The binding of acetic anhydride- and citraconic anhydride-modified human low-density lipoprotein to mouse peritoneal macrophages. The evidence for separate binding sites

Human plasma low-density lipoprotein (LDL) was modified chemically with either the monocarboxylic acid derivative, acetic anhydride, or the dicarboxylic acid derivative, citraconic anhydride, reagents which react principally with the lysine residues of protein. The modifications increased the net negative charge on the LDL particles, with citraconyl-LDL displaying a greater negative charge than acetylated LDL. Neither the antigenic reactivity nor the overall gross protein/lipid composition of the LDL were affected by the modification procedures, although a small reduction in the total cholesterol content was observed. The altered LDL species lost the ability to bind to the high-affinity cell surface B/E receptor but both bound to mouse peritoneal macrophages with saturable high-affinity kinetics. At 4 degrees C, the macrophages bound 125I-labelled citraconyl-LDL more avidly (K = 21 X 10(-3) ml/ng) than they bound labelled acetyl-LDL (K = 2 X 10(-3) ml/ng). Competitive inhibition studies indicated that acetyl-LDL and citraconyl-LDL were bound to non-identical sites on the macrophage monolayer surface and that the binding site for citraconyl-LDL was also different from that recognized by hypercholesterolaemic rabbit plasma VLDL (beta VLDL).     

Dioxin alters the human low-density and very low-density lipoprotein structure with evidence for specific quenching of Trp-48 in apolipoprotein C-II

The intercellular transport of cholesterol and triglycerides via lipoproteins interacting with their receptors is a critical component in human lipid metabolism. The delivery of cholesterol to cells is accomplished primarily through low-density lipoproteins (LDLs), while the transport of fatty acids to adipose and muscle tissue is accomplished primarily through the actions of very low-density lipoproteins (VLDLs). Disruption of lipoprotein structure leading to impaired binding between these lipoproteins and their obligate receptors is a known risk factor for cardiovascular disease. Because of recent investigations linking 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in humans with coronary artery disease, investigations have been carried out by fluorescence and circular dichroism to evaluate conformational changes in LDL and VLDL structure upon binding of TCDD. These studies demonstrate that, at a molar ratio of three TCDD molecules to one lipoprotein molecule, TCDD binds and disrupts the secondary and tertiary lipoprotein structure. Circular dichroism studies show that residues within the inner core of apoC-II, which compose a four-alpha-helix bundle when this apolipoprotein is associated with VLDL, are directly affected upon binding TCDD. Fluorescence also indicates the specific interaction of Trp-48 within apoC-II upon TCDD binding. We found that the TCDD/apoC-II complex suffers a 5-fold reduction in its ability to bind lipoprotein lipase compared to untreated apoC-II. The interaction of TCDD with LDL markedly altered the secondary structure of apoB reducing its alpha-helical content. These cumulative responses in lipoprotein structure may impair the LDL and VLDL cellular uptake leading to a buildup of serum lipoproteins and fats thus hastening the development of coronary artery disease.     

Lipolysis products promote the formation of complexes of very-low-density and low-density lipoproteins

In the course of lipolysis, surface lipid products may accumulate on very-low-density lipoproteins (VLDL). To investigate potential lipoprotein interactions mediated by such products, radiolabeled low-density lipoproteins (LDL) were incubated with VLDL and bovine milk lipoprotein lipase in the presence of limited free fatty acid acceptor. With partial VLDL degradation, association of radiolabeled LDL with VLDL remnants or larger aggregates of VLDL density was demonstrated by gradient gel electrophoresis, agarose chromatography, and density gradient ultracentrifugation. VLDL-LDL complex formation was also observed in incubations with lipid extracts from lipolyzed VLDL or with purified palmitic acid in the absence of lipolysis. Complex formation was inhibited by addition of increasing amounts of albumin as free fatty acid acceptor, but could be detected at molar ratios of free fatty acids/albumin that occur in vivo. Composition analysis of LDL reisolated following incubation with VLDL and lipase under conditions favoring partial complex formation revealed enrichment in glycerides and depletion of cholesterol. We conclude that lipolysis products can promote the formation of stable complexes of LDL and VLDL, and that physical interactions of this nature may play a role in the transfer of lipids and apolipoproteins between lipoprotein particles.     

