Biomedical subjects

S Lund-Katz

Publications and source records attributed to S Lund-Katz.

At least 37 records · Page 2Linked to original sources

PubMed1998

Dietary modification of high density lipoprotein phospholipid and influence on cellular cholesterol efflux.

African green monkeys fed fat-specific diets served as a model to investigate the effect of phospholipid acyl chain modification on high density lipoprotein (HDL)-mediated cellular cholesterol efflux. Diets enriched in saturated, monounsaturated, n-6 polyunsaturated, or n-3 polyunsaturated fats were provided during both low cholesterol and cholesterol-enriched stages; sera and HDL3 samples were obtained at specific points during the treatment period. Analysis of the HDL phospholipid composition revealed significant acyl chain modification, consistent with the respective fat-specific diet. Cholesterol efflux from mouse L-cell fibroblasts to HDL3 isolated from the specific diet groups was measured and revealed no differences in the abilities of the particles to accept cellular cholesterol; determination of the bidirectional flux of cholesterol between the cells and HDL3 species further demonstrated no effect of phospholipid acyl chain modification on this process. The effects of dietary modification of phospholipid acyl chains on cellular cholesterol efflux were directly examined by isolating the HDL phospholipid and combining it with human apolipoprotein A-I to form well-defined reconstituted HDL particles. These complexes did not display any differences with respect to their ability to stimulate cellular cholesterol efflux. Incubations with 5% sera further confirmed that the fat-specific diets do not influence cholesterol efflux. These results suggest that the established influences of specific dietary fats on the progression of atherosclerosis are due to effects on cholesterol metabolism other than the efflux of cellular cholesterol in the first step of reverse cholesterol transport.

Animals↗

PubMed1997

Truncation of the amino terminus of human apolipoprotein A-I substantially alters only the lipid-free conformation.

An amino-terminal deletion mutant (residues 1-43) of human apolipoprotein A-I (apo hA-I) has been produced from a bacterial expression system to explore the structural and functional role of these amino acids, encoded by exon 3, in apo hA-I. Lipid binding of apo delta (1-43)A-I and lipid binding of apo hA-I are very similar as assessed by surface activity, lipid association with palmitoyloleoylphosphatidylcholine (POPC) vesicles, and lipid association with plasma lipoproteins. Preliminary kinetic measurements appear to show that the reactivity of lecithin:cholesterol acyltransferase (LCAT) with the mutant is slightly decreased compared to wild-type apo hA-I. Collectively, these results indicate that the N-terminal region is not necessary for lipid binding or activation of LCAT. In contrast, there are significant structural differences between lipid-free apo delta (1-43)A-I and apo hA-I, as judged by denaturant-induced unfolding, binding of the fluorescent probe 1-anilinonaphthalene-8-sulfonate, surface balance measurements, and far- and near-ultraviolet circular dichroic spectroscopy. All spectral and physical measurements indicate apo delta (1-43)A-I has a folded, tertiary structure, although it is significantly less stable than that of apo hA-I. It is concluded that the N-terminal 43 residues are an important structural element of the lipid-free conformational state of apo hA-I, the absence of which induces a fundamentally different fold for the remaining carboxy-terminal residues, compared to those in native apo hA-I.

Anilino Naphthalenesulfonates↗

PubMed1997

Peroxidation of LDL from combined-hyperlipidemic male smokers supplied with omega-3 fatty acids and antioxidants.

The effects of marine omega-3 polyunsaturated fatty acids (FAs) and antioxidants on the oxidative modification of LDL were studied in a randomized, double-blind, placebo-controlled trial. Male smokers (n = 41) with combined hyperlipidemia were allocated to one of four groups receiving supplementation with omega-3 FAs (5 g eicosapentaenoic acid and docosahexaenoic acid per day), antioxidants (75 mg vitamin E, 150 mg vitamin C, 15 mg beta-carotene, and 30 mg coenzyme Q10 per day), both omega-3 FAs and antioxidants, or control oils. LDL and human mononuclear cells were isolated from the patients at baseline and after 6 weeks of supplementation. LDL was subjected to cell-mediated oxidation by the patients' own mononuclear cells, as well as to Cu(2+)-catalyzed and 2,2'-azobis-(2-amidinopropane hydrochloride) (AAPH)-initiated oxidation. Extent of LDL modification was measured as lag time, the formation rate of conjugated dienes (CDs), the maximum amount of CDs formed, formation of lipid peroxides, and the relative electrophoretic mobility of LDL on agarose gels. Dietary supplementation with omega-3 FAs increased the concentration of total omega-3 FAs in LDL and reduced the concentration of vitamin E in serum. The omega-3 FA-enriched LDL particles were not more susceptible to Cu(2+)-catalyzed, AAPH-initiated, or autologous cell-mediated oxidation than control LDL. In fact, enrichment with omega-3 FAs significantly reduced the formation rate of CDs when LDL was subjected to AAPH-induced oxidation. Supplementation with moderate amounts of antioxidants significantly increased the concentration of vitamin E in serum and increased the resistance of LDL to undergo Cu(2+)-catalyzed oxidation, measured as increased lag time, reduced formation of lipid peroxides, and reduced relative electrophoretic mobility compared with control LDL. Supplementation with omega-3 FAs/antioxidants showed oxidizability of LDL similar to that of control LDL and omega-3 FA-enriched LDL. In conclusion, omega-3 FAs neither rendered the LDL particles more susceptible to undergo in vitro oxidation nor influenced mononuclear cells' ability to oxidize autologous LDL, whereas moderate amounts of antioxidants protected LDL against oxidative modification.

