Composition and structure of lipopolysaccharide-human plasma low density lipoprotein complex. Analytical ultracentrifugation, 31P-NMR, ESR and fluorescence spectroscopy studies

Complexes of Salmonella typhimurium lipopolysaccharide toxin (LPS) with low density lipoproteins (LDL) prepared in vitro have been analyzed. LPS-LDL complexes were found to comprise approx. 0.24 mg LPS/mg LDL protein. The major protein of complexes was apolipoprotein apoB-100 (greater than or equal to 90-95%). Incorporation of LPS molecules into LDL was accompanied by small changes in lipid composition, i.e. the phosphatidylcholine content was diminished by approx. 11% and the free fatty acid concentration was raised 2-fold. Analytical ultracentrifugation showed that insertion of LPS into LDL results in the increase of a portion of particles with higher density (lower flotation coefficient) compared to initial LDL. As was evidenced by ESR, in LPS-LDL complexes, the phospholipid hydrocarbon chains are more ordered than in LDL. 31P-NMR spectra indicated that in LPS-LDL complexes the mobility of phospholipid polar headgroups is restricted in comparison with LDL. Application of the shift reagent (Pr3+) revealed that phospholipid molecules form a monolayer structure on the surface of complexes. Upon binding of LPS to LDL, a maximum of the apoB intrinsic fluorescence was slightly red-shifted (1-2 nm) which may testify that the localization of apoB remains nearly unchanged. For LPS-LDL complexes, the accessibility of apoB fluorophores to quenchers (I-, Cs+, acrylamide) did not dramatically differ from that of LDL. It is concluded that rather large amounts of LPS (about 9-10 molecules) can accommodate in one LDL particle without severely perturbing its original composition and structure. Moreover, in the LPS-LDL complexes, oligosaccharide chains of LPS screen notably neither phospholipid polar headgroups nor, what is very important, apoB. LPS-LDL complexes are suggested to be able in vivo to bind to cellular apoB/E receptors, possible LPS receptors and scavenger-receptors of macrophages (monocytes).     

Insoluble complex formation between low density lipoprotein and heparin. A 31P-NMR study

31P-NMR has been used to probe the motions of the phosphate moiety of phospholipid head-groups in samples of human low density lipoprotein (LDL) in which particle tumbling has been greatly reduced by increasing the viscosity of the medium, by forming an LDL gel by ultracentrifugation, or by precipitation with heparin. The 31P-NMR spectra of LDL gel give broad "powder-like" lineshapes, with the sign and magnitude of the anisotropy characteristic of the bilayer mesophase, which narrow as the temperature is raised from 5 to 45 degrees C. This narrowing occurs over the same temperature range as the core cholesteryl ester liquid-crystalline to liquid phase transition, suggesting interactions between the surface and core. The 31P lineshapes of LDL-heparin insoluble complexes are also "powder-like", but are broader than native LDL at all temperatures studied. The spectra were simulated assuming an axially-symmetric shielding tensor motionally narrowed by Brownian isotropic diffusion [Burnell et al. (1980) Biochim. Biophys. Acta 603, 63-69], allowing determination of the lateral diffusion coefficients, DT, and the chemical shift anisotropy, delta sigma, of the monolayer phospholipids. Relative to LDL gel, the temperature-dependence of DT was reduced in the LDL-heparin insoluble complexes, and delta sigma was increased from 50 to 60 ppm. The results suggest that insoluble complex formation slows phospholipid lateral diffusion in the LDL monolayer and alters the orientation and/or order of the head-group.     

Heterogeneity of human plasma low density lipoprotein.

Low density lipoprotein (LDL) was fractionated into subspecies by the use of DEAE-agarose column chromatography and the peptide compositions of the LDL subspecies which eluted at different NaCl concentrations were determined. LDL which elutes at low NaCl concentration has relatively less non-B apoprotein than does LDL which elutes at high salt concentration. The LDL subspecies which elute at high NaCl concentration contain more apo A-1 than do those which elute at the lower NaCl molarity. These results indicate that LDL consists of subfractions which differ in their peptide compositions.     