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.     

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.     

Remnant lipoprotein particles are taken up into myocardium through VLDL receptor--a possible mechanism for cardiac fatty acid metabolism

The VLDL (very low-density lipoprotein) receptor is a peripheral lipoprotein receptor expressing in fatty acid active tissues abundantly. In the Balb/c fasting mice, VLDL receptor as well as LPL (lipoprotein lipase), FAT (fatty acid translocase)/CD36, H-FABP (heart-type fatty acid-binding protein), ACS (acyl-CoA synthetase) and LCAD (long-chain acyl-CoA dehydrogenase) expressions increased. An electron microscopic examination indicated the lipid droplets that accumulated in the hearts of fasting Balb/c mice. During the development of SD (Sprague-Dawley) rats, VLDL receptor, LPL, FAT/CD36, H-FABP, ACS, and LCAD mRNAs concomitantly increased with growth. However, PK (pyruvate kinase) mRNA expression was negligible. In cultured neonatal rat cardiomyocytes, VLDL receptor expression increased with days in culture. Oil red-O staining showed that cardiomyocytes after 7 days in culture (when the VLDL receptor protein is present) accumulated beta-migrating VLDL. Thereby, we showed that the cardiac VLDL receptor pathway for delivery of remnant lipoprotein particles might be part of a cardiac fatty acid metabolism.     

Characterization and specificity of lipoprotein binding to term human placental membranes

The binding characteristics of very-low-density (VLDL), low-density (LDL) and high-density (HDL) lipoprotein fractions to a purified human term placental microvillous membrane preparation were determined. Binding of LDL was saturable with a maximal binding capacity of 270 ng LDL protein per mg of membrane protein. Scatchard analysis revealed the presence of a single population of 3.4 · 10¹¹ sites per mg of membrane protein and a mean affinity constant of 5.8 · 10⁻⁹ M. Binding of VLDL was also saturable but the maximal capacity was 4.5-times greater than that of LDL. The Scatchard analysis revealed the presence of 2.1 · 10¹¹ binding sites and an affinity constant nearly one order of magnitude greater than that of LDL. Binding of HDL showed less tendency to saturate. Scatchard analysis showed a similar number of receptor sites to that calculated for VLDL and LDL but the affinity constant for HDL was over 100-fold less than that of VLDL. Self- and cross-inhibition studies of VLDL and LDL binding revealed that VLDL was better at blocking the binding of LDL than was LDL itself. This preferential binding of VLDL suggests that this lipoprotein fraction could be an important source of cholesterol for placental progesterone production.     

The very low density lipoprotein (VLDL) receptor – a peripheral lipoprotein receptor for remnant lipoproteins into fatty acid active tissues

The VLDL (very low density lipoprotein) receptor is a member of the LDL (low density lipoprotein) receptor family. The VLDL receptor binds apolipoprotein (apo) E but not apo B, and is expressed in fatty acid active tissues (heart, muscle, adipose) and macrophages abundantly. Lipoprotein lipase (LPL) modulates the binding of triglyceride (TG)-rich lipoprotein particles to the VLDL receptor. By the unique ligand specificity, VLDL receptor practically appeared to function as IDL (intermediate density lipoprotein) and chylomicron remnant receptor in peripheral tissues in concert with LPL. In contrast to LDL receptor, the VLDL receptor expression is not down regulated by lipoproteins. Recently several possible functions of the VLDL receptor have been reported in lipoprotein metabolism, atherosclerosis, obesity/insulin resistance, cardiac fatty acid metabolism and neuronal migration. The gene therapy of VLDL receptor into the LDL receptor knockout mice liver showed a benefit effect for lipoprotein metabolism and atherosclerosis. Further researches about the VLDL receptor function will be needed in the future.     

Lipoprotein cholesterol in the blood plasma of rats with a bile circulation disorder

The experiments were carried out on two groups of rats with obstruction or cannulation of bile duct. Plasma was obtained from the tail venous blood before and 24 hours after operation. The bile duct obstruction caused hypercholesterolemia and considerable increase in the cholesterol content of low-density and very low-density lipoprotein (cholesterol bound to LDL and VLDL). Cannulation of total bile duct slightly increased CH-LDL+VLDL. The change in concentration of high-density lipoprotein cholesterol was insignificant in both models of bile disturbance while index of atherogenesis increased.     

