The recognition domain for the low density lipoprotein cellular receptor is expressed once on each lipoprotein particle

The stoichiometry of binding of monoclonal antibodies and Fab fragments to LDL was assessed. Increasing amounts of two [125I]-labelled antibodies which define epitopes at or near the LDL-receptor recognition domains of apoB were incubated with fixed amounts of LDL and antibody-LDL complexes were separated from free antibodies by heparin-MnCl2 precipitation. Saturation kinetics were obtained and data were analyzed according to Scatchard. One antibody or Fab fragment was bound per LDL particle. Homogeneity of binding was indicated by straight Scatchard lines and by the binding of virtually all LDL particles by an antibody affinity chromatographic column.     

Differential reactivity of human low density lipoproteins with monoclonal antibodies

The immunoreactivities of LDL (low density lipoprotein) samples obtained from a variety of subjects were analyzed by comparing their capacities to compete with 125I-labeled LDL for binding to various monoclonal anti-LDL antibodies in competitive binding assays. A marked variation in epitope expression was observed. In comparison to an LDL standard, different preparations exhibited immunoreactivities (expressed as apparent apoB content) ranging from 30 to 400% of the LDL standard. Some epitopes were much more uniformly expressed than others. The number of epitopes expressed in different LDL preparations appeared to be related to the percentage composition of various lipid constituents in LDL. The results support the hypothesis that the epitope expression of apoB is modulated by the composition of the lipids associated with it.     

Regional specificities of monoclonal anti-human apolipoprotein B antibodies

The usefulness of monoclonal antibodies as probes of protein structure is directly related to knowledge of the structures and locations of the epitopes with which they interact. In this report we provide a detailed map of 13 epitopes on apoB-100 defined by our anti-apoB monoclonal antibodies based on current information on the amino acid sequence of apoB-100. To localize antibody specificities to smaller regions along the linear sequence of the apoB-100 molecule we used a) thrombin- and kallikrein-generated fragments of apoB-100; b) beta-galactosidase- apoB fusion proteins; c) heparin; and d) antibody versus antibody competition experiments. Most of the monoclonal antibodies elicited by immunization with LDL were directed towards epitopes within the first 1279 amino terminal (T4/K2 fragments) or last 1292 carboxyl terminal amino acid residues (T2/K4 fragments) of apoB-100. One epitope localized to the mid-portion of apoB-100 was elicited by immunization with VLDL (D7.2). Saturating amounts of heparin bound to LDL did not inhibit the binding of any of the monoclonal antibodies to their respective epitopes on apoB-100, indicating that none of the antibody determinants is situated close to any of the reported heparin binding sites on LDL apoB. We examined the expression of apoB epitopes on VLDL subfractions and LDL isolated from a normolipidemic donor. The apparent affinities with which the antibodies interacted with their respective epitopes on the VLDL subfractions and LDL uniformly increased as follows: LDL greater than VLDL3 greater than VLDL2 greater than VLDL1, suggesting that each of the major regions of apoB-100 is progressively more exposed as normal VLDL particles become smaller in size and epitopes are most exposed in LDL. Previous experiments utilizing hypertriglyceridemic VLDL subfractions yielded similar results, but the rank order of VLDL subfractions and LDL was not the same for all antibodies tested. Thus, differences in apoB epitope expression on VLDL particles of differing sizes is a general phenomenon, but the expression of apoB epitopes in hypertriglyceridemic VLDL appears to be more heterogeneous than is the case for VLDL from normolipidemic donors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunochemical properties of human low density lipoproteins as explored by monoclonal antibodies. Binding characteristics distinct from those of conventional serum antibodies

Spleen cells obtained from mice immunized with human plasma low-density lipoproteins (LDL) were fused with mouse myeloma cells. The resulting hybridoma cells secreting immunoglobulin specific for LDL were screened and scored by radioimmunoassay and cloned by multiple limiting dilutions. Immunochemical properties of the monoclonal antibodies were compared with convential mouse serum antibodies. It was found that conventional antibodies precipitated LDL and bound more than 95% of 125I-labeled LDL and the maximal binding was independent of temperature. The monoclonal antibodies were incapable of precipitating LDL and bound a maximum of only 20% of the total 125I-labeled LDL. The maximal binding between monoclonal antibodies and LDL was extremely temperature-dependent. An optimal degree of binding was observed at 4 degrees C, whereas binding at 37 degrees C was only 30% of that achieved at 4 degrees C. Although the binding at 37 degrees C was low, the maximal binding could be re-established following a subsequent incubation at 4 degrees C, suggesting that the antigenic structure of LDL is reversibly modulated at temperatures between 4 and 37 degrees C. Since the orientation of apolipoprotein B in LDL is known to be dynamic at different temperatures, this result suggests that monoclonal antibodies, but not conventional antibodies, are capable of detecting subtle conformational changes in LDL. In addition, we have determined the binding affinity of LDL to monoclonal antibodies and to conventional antibodies. Only monoclonal antibodies showed a linear Scatchard plot, suggesting that the binding was to a single site with a single affinity. The monoclonal antibodies also possessed high specificity and failed to react with porcine LDL, while serum antibodies could recognize both human and porcine LDL.     

