A study on the selective binding of apoprotein B- and E-containing human plasma lipoproteins to immobilized rat serum phosphorylcholine-binding protein

Rat serum phosphorylcholine-binding protein (PCBP), a member of the pentraxin family of proteins, was previously shown to bind multilamellar liposomes prepared with egg phosphatidylcholine and lysophosphatidylcholine. The results suggested that the phosphorylcholine groups on the surface of liposomes play an important role in the binding process (Nagpurkar, A., Saxena, U., and Mookerjea, S. (1983) J. Biol Chem. 258, 10518-10523). A study on the binding of human plasma lipoproteins to PCBP immobilized on Sepharose has now been initiated. Very low density lipoproteins were partially bound to a Sepharose-PCBP column, and the bound fraction contained higher concentrations of apoprotein B and E. All the low density lipoproteins applied were bound to the column. In the case of high density lipoproteins, only a small fraction was retained on the column (based on protein analysis), and that bound fraction contained all the apoprotein E and Lp(a) lipoprotein. The binding of very low, low, and high density lipoproteins to Sepharose-PCBP was Ca2+-dependent, and the bound lipoproteins were quantitatively eluted by a phosphorylcholine gradient. Apoprotein B and E were also bound when whole human plasma was applied to Sepharose-PCBP. The effect of selective modification of lysine residues by acetoacetylation and of arginine residues by cyclohexanedione on the binding of low density lipoproteins to Sepharose-PCBP was examined. Modification of arginyl residues resulted in marked reduction of binding, whereas modification of lysine had no effect. Removal of sialic acid from PCBP also had no effect on the binding of low density lipoproteins to immobilized-desialylated PCBP column. The preferential binding of apoprotein B- and E-containing lipoproteins to Sepharose-PCBP indicates a possible physiological role of PCBP and other similar circulating phosphorylcholine-binding proteins of the pentraxin family in lipoprotein metabolism.     

Stimulation of cholesterol esterification in hepatic microsomes by lipoproteins from normal and hypercholesterolemic rabbit serum.

Incubation of plasma lipoproteins with rabbit hepatic microsomes enriched the microsomes with free cholesterol and stimulated cholesterol esterification. The rate of cholesterol esterification correlated well (r = 0.96) with the concentration of microsomal free cholesterol. Lipoproteins from normal and hypercholesterolemic serum varied in their propensity to stimulate cholesterol esterification. Among the normal lipoproteins, low density lipoproteins was more stimulatory than either high density lipoproteins or intermediate density lipoproteins. However, the intermediate density lipoproteins fraction from hypercholesterolemic serum was consistently more stimulatory than any of the normal lipoproteins. The augmentation of cholesterol content, when microsomes were exposed to mixed hyperlipidemic lipoproteins, was proportionately much greater than augementation of phospholipid or protein concentration.     

Stimulation of cholesterol ester exchange by lipoprotein-free rabbit plasma.

A fraction in normal and hypercholesterolemic rabbit plasma of density greater than 1.25 stimulates the exchange of cholesterol esters between very low density and low density lipoproteins from hypercholesterolemic rabbit plasma. The exchange does not result from lecithin:cholesterol acyltransferase activity. The active factor appears to be a high molecular weight globulin with an isolelectric point of 5.2.     

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.     

Characterization of monoclonal anti-rabbit apolipoprotein E antibodies and chemical composition of lipoproteins separated by anti-apolipoprotein E immuno-affinity chromatography

Six mouse monoclonal antibodies against rabbit apolipoprotein E (apo E) have been developed. Of these monoclonal antibodies, clone 5 revealed a high affinity for purified apo E, very low density lipoprotein (VLDL) and beta-VLDL. This monoclonal antibody was used to prepare an immunoaffinity column. Coupled to Sepharose 4B, this antibody allowed complete removal of lipoproteins containing apo E from plasma of New Zealand white (NZW) rabbits; 62, 46, 14, and 3% of VLDL-, IDL-, LDL-, and HDL-protein, respectively, were bound to the anti-apo E affinity column. The bound VLDL was significantly rich in free cholesterol (FC) and cholesteryl esters (CE) relative to the unbound VLDL, whereas bound IDL, LDL and HDL were significantly rich in FC only. All of the bound fractions were characterized by significantly increased ratios of FC/phospholipids (PL). These results indicate that the two lipoprotein populations with and without apo E have different lipid compositions. The relatively high content of cholesterol in lipoproteins containing apo E suggests a contribution of apo E to plasma cholesterol transport.     

