Clinical chemistry of common apolipoprotein E isoforms

Apolipoprotein E plays a central role in clearance of lipoprotein remnants by serving as a ligand for low-density lipoprotein and apolipoprotein E receptors. Three common alleles (apolipoprotein E₂, E₃ and E₄) give rise to six phenotypes. Apolipoprotein E₃ is the ancestral form. Common apolipoprotein E isoforms derive from nucleotide substitutions in codons 112 and 158. Resulting cysteine-arginine substitutions cause differences in: affinities for low-density lipoprotein and apolipoprotein E receptors, low-density lipoprotein receptor activities, distribution of apolipoprotein E among lipoproteins, low-density lipoprotein formation rate, and cholesterol absorption. Accompanying changes in triglycerides, cholesterol and low-density lipoprotein may promote atherosclerosis development. Over 90% of patients with familial dysbetalipoproteinaemia have apolipoprotein E₂/E₂. Apolipoprotein E₄ may promote atherosclerosis by its low-density lipoprotein raising effect. Establishment of apolipoprotein E isoforms may be important for patients with diabetes mellitus and several non-atherosclerotic diseases. Apolipoprotein E phenotyping exploits differences in isoelectric points. Isoelectric focusing uses gels that contain pH4–7 ampholytes and urea. Serum is directly applied, or prepurified by delipidation, lipoprotein precipitation or dialysation. Isoelectric focusing is followed by immunofixation/protein staining. Another approach is electro- or diffusion blotting, followed by protein staining or immunological detection with anti-apolipoprotein E antibodies and an enzyme-conjugated second antibody. Apolipoprotein E genotyping demonstrates underlying point mutations. Analyses of polymerase chain reaction products are done by allele-specific oligonucleotide probes, restriction fragment length polymorphism, single-stranded conformational polymorphism, the primer-guided nucleotide incorporation assay, or denaturating gradient gel electrophoresis. Detection with primers that either or not initiate amplification is performed with the amplification refractory mutation system. Disparities between phenotyping and genotyping may derive from isoelectric focusing methods that do not adequately separate apolipoprotein E posttranslational variants, storage artifacts or faint isoelectric focusing bands.     

The role of apolipoprotein E in neurodegeneration and cardiovascular disease

Apolipoprotein E (ApoE) is an abundant plasma protein that interacts with low density lipoprotein receptors and other proteins, participating in the transport of cholesterol and lipids. Research has revealed many other roles for this multifunctional protein. ApoE is polymorphic and exists in three major isoforms: ApoE2, ApoE3 (the most common isoform) and ApoE4, which differ by only one amino acid, at positions 112 and 158. The altered binding to lipids and receptors by ApoE isoforms E2 and E4 results in an elevated risk for neurological, cerebrovascular and cardiovascular pathologies. Most notably, ApoE4 is associated with an elevated risk (relative to E3) for Alzheimer’s disease. The application of mass spectrometry for genotyping and also direct measurement of ApoE protein isoforms is a recent development and is well suited to high-throughput applications. The precise quantification of protein isoforms will allow better characterization of effects resulting from heterozygous APOE genotypes.     

Apolipoprotein E: from atherosclerosis to Alzheimer's disease and beyond.

Apolipoprotein E is a key regulator of plasma lipid levels. Our appreciation of its role continues to expand as additional aspects of its function are discovered. Apolipoprotein E affects the levels of all lipoproteins, either directly or indirectly by modulating their receptor-mediated clearance or lipolytic processing and the production of hepatic very low density lipoproteins. Furthermore, it plays a critical role in neurobiology. The apolipoprotein E4 allele is the major susceptibility gene related to the occurrence and early age of onset of Alzheimer's disease. It is probable that one of the major functions of apolipoprotein E in the central nervous system is to mediate neuronal repair, remodeling, and protection, with apolipoprotein E4 being less effective than the E3 and E2 alleles. The isoform-specific effects of apolipoprotein E are currently being unraveled through detailed structure and function studies of this protein.     