Low density lipoprotein binding, internalization, and degradation in human adipose cells.

Human adipose tissue derives its cholesterol primarily from circulating lipoproteins. To study fat cell-lipoprotein interactions, low density lipoprotein (LDL) uptake and metabolism were examined using isolated human adipocytes. The 125I-labelled LDL (d = 1.025-1.045) was bound and incorporated by human fat cells in a dose-dependent manner with an apparent Km of 6.9 + 0.9 microgram LDL protein/mL and a Vmax of 15-80 microgram LDL protein/mg lipid per 2 h. In time-course studies, LDL uptake was characterized by rapid initial binding followed by a linear accumulation for at least 4 h. The 125I-labelled LDL degradation products (trichloroacetic acid soluble iodopeptides) accumulated in the incubation medium in a progressive manner with time. Azide and F- inhibited LDL internalization and degradation, suggesting that these processes are energy dependent. Binding and cellular internalization of 125I-labelled LDL lacked lipoprotein class specificity in that excess (25-fold) unlabelled very low density lipoprotein (VLDL) (d less than 1.006) and high density lipoprotein (HDL) (d = 1.075-1.21) inhibited binding and internalization of 125I-labelled LDL. On an equivalent protein basis HDL was the most potent. The 125I-labelled LDL binding to an adipocyte plasma membrane preparation was a saturable process and almost completely abolished by a three- to four-fold greater concentration of HDL. The binding, internalization, and degradation of LDL by human adipocytes resembled that reported by other mesenchymal cells and could account for a significant proportion of in vivo LDL catabolism. It is further suggested that adipose tissue is an important site of LDL and HDL interactions.     

Mechanism for Endogenously Expressed ApoE Modulation of Adipocyte Very Low Density Lipoprotein Metabolism: ROLE IN ENDOCYTIC AND LIPASE-MEDIATED METABOLIC PATHWAYS*

Triglyceride-rich lipoproteins distribute energy in the form of fatty acids to peripheral tissues. We have previously shown that the absence of endogenous adipocyte apoE expression impairs adipocyte triglyceride acquisition from apoE-containing triglyceride-rich lipoproteins in vitro and in vivo. Studies were performed to evaluate the mechanism(s) for this impairment. We excluded a role for secreted apoE in accounting for the difference in very low density lipoprotein (VLDL)-induced adipocyte triglyceride accumulation using cross-incubation studies to show that secreted apoE did not enhance triglyceride synthesis in apoE knockout (EKO) adipocytes incubated with apoE-containing VLDL. Subsequent experiments established that both endocytic and lipase-mediated pathways for lipid acquisition from VLDL were impaired in EKO adipocytes. Binding and internalization of VLDL to EKO adipocytes were significantly lower due to decreased expression or redistribution of low density lipoprotein receptor family proteins. An important role for the VLDL receptor for contributing to differences in VLDL binding between wild-type and EKO adipocytes was identified. Lipoprotein lipase-dependent adipocyte lipogenesis was also significantly decreased in EKO adipocytes even though they secreted as much or more lipolytic activity. This decrease was related to impaired fatty acid internalization in EKO cells. Evaluation of potential mechanisms revealed reduced caveolin-1 and plasma membrane raft expression in EKO adipocytes. Increasing caveolin expression in EKO adipocytes increased fatty acid internalization. Our results establish a role for endogenous adipocyte apoE in VLDL-induced adipocyte lipogenesis by impacting both endocytic and lipoprotein lipase-mediated metabolic pathways. Reduced adipocyte apoE expression, for example that accompanying obesity, will suppress adipocyte acquisition of lipid from apoE-containing VLDL.     

