Antigenic mapping of human low density lipoprotein with monoclonal antibodies

Monoclonal anti-LDL antibodies were produced in a mouse spleen-myeloma system and purified by affinity chromatography on insolubilized low density lipoprotein (LDL). Five antibodies with different specificities could be distinguished by their immunoreactivities with chemically modified LDL preparations, and by their competition for binding to LDL. One of the antibodies inhibited the binding of (125)I-labeled LDL to the apoB,E receptors of cultured human fibroblasts. The same degree of inhibition was achieved using isolated Fab fragments. This antibody may bind to an antigenic site located near the cellular binding site of LDL-apoB.-Tikkanen, M. J., R. Dargar, B. Pfleger, B. Gonen, J. M. Davie, and G. Schonfeld. Antigenic mapping of human low density lipoprotein with monoclonal antibodies.     

Evolution of low density lipoprotein structure probed with monoclonal antibodies

To assess the relationship of apoB structures in different species of animals, the expressions of apoB epitopes in the sera or plasmas of 23 different mammalian species and one marsupial, and on the low density lipoprotein (LDL) from three species of apes, six species of monkeys, and eight non-primates were measured in competitive radioimmunoassays. The abilities of the sera or LDL to compete with 125I-labeled human LDL for binding to seven monoclonal antihuman LDL antibodies immobilized on microtiter plates were determined. LDL of apes bound to most antibodies, while monkey LDL bound to two or three antibodies. Other mammalian LDL bound only weakly to two of the antibodies or to none. The two monoclonal antibodies binding the LDL of more species were those antibodies which also inhibited the binding to and degradation of LDL by human fibroblasts. The rank order of binding of the LDL of a given species to the antibodies correlated with the rank order inhibition of binding and degradation of 125I-labeled human LDL in the human fibroblast system. This suggests that epitopes spatially located near the recognition site of apoB for cellular receptors have a greater tendency to be conserved.     

Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.

The sulfated glycosaminoglycan, heparin, was found to release 125I-labeled low density lipoprotein (125I-LDL) from its receptor site on the surface of normal human fibroblasts. Measurement of the amount of 125I-LDL released by heparin permitted the resolution of the total cellular uptake of 125I-LDL at 37 degrees C into two components: first, an initial rapid, high affinity binding of the lipoprotein to the surface receptor, from which the 125I-LDL could be released by heparin, and second, a slower process attributable to an endocytosis of the receptor-bound lipoprotein, which rendered it resistant to heparin release. At 4 degrees C the amount of heparin-releasable 125I-LDL was similar to that at 37 degrees C, but interiorization of the lipoprotein did not occur at the lower temperature. The physiologic importance of the cell surface LDL receptor was emphasized by the finding that mutant fibroblasts from a subject with homozygous Familial Hypercholesterolemia, which lack the ability to take up 125I-LDL at 37 degrees C, did not show cell surface binding of 125I-LDL, as measured by heparin release, at either 4 degrees C or 37 degrees C. Although heparin released 125I-LDL from its binding site, it did not release 3H-concanavalin A from its surface receptor, and conversely, alpha-methyl-D-mannopyranoside, which released 3H-concanavalin A, did not release surface-bound 125I-LDL. When added to the culture medium simultaneously with LDL, heparin prevented the binding of LDL to its receptor and hence prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. The uptake of LDL by fibroblasts is proposed as a model of receptor-mediated adsorptive endocytosis of macromolecules in human cells.     

High density lipoprotein and low density lipoprotein catabolism by human liver and parenchymal and non-parenchymal cells from rat liver.

The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprotein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a 10-times higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.     

