Glycosaminoglycan-lipoprotein interactions: 3. Structure of dermatan sulphate variants with high affinity for low density lipoprotein

An iduronate-rich dermatan sulphate fraction was found to contain variants with high affinity for human low density lipoprotein. Digestion of a dermatan sulphate-lipoprotein complex with chondroitinase-ABC afforded fragments that contained between 10 and 20 disaccharide repeats. In this fragment half of the uronate residues were iduronate-sulphate. The non-sulphated iduronate-bearing repeats were interspersed among the repeats containing sulpho-iduronate. This feature is similar to that observed with heparin-related glycans having high affinity for lipoproteins.     

Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.     

Interactions between glycosaminoglycans and blood coagulation factors: 1. Affinity chromatography of glycosaminoglycans on thrombin-substituted agarose

Various glycosaminoglycans have been subjected to affinity chromatography on immobilized bovine thrombin. Chondroitin sulphate, dermatan sulphate and heparan sulphate variants with a sulphate-to-hexosamine molar ratio of ～ 1 exhibited weak affinities. Heparan sulphate/heparin fractions of higher sulphate content could be separated into material with high and low affinity for thrombin. Removal of N-sulphate followed by N-acetylation did not affect binding, whereas oxidation and cleavage of non-sulphated hexuronate abolished the interaction. Heparan-related molecules of high thrombin-affinity comprised sequences where large blocks of sulphated iduronate-containing repeats were joined via a few repeats carrying non-sulphated iduronate or glucuronate to form continuous segments that were larger than decasaccharide.     

Role of net charge of low density lipoproteins in high affinity binding and uptake by cultured cells.

Selective modification of arginine residues of LDL by cyclohexanedione or acetylation of lysine residues of LDL deminishes their high affinity binding and internalisation by human skin fibroblast up to 50% as compared with native LDL. The enhanced negative charge of the modified LDL particles results in an accelerated electrophoretic mobility towards the anode. Neuraminidase treatment of cyclohexanedione-modified LDL and acetyllysine-LDL normalizes not only their electrophoretic mobility, but also restores more than 80% of the original binding and uptake capacity, the specificity of this effect being indicated by using fibroblasts deficient in LDL receptor and by competitive binding and internalization experiments.     

Antioxidative activity on human low density lipoprotein (LDL) oxidation by pentagalloic acid

The aim of this study was to investigate the efficiency of the pentagalloic acid compound in inhibiting the metal ions and cell lines that mediate in low density lipoprotein (LDL) oxidation. Pentagalloic acid prolonged the lag time preceeding the onset of conjugated diene formation. In chemically induced LDL oxidation by Cu²⁺ plus hydrogen peroxide or peroxyl radical generated by 2, 2′-azo-bis (2-amidino propane) hydrochloride (AAPH), pentagalloic acid inhibited LDL oxidation as monitored by measuring the thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA), and gel electrophoretic mobility. The physiological relevance of the antioxidative activity was validated at the cellular level where pentagalloic acid inhibited mouse macrophage J774 and endothelial cell-mediated LDL oxidation. When compared with several other antioxidants, pentagalloic acid showed a much higher ability than naturally occuring antioxidants, α-tocopherol and ascorbic acid, and the synthetic antioxidant, probucol.     

Scavenger lipoprotein receptors are more effective in ligand internalization than low density lipoprotein receptors in human monocyte-macrophages

Human monocyte-macrophages in culture express specific receptors for low density lipoproteins (LDL receptor) and human acetylated LDL (AcLDL receptors or scavenger receptors). After 24 h in lipoprotein-deficient serum, the cells expressed 2-3 fold more AcLDL receptors than LDL receptors as measured by trypsin releasable radioactivity after exposure to 125I-LDL or 125I-AcLDL at 37 degrees C. The efficiency of intracellular ligand delivery by the two receptors was evaluated as an internalization index (defined as intracellular + degraded/bound ligand). This index was several fold greater for 125I-AcLDL than for 125I-LDL, in the same cells exposed to either ligand under identical conditions. These results suggest that the scavenger receptors recycle more rapidly than do LDL receptors.     

An x-ray small-angle-scattering study of the structure of trypsin treated low density lipoprotein from human serum.

X-ray small-angle-scattering curves were obtained from human serum low-density lipoprotein (LDL) from which some 20 % of the protein moiety had been removed by limited tryptic digestion, and the results are compared to those from the undigested lipoprotein. The basic structural features of LDL, i.e. a quasi-spherical shape and internal lipid compartmentation, were essentially unaltered by tryptic treatment. The trypsin-treated material exhibited the same thermotropic transition as the control. Our experiments indicate that proteolysis affected the outermost electrondense shell in a uniform manner, without causing any significant alterations in the radial symmetry of the internal molecular architecture. Since none of the structural regularities observed in the real-space pair distance distribution are affected, in particular the 3.6 nm periodicity below 15°C, it is concluded that these regularities do not relate to the protein arrangement in LDL.     

CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis

Atherosclerotic plaques result from the excessive deposition of cholesterol esters derived from lipoproteins and lipoprotein fragments. Tissue macrophage within the intimal space of major arterial vessels have been shown to play an important role in this process. We demonstrate in a transfection system using two human cell lines that the macrophage scavenger receptor CD36 selectively elicited lipid uptake from Cu(2+)-oxidized high density lipoprotein (HDL) but not from native HDL or low density lipoprotein (LDL). The uptake of oxHDL displayed morphological and biochemical similarities with the CD36-dependent uptake of oxidized LDL. CD36-mediated uptake of oxidized HDL by macrophage may therefore contribute to atheroma formation.     

Interaction between heparan sulphate chains II. Structural characterization of iduronate- and glucuronate-containing sequences in aggregating chains

A heparan sulphate fraction (uronic acid composition: 20% sulphated iduronate, 15% iduronate and 65% glucuronate of total uronate) was separated into aggregating and non-aggregating chains by gel chromatography. ¹³C-NMR analyses revealed that non-aggregating chains had a higher degree of sulphation than did aggregating chains. In aggregating chains, there was more N-acetyl-glucosamine than N-sulphamidoglucosamine; the extent of C-6 sulphation of the latter moiety was low and most of the iduronate residues were non-sulphated. In non-aggregating chains, the N-acetyl-to-N-sulphate ratio was approx. 2 : 1, the N-sulphated glucosamines were also largely C-6 sulphated and the sulphated iduronates were concentrated to these species.Both preparations were subjected to deaminative cleavage which produces fragments like uronate-(N-acetylglucosamine-uronate)_n-anhydromannose. Tetrasaccharides (n = 1) were further fractionated into non-, mono-, di- and trisulphated species by ion-exchange chromatography. The tetrasaccharides have the general carbohydrate structure uronate-N-acetylglucosamine-glucuronate-anhydromannose. Non-reducing terminal glucuronate was removed by β-glucuronidase. The results showed that saccharides containing glucuronate in both positions were more prevalent in the products of aggregating chains. The β-glucuronidase-resistant saccharides (carrying either sulphated or non-sulphated iduronate in non-reducing terminal position) were oxidised with periodate under conditions where non-sulphated residues are degraded, whereas sulphated residues are resistant. Mono-sulphated and di-sulphated tetrasaccharides from aggregating chains were extensively degraded indicating that iduronate-N-acetylglucosamine-glucuronate-anhydromannose was the major sequence.In saccharides from non-aggregating chains iduronate was frequently sulphated. The results of this and previous investigations (Fransson, L.-Å., Nieduszynski, I.A. and Sheehan, J.K. (1980) Biochim. Biophys. Acta 630, 287–300) indicate that an alternating arrangement of iduronate and glucuronate in aggregating chains is present both in N-sulphated block regions and in regionsthat carry alternating N-acetyl- and N-sulphated glucosamine.     

Interaction of very low density lipoprotein with chicken oocyte membranes

The interaction of hen 125I-VLDL (very low density lipoprotein) with chicken oocyte membranes was characterized using a rapid sedimentation assay. Equilibrium and kinetic studies showed an apparent dissociation constant (Kd) 8.7-9.1 x 10(-8) M or 43.5-45.5 micrograms VLDL protein/ml. Binding capacity was 2.0 micrograms VLDL protein/mg membrane homogenate protein. The apparent rate constants were k1 = 2.4 x 10(5) M-1 min-1 and k2 = 2.1 x 10(-2) min-1. Specific binding required the presence of divalent cations. Whereas binding was completely restored after treatment with EDTA by the addition of MN++, only 60% of binding was restored using Ca++.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

Molecular parameters that control the association of low density lipoprotein apo B-100 with chondroitin sulphate