Chylomicron remnant cholesteryl esters as the major constituent of very low density lipoproteins in plasma of cholesterol-fed rabbits.

Feeding rabbits 500 mg of cholesterol daily for 4 to 15 days greatly increased the concentration of esterified cholesterol in lipoproteins of d less than 1.006 g/ml. The origin of hypercholesterolemic very low density lipoproteins was investigated by monitoring the degradation of labeled lymph chyomicrons administered to normal and cholesterol-fed rabbits. Chylomicrons were labeled in vivo by feeding either 1) [3H]cholesterol and [14C]oleic acid or 2) [14C]cholesterol and [3H]retinyl acetate. After intravenous injection of labeled chylomicrons to recipient rabbits, [14C]triglyceride hydrolysis was equally rapid in normal and cholesterol-fed animals. Normal rabbits rapidly removed from plasma both labeled cholesteryl and retinyl esters, whereas cholesterol-fed rabbits retained nearly 50% of doubly labeled remnants in plasma 25 min after chylomicron injection. Ultracentrifugal separation of plasma into subfractions of very low density lipoproteins showed that chylomicron remnants in cholesterol-fed animals are found among all subclasses of very low density lipoproteins. Analysis of cholesteryl ester specific activity-time curves for the very low density lipoproteins subfraction from hypercholesterolemic plasma showed that nearly all esterified cholesterol in large very low density lipoproteins and approximately 30% of esterified cholesterol in small very low density lipoproteins was derived from chylomicron degradation. Apparently, nearly two-thirds of the esterified cholesterol in total very low density lipoproteins from moderately hypercholesterolemic rabbits is of dietary origin.     

Regulation of lipogenesis in isolated hepatocytes by triglyceride-rich lipoproteins.

Very low density lipoproteins, chylomicrons, and remnants caused, within an hour, significant inhibition of fatty acid synthesis but not cholesterol synthesis in hepatocytes isolated from meal-fed rats. In contrast, low density lipoproteins, high density lipoproteins, and the serum fraction of density greater than 1.21 failed to significantly inhibit either fatty acid or cholesterol synthesis within 1 h. The Scatchard plots of specific binding showed that rat and human very low density lipoproteins interact with the high affinity sites on the hepatocytes with the apparent dissociation constants of 64 and 106 nM, respectively. These data also indicated that each hepatocyte was capable of binding 6 X 10(5) molecules of very low density lipoproteins.     

The role of the female Syrian hamster protein on the interaction between serum lipoproteins and heparin

We have previously shown the effect of phosphorylcholine-binding proteins from rat (PCBP) and rabbit (CRP) on the precipitation of serum lipoproteins by heparin in presence of Ca2+. The present paper describes the effect of a phosphorylcholine-binding protein from the female Syrian hamster (FP) on the lipoprotein precipitation reaction. The precipitation of lipoproteins by heparin was lower in assays using female hamster serum in which FP is a prominent protein, compared with assays with male serum in which FP is present in very low concentration. Depletion of FP from female serum resulted in increased lipoprotein precipitation. The addition of purified FP to assays using human very low density lipoprotein (VLDL) inhibited the precipitation reaction. The precipitation of lipoproteins was also examined using serum from male hamsters treated with diethylstilbestrol and female hamsters treated with testosterone, treatments known to modulate the levels of FP. Results indicate an inverse relationship between serum FP levels from normal and hormone-treated hamsters and the precipitation of lipoproteins from their serum. The partially desialylated FP when added to precipitation assays using human VLDL resulted in reduced inhibition of VLDL precipitation.     

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.     