Sphingomyelinase activity associated with human plasma low density lipoprotein

Isolated human plasma low density lipoprotein (LDL) was observed to possess sphingomyelinase activity. Accordingly, the formation of ceramide was catalyzed by LDL at 37 degrees C using tertiary liposomes composed of sphingomyelin (mole fraction (x) = 0.2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (x = 0.7), 1, 2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (x = 0.1), and either the fluorescent sphingomyelin analog Bodipy-sphingomyelin or [(14)C]sphingomyelin as substrates. However, this activity was not present in either very low density lipoprotein or the high density lipoprotein subfractions HDL(2) and HDL(3). Oxidation of LDL abrogated its sphingomyelinase activity. Aggregation of the liposomes upon incubation with LDL was evident from the light scattering measurements. Microinjection of LDL to the surface of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-d-sphingomyelin (C16:0-sphingomyelin), and Bodipy-sphingomyelin as a fluorescent tracer (0.75:- 0.20:0.05, respectively) revealed the induction of vectorial budding of vesicles, resembling endocytosis.     

Protocol for efficient plasma sampling for low density lipoprotein turnover studies

Radiolabeled low density lipoprotein (LDL) is commonly used to study the turnover of LDL apolipoprotein B (apoB), the major protein component of LDL. Following an intravenous injection of radioiodinated LDL, typical sampling schedules have including 20-25 samples over a 14-day period with frequent sampling during the first 12 hr and daily samples thereafter. This is a burdensome task for subjects and investigators. To improve acceptance of the procedure, we have examined the effects of reduced sampling schedules upon the estimation of the fractional catabolic rate (FCR) for LDL apoB. Data from 36 different sets of LDL decay curves obtained from investigations of subjects with a variety of lipoprotein phenotypes have been used to test these schedules. Our results indicate that by choosing specific intervals over a 14-day period only 10 samples are sufficient to accurately determine the fractional catabolic rate for LDL in plasma. This reduced sampling schedule should facilitate the study of LDL turnover in large groups of subjects as outpatients.     

Comparison of plasma clearance of low density lipoprotein with beta-very low density lipoprotein or acetoacetylated low density lipoprotein in cholesterol-fed rabbits

The plasma clearance and tissue distribution of radioiodinated low-density lipoprotein (LDL), beta-very low density lipoprotein (beta-VLDL), and acetoacetylated LDL were studied in cholesterol-fed rabbits. Radioiodinated LDL ([125I]LDL) was cleared more slowly than either [125I]beta-VLDL or acetoacetylated-[125I]LDL and its fractional catabolic rate was one-half that of [125I]beta-VLDL and one-ninth that of acetoacetylated-[125I]LDL. Forty-eight hours after the injection of the labeled lipoproteins, the hepatic uptake was the greatest among the organs evaluated with the uptake of [125I]LDL being one-third that of either [125I]beta-VLDL or acetoacetylated-[125I]LDL. The reduction in the hepatic uptake of LDL due to a down-regulation of the receptors would account for this retarded plasma clearance.     

Interaction of cholesterol ester and triglyceride in human plasma very low density lipoprotein.

The properties of human plasma very low density lipoproteins (VLDL), low density lipoproteins (LDL), and their extracted lipids were compared using calorimetric, X-ray scattering, and polarizing microscopy techniques. Intact LDL, and cholesterol esters isolated from LDL and VLDL each undergo reversible changes in their physical state around body temperature. These transitions are associated with ordered liquid crystalline to liquid phase changes of the cholesterol esters. In contrast to LDL, VLDL has no reversible transitions and shows no evidence of ordered liquid crystalline structures between 10 and 45 degrees C. Therefore, unlike LDL, VLDL does not contain a separate cholesterol ester region capable of undergoing cooperative melting. Solubility studies at 37 degrees C of cholesterol esters and triglyceride isolated from VLDL show that even at a weight ratio of 1:1, which greatly exceeds the relative amount of cholesterol esters in VLDL, cholesterol ester is completely soluble in triglyceride. Thus, the cholesterol ester in VLDL is not sequestered in a separate domain within VLDL, but is dissolved in the liquid core of the particle.     

Interaction of human plasma low density lipoprotein with concanavalin A and with ricin.

Native human plasma low density lipoprotein (LDL) interacts with concanavalin A but not with ricin; apOLDL reacts with both lectins. Each reaction is inhibited by the appropriate lectin-specific carbohydrate. The "receptors" on LDL for these two lectins are not destroyed by digestion by proteolytic enzymes. Peptide hydrolysis does not influence the reactivity of LDL toward concanavalin A. It does, however, substantially enhance the ability of the lipoprotein to interact with ricin. The data strongly suggest that the carbohydrate protion of a glycoprotein component of LDL is bound at the saccharidespecific active site on the lectin.     

Structure of human high density lipoprotein reassembled in vitro. Radioimmunoassay studies.