The low density lipoprotein receptor

The study of familial hypercholesterolemia at the molecular level has led to its advancement from a clinical syndrome to a fascinating experimental system. FH was first described 50 years ago by Carl Müller who concluded that the disease produces high plasma cholesterol levels and myocardial infarctions in young people, and is transmitted as an autosomal dominant trait determined by a single gene. The existence of two forms of FH, namely heterozygous and homozygous, was recognized by Khachadurian and Fredrickson and Levy much later. The value of FH as an experimental model system lies in the availability of homozygotes, because mutant genes can be studied without interference from the normal gene. The first and most important breakthrough was the realization that the defect underlying FH could be studied in cultured skin fibroblasts. Rapidly, the LDL receptor pathway was conceptualized and its dysfunction in cells from FH homozygotes was demonstrates. Isolation of the normal LDL receptor protein and studies on the biosynthesis and structure of abnormal receptors in mutant cell lines provided essential groundwork for elucidation of defects at the DNA level. The power of the experimental system, FH, became nowhere more obvious than in work that correlated structural information at the protein level with the elucidation of defined defects in the LDL receptor gene. In addition to revealing important structure-function relationships in the LDL receptor polypeptide and delineating mutational events, studies of FH have established several more general concepts. First, the tight coupling of LDL binding to its internalization suggested that endocytosis was not a non-specific process as suggested from early observations. The key finding was that LDL receptors clustered in coated pits, structures that had been described by Roth and Porter 10 years earlier. These investigators had demonstrated, in electron microscopic studies on the uptake of yolk proteins by mosquito oocytes, that coated pits pinch off from the cell surface and form coated vesicles that transport extracellular fluid into the cell. Studies on the LDL receptor system showed directly that receptor clustering in coated pits is the essential event in this kind of endocytosis, and thus established receptor-mediated endocytosis as a distinct mechanism for the transport of macromolecules across the plasma membrane. Subsequently, many additional systems of receptor-mediated endocytosis have been defined, and variations of the overall pathway have been described.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of three monoclonal antibodies to human serum low density lipoprotein apoprotein B

Human serum low density lipoprotein (LDL) is a large (Mr = 2-3 X 10(6), complex particle composed of lipid, protein and carbohydrate. We obtained about 40 mouse spleen-myeloma hybrid cell lines which produce antibodies against LDL. Three of them, SC2, SC3 and SC10, have been cloned and subcloned and their antibody products characterized. They recognize three non-overlapping epitopes in native LDL. Two of them, SC3 and SC10, also are capable of recognizing very low density lipoprotein, (VLDL), whereas SC2 reacts only weakly with VLDL. All three antigenic determinants remain intact, and accessible to antibodies on the LDL protein apo B, prepared by delipidation in a 'non-denaturing' detergent, sodium deoxycholate. However, apo B prepared by organic solvent, ether-ethanol, or sodium dodecyl sulfate (SDS) delipidation, while reacting strongly with SC10, is only poorly recognized by SC2 or SC3. Proteolysis of LDL with trypsin, chymotrypsin, Staphylococcus aureus protease, papain or thermolysin gives, in each case, several non-identical protein fragments which are separable by SDS-polyacrylamide gel electrophoresis. Upon immunoblotting, some of these fragments are now recognized by either SC3 or SC10 but not SC2, some are recognized by both SC3 and SC10, and others are immunologically unreactive. The protein bands that are separated by SDS gel electrophoresis are composed of several non-identical fragments and contain the antigenic sites to differing degrees. Some of the immunologically reactive fragments do not appear to contain carbohydrate. Reduction and carboxymethylation do not destroy the immunoreactivity of LDL toward any of the antibodies; however, modification of lysine residues by citraconic anhydride markedly diminishes the reactivity of LDL toward SC3. It is likely that the two antibodies SC3 and SC10 are directed against different linear amino acid sequences or very stable domains, whereas the third, SC2, is directed against a more fragile conformational domain of apo B.     

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

Reconstitution of the low density lipoprotein receptor

The receptor for low density lipoprotein was purified from bovine adrenal cortex in the presence of the nonionic detergent octylglucoside. Receptors were incorporated into the bilayer of egg phosphatidylcholine vesicles by a detergent-dialysis method. Reconstituted receptors were functional in that they bound low density lipoprotein as well as a monoclonal antibody directed against the receptor in a specific, saturable fashion. Binding activity of reconstituted receptors was measured by a gel chromatography assay. The orientation of the receptor molecule within the phospholipid bilayer was investigated by binding assays following proteolytic digestion. Reconstituted receptors showed an orientation that was functionally indistinguishable from that of low density lipoprotein receptors in the plasma membrane of intact human fibroblasts.     

Low density lipoprotein receptor-related protein mediates endocytosis of monoclonal antibodies in cultured cells and rabbit liver

