Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts

Human apolipoprotein E (apo E) consists of two distinct domains, the lipid-associating domain (residues 192-299) and the globular domain (residues 1-191) which contains the LDL receptor (LDLR) binding site (residues 129-169). To test the hypothesis that an arginine-rich apo E receptor binding domain (residues 141-150) is sufficient to enhance low-density lipoprotein (LDL) uptake and clearance when covalently linked to a class A amphipathic helix, a peptide in which the receptor binding domain of human apo E, LRKLRKRLLR (hApoE[141-150]), is linked to 18A, a well-characterized high-affinity lipid-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApoE[141-150]-18A (hE18A) and its end-protected analogue, Ac-hE18A-NH(2). The importance of positively charged residues and the role of the hydrophobic residues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH(2) [Ac-hE(R)18A-NH(2)] and Ac-LRKMRKRLMR-18A-NH(2) (Ac-mE18A-NH(2)) contained an extended hydrophobic face, including the receptor binding region. Control peptides, Ac-LRLLRKLKRR-18A-NH(2) [Ac-hE(Sc)18A-NH(2)], had the amino acid residues of the apo E receptor binding domain scrambled to disrupt the extended hydrophobic face, and Ac-RRRRRRRRRR-18A-NH(2) (Ac-R(10)18A-NH(2)) had only positively charged Arg residues as the receptor binding domain. The effect of the dual-domain peptides on the uptake and degradation of human LDL by fibroblasts was determined in murine embryonic fibroblasts (MEF1). LDL internalization was enhanced 3-, 5-, and 7-fold by Ac-mE18A-NH(2), Ac-hE18A-NH(2), and Ac-hE(R)18A-NH(2), respectively, whereas the control peptides had no significant biological activity. All three active peptides increased the level of degradation of LDL by 100%. The LDL binding and internalization to MEF1 cells in the presence of these peptides was not saturable over the LDL concentration range that was studied (1-10 microgram/mL). Furthermore, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor-related protein (LRP), LDLR, or both. Pretreatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We conclude that the dual-domain peptides enhanced LDL uptake and degradation by fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.     

Evidence for a cholesterol-lowering gene in a French-Canadian kindred with familial hypercholesterolemia

We describe a four-generation kindred with familial hypercholesterolemia (FH) in which two of the eight heterozygotes for a 5-kb deletion (exons 2 and 3) in the low density lipoprotein (LDL) receptor gene were found to have normal LDL-cholesterol levels. In our search for a gene responsible for the cholesterol-lowering effect in this family, we have studied variation in the genes encoding the LDL receptor, apolipoprotein (apo) B, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, apoAI-CIII-AIV, and lipoprotein lipase. The analysis showed that it was unlikely that variation in any of these genes was responsible for the cholesterol-lowering effect. Expression of the LDL receptor, as assessed in vitro with measurements of activity and mRNA levels, was similar in normo and hyperlipidemic subjects carrying the deletion. Analysis of the apo E isoforms revealed that most of the e2 allele carriers in this family, including the two normolipidemic 5-kb deletion carriers, were found to have LDL-cholesterol levels substantially lower than subjects with the other apo E isoforms. Thus, this kindred provides evidence for the existence of a gene or genes, including the apo e2 allele, with profound effects on LDL-cholesterol levels.C. S. and M. G. contributed equally to this work.     

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.     

Site-directed mutagenesis of an apolipoprotein E mutant, apo E5(Glu3----Lys) and its binding to low density lipoprotein receptors.

Apo E5(Glu3----Lys) is a naturally occurring apolipoprotein E (apo E) mutant found in patients with hyperlipoproteinemia and atherosclerosis. It has been shown to have a high affinity for low density lipoprotein (LDL) receptors. In this study, mutant apo E5 was produced by Chinese hamster ovary cells by means of an in vitro site-directed mutagenesis technique, and its LDL receptor binding activity was assessed. The apo E5 obtained from gene expression bound more readily to the LDL receptor than did plasma apo E3. The concentrations required for 50% competitive binding of 125I-labeled LDL to the LDL receptors were 58.9 ng/ml for plasma apo E3 and 25.7 ng/ml for the expressed apo E5. The expressed apo E5 displayed 229% normal binding. This result is highly consistent with that obtained with plasma apo E5, which showed 217% normal binding. Although the experimental apo E isoproteins contained more sialic acid than plasma apo E, the extent of sialylation had no effect on the receptor binding of apo E.     

Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts.

