Mapping and characterization of the binding site for specific oxidized phospholipids and oxidized low density lipoprotein of scavenger receptor CD36

Recent studies have identified a novel family of oxidized phosphatidylcholines (oxPC(CD36)) that serve as highly specific ligands for scavenger receptor CD36. oxPC(CD36) accumulate in vivo and mediate macrophage foam cell formation as well as promote platelet hyper-reactivity in hyperlipidemia via CD36. The structural basis of oxPC(CD36) binding to CD36 has not been elucidated. We used liquid-phase binding to glutathione S-transferase fusion proteins containing various regions of CD36 to initially identify the region spanning CD36 amino acids 157-171 to contain a major binding site for oxPC(CD36). A bell-shaped pH profile and salt concentration dependence suggest an electrostatic mechanism of the binding. Two conserved, positively charged amino acids in the region 157-171 (lysines at positions 164 and 166) were identified as critical for oxPC(CD36) and oxidized low density lipoprotein (oxLDL) binding to CD36. Lysine neutralization with chemical modifier or site-directed mutagenesis of lysine 164/166 to alanine or glutamate, but not to arginine, abolished binding. Cells expressing full-length CD36 with mutated lysines (164 and 166) failed to recognize oxPC(CD36) and oxLDL. Synthetic peptides mimicking the CD36 binding site, but not mutated or scrambled peptides, effectively prevented: (i) oxLDL binding to CD36, (ii) macrophage foam cell formation induced by oxLDL, and (iii) platelet activation by oxPC(CD36). These data indicate that CD36 (160-168) represents the core of the oxPC(CD36) binding site with lysines 164/166 being indispensable for the binding.     

Macrophage binding and the uptake of oxidized low density lipoprotein are regulated by intracellular protein phosphorylation

The involvement of intracellular protein phosphorylation in macrophages in the binding and uptake of oxidized low density lipoprotein (oxLDL) was investigated. The treatment of fibronectin-unstimulated and stimulated mouse thioglycolate-induced macrophages with inhibitors of myosin light chain kinase, protein kinase C and protein tyrosine kinase resulted in decreased macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. The treatment of fibronectin-unstimulated and stimulated macrophages with inhibitors of protein serine/threonine and tyrosine phosphatases caused enhanced macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. Fibronectin, which stimulates macrophage activity, enhanced macrophage intracellular protein phosphorylation. Myosin light chain phosphorylation may be involved in the fibronectin stimulation of macrophages. Treatment of fibronectin-unstimulated and stimulated macrophages with thiophosphate, which forms thiophosphate esters of intracellular proteins that are not so susceptible to protein phosphatases, enhanced macrophage binding of oxLDL. The above results indicate that intracellular protein phosphorylation maintains and enhances macrophage binding and the uptake of oxLDL.     

Endocytosis of oxidized low density lipoprotein through scavenger receptor CD36 utilizes a lipid raft pathway that does not require caveolin-1

The scavenger receptor CD36 binds a diverse array of ligands, including thrombospondin-1, oxidized low density lipoprotein (OxLDL), fatty acids, anionic phospholipids, and apoptotic cells. CD36 has been reported to be present in lipid rafts/caveolae, but little is known about the membrane trafficking of this protein at baseline or following ligand binding. Here, we determined that expression of CD36 in Chinese hamster ovary (CHO) cells and endogenous expression of CD36 in C32 cells led to a homogeneous distribution of the protein on the plasma membrane, as judged by confocal fluorescence microscopy. This homogeneous pattern was observed both by anti-CD36 antibody staining and by live cell imaging of CHO cells expressing a chimeric CD36-green fluorescent protein construct. In contrast, caveolin-1 displayed its usual punctate surface distribution. Correspondingly, dual labeling of CD36 and caveolin-1 showed essentially no overlap, neither by immunofluorescence light microscopy nor by immunogold electron microscopy. Furthermore, isolation of lipid rafts by sucrose gradient ultracentrifugation of cold Triton X-100 cell lysates yielded both CD36 and caveolin-1, but immunoprecipitates of caveolin-1 did not contain CD36. Binding of Ox-LDL led to internalization of CD36 and OxLDL into endosomal structures that did not contain caveolin-1 or transferrin but that co-internalized the glycosyl-phosphatidylinositol-anchored protein decay accelerating factor, a lipid raft protein. Furthermore, expression of CD36 in the caveolin-1-negative KB cell line is sufficient for OxLDL-induced internalization of CD36, indicating that caveolin-1 is not required for this endocytic process. Taken together, these data demonstrate that at steady state, CD36 is localized in lipid rafts but not in caveolae, and that binding of OxLDL to CD36 leads to endocytosis through a lipid raft pathway that is distinct from the clathrin-mediated or caveolin internalization pathways.     