Monoclonal antibodies that bound to the external domain of the rabbit low density lipoprotein receptor-related protein (LRP) were taken into rabbit fibroblasts by receptor-mediated endocytosis. Uptake occurred in fibroblasts from Watanabe-heritable hyperlipidemic rabbits, which lack low density lipoprotein receptors, as well as in normal rabbit fibroblasts. The fate of the internalized antibodies differed, depending on the domain of LRP that was recognized. LRP is synthesized as a single polypeptide chain that is cleaved to form a heterodimer of two noncovalently bound proteins, 1) a 515-kDa subunit that contains the binding domain, and 2) an 85-kDa subunit that contains the membrane-spanning region and cytoplasmic tail. A monoclonal antibody directed against the 515-kDa subunit (anti-LRP 515) rapidly dissociated from LRP at pH 5.2. After uptake by cells this antibody dissociated from the receptor and was degraded in lysosomes. A second antibody directed against the external portion of the 85-kDa subunit (anti-LRP 85) failed to dissociate at acid pH. After uptake by cells this antibody was not degraded, but instead was released from the cells in an acid-precipitable form. When administered intravenously to rabbits, both 125I-labeled antibodies were rapidly cleared from the circulation, 75-95% of the uptake occurring in the liver. The anti-LRP 515 antibody was degraded and acid-soluble products appeared in the plasma. No significant acid-soluble products appeared when the anti-LRP-85 antibody was infused. We conclude that LRP can carry out receptor-mediated endocytosis and that its ligand-binding domain, like the similar domain of the low density lipoprotein receptor, undergoes an acid-dependent conformational change that ejects ligands within the endosome. We also conclude that in the body this endocytotic function is expressed primarily in the liver. Both of these conclusions lend support to the hypothesis that LRP may function in humans and animals as a receptor for apolipoprotein E-enriched lipoproteins, such as chylomicron remnants.     

A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.     

Macrophage-derived factors increase low density lipoprotein uptake and receptor number in cultured human liver cells.

Recent evidence suggests the possibility that macrophages can influence lipoprotein metabolism. Therefore we investigated the ability of cultured macrophages to alter low density lipoprotein (LDL) uptake in a human liver cell line (HepG2). Conditioned media from phlogogenic-induced mouse peritoneal macrophages or from a human macrophage cell line stimulated with endotoxin increased HepG2 LDL uptake by as much as 60-70%. The increase was due, in part, to a significant macrophage-induced 40% increase in the number of LDL receptors per cell. Although macrophage conditioned media inhibited HepG2 cholesterol synthesis, the LDL receptor up-regulation did not appear to be due to the effects on cholesterol synthesis. The LDL receptor stimulatory activity was sensitive to proteolysis and heat. Its molecular mass was approximately 20 kDa based on gel filtration. Several macrophage secretory proteins were tested in HepG2 cultures for LDL uptake stimulation. Of these, oncostatin M (approximately 18 kDa by gel filtration) gave the strongest response. The rank order for LDL uptake stimulation was oncostatin M much greater than interleukin 6 = interleukin 1 = transforming growth factor-beta 1. A neutralizing antibody directed against oncostatin M inhibited the ability of conditioned media to up-regulate LDL receptors by 85%. Thus, our results indicate that macrophages can secrete several proteins that up-regulate LDL receptors in HepG2 cells and that most of the up-regulatory activity in macrophage conditioned media appears to be due to oncostatin M.     

The low density lipoprotein receptor prevents secretion of dense apoB100-containing lipoproteins from the liver

The assembly and secretion of very low density lipoproteins (VLDL) require microsomal triglyceride transfer protein (MTP). Recent evidence also suggests a role for the low density lipoprotein (LDL) receptor in this process. However, the relative importance of MTP in the two steps of VLDL assembly and the specific role of the LDL receptor still remain unclear. To further investigate the role of MTP and the LDL receptor in VLDL assembly, we bred mice harboring "floxed" Mttp alleles (Mttpflox/flox) and a Cre transgene on a low-density lipoprotein receptor-deficient background to generate mice with double deficiency in the liver (Ldlr-/- MttpDelta/Delta). In contrast to the plasma of Ldlr+/+ MttpDelta/Delta mice, the plasma of Ldlr-/- MttpDelta/Delta mice contained apoB100. Accordingly, Ldlr-/- MttpDelta/Delta but not Ldlr+/+ MttpDelta/Delta hepatocytes secreted apoB100-containing lipoprotein particles. The secreted lipoproteins were of LDL and HDL sizes but no VLDL-sized lipoproteins could be detected. These findings indicate that hepatic LDL receptors function as "gatekeepers" targeting dense apoB100-containing lipoproteins for degradation. In addition, these results suggest that very low levels of MTP are insufficient to mediate the second step but sufficient for the first step of VLDL assembly.     