Large lipoproteins are excluded from the arterial wall in diabetic cholesterol-fed rabbits

In diabetic hypercholesterolemic rabbits at plasma triglyceride concentrations of approximately 5000 mg/dl, 55% of plasma cholesterol (1400 mg/dl) was in lipoproteins with diameters larger than 75 nm (Sf greater than 400), 40% in smaller very low density and intermediate density lipoproteins, 4% in low density lipoproteins, and 1% in high density lipoproteins. Specific intimal clearance (nl/h.mg aortic cholesterol) of the giant Sf greater than 400 lipoproteins was about 4% of that of the low density lipoproteins. The data suggest that even very low density lipoproteins with diameters smaller than 75 nm were practically excluded from entering the arterial wall. Specific intimal clearance of low density lipoproteins in hypertriglyceridemic, diabetic cholesterol-fed rabbits was similar to that in normal cholesterol-fed rabbits, but low density lipoprotein concentrations in diabetic rabbits were low. Thus, at plasma triglyceride concentrations of approximately 5000 mg/dl, only 5% of plasma cholesterol may be readily available for infiltration of arteries. These results add further support to the hypothesis that hypertriglyceridemic, diabetic cholesterol-fed rabbits are protected against atherogenesis because the major part of plasma cholesterol is carried in large lipoproteins to which the artery is not very permeable.     

Intestinal lipoprotein synthesis. Comparison of nascent Golgi lipoproteins from chow-fed and hypercholesterolemic rats

Hypercholesterolemia, induced by a cholesterol-enriched diet, is associated with distinctive modifications in the serum lipoproteins of a variety of species. Present in the serum of these animals are several classes of lipoproteins enriched in cholesteryl esters and apolipoprotein E. To investigate the role of intestinal lipoprotein synthesis in diet-induced hypercholesterolemia, we characterized nascent lipoproteins retrieved from Golgi apparatus-rich fractions of intestinal epithelial cells from chow-fed control and hypercholesterolemic rats. To eliminate chylomicrons from the preparations, rats were fasted overnight prior to the experiments. Golgi very low density lipoproteins (d less than 1.006 g/ml) from control rats were triglyceride-rich lipoproteins that migrated slightly slower than pre-beta migrating serum very low density lipoproteins. These particles contained apoproteins B-240, A-IV, and A-I. Golgi very low density lipoproteins from hypercholesterolemic rats were likewise triglyceride-rich lipoproteins migrating electrophoretically like control Golgi very low density lipoproteins and they contained apoproteins B-240, A-IV, and A-I. However, these latter particles contained less triglyceride and more cholesterol compared to control Golgi very low density lipoproteins. In addition, by radioisotope incorporation studies, Golgi very low density lipoproteins from hypercholesterolemic rats contained relatively more apoprotein A-IV (21.6 vs. 11.0%) and less apoprotein B-240 (17.0 vs. 27.0%) than found in control Golgi very low density lipoproteins. Approximately 60% of the total apoprotein radioactivity was found in apoprotein A-I in both preparations. We conclude that intestinal lipoprotein synthesis is modified by diet-induced hypercholesterolemia. The significance of these modifications with respect to the marked hypercholesterolemia observed in these animals remains to be determined.     

Transport of poly-β-hydroxybutyrate in human plasma

Poly-β-hydroxybutyrate (PHB) is an amphiphilic lipid that has been found to be a ubiquitous component of the cellular membranes of bacteria, plants and animals. The distribution of PHB in human plasma was investigated using chemical and immunological methods. PHB concentrations proved highly variable; in a random group of 24 blood donors, total plasma PHB ranged from 0.60 to 18.2 mg/l, with a mean of 3.5 mg/l. In plasma separated by density gradient ultracentrifugation, lipoproteins carried 20–30% of total plasma PHB; 6–14% in the very low density lipoproteins (VLDL), 8–16% in the low density lipoproteins (LDL), and < 3% in the high density lipoproteins (HDL). The majority of plasma PHB (70–80%) was found in protein fractions of density > 1.22 g/ml. Western blot analysis of the high density fractions with anti-PHB F(ab')₂ identified albumin as the major PHB-binding protein. The affinity of albumin for PHB was confirmed by in vitro studies which demonstrated transfer of ¹⁴C-PHB from chloroform into aqueous solutions of human and bovine serum albumins. PHB was less tightly bound to LDL than to other plasma components; the polymer could be isolated from LDL by extraction with chloroform, or by digestion with alkaline hypochlorite, but it could not similarly be recovered from VLDL or albumin. PHB in the LDL correlated positively with total plasma cholesterol and LDL cholesterol, and negatively with HDL cholesterol. The wide concentration range of PHB in plasma, its presence in VLDL and LDL and absence in HDL, coupled with its physical properties, suggest it may have important physiological effects.     

Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma

Clusterin/human complement lysis inhibitor (CLI) is incorporated stoichiometrically into the soluble terminal complement complex and inhibits the cytolytic reaction of purified complement components C5b-9 in vitro. Using an anti-clusterin affinity column, we found that an additional protein component with a molecular mass of 28-kDa co-purifies with clusterin from human plasma. We show by immunoblotting and amino acid sequencing that this component is apolipoprotein A-I (apoA-I). By using physiological salt buffers containing 0.5% Triton X-100, apoA-I is completely dissociated from clusterin bound to the antibody column. Free clusterin immobilized on the antibody-Sepharose selectively retains apoA-I from total human plasma. Delipidated apoA-I and to a lesser extent ultracentrifugation-purified high density lipoproteins (HDL) adsorbed to nitrocellulose also have a binding affinity for purified clusterin devoid of apoA-I. The isolated apoA-I-clusterin complex contains approximately 22% (w/w) lipids which are composed of 54% (mole/mol) total cholesterol (molar ratio of unesterified/esterified cholesterol, 0.58), 42% phospholipids, and 4% triglycerides. In agreement with the low lipid content, apoA-I-clusterin complexes are detected only in trace amounts in HDL fractions prepared by density ultracentrifugation. In free flow isotachophoresis, the purified apoA-I-clusterin complex has the same mobility as the native clusterin complex in human plasma and is found in the slow-migrating HDL fraction of fasting plasma. Our data indicate that clusterin circulates in plasma as a HDL complex, which may serve not only as an inhibitor of the lytic terminal complement cascade, but also as a regulator of lipid transport and local lipid redistribution.     

Immobilization of L-glyceryl phosphorylcholine: isolation of phosphorylcholine-binding proteins from seminal plasma

The preparation of an affinity sorbent containing immobilized L-glyceryl phosphorylcholine for affinity chromatography of phosphorylcholine-binding proteins from seminal plasma is described. The ligand was coupled either after its maleinylation to poly(acrylamide-allyl amine) copolymer or directly to divinyl sulfone-activated Sepharose. The prepared phosphorylcholine derivative coupled to Sepharose was used for affinity chromatography of phosphorylcholine-binding proteins from bull and boar seminal plasma. Adsorbed proteins were specifically eluted with phosphorylcholine solution. Isolated phosphorylcholine-binding proteins were characterized by SDS electrophoresis and HPLC with reversed phase. Composition of the boar phosphorylcholine-binding fraction obtained by affinity chromatography on immobilized L-glyceryl phosphorylcholine was compared with that eluted from immobilized heparin by the phosphorylcholine solution. No phosphorylcholine-binding proteins were found in human seminal plasma.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.     

The levels of apolipoprotein-E in hypercholesterolemic rat serum.

The levels of apolipoprotein-C (apo-E) in serum and isolated liproproteins from diet-induced hypercholesterolemic, and to some extent hypertriglycerdemic rats were measured by electroimmunoassay. The hypocholesterolemia was accompanied by a mild hypertriglyceridemia. The apo-E was increased by 60% in the hypercholesterolemic serum with a 5- and 50-fold increase in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) respectively. However, the proportion of apo-E in nascent VLDL isolated from the hepatic Golgi apparatus of hypercholesterolemic rats was significantly decreased. In control serum, 40--50% of the apo-E is found in the density greater than 1.21 g/ml fraction, although this is at least partially due to ultracentrifugation. The aproprotein is absent from the density greater than 1.21 g/ml fraction from hypercholesterolemic serum, suggesting that it is bound more firmly to the lipoprotein complex. It is concluded that the large increases in apo-E in the VLDL and LDL density ranges of serum from hypercholesterolemic rats may in part be accounted for by the utilization of apo-E normally found at higher densities.     