A change in apolipoprotein B expression is required for the binding of apolipoprotein E to very low density lipoprotein

Factors affecting the association of apolipoprotein E (apoE) with human plasma very low density lipoprotein (VLDL) were investigated in experiments in which the lipid content of the lipoprotein was modified either by lipid transfer in the absence of lipolysis or through the action of lipoprotein lipase. In both cases, lipoprotein particles initially containing no apoE (VLDL-E), isolated by heparin affinity chromatography, were modified until they had the same lipid composition as native apoE-containing VLDL (VLDL+E) from the same plasma. Transfer-modified lipoproteins, unlike native VLDL+E, did not bind apoE or interact with heparin. In contrast, VLDL-E, whose lipid composition was modified to the same extent by lipase, bound apoE and bound to heparin under the same conditions as native VLDL+E. A structural protein (apolipoprotein B) epitope characteristic of VLDL+E was expressed during lipolysis prior to ApoE or heparin binding. The data suggest that the reaction of apoE with VLDL-E is a two-step reaction. The appearance of apoB is modified during lipolysis, with expression of a major heparin-binding site. The modified VLDL then becomes competent to bind apoE. The lipid composition of VLDL appears not to be a major factor in the ability of VLDL to bind apoE or to bind to heparin.     

Very fast ultracentrifugation of serum lipoproteins: influence on lipoprotein separation and composition

A very short run time and small sample volumes in the separation of lipoproteins by preparative ultracentrifugation are needed for several investigations. Recently, a very fast sequential separation method was described that needs only 100 min for one run in a centrifugal field of 625 000 × g. We studied the influence of centrifugal fields of this dimension on lipoprotein separation and lipoprotein particle integrity using a Beckman Optima^TM TLX ultracentrifuge with a TLA-120.2 rotor. Rotor speed (120/90/60/30 · 10³ rev./min) and run time (100 min/3 h/6.7 h/27 h) were selected in such a way that the product of centrifugal field and run time remained constant. The first conditions correspond to the very fast ultracentrifugation (VFU) procedure with a centrifugal field of 625 000 × g. Thirty different plasma samples covering a wide range of lipid and protein concentrations were separated in the course of two centrifugal runs at densities of 1.006 and 1.063 kg/l which yielded very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and the subnatant of low-density lipoproteins, including high-density lipoproteins (HDL) and concomitant sedimented plasma proteins. The major lipid components of the lipoproteins, triacylglycerols, free and esterified cholesterol, phospholipids and the apolipoproteins B and A-I, were estimated considering the masses of the tube contents after a slicing procedure. Measurements of lipids and proteins showed a very good recovery of better than 94% and 91%, respectively, and precision-within-series (coefficient of variation) of better than 4.2% and 6.5%, respectively. The effects of the rotor speed on the lipoprotein structure appeared to be weak. With increasing rotor speed, VLDL and LDL lipid constituents principally tended to decrease, whereas they increased in the subnatant of the LDL-run. The mean lipoprotein mass composition, considering the mass percentage of each measured particle constituent, did not show significant alterations. Total protein decreased in VLDL and in LDL and increased in the subnatant of the LDL-run. As checked by an enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein effects were due to nearly complete disappearence of contaminating plasma proteins, especially albumin as the major contamination of VLDL and LDL. The apolipoproteins (apo) B-100, A-I, E and C-I to C-III remained nearly unaffected. The main advantages of VFU were the very short run time (cumulative flotation time is 3.4 h) and the elimination of albumin without repeated runs. The procedure was suitable for the assessment of lipid and protein constituents in lipoproteins from very small plasma samples (500 μl).     

A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands

The low-density lipoprotein (LDL) receptor transports two different classes of cholesterol-carrying lipoprotein particles into cells: LDL particles, which contain a single copy of apolipoprotein B-100 (apoB-100), and beta-migrating very low-density lipoprotein (beta-VLDL) particles, which contain multiple copies of apolipoprotein E (apoE). The ligand-binding domain of the receptor lies at its amino-terminal end within seven adjacent LDL-A repeats (LA1-LA7). Although prior work clearly establishes that LA5 is required for high-affinity binding of particles containing apolipoprotein E (apoE), the number of ligand-binding repeats sufficient to bind apoE ligands has not yet been determined. Similarly, uncertainty exists as to whether a single lipid-activated apoE receptor-binding site within a particle is capable of binding to the LDLR with high affinity or whether more than one is required. Here, we establish that the LA4-5 two-repeat pair is sufficient to bind apoE-containing ligands, on the basis of binding studies performed with a series of LDLR-derived "minireceptors" containing up to four repeats. Using single chain multimers of the apoE receptor-binding domain (N-apoE), we also show that more than one receptor-binding site in its lipid-activated conformation is required to bind to the LDLR with high affinity. Thus, in addition to inducing a conformational change in the structure of N-apoE, lipid association enhances the affinity of apoE for the LDLR in part by creating a multivalent ligand.     