First cysteine-rich repeat in ligand-binding domain of low density lipoprotein receptor binds Ca2+ and monoclonal antibodies, but not lipoproteins

The ligand binding domain of the low density lipoprotein receptor consists of seven cysteine-rich repeats of approximately 40 amino acids each. These repeats, which are located at the NH2 terminus of the protein, are homologous to sequences in complement components C8 and C9. To determine the role of the first repeat (amino acids 2-42), we prepared two plasmids containing expressible low density lipoprotein receptor cDNAs. The first plasmid, p delta R1, lacks only the nucleotides encoding the first repeat. It produced a receptor that bound and internalized lipoproteins and recycled to the cell surface with the same efficiency as the normal receptor. This deleted receptor failed to bind two monoclonal antibodies, IgG-C7 and IgG-15C8, which were shown previously to react with the ligand-binding domain. The second plasmid, pR1, encodes a markedly truncated protein whose extracellular domain consists of the first repeat joined to the transmembrane and cytoplasmic domains. This protein bound the two monoclonal antibodies with the same affinity as the normal receptor, but failed to bind lipoproteins. Binding of IgG-15C8 to the normal receptor and the pR1-encoded protein was Ca2+-dependent, indicating that the first repeat binds Ca2+. We conclude that repeats 2-6 in the ligand-binding domain are sufficient for binding lipoproteins and that the first repeat is highly immunogenic, but is not required for lipoprotein binding.     

The binding of low density lipoprotein by liver membranes in the pig.

Porcine liver membranes are capable of high affinity binding of homologous low density lipoproteins (LDL). Binding is time and temperature dependant and substrate saturable. High affinity binding sites are half saturated at 11 μg/ml lipoprotein-protein. The binding of ¹²⁵I-LDL is inhibited by unlabelled homologous LDL, very low density lipoproteins (VLDL) and high density lipoproteins (HDL) and also be human LDL and HDL, but not by unrelated proteins tested. The binding and displacement patterns with membranes from several other porcine tissues are similar to those of liver membranes. These results suggest the presence of “lipoprotein binding sites” in liver membranes which recognize structural features common to the lipoproteins and further indicate that liver membranes are not unique in their ability to bind LDL.     

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.     

Purification of catalytic subunit of low density lipoprotein receptor kinase and identification of heat-stable activator protein

Bovine adrenal cortex contains a high molecular weight casein kinase II-like enzyme (Mr 500,000) that phosphorylates a specific serine residue in the cytoplasmic domain of the low density lipoprotein (LDL) receptor (Kishimoto, A., Brown, M. S., Slaughter, C. A., and Goldstein, J. L. (1987) J Biol. Chem. 262, 1344-1351). In the current paper, we provide evidence to suggest that this 500-kDa kinase can be dissociated into two subunits, a catalytic subunit and an activator subunit, by treatment with 1 M NaCl. The catalytic subunit was purified to homogeneity (greater than 100,000-fold) using affinity chromatography on GTP-agarose plus several other chromatography steps. It had an Mr of 50,000 by gel filtration and 35,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The catalytic subunit phosphorylated casein actively, but it phosphorylated the LDL receptor with only low affinity. The affinity for the LDL receptor was increased 10-fold (saturation at 10 nM LDL receptor) by addition of a second protein that was released from a high molecular weight 500-kDa complex by 1 M NaCl. This activator protein (Mr 120,000 by gel filtration) was extremely heat stable but was destroyed by trypsin. It appeared to be required in stoichiometric amounts with relation to the LDL receptor. It did not increase the ability of the 50-kDa subunit to phosphorylate casein nor did it activate phosphorylation of the LDL receptor or casein by classic casein kinase II. The current data raise the possibility that the specificity of the 500-kDa LDL receptor kinase is attributable to a heat-stable activator subunit that binds to the LDL receptor and thereby renders it a better substrate for the catalytic subunit of the kinase.     

Apolipoprotein B production and very low density lipoprotein secretion by calf liver slices.

Secretion of triglycerides by the liver in ruminants as components of very low density lipoproteins particles is low as compared with that in primates or rodents. The rate-limiting steps for the hepatic export of very low density lipoproteins have been studied in liver slices to determine the origin of the low lipotropic capacity of calf liver compared to that of rat liver. The rates of production of apolipoprotein B (apo B) and albumin as well as the rate of secretion of VLDL-apolipoproteins were measured during 12-h incubation of liver slices in organo-culture using [35S]methionine-cysteine labeling. Hepatic apo B production was similar in the two animal species but the VLDL-apolipoprotein secretion rate for calf liver slices amounted to only 20% of that observed for rat liver slices. Although calf and rat liver slices synthesized similar amounts of total protein, the hepatic production of albumin, measured in cells and media, was much higher in calf than rat liver slices (around 2.7-fold), whereas the rate secretion of albumin was similar in the two species. Our results showed that the slow rate of secretion of VLDL by calf liver cells was not consecutive to a low rate of synthesis of apo B but rather to a defect in VLDL assembly and/or secretion.