Crystallization and preliminary x-ray diffraction study of synthetic apolipoprotein E fragment (residues 129-169)

Apolipoprotein E is a plasma protein comprised of a lipid binding region (which together with other apoproteins maintains the structure of lipoprotein particles) and a receptor binding domain (which interacts with cellular receptors for control of triglyceride and cholesterol metabolism). A peptide, comprising residues 129-169 of human apolipoprotein E, which contains both a putative lipid-binding region and receptor binding domain, has been synthesized by solid phase techniques. Diffraction quality crystals of the synthetic apolipoprotein E fragment129-169 have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octylglucoside. The crystals have been characterized with x-radiation as orthorhombic, space group I222 or I2(1)2(1)2(1), with unit cell dimensions a = 61.91, b = 30.84, and c = 42.79 A. There are eight molecules per unit cell, with one molecule (Mr = 4771) in each asymmetric unit. Precession photographs show that crystals diffract beyond 2.7-A resolution and are stable in the x-ray beam at room temperature for at least 200 h; thus, they can be used to collect three-dimensional data for a detailed crystallographic analysis.     

Intracellular metabolism of triglyceride-rich lipoproteins

Over the past 10 years, many advances have been made in our understanding of the intravascular metabolism of triglyceride-rich lipoproteins. It is now known that the complex extracellular interactions of triglyceride-rich lipoprotein-associated apolipoprotein E, lipoprotein lipase and hepatic lipase with heparan sulfate proteoglycans and lipoprotein receptors facilitate the hepatocellular uptake of triglyceride-rich lipoproteins. Recent studies have also revealed that the intracellular fate of internalized triglyceride-rich lipoproteins is highly complex. The dissociation of triglyceride-rich lipoprotein components within intracellular endosomal compartments involves the recycling of apolipoprotein E, whereas the remaining lipid core associated with apolipoprotein B is susceptible to lysosomal degradation. Apolipoprotein E recycling is an important newly discovered feature of lipoprotein metabolism, and will be discussed in the context of its intracellular transport mechanisms and cholesterol efflux. Current concepts concerning its potential relevance with regard to lipoprotein metabolism and atherosclerosis will also be discussed.     

Apolipoprotein A-V interaction with members of the low density lipoprotein receptor gene family

Apolipoprotein A-V is a potent modulator of plasma triacylglycerol levels. To investigate the molecular basis for this phenomenon we explored the ability of apolipoprotein A-V, in most experiments complexed to disks of dimyristoylphosphatidylcholine, to interact with two members of the low density lipoprotein receptor family, the low density lipoprotein receptor-related protein and the mosaic type-1 receptor, SorLA. Experiments using surface plasmon resonance showed specific binding of both free and lipid-bound apolipoprotein A-V to both receptors. The binding was calcium dependent and was inhibited by the receptor associated protein, a known ligand for members of the low density lipoprotein receptor family. Preincubation with heparin decreased the receptor binding of apolipoprotein A-V, indicating that overlap exists between the recognition sites for these receptors and for heparin. A double mutant, apolipoprotein A-V (Arg210Glu/Lys211Gln), showed decreased binding to heparin and decreased ability to bind the low density lipoprotein receptor-related protein. Association of apolipoprotein A-V with the low density lipoprotein receptor-related protein or SorLA resulted in enhanced binding of human chylomicrons to receptor-covered sensor chips. Our results indicate that apolipoprotein A-V may influence plasma lipid homeostasis by enhancing receptor-mediated endocytosis of triacylglycerol-rich lipoproteins.     