Effects of dietary sea squirt (Halocynthia roretzi) on lipid metabolism in rats

The effect of dietary sea squirt ( Halocynthia roretzi) on lipid metabolism in rats was investigated. Rats were fed sea squirt muscle (Experiment 1); sea squirt muscle, defatted sea squirt muscle and its hexane extract (Experiment 2); and whole body sea squirt and its parts as muscle or viscera (Experiment 3). All of the diets contained the same levels of protein (20%) and lipid (7%). In experiment 1, serum total cholesterol (T-Ch), very-low-density lipoprotein plus low-density lipoprotein (VLDL+LDL)-Ch, triglyceride (TG), phospholipid (PL) and nonesterified fatty acid (NEFA) levels were reduced by 20% dietary sea squirt muscle ingestion; steroid excretions into feces were enhanced by the same diet. In experiment 2, serum T-Ch, (VLDL+LDL)-Ch, TG, PL and NEFA levels were significantly reduced and steroid excretions into feces were significantly enhanced by ingestion of the sea squirt muscle hexane extract. Ingestion of defatted sea squirt muscle also reduced these serum lipid levels, but not as much as did that of whole sea squirt muscle. In experiment 3, serum T-Ch and HDL-Ch levels were significantly elevated by the 10% sea squirt viscera ingestion.     

Effects of triacylglycerol on the structural remodeling of human plasma very low- and low-density lipoproteins

Very low-density lipoprotein (VLDL) is the main plasma carrier of triacylglycerol that is elevated in pathological conditions such as diabetes, metabolic syndrome, obesity and dyslipidemia. How variations in triacylglycerol levels influence structural stability and remodeling of VLDL and its metabolic product, low-density lipoproteins (LDL), is unknown. We applied a biochemical and biophysical approach using lipoprotein remodeling by lipoprotein lipase and cholesterol ester transfer protein, along with thermal denaturation that mimics key aspects of lipoprotein remodeling in vivo. The results revealed that increasing the triacylglycerol content in VLDL promotes changes in the lipoprotein size and release of the exchangeable apolipoproteins. Similarly, increased triacylglycerol content in LDL promotes lipoprotein remodeling and fusion. These effects were observed in single-donor lipoproteins from healthy subjects enriched in exogenous triolein, in single-donor lipoproteins from healthy subjects with naturally occurring differences in endogenous triacylglycerol, and in LDL and VLDL from pooled plasma of diabetic and normolipidemic patients. Consequently, triacylglycerol-induced destabilization is a general property of plasma lipoproteins. This destabilization reflects a direct effect of triacylglycerol on lipoproteins. Moreover, we show that TG can act indirectly by increasing lipoprotein susceptibility to oxidation and lipolysis and thereby promoting the generation of free fatty acids that augment fusion. These in vitro findings are relevant to lipoprotein remodeling and fusion in vivo. In fact, fusion of LDL and VLDL enhances their retention in the arterial wall and, according to the response-to-retention hypothesis, triggers atherosclerosis. Therefore, enhanced fusion of triacylglycerol-rich lipoproteins suggests a new causative link between elevated plasma triacylglycerol and atherosclerosis.     

The carboxyl-terminal domain of receptor-associated protein facilitates proper folding and trafficking of the very low density lipoprotein receptor by interaction with the three amino-terminal ligand-binding repeats of the receptor.