Immunologic approaches to studying lipoprotein structure have been limited because the methods have not been quantitative enough. Recently we reported (Schonfeld, G., and Pfleger, B. (1974) J. Clin. Invest. 54, 236-246) a radioimmunoassay for human apoprotein A-1 (ApoA-I). Only 8% of the ApoA-I of high density lipoprotein (HDL) reacted in the radioimmunoassay system consisting of rabbit anti-human ApoA-I, 125I-ApoA-I, and unlabeled ApoA-I. We suggested that the ApoA-I in HDL were poorly reactive in the radioimmunoassay because they were "masked" by lipid- or protein-protein interactions. To test this, "lipoproteins" were reconstituted from lipids and apoproteins and assayed for their reactivity in the radioimmunoassay. Apo-HDL, ApoA-I alone, or ApoA-I + ApoA-II were sonified with lecithin or with lipids extracted from HDL. Sonicates were fractionated by ultracentrifugation or by Sepharose 4B chromatography. HDLs were also made by incubating dispersed lecithin or lecithin + cholesterol with Apo-HDL, ApoA-I, or ApoA-II. The lipoproteins were analyzed for lipids and protein chemically. Apoprotein compositions were determined by polyacrylamide disc gel electrophoresis. ApoA-I content by radioimmunoassay then was compared with the ApoA-I content obtained by disc gel electrophoresis. Most reconstituted "lipoproteins" had less than the expected ApoA-I contents. Discrepancies between ApoA-I contents were greatest for lipoproteins prepared from Apo-HDL and HDL-lipids (20 to 30% of expected contents). Discrepancies were smaller for particles prepared with lecithin, with ApoA-I alone or with ApoA-I + ApoA-II (40 to 85% of expected). HDLs made by incubation were less reactive than those prepared by sonication. Thus, the reactivity of ApoA-I in the radioimmunoassay could be diminished by causing it to interact with lipids or their apoproteins, or both, suggesting that antigenic sites became masked. From this one can extrapolate that the poor reactivity of the ApoA-I in HDL isolated from plasma also may have been due to the masking of some of its antigenic determinants. The identification of the determinants involved awaits the development of radioimmunoassays for specific regions of ApoA-I.     

Structure and interactions of lipids in human plasma low density lipoproteins.

Temperature-dependent techniques (differential scanning calorimetry, polarizing microscopy, and x-ray scattering and diffraction techniques) were used to compare the properties of human plasma low density lipoproteins (LDL) with its extracted lipid classes. Three types of thermal transitions were characterized: (a) a reversible transition in intact LDL near body temperature associated with a liquid crystalline order-disorder phase change of cholesterol esters within the particles; (b) an irreversible high temperature transition (approximately 70-90 degrees) associated with LDL denaturation and release of cholesterol esters from the disrupted particles; and (c) low temperature transitions related to liquid crystalline and crystalline phase changes in these released esters. The temperature of the reversible transition in intact LDL varies among individual donors. Correlation analysis shows that the temperature of this transition negatively correlates with the amount of triglyceride relative to cholesterol ester in LDL. Studies on mixtures of cholesterol esters and triglycerides isolated from LDL show a similar effect, increasing amounts of triglycerides decreasing the temperature of the liquid leads to smectic liquid crystalline transition of the isolated esters. Thus, the amount of triglyceride in LDL influences the fluidity of the cholesterol esters in LDL. The enthalpy of the reversible transition in intact LDL is 0.69 cal/g of LDL cholesterol ester. This compares with 0.89 cal/g for the liquid leads to liquid crystalline transition of the cholesterol esters released from denatured LDL and 1.01 cal/g for the same transition in the extracted esters. Unlike the cholesterol esters released from denatured LDL, or isolated LDL esters, cholesterol ester in the intact LDL particle does not crystallize. These findings suggest that the behavior of cholesterol esters in intact LDL is constrained relative to their behavior when freed from the restrictions of the particle. These results together with experiments on partitioning of the individual lipid classes of LDL allow us to define the distribution and interaction of lipids in the intact LDL particle.     

Phospholipid-protein interactions in human low density lipoprotein detected by 31P nuclear magnetic resonance.

31P nuclear magnetic resonance (NMR) spectra of human low density lipoprotein (LDL) has been obtained and the major phospholipid components identified. Analysis of the spectra revealed two phospholipid environments: one occupied by 4/5 of the phospholipid with high resolution resonances possessing properties similar to phospholipids in vesicles, and a second occupied by 1/5 of the phospholipid with broad lines indicative of immobilization. Limited trypsin treatment of the particle cleaved all of the B peptide into smaller molecular weight peptides which remained with the particle. Trypsin-treated LDL eluted from a Sepharose CL-6B column similarly to native LDL so that the modified particle remained intact. 31P NMR spectra of trypsin-treated LDL showed little or no immobilized phospholipid. The immobilization in the native LDL particle is attributed to lipid-protein interactions between 1/5 of the phospholipid and the B peptide.     