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.     

Abnormal high density lipoproteins from patients with liver disease regulate cholesterol metabolism in cultured human skin fibroblasts

Apolipoprotein B (apoB) of plasma low density lipoproteins (LDL) binds to high affinity receptors on many cell types. A minor subclass of high density lipoproteins (HDL), termed HDL1, which contains apoE but lacks apoB, binds to the same receptor. Bound lipoproteins are engulfed, degraded, and regulate intracellular cholesterol metabolism and receptor activity. The HDL of many patients with liver disease is rich in apoE. We tested the hypothesis that such patient HDL would reduce LDL binding and would themselves regulate cellular cholesterol metabolism. Normal HDL had little effect on binding, uptake, and degradation of 125I-labeled LDL by cultured human skin fibroblasts. Patient HDL (d 1.063-1.21 g/ml) inhibited these processes, and in 15 of the 25 samples studied there was more than 50% inhibition at 125I-labeled LDL and HDL protein concentrations of 10 micrograms/ml and 25 micrograms/ml, respectively. There was a significant negative correlation between the percentage of 125I-labeled LDL bound and the apoE content of the competing HDL (r = -0.54, P less than 0.01). Patient 125I-labeled HDL was also taken up and degraded by the fibroblasts, apparently through the LDL-receptor pathway, stimulated cellular cholesterol esterification, increased cell cholesteryl ester content, and suppressed cholesterol synthesis and receptor activity. We conclude that LDL catabolism by the receptor-mediated pathway may be impaired in liver disease and that patient HDL may deliver cholesterol to cells.     

Monoclonal antibodies that inhibit IgE binding

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.     

Characterization of lipoprotein receptors on rat Fu5AH hepatoma cells

The rat hepatoma cell line Fu5AH has the unusual property of accumulating massive amounts of cholesteryl ester upon incubation with hypercholesterolemic serum, and especially when incubated with beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs. The present study was designed to identify and characterize the lipoprotein receptors that mediate the cholesteryl ester accumulation. The beta-VLDL and cholesterol-induced apolipoprotein (apo) E-containing high density lipoproteins (apoE HDLc) bound to Fu5AH cells with very high affinity (Kd approximately equal to 10(-10) M), whereas low density lipoproteins (LDL) bound with unusually low affinity (Kd approximately equal to 10(-8) M). Receptor binding activity of 125I-labeled beta-VLDL, 125I-labeled apoE HDLc, and 125I-labeled LDL was abolished by incubation in the presence of an excess of unlabeled LDL or of a polyclonal antibody to the bovine adrenal apoB,E(LDL) receptor. The receptors were completely down-regulated by preincubating Fu5AH cells with beta-VLDL, but much higher levels of beta-VLDL were required than for down-regulation of fibroblast apoB,E(LDL) receptors. Receptor binding was abolished by reductive methylation of the lysyl residues of the apolipoprotein of the beta-VLDL and by an apoE monoclonal antibody (1D7) that blocks receptor binding. The Fu5AH receptor was further characterized by using the bovine adrenal apoB,E(LDL) receptor antibody. A single protein (Mr approximately equal to 130,000) was identified in Triton extracts of whole cells, and two proteins (Mr approximately equal to 130,000 and 115,000) were found in Fu5AH cell membranes disrupted by homogenization. The Mr approximately equal to 115,000 protein was released from the membranes and did not react with an antibody to the carboxyl-terminal (cytoplasmic) domain of the apoB,E(LDL) receptors. These studies indicate that Fu5AH cells express apoB,E(LDL) receptors that have unusually low affinity for apoB-continuing lipoproteins, require large amounts of cholesterol to induce down-regulation, and are susceptible to specific proteolysis in cell homogenates. These apoB,E(LDL) receptors are responsible for the receptor-mediated uptake of beta-VLDL and chylomicron remnants by Fu5AH cells.     

Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.

Apolipoprotein B transports cholesterol in plasma as low density lipoprotein (LDL) and targets its delivery to cells by binding to a specific plasma membrane receptor. The cellular consequences of apoB binding to its receptor were investigated to determine whether it suppresses cholesterol biosynthesis and reduces the number of cellular receptors for the apoprotein. Upon preincubation of fibroblasts with lipoprotein-deficient medium alone or supplemented with either LDL or apoB complexed to BSA (apoB-BSA), LDL suppressed cholesterol biosynthesis, but apoB enhanced it. Similarly, fibroblasts preincubated in medium supplemented with LDL bound decreased amounts of either (125)I-labeled LDL or (125)I-labeled apoB-BSA to their receptors, while preincubation with apoB-BSA increased the binding relative to the controls. These latter results occurred in association with a decrease in cellular cholesterol content, indicating that apoB in the medium bound cholesterol and removed it from the cells, thus stimulating both cholesterol synthesis and cellular binding of apoB. Accordingly, fibroblast cholesterol synthesis and the number of functional LDL receptors are not suppressed by the binding of the apoprotein to the receptor, and the known role of apoB remains that of transporting cholesterol in plasma and delivering it to the cell. A possible physiologic role for apoB in depleting cells of cholesterol is presently unknown since apoB is not known to exist free in plasma; however, these findings demonstrate such a functional capability for this apoprotein.-Shireman, R. B., and W. R. Fisher. Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.     

Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia

Monoclonal antibodies directed against the low density lipoprotein (LDL) receptor have been prepared by immunization of mice with a partially purified receptor from bovine adrenal cortex. Spleen cells from the mice were fused with the Sp2/0-Ag14 line of mouse myeloma cells. The most extensively studied monoclonal antibody, designated immunoglobulin-C7, reacts with the human and bovine LDL receptor, but not with receptors from the mouse, rat, Chinese hamster, rabbit, or dog. 125I-labeled monoclonal antibody binds to human fibroblasts in amounts that are equimolar to 125I-LDL. In fibroblasts from 6 of 8 patients with the receptor-negative form of homozygous familial hypercholesterolemia, which have less than 5% of normal LdL binding, the amount of monoclonal antibody binding was also less than 5% of normal. Fibroblasts from the other two receptor-negative homozygotes bound an amount of monoclonal antibody that was much greater than expected on the basis of LDL binding, suggesting that these two patients produce a structurally altered receptor that binds the antibody, but not LDL. In normal fibroblasts, the receptor-bound monoclonal antibody was taken up and degraded at 37 degrees C at rapid rate similar to that for LDL. Fibroblasts from a patient with the internalization defective form of familial hypercholesterolemia bound the monoclonal antibody, but did not internalize or degrade it. The current data demonstrate the usefulness of monoclonal antibodies as probes for the study of the cellular and genetic factors involved in receptor-mediated endocytosis.     

Interaction of LDL, Lp[a], and reduced Lp[a] with monoclonal antibodies against apoB

Five monoclonal antibodies (2A, 9A, 6B, L3, L7) produced in mice against human apolipoprotein B were investigated by competitive and inhibitive electroimmunoassay (EIA) for their reactivity with low density lipoprotein (LDL), lipoprotein[a] (Lp[a]), and reduced Lp[a]. All of the antibodies reacted with apoB of the different lipoproteins indicated by very similar slopes of the binding curves. None of them gave a positive reaction with apolipoprotein[a]. The amount of apoB required for 50% inhibition of antibody binding varied for the different antibodies and lipoproteins. Antibody 9A showed almost the same affinity for LDL, Lp[a], and reduced Lp[a]. Antibodies 2A and 6B bound about twofold better to LDL and reduced Lp[a] than to untreated Lp[a]. Antibodies L3 and L7 needed nearly threefold higher amounts of Lp[a]-apoB for 50% inhibition of antibody binding than of apoB of LDL and reduced Lp[a]. The amount of apoB required for 50% inhibition of antibody binding was somewhat higher in inhibitive assay than in competitive assay. We suggest that apo[a] covers certain epitopes of apoB in native Lp[a] leading to a reduced reaction with the monoclonal antibodies. However, it could also be that the binding of the [a]antigen to apoB via disulfide bridges causes profound conformational changes of the apoB region exposed to the surface.     