The association of low density lipoprotein (LDL) with proteoglycans of the intima, in particular chondroitin 6-sulphate proteoglycans, may contribute to LDL accumulation during atherogenesis. We studied the interactions of apolipoprotein B-100 (apo B-100) peptide segments and model peptides with chondroitin 6-sulphate. The ability of these peptides to inhibit complex formation between LDL and chondroitin 6-sulphate was used as a measurement of the interaction. Results from earlier studies suggest that surface located segments of apo B-100 are responsible for the interaction of LDL with heparin and chondroitin sulphate-rich arterial proteoglycans. Therefore 16 hydrophilic apo B-100 peptides were selected for studies and synthesized with a peptide synthesizer. These synthetic peptides were 7 to 26 amino acids long. Four of the peptides inhibited the association of LDL with chondroitin 6-sulphate, namely apo B segments 4230–4254, 3359–3377, 3145–3157 and 2106–2121. The 3359–3377 segment was the most efficient. A common feature betweeb the interacting peptides was an excess of positively charged side chains and based on these results we synthesized nine model peptides that shared sequence characateristics with the interacting apo B-100 peptides. Five of these: RSGRKRSGK, RSSRKRSGK, RGGRKRGGK, RSRSRSRSR AND RGRGRGRGR were shown to block the LDL-chrondroitin-6-sulphate association, RSRSRSRSR being the most effective. The results suggest that the optimal association of the peptides with chrondroitin 6-sulphate is obtained with a minimal chain length of nine amino acids and a minimum of five positive charges and that flexibility in the binding region is important.     

Release of low density lipoprotein receptors from human fibroblasts or HeLa cells by tryptic digestion.

Trypsin treatment of cultured normal human skin fibroblasts or Hela cells releases material which is retained on a low density lipoprotein (LDL)-Sepharose affinity column, may be eluted from it with 2.5 M KI and, after dialysis, agglutinates LDL or apo-B-coated formocells. Such agglutination is prevented by preincubation of the receptor with LDL in solution or with arginine-rich protamine. Trypsin treatment of “receptor defective” or “receptor negative” mutant fibroblasts releases material which is retained on LDL-Sepharose column but fails to agglutinate LDL-coated formocells. The receptor may be labeled with $\text{6-[}{}^3\text{H]-glucosamine·HCl}$ and [${}^3\text{H}$]-leucine, it is inactivated by heating at 80°C for 10 min and may be obtained from normal fibroblasts or Hela cells, whether they were cultured in presence or in absence of lipoprotein-containing fetal calf serum.     

Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts

The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion.Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of ¹²⁵I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on ¹²⁵I-labelled LDL metabolism.Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of ¹²⁵I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.     

The carbohydrate content of apolipoprotein E from human very low density lipoproteins.

The carbohydrate content of the E protein of human very low density lipoprotein (VLDL) was evaluated both by colorimetric methods and by gas liquid chromatography of the trifluoroacetylated 0-methyl glycosides. The major unmodified hexose was noted to be galactose with a mole ratio with respect to protein which ranged from 0.81 to 1.54. N-acetyl glucosamine (molar ratios from 0.52 to 1.76) and N-acetyl galactosamine (molar ratios from 0.73 to 1.59) and the respective unacetylated amino sugars were noted for all of the apoproteins evaluated. Sialic acid (molar ratios from 0.79 to 1.69) was a prominent carbohydrate for each of the E protein preparations. When the apoprotein was exposed to neuraminidase with a resultant loss of two-thirds of the sialic acid, the isoelectric focus behavior was found to be unchanged. The E protein isolated from the very low density lipoproteins of Type III patients (dysbetalipoproteinemia) revealed a carbohydrate content similar to the normals or Type IV patients.     

Identification of nascent high density lipoproteins containing arginine-rich protein in human plasma.

A high density lipoprotein fraction accumulates in the plasma of patients with alcoholic hepatitis when a severe lecithin:cholesterol acyltransferase (EC 2.3.1.43) deficiency is present. The major apoprotein present in this fraction is arginine-rich protein, the fraction is a preferred substrate for lecithin:cholesterol acyltransferase, and by electron microscopy appears as stacked bilayer discs. It is proposed that the lipoprotein represents the accumulation of nascent high density lipoprotein and is the principal pathway through which arginine-rich protein is secreted by the liver in man. The results also suggest that apoprotein AI is acquired by normal high density lipoprotein during the course of lipoprotein metabolism.     

Comparative study of Ca2+ binding to lipoprotein(a) and low density lipoprotein by spin labeling

The effect of Ca2+ binding on the dynamic properties of various spin labeled fatty acids in lipoprotein(a) (Lp(a)) was studied in comparison with low density lipoprotein (LDL) isolated from human plasma. In contrast to LDL, binding of Ca2+ to Lp(a) induced broadening of the lines in the ESR spectra of the spin labeled stearic acids. In 1.6 M NaBr solutions the thermotropic change in the surface structure was observed in both lipoproteins at similar temperatures. Ten millimolar concentration of Ca2+ shifted the temperature of the thermotropic change in the surface structure of Lp(a) to considerably higher values. We conclude that Ca2+ binding to Lp(a) induces changes in the lipid structure of the particle surface.