Binding of [3H]heparin to human plasma low density lipoprotein.

The binding of [³H]heparin to human plasma lipoproteins was measured using a gel filtration assay on columns of Ultrogel AcA 54. [³H]Heparin formed a soluble complex with low density lipoprotein (LDL) as evidenced by the appearance of a new radioactive peak emerging at the void volume where the lipoproteins elute. Free heparin on the other hand was retarded on this column and eluted at a later volume. Heparin binding to LDL could also be demonstrated on columns of Sepharose 4B, in which case two included peaks of ³H were observed to elute in the area of LDL and of heparin. [³H]Heparin did not bind to either high or very low density lipoproteins as determined by the gel filtration assay. The binding of the [³H]heparin to LDL was proportional to both the concentration of LDL and of heparin and both showed saturation kinetics. Cations were not necessary for binding, nor was binding inhibited by EDTA. LDL showed a marked specificity for heparin. Thus, the binding of [³H]heparin to LDL was strongly inhibited by the addition of unlabeled heparin, while other glycosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, and dermatan sulfate were not effective inhibitors except at very high concentrations. Salts, especially K₂HPO₄ and (NH₄)₂SO₄, also inhibited binding when added at concentrations of 10 mm or higher suggesting an ionic interaction between heparin and LDL. The pH optimum for binding was between 7.5 and 8.5 but binding fell off markedly above pH 9.0. The [³H]heparin was heterogeneous and could be separated into four fractions on columns of Sephadex G-75. When these fractions were tested for binding to LDL, only the high molecular weight fraction bound to any significant extent. LDL was treated with reagents used to selectively modify basic amino acid residues, and the effect of these treatments on heparin binding was examined. Thus, ethoxyformic anhydride was used for histidine modification, acetic anhydride and succinic anhydride for lysines and cyclohexanedione for arginine residues. In each case there was a significant loss in heparin binding suggesting that various basic amino acids are involved in binding and/or that basic amino acids are necessary to maintain the proper conformation of LDL.     

Structural aspects of thiol-specific spin labeling of human plasma low density lipoprotein

A novel thiol-specific spin labeling procedure for the protein component (apoprotein B, apoB) of low density lipoproteins (LDLs) is presented. A methanethiosulfonate spin label was used to probe the free cysteine residues of apoB with electron paramagnetic resonance (EPR) spectroscopy. The results indicated that the spin labeled sites are predominantly buried in the LDL particle in two distinct environments that differ in their mobility restrictions. The suitability of thiol-specific labeling for the study of the stability and conformation of apoB was demonstrated in experiments with denaturing agents. The results presented in this work offer a new approach for the matching of EPR data with the primary structure of apoB.     

Lipoxygenase-mediated transformation of human low density lipoprotein to an oxidized and cytotoxic complex

We have been studying the mechanisms involved in the oxidative modification of low density lipoprotein (LDL) that lead to its transformation to a cytotoxic complex. Here we examine the direct effect-of soybean lipoxygenase (SLO), a 15-lipoxygenase, on normal human LDL. SLO oxidized LDL and rendered it cytotoxic; agents known to interfere with lipoxygenase activity inhibited this reaction. Enhancement of both the SLO-mediated LDL oxidation and the conversion of LDL to a cytotoxin was observed when either superoxide dismutase or copper (II) (3,5,-diisopropylsalicylic acid)2, both of which dismute superoxide anion, were included during the incubation of SLO with LDL. In contrast, catalase inhibited this reaction in the presence or absence of agents that dismute superoxide anion. Thus, purified lipoxygenase can mediate LDL modification and superoxide anion inhibits this reaction, Furthermore, H2O2 is essential for SLO-mediated LDL oxidation and conversion of LDL to a cytotoxin.     