Expression of a monoclonal antibody-defined aminoterminal epitope of human apoC-I on native and reconstituted lipoproteins

Nine distinct mouse monoclonal antibodies were produced in two fusions using holo-human very low density lipoprotein (VLDL) as antigen. On immunoblotting first with human VLDL and then with isolated human apoC-I, seven of the antibodies, representing three isotypes, manifested specificity for apoC-I. Two antibodies were directed against apoB. To assess whether the seven anti-apoC-I antibodies were directed against the same or distinctively different epitopes, cross-competition assays were performed wherein 125I-labeled monoclonal antibodies were made to compete with unlabeled antibodies for occupancy on immobilized VLDL-associated apoC-I. All antibodies cross-competed to varying extents implying that they were directed against closely spaced epitopes, but based on these experiments three different epitopes were defined. On immunoblotting with CNBr fragments, all of the epitopes were assigned to the CNBr I fragment of human apoC-I (amino acids 1-38) suggesting that the NH2-terminal region of apoC-I is more immunogenic in mice than other parts of the molecule when apoC-I is associated with VLDL. A competitive solid-phase radioimmunoassay (RIA) was developed employing one of the anti-apoC-I antibodies (A3-4). VLDL was adsorbed to plastic microtiter wells, and a limiting amount of the antibody was reacted with the adsorbed VLDL. The amount of monoclonal antibody that bound to the immobilized VLDL-apoC-I was determined with a 125I-labeled goat anti-mouse IgG antibody. The addition of competitor apoC-I complexed with lipids resulted in reduced binding of the anti-apoC-I antibody to the immobilized VLDL-apoC-I. Competitor complexes consisted of an artificial lipid emulsion (Intralipid) incubated with apoC-I at phospholipid/apoC-I ratios of 1:1 to 60:1 (w/w). As the lipid/protein ratios were increased, the competitive displacement curves produced by the complexes become progressively steeper, while isolated lipid-free apoC-I produced curves with very shallow slopes, suggesting that a conformation-dependent epitope was being probed. Other apoproteins (C-II, C-III, A-I, A-II, and E) whether lipid-free or complexed with lipids did not compete. Fractionation of the 30:1 apoC-I-Intralipid complex by gel permeation chromatography suggested that apoC-I bound to phospholipids was the most effective competitor. This was confirmed by testing of apoC-I-DMPC complexes, which yielded curves that paralleled those produced by apoC-I-Intralipid.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoprotein-deficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins2 (HDL2). The rate of degradation of HDL2 was constant in incubations from 3 to 25 h. As the concentration of HDL2 in the incubation medium was increased, binding reached saturation. At 37 degrees C, half-maximal binding (Km) was achieved at a concentration of 7.3 micrograms of HDL2 protein/ml (4.06 X 10(-8)M) and the maximum amount bound was 476 ng of HDL2 protein/mg of cell protein. At 4 degrees C, HDL2 had a Km of 18.6 micrograms protein/ml (1.03 X 10(-7)M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL2. Quantification of 125I-labeled HDL2 binding to a specific receptor (based on incubation of cells at 4 degrees C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45 X 10(-7)M. Excess HDL2 inhibited the binding of both 125I-labeled HDL2 and 125I-labeled HDL3, but excess HDL3 did not affect the binding of 125I-labeled HDL3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL2 and LDL from control and hypercholesterolemic rabbits inhibited the degradation of 125I-labeled HDL2, but HDL3 did not. Treatment of HDL2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4 degrees C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL2 or HDL3 to isolated liver membranes. Hepatocytes incubated with [2-14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2-14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.     

Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages

The aim of this work was to investigate the possible mechanisms for uptake by human monocyte-derived macrophages (HMDM) of low density lipoprotein (LDL) pretreated with human arterial chondroitin-6-SO4-rich proteoglycan (LDL-PG). HMDM were incubated with 125I-labeled tyramine cellobiose-labeled LDL-PG, native LDL, and acetylated LDL (Ac-LDL). The results showed that two to four times more LDL-PG than LDL was bound and internalized by the HMDM. Competition experiments showed that LDL-PG competed with native LDL for the apoB,E (LDL) receptor, but not for the Ac-LDL scavenger receptor. Both the LDL and LDL-PG uptake were reduced after preincubation of the macrophages with unlabeled native LDL, though to a lesser extent with LDL-PG. The specific binding of 125I-labeled LDL and 125I-labeled LDL-PG at 4 degrees C was both saturable and concentration-dependent. The dissociation constant (Kd) for binding was 8.6 x 10(-9) M for LDL and 9.4 x 10(-9) M for LDL-PG, but the maximum binding (Bmax) was 1.5-times higher for LDL-PG. Cholesterol derived from LDL-PG was less effective than native LDL in suppressing HMG-CoA reductase activity. The results indicate that the uptake of LDL-PG is mediated not only by the LDL-receptor, but also by another unspecific pathway, which may not be subjected to regulation. These results provide further support for the hypothesis that LDL modifications induced by arterial PG may contribute to the formation of foam cells.     