Structure-function relationships of apolipoprotein A-I: a flexible protein with dynamic lipid associations

PURPOSE OF REVIEW: Apolipoprotein A-I is the major structural protein of HDL. Its physicochemical properties maintain a delicate balance between maintenance of stable lipoproteins and the ability to associate with and dissociate from the lipid transported. Here we review the progress made in the last 2-3 years on the structure-function relationships of apolipoprotein A-I, including elements related to the ATP binding cassette transporter A1. RECENT FINDINGS: Current evidence now supports the so-called 'belt' or 'hairpin' models for apolipoprotein A-I conformation when bound to discoidal lipoproteins. In-vivo expression of apolipoprotein A-I mutant proteins has shown that both the N- and C-terminal domains are important for lipid association as well as for the esterification reaction, particularly binding of cholesteryl esters and formation of mature alpha-migrating lipoproteins. This property is apparently quite distinct from the activation of the enzyme lecithin cholesterol acyl transferase, which requires interaction with the central helix 6. The interaction of apolipoprotein A-I with the ATP binding cassette transporter A1 has been shown to require the C-terminal domain, which is proposed to mediate the opening of the helix bundle formed by lipid-free or lipid-poor apolipoprotein A-I and allow its association with hydrophobic binding sites. SUMMARY: Significant progress has been made in the understanding of the molecular mechanisms controlling the folding of apolipoprotein A-I and its interaction with lipids and various other protein factors involved in HDL metabolism.     

Isolation and lipid-binding properties of rat apolipoprotein A-IV

Apolipoprotein A-IV was isolated from the d less than 1.21 g/ml fraction of rat serum by gel filtration followed by heparin-Sepharose affinity chromatography; this method also facilitated the preparation of apolipoprotein A-I and apolipoprotein E. The apolipoprotein A-IV preparation was characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and immunodiffusion. The lipid-binding properties of this protein were studied. Apolipoprotein A-IV associated with dimyristoylphosphatidylcholine (DMPC) to form recombinants which contained two molecules of apolipoprotein A-IV and had a lipid/protein molar ratio of 110. The density of the DMPC/apolipoprotein A-IV particles was determined to be 1.08 g/ml and the particles were visualized by electron microscopy as discs which were 5.8 nm thick and 18.0 nm in diameter. The stability of the DMPC/apolipoprotein A-IV recombinants, as determined by resistance to denaturation, was comparable to the stability of DMPC/apolipoprotein A-I complexes. However, by competition studies it was found that apolipoprotein A-I competed for the binding to DMPC more effectively than did apolipoprotein A-IV. It is concluded that, while rat apolipoprotein A-IV resembles other apolipoproteins in its lipid-binding characteristics, it may be displaced from lipid complexes by apolipoprotein A-I.     

Activation of lecithin: cholesterol acyltransferase by apolipoproteins E-2, E-3, and A-IV isolated from human plasma

Apolipoprotein A-IV, apolipoprotein E-2 and apolipoprotein E-3 were individually incorporated into defined phosphatidylcholine/cholesterol liposomes for study of lecithin:cholesterol acyltransferase activation. Enzyme activities obtained with these liposomes were compared with that from liposomes containing purified apolipoprotein A-I. Apolipoprotein A-IV, apolipoprotein E-2, and apolipoprotein E-3 all activated lecithin:cholesterol acyltransferase. With purified enzyme and with egg yolk phosphatidylcholine as the acyl donor, maximal activation was obtained at a concentration of approximately 0.5 nmol for apolipoprotein A-IV and 0.4 nmol for the apolipoprotein E isoforms. Apolipoprotein A-IV was approximately 25% as efficient as apolipoprotein A-I for the activation of purified enzyme; apolipoprotein E-2 was 40% as efficient, and apolipoprotein E-3, 30%. Similar activation results were obtained using plasma as the enzyme source. Analysis of the plasma of patients with absence of apolipoprotein A-I or with only trace amounts of apolipoprotein A-I exhibited a reduced rate of cholesterol esterification and lecithin:cholesterol acyltransferase activity that was proportional to the reduced level of the enzyme's mass. These results indicate that apolipoprotein A-IV and apolipoprotein E may serve as physiological cofactors for the enzyme reaction.     