The surface lipid layer of human low density lipoprotein probed by dipyrenyl phospholipids

Properties of the surface lipid-protein layer of human low density lipoprotein (LDL) have been studied with fluorescent phosphatidylcholine analogues containing a pyrenyl fatty acid of variable length at both sn-1 and sn-2 position of the glycerol moiety. Only intramolecular excimer formation takes place at low concentrations, as indicated by the independence of the ratio of excimer to monomer fluorescence intensities (E/M) on the amount of the incorporated dipyrenyl phospholipid. The E/M parameter which depends on the fluidity of the probe's environment were measured for a series of dipyrenyl phospholipids in three systems, i.e. in LDL, LDL-like lipid particles (LDp) and small unilamellar phosphatidylcholine/sphingomyelin/cholesterol vesicles (SUV). The data indicate that the fluidity of the phospholipid acyl chain region decreases in the order: SUV greater than LDp greater than LDL. This suggests that interactions with both the core lipids and the protein moiety (apoB-100) contribute to the rigidity of the surface lipid layer of LDL. Dipyrenyl phospholipids also detect the thermotropic transition of the core lipids of both LDL and LDp, suggesting that this transition influences the fluidity of the surface lipid layer.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

Macrophage mediated protein hydroperoxide formation and lipid oxidation in low density lipoprotein are inhibited by the inflammation marker 7,8-dihydroneopterin

The formation of oxidised low density lipoprotein (LDL) within the atherosclerotic plaque appears to be a factor in the development of advanced atherosclerotic plaques. LDL oxidation is dependent on the balance of oxidants and antioxidants within the intima. In addition to producing various oxidants, human macrophages release 7,8-dihydroneopterin which in vivo is oxidised to the inflammation marker neopterin. Using macrophage-like THP-1 cells and human monocyte-derived macrophages, we demonstrate that 7,8-dihydroneopterin is a potent inhibitor of cell-mediated LDL oxidation. 7,8-Dihydroneopterin scavenges the chain propagating lipid peroxyl radical, inhibiting both lipid and protein hydroperoxide formation. A significant amount of the hydroperoxide formed during cell-mediated LDL oxidation was protein hydroperoxide. 7,8-Dihydroneopterin oxidation to 7,8-dihydroxanthopterin was only observed in the presence of both cells and LDL, showing that 7,8-dihydroneopterin had no effect on initiating oxidant generation by the cells. 7,8-Dihydroneopterin did not regenerate alpha-tocopherol but competed with it for the lipid peroxyl radical. Although stimulation of both cell types with gamma-interferon failed to produce sufficient 7,8-dihydroneopterin to inhibit LDL oxidation in tissue culture, analysis of advanced atherosclerotic plaque removed from patients showed that total neopterin levels could reach low micromolar concentrations. This suggests that 7,8-dihydroneopterin synthesis by macrophages could play a significant role in the development of atherosclerotic plaques.     

CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis

Atherosclerotic plaques result from the excessive deposition of cholesterol esters derived from lipoproteins and lipoprotein fragments. Tissue macrophage within the intimal space of major arterial vessels have been shown to play an important role in this process. We demonstrate in a transfection system using two human cell lines that the macrophage scavenger receptor CD36 selectively elicited lipid uptake from Cu(2+)-oxidized high density lipoprotein (HDL) but not from native HDL or low density lipoprotein (LDL). The uptake of oxHDL displayed morphological and biochemical similarities with the CD36-dependent uptake of oxidized LDL. CD36-mediated uptake of oxidized HDL by macrophage may therefore contribute to atheroma formation.     

Apoptosis induced by oxidized low density lipoprotein in human monocyte-derived macrophages involves CD36 and activation of caspase-3.

Macrophage death may play a crucial role in the progression of atherosclerotic lesions. Here we present evidence that CD36 is involved in oxidized LDL (OxLDL)-induced apoptosis in human monocyte-derived macrophages. Anti-CD36 mAb SMO and OKM-5 reduced the number of apoptotic cells in OxLDL-treated macrophages by more than 94%, but they did not block ceramide-triggered apoptosis. Thrombospondin inhibited the induction of apoptosis by OxLDL in a dose-dependent manner with an IC50 of 10-30 microM. OxLDL did not induce apoptosis in CD36-negative macrophages, demonstrating the essential role of this scavenger receptor in OxLDL-triggered programmed cell death. Neither anti-CD36 Ig nor thrombospondin triggered programmed cell death suggesting that binding to CD36 alone is not sufficient to initiate apoptosis. However, inhibitors of OxLDL-induced apoptosis did not block the uptake of 3H-labeled OxLDL. In contrast, acetylated LDL and polyinosinic acid, ligands of scavenger receptor A (SRA), inhibited uptake of 3H-labeled OxLDL by 65 and 49%, respectively, but did not block OxLDL-induced apoptosis, indicating that SRA is not involved in this process. OxLDL also stimulated caspase-3 activity in human macrophages. Activation of caspase-3 was blocked by anti-CD36 Ig and the caspase-3 inhibitor Z-DEVD-FMK. These results suggest that binding of OxLDL to CD36 initiates a yet unknown OxLDL-specific signaling event, which leads to the rapid activation of caspase-3 resulting in apoptosis of human macrophages. Our data demonstrate a novel role for CD36 in macrophage biology with likely consequences for the development of atherosclerotic lesions.     