Administration, Oral↗

PubMed1997

Interaction of class A amphipathic helical peptides with phospholipid unilamellar vesicles.

The exchangeable apolipoproteins are important in determining the structure/function properties of lipoproteins. These proteins typically contain varying amounts of amphipathic helices. Five model peptides, 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA, have been designed to investigate variations of the amphipathic alpha-helix structural motif on their lipid-binding properties. These include the 18-residue peptides, 18A and Ac-18A-NH2, examples of class A helices, and Ac-18R-NH2, which has the positions of acidic and basic residues interchanged relative to 18A. Three larger peptides were also studied: 36A, a dimer of 18A, 37pA and 37aA, dimers of 18A coupled by Pro (18A-Pro-18A) and Ala (18A-Ala-18A), respectively. We report here the results of a thermodynamic characterization of the binding properties of these peptides to small unilamellar vesicles of POPC. Partition coefficients, Kp, were determined by fluorescence spectroscopy and binding enthalpies, deltaH, by titration calorimetry. These parameters were used to obtain the free energies, deltaG0, and entropies, deltaS0, of binding. The results of this study indicate Kp values on the order of 10(5), with interactions being enthalpically but not entropically favored in all cases. The presence of positively charged residues at the interface (18A and Ac-18A-NH2) enhances binding but has little effect on the extent of bilayer penetration. The presence of tandem repeats decreases lipid affinities for these small, highly curved bilayers. Our results are consistent with the idea that interaction appears to be confined largely to the surface, with some degree of penetration of the hydrophobic face of the helix into the interior of the bilayer.

Calorimetry↗

PubMed1997

Effect of vesicle size on their interaction with class A amphipathic helical peptides.

Throughout the life span of a lipoprotein particle, the type and number of exchangeable apolipoproteins on its surface varies with particle size, suggesting a role of surface curvature on the lipid-binding properties of these proteins. Peptides 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA have been designed to investigate the lipid-binding properties of the amphipathic alpha-helix structural motif that appears to modulate the lipid-binding properties of the exchangeable plasma apolipoproteins. We report here the results of a quantitative thermodynamic characterization of the effects of modifying helix length and of varying both the location of charged residues about the polar face of the peptides and vesicle size on the lipid affinity and depth of bilayer penetration for model amphipathic alpha-helices. Partition coefficients, Kp, were determined by fluorescence spectroscopy, and binding enthalpies, deltaH, by titration calorimetry. The results indicate that Kp values are on the order of 10(5), with similar deltaG(o) values for the interactions of the peptides with vesicles of various sizes. It appears that a class A motif and increased alpha-helical content optimize binding for 18-residue peptides. The interactions of the model peptides with 20 nm SUV are enthalpically driven with small, negative entropy changes; however, interactions for larger vesicles are entropically driven, likely due to disordering of bilayer hydrocarbon chains. Thermodynamic data indicate that 37pA and 37aA induce greater disordering of bilayer hydrocarbon chains than Ac-18A-NH2. The results of this study suggest that the type of interaction, i.e., enthalpically or entropically driven, may be modulated by the lateral compressibility of the bilayer membrane.

Acrylamide↗

PubMed1996

Apolipoprotein A-I structural modification and the functionality of reconstituted high density lipoprotein particles in cellular cholesterol efflux.