Mapping apolipoprotein B on the low density lipoprotein surface by immunoelectron microscopy

Apolipoprotein B (apoB) was mapped using electron microscopy to visualize pairs of monoclonal antibodies binding to the low density lipoprotein (LDL) surface. The sites at which these monoclonals bind the apoB polypeptide sequence had already been established. The angular distances between all possible pairs of binding sites except one allowed the relative placement of six epitopes on the LDL sphere. We conclude that apoB extends over at least a hemisphere of the LDL surface since four epitopes are located in the Northern Hemisphere at sites arbitrarily designated as the North Pole, the Aleutian Islands, Bogotá, and in the Atlantic Ocean, while two are found in the Southern Hemisphere at Buenos Aires and at Madagascar. ApoB appears to possess a restricted flexibility, since these relative epitope locations show a substantial standard deviation in latitude and longitude. Mapping of additional epitopes may provide an answer to the question of whether apoB circumnavigates the LDL sphere.     

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.     

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.     

Radioimmunoassay of rat apolipoprotein B peptides in lipoproteins and tissues

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) separates rat apolipoprotein B (apoB) into one lower and two higher molecular weight components. Of the latter, peptide I (PI) corresponds to human B-100, while the slightly faster-migrating peptide II (PII) lacks a human counterpart; the smaller species peptide III (PIII) corresponds to human B-48. We describe here a competitive radioimmunoassay which separately measures the amounts of total (i.e., PI + PII + PIII) and larger (i.e., PI + PII) rat apoB peptides, with the amounts of PIII obtained by difference. Standard rat PIII and combined PI + PII (PI,II) were isolated by high-pressure gel filtration liquid chromatography in the presence of SDS, and the PI,II was used as an immunogen to raise rabbit antisera which were capable of binding all three forms of rat apoB. However, Scatchard analysis showed this binding to represent two distinct types of antibodies: one high-affinity class which bound only PI,II and a second class which bound all apoB peptides with equal but lower affinity. Thus, since 125I-labeled PIII was displaced equally effectively by PI,II and PIII, but 125I-labeled PI,II was displaced only by PI,II, the unabsorbed antiserum could be used to measure either total apoB or PI,II alone, depending on the choice of labeled ligand. The validity of the assay for apoB peptides in very-low-density and low-density lipoproteins and in liver microsomes was verified by comparison with peptide determinations by SDS-PAGE.     

Glycosylation of LDL decreases its ability to interact with high-affinity receptors of human fibroblasts in vitro and decreases its clearance from rabbit plasma in vivo

Incubation of human LDL in vitro at 37 degrees C for 48 h with [14C]glucose at concentrations from 5 to 200 mM resulted in a glycosylated LDL, containing 0.4-20 mol of glucose incorporated per apolipoprotein B of 250 000 daltons. The extent of glucose incorporated was proportional to the time of incubation and concentration of glucose. Glycosylation of LDL abolished its uptake and degradation by the high-affinity process for LDL in normal human skin fibroblasts. 125I-labeled glycosylated LDL was bound, internalized and degraded by the fibroblasts via a nonspecific low-affinity process. The 125I-labeled glycosylated LDL and 125I-labeled LDL were taken up and degraded at similar rates in a non-saturable, low-affinity process by peritoneal macrophages isolated from mice. When 125I-labeled glycosylated LDL or 125I-labeled LDL were injected into rabbits, the glycosylated LDL had a delayed plasma clearance in comparison to the LDL. The mean fractional catabolic rates were 0.67 day-1 and 1.70 day-1 for 125I-labeled glycosylated LDL and 125I-labeled LDL, respectively. The uptake and degradation of 125I-labeled LDL by human skin fibroblasts was decreased as the concentration of free carbohydrate, glucose, sucrose or sorbitol, in the medium was increased from 10 mM to 1 M. It is speculated that pathologic levels of plasma glucose in vivo could result in a decrease in LDL uptake as a result of glycosylation of LDL. A decrease in uptake of native or modified LDL in vivo could contribute to hypercholesterolemia and its pathophysiology.