Apolipoprotein E activates Akt pathway in neuro-2a in an isoform-specific manner

Apolipoprotein E (apoE) is a ligand for members of the low density lipoprotein (LDL) receptor family, receptors highly expressed in neurons. A study of one of the mechanisms by which apoE might affect neuronal cell metabolism is reported herein. ApoE can induce Akt/protein kinase B phosphorylation in Neuro-2a via two different pathways. Both pathways are mediated by phosphatidylinositol 3-kinase and cAMP-dependent protein kinase. The first pathway is stimulated by apoE3 and E4, but not by E2, after a 1-h incubation. The process requires the binding of apoE to the heparan sulfate proteoglycan/LDL receptor-related protein complex. The second pathway is activated after a 2-h incubation of the cells, in another isoform-dependent manner (E2 = E3 dbl greater-than sign E4) and is mediated by calcium. Our results suggest that apoE might affect cell metabolism and survival in neurons in an isoform-specific manner by inducing novel signaling pathways.     

Apolipoprotein A-I decreases neutrophil degranulation and superoxide production.

Neutrophils participate in the acute phase response and are often associated with tissue injury in a number of inflammatory disorders. The acute phase response is accompanied by alterations in the metabolism of apolipoprotein A-I and high density lipoprotein (HDL). Structural considerations led to studies investigating the effect of purified HDL and apolipoprotein A-I on neutrophil degranulation and superoxide production. Apolipoprotein A-I but not HDL inhibited IgG-induced neutrophil activation by about 60% as measured by degranulation and superoxide production. This suggests that the lipid-associating amphipathic helical domains of apolipoprotein A-I mediate this effect. In support of this was finding inhibitory effects with two synthetic model lipid-associating amphipathic helix peptide analogs. Apolipoprotein A-I, containing tandem repeating amphipathic helical domains, was approximately ten times more effective than the two peptide analogs and inhibited neutrophil activation at well below physiologic concentrations. Competitive binding studies indicate that resting neutrophils have approximately 190,000 (Kd = 1.7 x 10(-7)) binding sites per cell for apolipoprotein A-I, consistent with a ligand-receptor interaction. These observations suggest that apolipoprotein A-I may play an important role in regulating neutrophil function during the inflammatory response.     

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.     

Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli.

Apolipoprotein (apo) E plays an important role in lipid metabolism, and the major isoforms of apoE (apoE2, apoE3, and apoE4) have significantly different metabolic effects. Apolipoprotein E4 is associated with a higher risk of both heart disease and Alzheimer's disease (AD). Patients homozygous for apolipoprotein E2 are predisposed to type III hyperlipoproteinemia, and apoE2 may be protective against AD. Structure/function studies have proved to be a useful tool in understanding how the different apoE isoforms result in different pathological consequences. As these studies continue, it is essential to have a reliable method to produce large quantities of apoE and mutants of apoE. We describe here a method of apoE production in Escherichia coli strain BL21(DE3). The cDNA from apoE isoforms was inserted into a pET32a vector with a T7 promoter and a fusion partner (thioredoxin). The T7 promoter results in high expression of an easily purified His-tagged fusion protein. A thrombin recognition site was positioned in the expression vector so that only two novel amino acids (Gly-Ser) are added to the amino terminus of apoE following the removal of thioredoxin. Approximately 20 mg of apoE is obtained from a 1-liter culture. The major isoforms of apoE produced with this system were extensively characterized for their ability to bind the low-density lipoprotein (LDL) receptor, for their characteristic lipid association preferences, and for their stability as measured by guanidine denaturation. The recombinant proteins behaved identically to plasma-derived apoE isoforms.     