CD9 tetraspanin interacts with CD36 on the surface of macrophages: a possible regulatory influence on uptake of oxidized low density lipoprotein

CD36 is a type 2 scavenger receptor with multiple functions. CD36 binding to oxidized LDL triggers signaling cascades that are required for macrophage foam cell formation, but the mechanisms by which CD36 signals remain incompletely understood. Mass spectrometry analysis of anti-CD36 immuno-precipitates from macrophages identified the tetraspanin CD9 as a CD36 interacting protein. Western blot showed that CD9 was precipitated from mouse macrophages by anti-CD36 monoclonal antibody and CD36 was likewise precipitated by anti-CD9, confirming the mass spectrometry results. Macrophages from cd36 null mice were used to demonstrate specificity. Membrane associations of the two proteins on intact cells was analyzed by confocal immunofluorescence microscopy and by a novel cross linking assay that detects proteins in close proximity (<40 nm). Functional significance was determined by assessing lipid accumulation, foam cell formation and JNK activation in wt, cd9 null and cd36 null macrophages exposed to oxLDL. OxLDL uptake, lipid accumulation, foam cell formation, and JNK phosphorylation were partially impaired in cd9 null macrophages. The present study demonstrates that CD9 associates with CD36 on the macrophage surface and may participate in macrophage signaling in response to oxidized LDL.     

Covalent binding of oxidized cholesteryl esters to protein: implications for oxidative modification of low density lipoprotein and atherosclerosis

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apoB). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apoB. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu2+ produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-oxovaleroylcholesterol peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde levels was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester core aldehydes and (ii) an increase in the amounts of apoB-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester core aldehydes were further converted to their 7-ketocholesterol- and apoB-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody 2A81, directed against 9-ONC-modified protein, and found that it extensively recognized protein-bound cholesteryl ester core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins.     

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.     

CD36 N-terminal cytoplasmic domain is not required for the internalization of oxidized low-density lipoprotein

The uptake of OxLDLs (oxidized low density lipoproteins) by CD36-expressing macrophages in the arterial intima and the subsequent 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. The present study has addressed the function of the CD36 N-terminal cytoplasmic domain in the binding and internalization of OxLDL. A selection of CD36 N-terminal cytoplasmic domain mutants were generated and stably expressed in HEK-293 (human embryonic kidney) cells. The capacity of three mutants [CD36_C3/7-A (CD36-C3A/C7A), CD36_D4/R5-A (CD36-D4A/R5A) and CD36_nCPD(-) (CD36 lacking the N-terminal cytoplasmic domain)] to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluorimetry. Each of the CD36 constructs was expressed at differing levels at the cell surface, as measured by flow cytometry and Western blotting. Following incubation with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-OxLDL, cells bearing the CD36_wt (wild-type CD36), CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD(-) constructs all internalized DiI-OxLDL into endosomal structures, whereas empty-vector-transfected cells failed to do so, indicating that, unlike the C-terminal cytoplasmic domain, the N-terminal cytoplasmic domain is not essential for the endocytosis of OxLDL. In conclusion, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 N-terminal cytoplasmic domain. However, the N-terminal cytoplasmic domain may conceivably be implicated in the maturation of CD36.     

Endocytosis of hepatic lipase and lipoprotein lipase into rat liver hepatocytes in vivo is mediated by the low density lipoprotein receptor-related protein

