Heparin binding to lipoprotein lipase and low density lipoproteins

Heparin was fractionated on an affinity column of bovine milk lipoprotein lipase (LpL) immobilized to Affi-Gel-15. The bound heparin, designated high-reactive heparin (HRH), enhanced LpL activity, presumably by stabilizing the enzyme against denaturation. The unbound heparin fraction had no observable effect on the initial rate of enzyme activity. However, at longer times of incubation there was inhibition of LpL activity. LpL-specific HRH also showed a high, Ca²⁺-dependent precipitating activity towards human plasma low density lipoproteins (LDL). Since LpL and LDL both bind to heparin-like molecules at the surface of the arterial wall, we suggest that their similar heparin-binding specificity may have physiological consequences as it relates to the development of atherosclerosis.

Heparin binding Lipoprotein lipase LDL Apolipoprotein Lipolysis     

Dermatan sulfate and chondroitin 6-sulfate conformations

X-ray diffraction patterns show that dermatan sulfate in oriented, crystalline films occurs as two or three or eight-fold helices. The two-fold helix has a greater axial rise per disaccharide residue $\text{[}\text{h}\text{= 9.6}\text{A}\text{?}\text{]}$ than the corresponding chondroitin 6-sulfate helix $\text{[}\text{h}\text{= 9.3}\text{A}\text{?}\text{]}$. Three-fold dermatan sulfate and chondroitin 6-sulfate helices both have $\text{h}\text{= 9.5}\text{A}\text{?}$. Consequently the α-L-iduronate residues in dermatan sulfate helices have the C1 chair conformation like β-D-glucuronate in chondroitin 6-sulfate. Since the eight-fold dermatan sulfate helix has $\text{h}\text{= 9.3}\text{A}\text{?}$ rather less than the eight-fold chondroitin 6-sulfate helix $\text{[}\text{h}\text{= 9.8}\text{A}\text{?}\text{]}$ the possibility of α-L-iduronate 1C chairs cannot be ruled out for it. Computer methods have been used to produce molecular models. In these the polysaccharide chains are almost linear. Each backbone conformation can accommodate a variety of arrangements of charged side groups.     

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.     

Very low density lipoprotein binding to cultured aortic endothelium

Because of very low density lipoprotein's (VLDL) potential atherogenicity and the demonstration that VLDL can bind to other cells, we examined the interaction of human VLDL with cultured porcine aortic endothelium. The lipoprotein-cell interaction had many properties similar to those seen with the binding of a ligand to a cell surface receptor. It was time and temperature dependent, saturable, and reversible. Scatchard analysis of competition data suggested that there may be more than one class of binding site. The affinity of the low affinity site was similar to that for low density lipoprotein (LDL). Also, the capacity of endothelial cells to bind VLDL was similar to that for LDL, when related to apo B (i.e., particle) concentration. Not only was unlabelled VLDL able to compete for VLDL binding sites, but so was LDL and high density lipoprotein (HDL). The maximal competition either by LDL or by HDL was less than that by VLDL. The maximal competition by HDL was more than by LDL. The VLDL binding was dependent on Ca2+. It was not changed by the content of lipoprotein in the medium in which cells were grown prior to the binding studies. These observations suggest that VLDL binding to endothelial cells is similar in some respects, but not in all, to the binding of LDL. Comparison of the data with endothelial cells to previous data with adipocytes also indicated differences between the interaction of these two cell types with VLDL. It is possible that this binding process may be involved in the formation of atherogenic remnants of triglyceride-rich lipoproteins on the endothelial surface of large blood vessels.     

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.     

The turnover of human plasma very low density lipoprotein protein.

The plasma decay of three groups of iodinated apoproteins on human very low density lipoproteins were evauluated in two normals, two subjects with endogenous hypertriglyceridemia and another two with dysbetalipoproteinemia. The apo beta decay was more rapid than that of the C apoproteins in all patients. The apo beta decay was more rapid for the normals than for either the subjects with hypertriglyceridemia or dysbetalipoprotenemia. The apo C protein had an irregular decay in the normals but decayed less irregularly for the hypertriglyceridemics. The arginine rich apoprotein had a decay somewhat similar to apo C protein in the normals. The apo beta protein of the alpha2 very low density lipoprotein of a dysbetalipoproteinemic was consistent with a precursor relationship to the apo beta of beta very low density lipoprotein of this subject, but the arginine rich apoprotein was not.     