The low density lipoprotein (LDL) cell surface receptors on human fibroblasts grown in culture bind specific plasma lipoproteins, initiating a series of events which regulate intracellular cholesterol metabolism. Specificity for the interaction with the receptors resides with the protein moieties of the lipoproteins, specifically with the B and E apoproteins of LDL and certain high density lipoproteins (HDLc HDLl), respectively. It was previously established that the amino acid arginine is a functionally significant residue in or near the recognition sites on the B and E apoproteins and that modification of this residue abolishes the ability of these apolipoproteins to bind to the receptor. The present study indicates that lysine residues are also involved in the lipoprotein-receptor interaction. Chemical modification of 15% of the lysine residues of LDL by carbamylation with cyanate or 20% by acetoacetylation with diketene prevents the LDL from competitively displacing unmodified 125I-LDL from the high affinity receptor sites or from binding directly to the receptor. Moreover, quantitative reversal of the aceto-acetylation of the lysine residues of LDL by hydroxylamine treatment regenerates the lysyl residues and reestablishes greater than 90% of the original binding activity of the LDL. The reversibility of this reaction establishes that the loss of binding activity which follows lysine modification is not due to an irreversible alteration of the LDL or HDLc but is probably due to an alteration of a property of the recognition site associated with specific lysine residues. While acetoacetylation and carbamylation neutralize the positive charge on the epsilon-amino group of lysine, reductive methylation selectively modifies lysine residues of LDL and HDLc without altering the positive charge, yet abolishes their ability to bind to the receptor. Preservation of the charge but loss of binding activity following reductive methylation of the lipoproteins suggests that the specificity of the recognition site does not reside simply with the presence of positive charges but depends on other more specific properties of the site determined by the presence of a limited number of the lysine (and arginine) residues. The precise role of lysine remains to be defined, but its function may be to establish and maintain the conformation of the recognition site or the alignment of reactive residues, or both, or to chemically react, through its epsilon-amino group, with the receptor (hydrogen bond formation would be such a possibility).     

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.     

The composition, structural properties and binding of very-low-density and low-density lipoproteins to the LDL receptor in normo- and hypertriglyceridemia: relation to the apolipoprotein E phenotype

The composition, apolipoprotein structure and lipoprotein binding to the LDL receptor were studied for very-low-density (VLDL) and low-density lipoprotein (LDL) particles isolated from subjects with apoE phenotype E3/3 (E3), E2/2 or E2/3 (E2+) and E3/4 or E4/4 (E4+) and a wide range of plasma triglyceride (TG) contents. The data combined for all three phenotype groups can be summarized as follows. (i) A decrease in accessibility of VLDL tryptophan residues to I- anions with a decrease in tryptophan surface density, concomitant with an increase in VLDL dimensions, reflects the increased efficiency of protein-protein interactions. (ii) A gradual increase in the quenching constant for LDL apoB fluorescence with an increase in TG/cholesterol (Chol) ratio reflects the 'freezing' effect of Chol molecules on apoB dynamics. (iii) Different mechanisms specific for a particular lipoprotein from E3/3 or E2/3 subjects are responsible for apoE-mediated VLDL binding and apoB-mediated LDL binding to the LDL receptor in a solid-phase binding assay. (iv) The 'spacing' effect of apoC-III molecules on apoE-mediated VLDL binding results in a decrease in the number of binding sites. (v) The maximum of the dependence of the LDL binding affinity constant on relative tryptophan density corresponds to LDL intermediate size. VLDL particles from hypertriglyceridemic E2/3 heterozygotic individuals had remnant-like properties (increased cholesterol, apoE and decreased apoC-III content) while their binding efficiency was unchanged. Based on the affinity constant value and LDL-Chol content, increased competition between VLDL and LDL for the binding to the LDL receptor upon increase in plasma TG is suggested, and LDL from hypertriglyceridemic E3/3 homozygotic individuals is the most efficient competitor.     

A potential complication in the use of monoclonal antibodies: inhibition of apoB-mediated receptor binding by an anti-apoE antibody

Monoclonal antibody (Mab) 1D7 is specific for human apolipoprotein (apo) E and blocks binding of lipid-associated apoE to the low density lipoprotein (LDL) receptor. We report here that 1D7 can also block the binding of apoE-free LDL to the LDL receptor. The inhibition of LDL-receptor binding is not due to immunological cross-reactivity between the anti-apoE Mab and apoB, the ligand responsible for the interaction of LDL with the LDL receptor: 1) Mab 1D7 did not react with apoE-depleted LDL; 2) the LDL receptor binding inhibitory activity of 1D7 immunoglobulin G (IgG) preparations could be dissociated from the anti-apoE activity; 3) the inhibition was maintained when the fibroblasts were preincubated with the 1D7 IgG, extensively washed, and only then exposed to 125I-labeled LDL. Rather, it appears that 1D7 recognizes mouse apoE, that mouse apoE-1D7 immune complexes contaminate 1D7 IgG preparations and that the contaminating mouse apoE can compete with 125I-labeled LDL for the LDL receptor. We have demonstrated mouse apoE in IgG preparations of 1D7 but not in those of other anti-apoE Mabs that do not influence LDL-receptor binding. Precipitation of 1D7 IgG with NH4SO4 eliminates both apoE and the capacity of 1D7 to block LDL receptor binding. Finally, mouse apoE can be isolated by immunoaffinity chromatography of mouse serum on immobilized 1D7 Mab. As this is probably not a unique case, the observation has important implications for the use of Mabs as structural probes.     