In isolated cell studies, the internalization and degradation of hepatic lipase (HL) has been linked to its binding to the low density lipoprotein receptor-related protein (LRP). We have utilized the receptor-associated protein (RAP), a universal inhibitor of high affinity ligand binding to LRP, to evaluate the participation of LRP in the endocytosis of HL and lipoprotein lipase (LPL). We isolated a total endosome fraction from rat livers after a 30-min infusion of recombinant RAP, administered as a glutathione S-transferase conjugate (GST-RAP). GST-RAP infusion had no effect on the concentration of HL in liver homogenates, but its concentration in blood plasma increased progressively by 20%, and enrichment over homogenate of HL in endosomes was reduced by 50% as compared with infusion of GST alone. The concentrations of LPL in liver and plasma were 1.4 and 0.5%, respectively, those of HL, but endosomal enrichment of the two enzymes was similar ( approximately 10-fold). GST-RAP infusion had no effect on the concentration of LPL in liver but increased its concentration in blood plasma by 250% and reduced its endosomal enrichment by 95% or greater. GST-RAP infusion also reduced endosomal enrichment of LRP by 40%, but enrichment of several other endocytic receptors was unaffected. Endosomal enrichment of several membrane trafficking proteins associated with the endocytic pathway in hepatocytes was unaffected by GST-RAP with the exception of early endosome endosome antigen 1, which was reduced by 85%. We conclude that HL is partially and LPL almost exclusively taken up into rat hepatocytes after binding to the endocytic receptor LRP.     

Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.     

Cholesterol delivered to macrophages by oxidized low density lipoprotein is sequestered in lysosomes and fails to efflux normally

Oxidized low density lipoprotein (LDL) has been found to exhibit numerous potentially atherogenic properties, including transformation of macrophages to foam cells. It is believed that high density lipoprotein (HDL) protects against atherosclerosis by removing excess cholesterol from cells of the artery wall, thereby retarding lipid accumulation by macrophages. In the present study, the relative rates of HDL-mediated cholesterol efflux were measured in murine resident peritoneal macrophages that had been loaded with acetylated LDL or oxidized LDL. Total cholesterol content of macrophages incubated for 24 h with either oxidized LDL or acetylated LDL was increased by 3-fold. However, there was no release of cholesterol to HDL from cells loaded with oxidized LDL under conditions in which cells loaded with acetylated LDL released about one-third of their total cholesterol to HDL. Even mild degrees of oxidation were associated with impairment of cholesterol efflux. Macrophages incubated with vortex-aggregated LDL also displayed impaired cholesterol efflux, but aggregation could not account for the entire effect of oxidized LDL. Resistance of apolipoprotein B (apoB) in oxidized LDL to lysosomal hydrolases and inactivation of hydrolases by aldehydes in oxidized LDL were also implicated. The subcellular distribution of cholesterol in oxidized LDL-loaded cells and acetylated LDL-loaded cells was investigated by density gradient fractionation, and this indicated that cholesterol derived from oxidized LDL accumulates within lysosomes. Thus impairment of cholesterol efflux in oxidized LDL-loaded macrophages appears to be due to lysosomal accumulation of oxidized LDL rather than to impaired transport of cholesterol from a cytosolic compartment to the plasma membrane.     

Receptor-associated protein binding blocks ubiquitinylation of the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) consists of two subunits, M(r) approximately 515,000 and 85,000. LRP is a receptor for activated alpha2-macrogobulin (alpha2M*), Pseudomonas exotoxin A, and many other proteins. We now report that ubiquitinylation of the LRP heavy chain occurred when either Pseudomonas exotoxin A or alpha2M* bound to LRP on macrophages. Ubiquitinylation was dose-dependent and maximal about 30 min after ligation of the receptor. Addition of the proteosome inhibitor MG-132 sustained the level of ubiquitin-LRP for longer time intervals in macrophages treated with either alpha2M* or Pseudomonas exotoxin A. By contrast, when receptor associated protein (RAP) bound to LRP, ubiquitinylation did not occur. While RAP is not found in the extracellular environment it binds to LRP and is believed to function as an intracellular chaperone. The presence of RAP within the cell may, therefore, contribute to the recycling of intact LRP which has ligated and internalized its ligands.     

Mutation in apolipoprotein B associated with hypobetalipoproteinemia despite decreased binding to the low density lipoprotein receptor

Mutations in apolipoprotein B (APOB) may reduce binding of low density lipoprotein (LDL) to the LDL receptor and cause hypercholesterolemia. We showed that heterozygotes for a new mutation in APOB have hypobetalipoproteinemia, despite a reduced binding of LDL to the LDL receptor. APOB R3480P heterozygotes were identified among 9,255 individuals from the general population and had reduced levels of apoB-containing lipoproteins. Most surprisingly, R3480P LDL bound with lower affinity to the LDL receptor than non-carrier LDL in vitro, and these results were confirmed by turnover studies of LDL in vivo. In very low density lipoprotein (VLDL) turnover studies, the amount of VLDL converted to LDL in R3480P heterozygotes was substantially reduced, suggesting that this was the explanation for the hypobetalipoproteinemia observed in these individuals. Our findings emphasized the importance of combining in vitro studies with both human in vivo and population-based studies, as in vitro studies often have focused on very limited aspects of complex mechanisms taken out of their natural context.