The low density lipoprotein receptor

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

Characterization of low density lipoprotein binding to human adipocytes and adipocyte membranes

125I-labeled low density lipoprotein (LDL) binding to purified plasma membranes prepared from freshly isolated human adipocytes was saturable, specific, and displaceable by unlabeled ligand. The maximum specific binding capacity measured at saturating concentrations of 125I-LDL was 1.95 +/- 1.17 micrograms of LDL bound/mg of membrane protein (mean +/- S.D., n = 16). In contrast to cultured fibroblasts, specific binding of LDL to adipocyte membranes was calcium-independent, was not affected by EDTA or NaCl, and was not destroyed by pronase. Plasma membranes purified directly from homogenized adipose tissue also showed calcium-independent LDL specific binding (0.58 +/- 0.33 micrograms of LDL bound/mg of membrane protein, mean +/- S.D. n = 11). Specific binding, internalization, and degradation of 125I-methylated LDL was demonstrated in isolated adipocytes and competition experiments showed that native and methylated LDL interacted with adipocytes through some common recognition mechanism(s). Compared to native LDL, specific binding of methylated LDL to adipocyte membranes was significantly reduced (43%), indicating that interaction of LDL with adipocyte was dependent in part on the lysine residues of apolipoprotein B. LDL binding to adipocyte plasma membranes was also competitively inhibited by human high density lipoprotein subfractions HDL2 and HDL3. Thus, LDL metabolism in mature adipocytes appears to be regulated by mechanisms distinctly different from a variety of cultured mesenchymal cells. In addition, the ability of adipocytes to bind, internalize, and degrade significant amounts of methylated LDL supports the view that adipose tissue is involved in the metabolism of modified lipoproteins in vivo.     

Unesterified fatty acids inhibit the binding of low density lipoproteins to the human fibroblast low density lipoprotein receptor

Micromolar concentrations of oleate were found to inhibit reversibly the binding of low density lipoprotein (LDL) to the human fibroblast LDL receptor. The decrease in LDL binding caused a parallel reduction of both 125I-LDL uptake and degradation at 37 degrees C. At 4 degrees C, oleate was also found to displace 125I-LDL already bound to the LDL receptor. The effect of oleate was rapid, reaching 70-80% of maximum displacement with 5-10 min of incubation, and was closely correlated to oleate-albumin molar ratios. Partition analysis of unesterified fatty acids between cells and LDL showed that the inhibitory effect of oleate resulted mainly from an interaction of unesterified fatty acids with the cell surface rather than with the LDL particles. Using different unesterified fatty acids and fatty acid analogs, we found that the inhibitory effect was modulated by both the length and the conformation of the monomeric carbon chain and was directly dependent on the presence of a negative charge on the carboxylic group. At 4 degrees C, the inhibitory effect of oleate never exceeded half of maximum binding capacity. This limitation was associated with the ability of oleate to interact only with part of the population of LDL receptors which spontaneously recycles in the absence of ligand, as demonstrated by the fact that oleate did not induce any reduction of LDL binding after cell treatment with monensin in the absence of LDL. Our results indicate that unesterified fatty acids could participate in the control of LDL catabolism in vivo by direct modulation of the ability of LDL receptor to bind LDL.     

Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids

The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins.     

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.     

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.     

The beta very low density lipoprotein present in hepatic lipase deficiency competitively inhibits low density lipoprotein binding to fibroblasts and stimulates fibroblast acyl-CoA:cholesterol acyltransferase

Beta very low density lipoprotein (VLDL) was isolated from a patient with hepatic lipase deficiency. The particles were found to contain apolipoprotein B-100 (apoB) and apolipoprotein E (apoE) and were rich in cholesterol and cholesteryl ester relative to VLDL with pre beta electrophoretic mobility. These particles were active in displacing human low density lipoprotein (LDL) from the fibroblast apoB,E receptor and produced a marked stimulation of acyl-CoA:cholesterol acyltransferase. Treatment of intact beta-VLDL with trypsin abolished its ability to displace LDL from fibroblasts. Incubation of trypsin treated beta-VLDL with fibroblasts resulted in a significant stimulation of acyl-CoA:cholesterol acyltransferase activity. beta-VLDL isolated from a patient with Type III hyperlipoproteinemia and an apoE2/E2 phenotype had a higher cholesteryl ester/triglyceride ratio than the beta-VLDL of hepatic lipase deficiency and contained apoB48. It displaced LDL from fibroblasts to a small but significant extent. The Type III beta-VLDL stimulated acyl-CoA:cholesterol acyltransferase to a level similar to that of trypsin-treated beta-VLDL isolated from the hepatic lipase-deficient patient. These results demonstrate that the cholesterol-rich beta-VLDL particles present in patients with hepatic lipase deficiency are capable of interacting with fibroblasts via the apoB,E receptor and that this interaction is completely due to trypsin-sensitive components of the beta-VLDL. These particles were very effective in stimulating fibroblast acyl-CoA:cholesterol acyltransferase. This stimulation was due to both trypsin-sensitive and trypsin-insensitive components.     