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.     

Very low density lipoprotein binding to cultured aortic endothelium

Because of very low density lipoprotein's (VLDL) potential atherogenicity and the demonstration that VLDL can bind to other cells, we examined the interaction of human VLDL with cultured porcine aortic endothelium. The lipoprotein-cell interaction had many properties similar to those seen with the binding of a ligand to a cell surface receptor. It was time and temperature dependent, saturable, and reversible. Scatchard analysis of competition data suggested that there may be more than one class of binding site. The affinity of the low affinity site was similar to that for low density lipoprotein (LDL). Also, the capacity of endothelial cells to bind VLDL was similar to that for LDL, when related to apo B (i.e., particle) concentration. Not only was unlabelled VLDL able to compete for VLDL binding sites, but so was LDL and high density lipoprotein (HDL). The maximal competition either by LDL or by HDL was less than that by VLDL. The maximal competition by HDL was more than by LDL. The VLDL binding was dependent on Ca2+. It was not changed by the content of lipoprotein in the medium in which cells were grown prior to the binding studies. These observations suggest that VLDL binding to endothelial cells is similar in some respects, but not in all, to the binding of LDL. Comparison of the data with endothelial cells to previous data with adipocytes also indicated differences between the interaction of these two cell types with VLDL. It is possible that this binding process may be involved in the formation of atherogenic remnants of triglyceride-rich lipoproteins on the endothelial surface of large blood vessels.     

Low-density-lipoprotein receptors in different rabbit liver cells.

Receptor-dependent uptake mechanisms for low-density lipoprotein (LDL) were studied in rabbit liver parenchymal and non-parenchymal cells. Hybridization studies with a cDNA probe revealed that mRNA for the apo (apolipoprotein) B,E receptor was present in endothelial and Kupffer cells as well as in parenchymal cells. By ligand-blotting experiments we showed that apo B,E-receptor protein was present in both parenchymal and non-parenchymal cells. Studies of binding of homologous LDL in cultured rabbit parenchymal cells suggested that about 63% of the specific LDL binding was mediated via the apo B,E receptor. Approx. 47% of the specific LDL binding was dependent on Ca2+, suggesting that specific Ca2+-dependent as well as Ca2+-independent LDL-binding sites exist in liver parenchymal cells. Methylated LDL bound to the parenchymal cells in a saturable manner. Taken together, our results showed that apo B,E receptors are present in rabbit liver endothelial and Kupffer cells as well as in the parenchymal cells, and that an additional saturable binding activity for LDL may exist on rabbit liver parenchymal cells. This binding activity was not inhibited by EGTA or reductive methylation of lysine residues in apo B. LDL degradation in parenchymal cells was mainly mediated via the apo B,E receptor.     

Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction

The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.     

RAP,a specialized chaperone,prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors.

The multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) forms a complex with a receptor-associated protein (RAP) within the secretory pathway. RAP inhibits ligand binding to LRP and is required for normal functional expression of LRP in vivo, suggesting a physiological function as a specialized chaperone. We have used RAP-deficient mice, generated by gene targeting, to investigate the role of RAP in the biosynthesis and biological activity of LRP and other members of the LDL receptor gene family in various organs and in embryonic fibroblasts. Our results demonstrate that RAP is required for the proper folding and export of the receptors from the endoplasmic reticulum (ER) by preventing the premature binding of co-expressed ligands. Overexpression of apolipoprotein E (apoE), a high affinity ligand for LRP, results in dramatically reduced cellular LRP expression, an effect that is prevented by co-expression of RAP. RAP thus defines a novel class of molecular chaperones that selectively protect endocytic receptors by binding to newly synthesized receptor polypeptides, thereby preventing ligand-induced aggregation and subsequent degradation in the ER.     