Provision of cholesterol to lymphocytes by high density and low density lipoproteins. Requirement for low density lipoprotein receptors

The capacity of lipoprotein fractions to provide cholesterol necessary for human lymphocyte proliferation was examined. When endogenous synthesis of cholesterol was blocked, proliferation of mitogen-stimulated normal human lymphocytes was markedly inhibited unless an exogenous source of sterol was supplied. All lipoprotein fractions with the exception of high density lipoprotein subclass 3 were able to provide cholesterol for lymphocyte proliferation. Each of the lipoprotein subfractions capable of providing cholesterol was also able to regulate endogenous sterol synthesis in cultured human lymphocytes. Provision of cholesterol by lipoproteins required the interaction of apolipoprotein B or apolipoprotein E with specific receptors on normal lymphocytes. Apolipoprotein modification by acetylation or methylation, which markedly reduced the ability to regulate sterol biosynthesis, also diminished the capacity of lipoproteins to provide cholesterol. In addition, depletion of apolipoprotein B- and apolipoprotein E-containing particles from high density lipoprotein decreased its ability to suppress cholesterol synthesis and prevented it from providing cholesterol to proliferating lymphocytes. Monoclonal antibodies directed against the receptor-recognition sites on apolipoprotein B and apolipoprotein E were used to define the specific apolipoproteins required for the provision of cholesterol to lymphocytes by the various lipoprotein fractions. The antibody to apolipoprotein B inhibited cholesterol provision by both low density lipoprotein (LDL) and other lipoprotein fractions. The antibody to apolipoprotein E did not decrease provision of cholesterol by LDL but did inhibit the capacity of other fractions to provide cholesterol. In addition, a monoclonal antibody against the ligand binding site on the LDL receptor inhibited provision of cholesterol to normal lymphocytes by all lipoproteins. Finally, lymphocytes lacking LDL receptors were unable to obtain cholesterol from any lipoprotein fraction. These studies demonstrate that LDL receptor-mediated interaction with apolipoprotein B or apolipoprotein E is essential for the provision of cholesterol to normal human lymphocytes from all lipoprotein sources.     

Apolipoprotein A-I directly interacts with amyloid precursor protein and inhibits A beta aggregation and toxicity

Amyloid precursor protein (APP) is the source of the neurotoxic amyloid beta (Abeta) peptide associated with Alzheimer's disease. Apolipoprotein A-I (apoA-I), a constituent of high-density lipoprotein complexes, was identified by a yeast two-hybrid system as a strong and specific binding partner of full-length APP (APPfl). This association between apoA-I and APPfl was localized to the extracellular domain of APP (APPextra). Furthermore, the interaction between apoA-I and APPfl was confirmed by coprecipitation using recombinant epitope-tagged APPextra and purified apoA-I. Several functional domains have been identified in APPextra, and we focused on a possible interaction between apoA-1 and the pathologically important Abeta peptide, because APPextra contains the nontransmembrane domain of Abeta. The binding between apoA-I and Abeta was saturable (K(d) = 6 nM), specific, and reversible. APPextra also competed with apoA-I for binding to Abeta. Direct evidence for this interaction was obtained by the formation of an SDS-resistant Abeta-apoA-I complex in polyacrylamide gels. Competitive experiments with apolipoprotein E (isoforms E2 and E4) showed that apoA-I had a higher binding affinity for Abeta. We also found that apoA-I inhibited the beta-sheet formation of Abeta with a mean inhibitory concentration close to that of alpha2-macroglobulin. Finally, we demonstrated that apoA-I attenuated Abeta-induced cytotoxicity. These results suggest apoA-I binds to at least one extracellular domain of APP and has a functional role in controlling Abeta aggregation and toxicity.     

Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins

We have previously demonstrated that astrocytes synthesize and secrete apolipoprotein E in situ. In the present work, primary cultures of rat brain astrocytes were used to study apolipoprotein E synthesis, secretion, and metabolism in vitro. The astrocytes in culture contained immunoreactive apolipoprotein E in the area of the Golgi apparatus. Incubation of the astrocytes with [35S]methionine resulted in the secretion of labeled immunoprecipitable apolipoprotein E, which constituted 1-3% of the total secreted proteins. The apolipoprotein E secreted in culture and the apolipoprotein E in rat brain extracts differed from serum apolipoprotein E in two respects: both had a slightly higher apparent molecular weight (approx. 36,000) and more acidic isoforms than serum apolipoprotein E. Sialylation of the newly secreted apolipoprotein accounted for the difference in both the apparent molecular weight and isoelectric focusing pattern of newly secreted apolipoprotein E and plasma apolipoprotein E. The astrocytes possessed apolipoprotein B,E(LDL) receptors capable of binding and internalizing apolipoprotein E-containing lipoproteins. The uptake of lipoproteins by the cells led to a reduction in the number of cell surface receptors and to the intracellular accumulation of cholesteryl esters. Since apolipoprotein E is present within the brain, and since brain cells can express apolipoprotein B,E(LDL) receptors, apolipoprotein E-containing lipoproteins may function to redistribute lipid and regulate cholesterol homeostasis within the brain.     

Genetic evidence that the multiple apolipoprotein A-1 isoforms are encoded by a common structural gene

We have used a genetic structural variation of apolipoprotein A-I in mice to examine the origin of the multiple charge isoforms of the plasma protein. Apolipoprotein A-I translated in vitro from hepatic or enteric mRNA revealed that the genetic variation simultaneously alters the charge of the protein produced by both tissues. The variation also shifted the charge of the entire family of isoforms found in plasma or translated in vitro. These results indicate that the protein produced by different tissues as well as the multiple isoforms are all derived from a common structural gene by processing.     

Genotyping and sequence analysis of apolipoprotein E isoforms

Apolipoprotein E (apoE), a polymorphic plasma protein, is essential for catabolism of lipoproteins by receptor-mediated endocytosis. One of the apoE isoforms (E2) differs in its binding affinity to specific receptors and contributes to variations in lipoprotein metabolism. Diagnosis of apoE isoforms is done by isoelectric focusing, but it is hindered by various degrees of post-translational sialylation of the apoE protein. Electrophoretically silent structural variations may also escape detection by this technique. We describe a method for genotyping apoE based on hybridization of allele-specific oligonucleotides with enzymatically amplified genomic DNA, which permits unambiguous diagnosis of six common apoE phenotypes within 24 h. Among 100 E2 alleles present in 81 unrelated individuals genotyped by this technique, we found two rare structural mutants of apoE in addition to the common E2 form, E2(158Arg----Cys). Automated sequencing of amplified DNA identified the rare mutants as E2(136Arg----Ser) and E2(145Arg----Cys). The genotypic method may complement or even replace isoelectric focusing for routine determination of apoE phenotypes and for identification of rare structural variants.     

Cholesteryl ester and apolipoprotein E transfer between human high density lipoproteins and chylomicrons

The transfer of cholesteryl esters and apolipoprotein E has been studied between plasma HDL and chylomicrons isolated either from ascitic fluid or from the plasma of a patient with type V hyperlipoproteinemia. Whereas apolipoprotein E transfer was rapid and occurred at low temperature, cholesteryl ester transfer was suppressed at 4 degrees C. Apolipoprotein E transfer did not depend upon the presence of cholesteryl ester transfer protein and was in fact inhibited by the partially purified preparation of this protein. Apolipoprotein E transfer was not increased by reduction with dithiothreitol. The transfer of cholesteryl esters increased sharply at a chylomicron to HDL ratio of cholesteryl ester above 1/10, a value which may be of physiological significance at the peak of postprandial lipemia. At this ratio, the transfer of apolipoprotein E was minimal and increased only at ratios above 2/1. From these results, it is concluded that there is no connection between apolipoprotein E and cholesteryl ester transfer from HDL to chylomicrons. It is, therefore, proposed that whereas chylomicron apolipoprotein E is acquired rapidly and mostly in the lymphatic system, the concentration of chylomicron cholesteryl esters increases significantly and independently in the circulation.     

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.