic anchor. In mice, silencing of expression in the liver with siRNA led to disappearance of pre-beta high-density lipoprotein and appearance of unusually large high-density lipoproteins. This suggests that apolipoprotein M is important for the formation of pre-beta high-density lipoprotein and reverse cholesterol transport. In accordance with this idea, hepatic overexpression of apolipoprotein M with an adenovirus in low-density lipoprotein-receptor deficient mice led to an approximately 70% reduction of atherosclerosis. In addition to the liver, apolipoprotein M is also expressed in the kidney. Kidney-derived apolipoprotein M binds to megalin, a member of the low-density lipoprotein-receptor family, which interacts with many lipocalins in renal tubuli. Apolipoprotein M is excreted in the urine of mice with a kidney-specific megalin deficiency but not in the urine of normal mice, suggesting megalin-mediated uptake of apolipoprotein M in the tubular epithelium of normal mice. SUMMARY: Apolipoprotein M is a novel apolipoprotein with unusual structural features that appears to play important roles in high-density lipoprotein metabolism and prevention of atherosclerosis.     

Total and high-density lipoprotein cholesterol measurements. Hazards in clinical interpretation.

Duplicate plasma specimens from 24 persons were sent to a community hospital, a commercial laboratory, and a university lipid research laboratory at two separate times to assess the intralaboratory and interlaboratory variations of total and high-density cholesterol measurements. For all three laboratories, the 95% confidence limits for the reproducibility of total cholesterol levels are about +/- 5.5%. For high-density lipoprotein (HDL) values, they are +/- 7%, +/- 14.45%, and +/- 9%. Interlaboratory differences for total cholesterol at the 95% confidence level are +/- 7.7% and for HDL +/- 18.4%. Construction of a "cardiac risk ratio" of total to HDL cholesterol levels is subject to confusion because small errors in HDL cholesterol levels produce large errors in the ratio.     

Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells

Binding of high-density lipoproteins to cultured mouse Ob1771 adipose cells was studied, using labeled human HDL3, mouse HDL and apolipoprotein AI- or AII-containing liposomes. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 18, 42, 30 and 3.4 micrograms/ml, whereas the maximal binding capacities were found to be 160, 100, 90 and 21 ng/mg of cell protein. Apoprotein AI not inserted into liposomes did not bind. The binding of 125I-HDL3 was competitively inhibited by apolipoprotein AI-containing liposomes greater than mouse HDL greater than HDL3. The binding of 125I-labeled apolipoprotein AI- and 125I-labeled apolipoprotein AII-containing liposomes was competitively inhibited by HDL3, apolipoprotein AI- and apolipoprotein AII-containing liposomes. Dimyristoylphosphatidylcholine liposomes containing or not cholesterol did not interfere with the binding of labeled HDL3 or apolipoprotein-containing liposomes. Binding studies on crude membranes of Ob1771 adipose cells revealed the presence of intracellular binding sites for LDL and HDL3. Thus, adipose cells have specific binding sites for apolipoprotein E-free HDL and apolipoprotein AI (or AII) is the ligand for these binding sites. Long-term exposure of adipose cells to LDL cholesterol as a function of LDL concentration led to an accumulation of cellular unesterified cholesterol. This process was saturable and reversible as a function of time and concentration by exposure to HDL3 or apolipoprotein AI-containing liposomes, whereas apolipoprotein AII-containing liposomes did not promote any cholesterol efflux. Since long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apolipoprotein B,E receptors and apolipoprotein E-free binding sites, respectively, it appears that adipose cells do not show efficient cholesterol homeostasis and thus could accumulate or mobilize unesterified cholesterol.     

Mechanism of the hepatic lipase induced accumulation of high-density lipoprotein cholesterol by cells in culture

Hepatic lipase can enhance the delivery of high-density lipoprotein (HDL) cholesterol to cells by a process which does not involve apoprotein catabolism. The incorporation of HDL-free (unesterified) cholesterol, phospholipid, and cholesteryl ester by cells has been compared to establish the mechanism of this delivery process. Human HDL was reconstituted with 3H-free cholesterol and [14C]sphingomyelin, treated with hepatic lipase in the presence of albumin to remove the products of lipolysis, reisolated, and then incubated with cultured rat hepatoma cells. Relative to control HDL, modification of HDL with hepatic lipase stimulated both the amount of HDL-free cholesterol taken up by the cell and the esterification of HDL-free cholesterol but did not affect the delivery of sphingomyelin. Experiments utilizing HDL reconstituted with 14C-free cholesterol and [3H]cholesteryl oleoyl ether suggest that hepatic lipase enhances the incorporation of HDL-esterified cholesterol. However, the amount of free cholesterol delivered as a result of treatment with hepatic lipase was 4-fold that of esterified cholesterol. On the basis of HDL composition, the cellular incorporation of free cholesterol was about 10 times that which would occur by the uptake and degradation of intact particles. The preferential incorporation of HDL-free cholesterol did not require the presence of lysophosphatidylcholine. To correlate the events observed at the cellular level with alterations in lipoprotein structure, high-resolution, proton-decoupled 13C nuclear magnetic resonance spectroscopy (90.55 MHz) was performed on HDL3 in which the cholesterol molecules were replaced with [4-13C]cholesterol by particle reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

The paradox of dysfunctional high-density lipoprotein

PURPOSE OF REVIEW: This review addresses how, in atherosclerosis or systemic inflammation, HDL can lose its usual atheroprotective characteristics and even paradoxically assume proinflammatory properties. RECENT FINDINGS: Specific chemical and structural changes within HDL particles can impede reverse cholesterol transport, enhance oxidation of LDL, and increase vascular inflammation. HDL may be viewed as a shuttle that can be either anti-inflammatory or proinflammatory, depending on its cargo of proteins, enzymes, and lipids. Some therapeutic approaches that reduce coronary risk, such as statins and therapeutic lifestyle changes, can favorably moderate the characteristics of proinflammatory HDL. In addition, apolipoprotein A-I mimetic peptides and other compounds that target functional aspects of HDL may offer novel approaches to reduction in cardiovascular risk. SUMMARY: Current data suggest that under some conditions HDL can become dysfunctional and even proinflammatory, but this characterization can change with resolution of systemic inflammation or use of certain treatments.     

Verapamil enhances high-density lipoprotein processing in Hep G2 cells preloaded with cholesterol

The effects of the calcium channel blocker of the arylalkylamine series verapamil have been investigated on high-density lipoprotein (HDL3) catabolism in the human hepatoma cell line Hep G2. It was found that verapamil markedly enhanced HDL3 binding, uptake and degradation in Hep G2 cells preloaded with nonlipoprotein cholesterol. This effect was dose-dependent, and a 1.5-2-fold increase of the three studied parameters was observed in cells pretreated 24 h with 100 microM verapamil. No significant effect of the drug was found in cells not preincubated with cholesterol. Verapamil induced an increase in the cellular cholesterol content in preloaded cells. Other calcium antagonists such as diltiazem, nifedipine, nitrendipine or amphiphilic drugs such as phenothiazines and propranolol also enhanced HDL3 uptake by Hep G2 cells. These effects of verapamil on HDL3 metabolism could be related to its amphiphilic characteristics, and to its calcium antagonist properties.