Oxidation-labile subfraction of human plasma low density lipoprotein isolated by ion-exchange chromatography

We isolated subfractions of human plasma low density lipoprotein (LDL) using ion-exchange chromatography. Plasma LDL from normolipidemic subjects were applied to a DEAE Sepharose 6B column. After elution of the bulk of LDL at 150 mM NaCl (the major fraction), the residual LDL was eluted at 500 mM NaCl and designated as the minor fraction. The minor fraction, only less than 1% of total LDL, tended to be somewhat similar in certain properties to oxidized LDL, e.g., an increased negative charge, higher protein/cholesterol ratio, and a higher flotation density than native LDL. These results were consistent with data reported by Avogaro et al. (1988. Arteriosclerosis. 8: 79-87). However, assays of 125I-labeled LDL binding activity for LDL receptors equal to that of the major fraction. Incorporation of [14C]oleate into cholesteryl ester [acyl-CoA:cholesterol acyltransferase (ACAT) activity] in mouse peritoneal macrophages incubated with the minor fraction was only slightly greater than that with the major fraction. Incubation of the minor fraction with 0.5 microM Cu2+ caused a remarkable stimulation of ACAT activity, while stimulation by the major fraction required incubation with 5 microM Cu2+, suggesting that the minor fraction was relatively labile to oxidation. The minor but definite presence of a plasma LDL subfraction more negative and susceptible to oxidation implicates the possibility of its association with atherogenesis.     

Interaction of rat plasma very low density lipoprotein with lipoprotein lipase-rich (postheparin) plasma.

Incubation of 125I-labeled very low density lipoprotein (VLDL) with lipoprotein lipase-rich (postheparin) plasma obtained from intact or supradiaphragmatic rats resulted in the transfer of more than 80% of apoprotein C from VLDL to high density lipoprotein (HDL), whereas apoprotein B was associated with lipoprotein of density less than 1.019 g/ml (intermediate lipoprotein). The transfer of 125I-labeled apoprotein C from VLDL to HDL increased with time and decreased in proportion to the amount of VLDL in the incubation system. A relationship was established between the content of triglycerides and apoprotein C in VLDL, whereas the amount of apoprotein C in VLDL was independent of that of other apoproteins, especially apoprotein B. The injection of heparin to rats preinjected with 125I-labeled VLDL caused apoprotein interconversions similar to those observed in vitro. The intermediate lipoprotein was relatively rich in apoprotein B, apoprotein VS-2, cholesterol, and phospholipids and poor in triglycerides and apoprotein C. The mean diameter of intermediate lipoprotein was 269 A (compared with 427 A, the mean Sf rate was 30.5 (compared with 115), and the mean weight was 7.0 X 10(6) daltons (compared with 23.1 X 10(6)). From these data it was possible to calculate the mass of lipids and apoproteins in single lipoprotein particles. The content of apoprotein B in both particles was virtually identical, 0.7 X 10(6) daltons. The relative amount of all other constituents in intermediate lipoprotein was lower than in VLDL: triglycerides, 22%; free cholesterol, 37%; esterified cholesterol, 68%; phospholipids, 41%; apoprotein C, 7%, and VS-2 apoprotein, 60%. The data indicate that (a) one and only one intermediate lipoprotein is formed from each VLDL particle, and (b) during the formation of the intermediate lipoprotein all lipid and apoprotein components other than apoprotein B leave the density range of VLDL to a varying degree. Whether these same changes occur during the clearance of VLDL in vivo is yet to be established.     

Structure of egg yolk very low density lipoprotein. Polydispersity of the very low density lipoprotein and the role of lipovitellenin in the structure

Egg yolk very low density lipoprotein contained on the average 75% of lipid which could be extracted by ether and 25% of a residual lipoprotein, the classical lipovitellenin. The ether-extracted lipid was composed of 75% triglycerides, 7% sterols, 2% mono- and diglycerides, and 16% phospholipids. Lipovitellenin contained 48% lipid composed of 87% phospholipids, 11% triglycerides, and 2% sterols. The protein vitellenin was composed for the most part of units of 74,000 and 270,000 daltons molecular weight.Egg yolk very low density lipoprotein is polydisperse. Preparative ultracentrifugation separated it into six fractions of different average molecular size, and gel chromatography separated it into five. The fractions of larger molecular size contained more lipid and triglyceride than did the fractions of smaller molecular size. The proteins of the fractions appeared to be similar.Egg yolk very low density lipoproteins appear to be a series of molecules composed of cores of lipid of varying sizes with each core surrounded by a layer of lipovitellenin, which is composed principally of glycoprotein and phospholipid.     

An apo-E-free very low density lipoprotein enriched in phosphatidylethanolamine in human plasma

Normal human plasma contains a fraction of very low density lipoprotein (VLDL) which, unlike most VLDL, contains no apolipoprotein E and, unlike apo-E-containing VLDL, is enriched in phosphatidylethanolamine. This fraction made up 0.28 +/- 0.09 of total VLDL triglyceride. Interconversion of the two isolated VLDL fractions was not detected during incubation (2 h, 37 degrees C) and they may represent the physical forms of apo-B corresponding to distinct metabolic pathways in plasma.