The 39-kDa receptor-associated protein (RAP) is a specialized antagonist that inhibits all known ligand interactions with receptors that belong to the low density lipoprotein (LDL) receptor gene family. Recent studies have demonstrated a role for RAP as a molecular chaperone for the LDL receptor-related protein during receptor folding and trafficking within the early secretory pathway. In the present study, we investigated a potential role for RAP as a chaperone for the very low density lipoprotein (VLDL) receptor, another member of the LDL receptor gene family. Using intracellular cross-linking techniques, we found that RAP is associated with newly synthesized VLDL receptor. In the absence of RAP co-expression, newly synthesized VLDL receptor exhibited slower trafficking along the early secretory pathway, most likely due to misfolding of the receptor. The role of RAP in the folding of the VLDL receptor was further studied using an anchor-free, soluble VLDL receptor. Metabolic pulse-chase labeling experiments showed that while only 3% of the soluble VLDL receptor was folded and secreted in the absence of RAP co-expression, over 50% of the soluble receptor was secreted in the presence of RAP co-expression. The functions of RAP in VLDL receptor folding and trafficking were mediated by its carboxyl-terminal repeat but not by the amino-terminal and central repeats. Using truncated VLDL receptor constructs, we identified the RAP-binding site within the first three ligand-binding repeats of the VLDL receptor. Thus, our present study demonstrates that RAP serves as a folding and trafficking chaperone for the VLDL receptor via interactions of its carboxyl-terminal repeat with the three amino-terminal ligand-binding repeats of the VLDL receptor.     

The effect of an HMG-CoA reductase inhibitor on arteriosclerotic nanoplaque formation and size in a biosensor model

Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic glycosaminoglycan side chains are stretched out into the blood substitute solution, thereby representing a receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques. Low-density lipoprotein (LDL) was found to deposit strongly at the proteoheparan sulfate-coated surface, particularly in the presence of Ca(2+), apparently through complex formation 'proteoglycan-LDL-calcium'. This ternary complex build-up may be interpreted as arteriosclerotic nanoplaque formation on the molecular level responsible for the arteriosclerotic primary lesion. HDL bound to heparan sulfate proteoglycan protected against LDL deposition and completely suppressed calcification of the proteoglycan-lipoprotein complex. In addition, HDL was able to decelerate the ternary complex deposition and to disrupt newly formed nanoplaques. Therefore, HDL attached to its proteoglycan receptor sites is thought to raise a multidomain barrier, selection and control motif for transmembrane and paracellular lipoprotein uptake into the arterial wall. The molecular arteriosclerosis model was tested on its reliability in a biosensor application in order to unveil possible acute pleiotropic effects of the lipid lowering drug fluvastatin. The very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL)/LDL and VLDL/IDL/LDL/HDL plasma fractions from a high-risk patient with dyslipoproteinemia and type 2 diabetes mellitus showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca(2+) concentration, with a strong increase at higher Ca(2+) concentrations. Nanoplaque formation and size of the HDL-containing lipid fraction remained well below that of the LDL-containing lipid fraction. Fluvastatin, whether applied acutely to the patient (one single 80 mg slow release matrix tablet) or in a 2-months medication regimen, markedly slowed down this process of ternary aggregational nanoplaque build-up and substantially inhibited nanoplaque size development at all Ca(2+) concentrations used. The acute action resulted without any significant change in lipid concentrations of the patient. Furthermore, after nanoplaque generation, fluvastatin, similar to HDL, was able to reduce nanoplaque formation and size. These immediate effects of fluvastatin have to be taken into consideration while interpreting the clinical outcome of long-term studies.     

Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid

The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid.     

Acute inflammation and infection maintain circulating phospholipid levels and enhance lipopolysaccharide binding to plasma lipoproteins

Circulating lipoproteins are thought to play an important role in the detoxification of lipopolysaccharide (LPS) by binding the bioactive lipid A portion of LPS to the lipoprotein surface. It has been assumed that hypocholesterolemia contributes to inflammation during critical illness by impairing LPS neutralization. We tested whether critical illness impaired LPS binding to lipoproteins and found, to the contrary, that LPS binding was enhanced and that LPS binding to the lipoprotein classes correlated with their phospholipid content. Whereas low serum cholesterol was almost entirely due to the loss of esterified cholesterol (a lipoprotein core component), phospholipids (the major lipoprotein surface lipid) were maintained at near normal levels and were increased in a hypertriglyceridemic subset of septic patients. The levels of phospholipids found in the LDL and VLDL fractions varied inversely with those in the HDL fraction, and LPS bound predominantly to lipoproteins in the LDL and VLDL fractions when HDL levels were low. Lipoproteins isolated from the serum of septic patients neutralized the bioactivity of the LPS that had bound to them. Our results show that the host response to acute inflammation and infection tends to maintain lipoprotein phospholipid levels and that, despite hypocholesterolemia and reduced HDL levels, circulating lipoproteins maintain their ability to bind and neutralize an important bacterial agonist, LPS.