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.     

Apolipoprotein E-mediated binding of hypertriglyceridemic very low density lipoproteins to isolated low density lipoprotein receptors detected by ligand blotting

HTG-VLDL1, like LDL, bind with high affinity to electrophoretically transferred, isolated LDL receptors partially purified from bovine adrenal glands. Ligand blotting techniques show that binding is calcium dependent; little or no binding of LDL or HTG-VLDL1 is observed in the presence of 10 mM EDTA. HTG-VLDL1 does not bind in the presence of 7 mM suramin, an inhibitor of LDL binding to the LDL receptor. Pretreatment of LDL with either thrombin or trypsin does not affect apoB-mediated LDL binding to the LDL receptor. ApoE-mediated binding of HTG-VLDL1 to the blotted LDL receptor is abolished or greatly decreased by thrombin treatment of HTG-VLDL1; trypsin treatment of HTG-VLDL1 abolishes binding. Reincorporation of apoE into trypsinized HTG-VLDL1 restores binding. These studies demonstrate unequivocally that HTG-VLDL1 bind to the LDL receptor, that the binding of HTG-VLDL1 to the isolated LDL receptor is mediated through the thrombin-accessible apoE, and that HTG-VLDL1 which bind via potentially dissociable apoE rather than non-transferable apoB can be used for ligand blotting.     

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

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

Low density lipoprotein binding induces asymmetric redistribution of the low density lipoprotein receptors in endothelial cells.

The uptake and transport of cholesterol-carrying low density lipoprotein (LDL) by the arterial wall is a continuous dynamic process, contributing to the cholesterol homeostasis in the plasma and in the cellular components of the vessel wall. Upon exposure to endothelial cells (EC), LDL interacts in part, with specific surface receptors (LDL-R). In this study we questioned: (i) the distribution of LDL receptors on the apical and basal cell membranes in endothelial cells; (ii) the role of LDL receptors in the control of cholesterol homeostasis and (iii) the translocation of LDL receptor across the EC. To this purpose bovine aortic EC were cultured on filters in a double-chamber system, in Dulbecco's medium supplemented either with 10% fetal calf serum (FCS) or with 10% lipoprotein-deficient serum (LPDS). The cells were exposed for 3h to 13H]acetate (40 microCi) added to both compartments of the cell culture inserts. The newly synthesized [3H]cholesterol was detected by thin layer chromatography and quantified by liquid scintillation counting. The LDL-R were detected in EC protein homogenates by immunoblotting using a monoclonal antibody against LDL-R (IgG-C7); the intracellular pathway of LDL-R was examined by electron microscopy using a complex made of protein A 5 nm or 20 nm colloidal gold particles and an anti-LDL receptor antibody (Au-PA-C7). To evaluate the distribution and the transport of LDL-R from one cell surface to the other, EC grown in LPDS were radioiodinated either on the apical or on the basolateral surface, incubated on the same surface with LDL, and subsequently biotinylated on the opposite non-radiolabeled surface. The EC were further solubilized and the protein extract immunoprecipitated with anti-LDL-R antibody or with mouse IgG (as control). The eluted antigen-antibody complexes were precipitated with streptavidin-agarose beads, solubilized, and subjected to SDS-PAGE. The results showed that: (a) the LDL-R were present on both endothelial cell fronts; (b) using the complex Au-PA-C7, the LDL-R were localized in endothelial plasmalemmal vesicles as well as coated pits and coated vesicles in multivesicular bodies and lysosomes, irrespective of the cell surface exposed to the complex; (c) biochemical assays indicated that upon ligand binding, the LDL-R were translocated preferentially from the apical to the basal plasma membrane.