ApoD Mediates Binding of HDL to LDL and to Growing T24 Carcinoma

Apolipoprotein (Apo) D is an important protein produced in many parts of the body. It is necessary for the development and repair of the brain and protection from oxidative stress. The purpose of this study was to investigate the extent to which apoD interacts with lipoproteins in human plasma. By using detergent-free ELISA, we show that immobilized monoclonal antibodies against apoD very efficiently bind to low density lipoprotein (LDL) from plasma; this binding is as equally efficient as binding to an anti-apoB monoclonal antibody. Adding detergent to the plasma inhibited the binding, suggesting that the binding is dependent on the presence of intact lipoprotein particles. Reversing the system by using immobilized anti-apoB revealed that the affinity of apoD for LDL is rather low, suggesting that multiple bindings are needed for a durable connection. Biosensor experiments using purified lipoproteins also showed that purified apoD and high density lipoprotein 3 (HDL3), a lipoprotein fraction rich in apoD, were both able to bind LDL very efficiently, indicating that the HDL3-LDL interaction may be a physiological consequence of the affinity of apoD for LDL. Furthermore, we found that apoD increases the binding of HDL to actively growing T24 bladder carcinoma cells but not to quiescent, contact-inhibited, confluent T24 cells. This result is especially intriguing given that the T24 supernatant only contained detectable levels of apoD after growth inhibition, raising the possibility that alternating the expression of apoD and a putative apoD-receptor could give direction to the flow of lipids. In the current paper, we conclude that apoD mediates binding of HDL to LDL and to growing T24 carcinomas, thereby highlighting the importance of apoD in lipid metabolism.     

Deletion of exon encoding cysteine-rich repeat of low density lipoprotein receptor alters its binding specificity in a subject with familial hypercholesterolemia

The proposed ligand binding domain of the low density lipoprotein (LDL) receptor consists of a 40-amino acid cysteine-rich unit that is repeated with some variation seven times. We describe here a mutant allele at the LDL receptor locus in which one of the seven repeats has been deleted. This mutation was found in a patient with the clinical syndrome of homozygous familial hypercholesterolemia. By molecular cloning, we show that the deletion arose by homologous recombination between repetitive Alu sequences in intron 4 and intron 5 of the gene. The deletion removes exon 5, which normally encodes the sixth repeat of the ligand binding domain. In the resultant mRNA, exon 4 is spliced to exon 6, preserving the reading frame. This mRNA produces a shortened protein that reaches the cell surface and reacts with anti-receptor antibodies but does not bind LDL, which contains apoprotein B-100 as its major protein component. Surprisingly, the deleted protein retains the ability to bind and internalize beta-migrating very low density lipoprotein, a lipoprotein that contains apoprotein E as well as apoprotein B-100. These data support the hypothesis that the seven repeated sequences in the receptor constitute the LDL binding domain. The data further indicate that the sixth repeat is required for binding of LDL, but not beta-migrating very low density lipoprotein, and that deletion of a single cysteine-rich repeat can alter the binding specificity of the LDL receptor.     

Structural characterization of a low density lipoprotein receptor-active apolipoprotein E peptide, ApoE3-(126-183)

Apolipoprotein E (apoE) plays a critical role in lipoprotein particle clearance from blood plasma through its interaction with the low density lipoprotein (LDL) receptor and other related receptors. Here, we studied a 58-residue peptide encompassing the receptor binding region of apoE. ApoE3-(126-183) was generated by cyanogen bromide cleavage of recombinant apoE3-(1-183), purified by reversed-phase high pressure liquid chromatography, and characterized by mass spectrometry. Far UV CD spectroscopy of the peptide showed that it is unstructured in aqueous solution. The addition of trifluoroethanol or dodecylphosphocholine induces the peptide to adopt an alpha-helical conformation. ApoE3-(126-183) efficiently transforms dimyristoylphosphatidylglycerol (DMPG) vesicles into peptide-lipid complexes. Analysis of apoE3-(126-183). DMPG complexes by electron microscopy revealed disc-shaped particles with an average diameter of 13 +/- 3 nm. Flotation equilibrium analysis yielded a particle molecular mass of 252 kDa. Far UV CD analysis of apoE3-(126-183).DMPG discs provided evidence that the peptide adopts a helical conformation. Competition binding experiments with (125)I-labeled low density lipoprotein (LDL) were conducted to assess the ability of apoE3-(126-183).DMPG complexes to bind to the LDL receptor. Both N-terminal apoE and the peptide, when complexed with DMPG, competed with (125)I-LDL for binding sites on the surface of cultured human skin fibroblasts. Under the conditions employed, apoE3-(126-183).DMPG complexes were similar to apoE3-(1-183).DMPG discs in their ability to bind to the receptor, demonstrating that the peptide represents a good model to study the interaction between apoE and the LDL receptor. Preliminary NMR results indicated that a high resolution structure of the apoE3-(126-183) peptide is obtainable.     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.     

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.