A novel explanation for the reduced LDL cholesterol in severe hypertriglyceridemia

When [3H]cholesteryl ester-labeled low density (LDL) and intermediate density lipoproteins (IDL) from a normotriglyceridemic, hypercholesterolemic rabbit were injected into severely hypertriglyceridemic, hypercholesterolemic rabbits, 60% of the label appeared in very low density lipoproteins (VLDL) at 3 hr. A similar experiment showed that 40% of injected 131I-protein-labeled LDL appeared in the IDL fraction at 4 hr. Taken together, these data suggest that the exchange of LDL cholesteryl ester for VLDL triglyceride results in a density shift of injected LDL to the IDL density range. Furthermore, the percent of injected 131I-labeled LDL from normotriglyceridemic rabbits that appeared in the IDL fraction increased in rabbits with increasing levels of plasma triglyceride. This LDL density shift was reproduced in vitro by incubating iodinated LDL from normotriglyceridemic, hypercholesterolemic rabbits with concentrations of VLDL from hypertriglyceridemic, hypercholesterolemic rabbits similar to those in plasma. With such a system, it was shown that the percentage of LDL that appeared in the IDL fraction increased with time, was enhanced fourfold by the addition of plasma lipid transfer protein, increased with increasing molar ratio of triglyceride to cholesteryl ester in VLDL, but apparently did not increase with increasing VLDL particle number. These studies suggest that a pronounced decrease in density of lipoproteins that would normally appear in the LDL density range, resulting from loss of cholesteryl ester in exchange for VLDL triglyceride, may explain, at least in part, the reduced LDL levels in severe hypertriglyceridemia.     

Metabolism by cells in culture of low-density lipoproteins of abnormal composition from non-human primates with diet-induced hypercholesterolemia

Diet-induced hypercholesterolemia in non-human primates results in the production of a low-density lipoprotein (LDL) of abnormal size and composition. This LDL from hypercholesterolemic monkeys has been shown to be more atherogenic than the same amount of LDL from normocholesterolemic animals. Previous studies have demonstrated that hypercholesterolemic LDL is approximately twice as effective as normal LDL in stimulating cholesterol accumulation and esterification in arterial smooth muscle cells in culture. The purpose of the present study was determine whether this effect was secondary to differences in metabolism of the normal and hypercholesterolemic LDL. for this, the metabolism of 125I-labeled normal and hypercholesterolemic LDL from rhesus and cynomolgus monkeys was compared in several lines of skin fibroblasts and smooth muscle cells. Both normal and hypercholesterolemic LDL bound with high affinity to the same cell surface receptor. However, the affinity for binding of hypercholesterolemic LDL was about twice that of normal LDL (apparent dissociation constant for binding, Kd, was 2.63 micrograms protein/ml and 4.35 micrograms protein/ml, respectively). Conversely, only about 50% as many particles of hypercholesterolemic were able to bind to the receptor, compared with normal LDL. Those cells with the greatest capacity to metabolize LD generally accumulated the most cholesterol with either hypercholesterolemic or normal LDL. In all cell lines, nearly twice as much cholesterol accumulated in cells incubated with hypercholesterolemic LDL compared with normal LDL, and this differential could not be explained by differences in metabolism of the two lipoproteins, suggesting that some cholesterol entered the cells independent of the uptake of the intact LDL molecule. LDL receptors appear necessary for this to occur, since no difference in cholesterol accumulation was observed in cells genetically deficient in LDL receptors.     

C-reactive protein binds to the 3beta-OH group of cholesterol in LDL particles

C-reactive protein (CRP) has been suggested to contribute to the development of atherosclerosis. We previously found binding of CRP to cholesterol in modified low density lipoprotein (LDL) particles. Here, we characterize the interaction between CRP and cholesterol in more detail. When lipids of native LDL were separated by thin-layer chromatography, CRP bound only to cholesterol. When various cholesterol analogues were compared for their ability to bind CRP, we found that any modification of the 3beta-OH group blocked binding of CRP to cholesterol. Similarly, enrichment of LDL with cholesterol but not with its analogues triggered the binding of CRP to LDL. Finally, with the aid of anti-CRP monoclonal antibodies and by molecular modeling, we obtained evidence for involvement of the phosphorylcholine-binding site of CRP in cholesterol binding. Thus, CRP can bind to cholesterol, and the interaction is mediated by the phosphorylcholine-binding site of CRP and the 3beta-hydroxyl group of cholesterol.