The role of HDL and its major protein constituent, apolipoprotein (apo) A-I, in promoting the removal of excess cholesterol from cultured cells has been well established; however, the mechanisms by which this occurs are not completely understood. To address the effects of apoA-I modification on cellular unesterified (free) cholesterol (FC) efflux, three recombinant human apoA-I deletion mutants and plasma apoA-I were combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal complexes. These particles were characterized structurally and for their efficiency as acceptors of mouse L-cell fibroblast cholesterol. The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta44-126)), central (apoA-I (Delta139-170)), or COOH-terminal (apoA-I (Delta190-243)) domains of apoA-I. The three deletion mutants all displayed lipid-binding abilities and formed discoidal complexes that were similar in major diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when reconstituted at a 100:5:1 (POPC:FC:protein) mole ratio. Gel filtration profiles indicated unreacted protein in the preparation made with apoA-I (Delta190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding. Measurements of the percent alpha-helix content of the proteins, as well as the number of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexes. The rHDL particles containing the deletion mutants had more molecules of protein present than particles containing intact apoA-I, to the extent that a similar number of helical segments was incorporated into each of the discoidal species. Comparison of the experimentally determined number of helical segments with an estimate of the available space indicated that the deletion mutant proteins are probably more loosely arranged than apoA-I around the edge of the rHDL. The abilities of the complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts. All four discoidal complexes displayed similar abilities to remove FC from the plasma membrane of L-cells when compared at an acceptor concentration of 50 microg of phospholipid/ml. Thus, none of the deletions imposed in this study notably altered the ability of the rHDL particles to participate in cellular FC efflux. These results suggest that efficient apoA-I-mediated FC efflux requires the presence of amphipathic alpha-helical segments but is not dependent on specific helical segments.

Animals↗

PubMed1996

Comparison of the structural and functional effects of monomeric and dimeric human apolipoprotein A-II in high density lipoprotein particles.

High density lipoprotein (HDL) is thought to play a significant role in the process of reverse cholesterol transport. It has become clear that the apolipoprotein (apo) composition of HDL is important in determining the metabolic fate of this particle. The major proteins of human HDL are apoAI and APOAII; the latter protein is a disulfide-linked dimer in humans and higher primates but monomeric in the other species. The consequences of the apo Cys6-Cys6 disulfide bridge in apoAII for human HDL structure and function are not known. To address this issue, the influence of the Cys6-Cys6 disulfide bridge on the interaction of human apoAII with palmitoyl-oleoyl phosphatidylcholine has been studied. The size and valence of a series of homogeneous discoidal complexes containing either monomeric (reduced and carboxymethylated) or dimeric apoAII have been determined, and their ability to remove cholesterol from rat Fu5AH hepatoma cells grown in culture has been compared. The apoAII dimer and monomer form discoidal complexes of similar size, with twice as many of the latter molecule required per disc. Removal of the disulfide bond influences the stability of the helical segments around the edge of the disc as seen by a decrease in alpha-helix content of the monomeric protein. The discoidal particles containing the monomeric form of apoAII are somewhat more effective than particles containing either dimeric apoAII or apoAI in removing cellular cholesterol. Overall, reduction of the disulfide bridge of apoAII probably does not have a major effect in the determination of HDL particle size in vivo. It follows that the evolution of the Cys6-Cys6 disulfide bond in higher primates probably has not had a major effect on the function of the apoAII molecule.

Animals↗

PubMed1996

Only the two end helixes of eight tandem amphipathic helical domains of human apo A-I have significant lipid affinity. Implications for HDL assembly.

Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid-associating properties of apo A-I, the first step is to understand the lipid-associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220-241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation.

Apolipoprotein A-I↗

PubMed1995

Effects of the neutral lipid content of high density lipoprotein on apolipoprotein A-I structure and particle stability.

Alterations in high density lipoprotein (HDL) composition that occur in dyslipidemic states may modulate a number of events involved in cholesterol homeostasis. To elucidate the details of how HDL-core composition can affect the molecular structure of different kinds of HDL particles, the conformation and stability of apoA-I have been investigated in homogeneous recombinant HDL particles (LpA-I) containing palmitoyloleoyl phosphatidylcholine (POPC), triolein (TG), and/or cholesteryl linoleate (CE). In a discoidal particle containing two molecules of apoA-I and 85 molecules of POPC, apoA-I exhibits an alpha-helix content of 70% and a free energy of stability of its alpha-helical segments (delta G0D) of 2.2 kcal/mol. Inclusion of eight molecules of TG into the complex significantly reduces the alpha-helix content and stability of apoA-I, whereas inclusion of four molecules of CE into the complex has an opposite effect in that the alpha-helix content is significantly reduced and the stability of the remaining alpha-helical structure of apoA-I is increased. Neutral lipids have a different effect on apoA-I conformation in spherical LpA-I particles. In a sonicated-spherical LpA-I particle containing two molecules of apoA-I and 70 molecules of POPC, apoA-I exhibits an alpha-helix content of about 60% and a delta G0D of 1.2 kcal/mol apoA-I. Inclusion of either 10 molecules of TG or six molecules of CE into such a particle increases both the alpha-helix content and stability of apoA-I. Increasing the CE/TG ratio in LpA-I particles that contain both neutral lipids enhances the stability of the alpha-helical segments. ApoA-I molecules tend to dissociate and cause particle instability when delta G0D for the lipid-bound alpha-helices is less than that for helices in the lipid-free state. The stabilities of both discoidal and spherical LpA-I particles are relatively low when the only neutral lipid present is TG but the particle stability is enhanced by the presence of CE molecules. Such dissociation of apoA-I molecules from LpA-I particles that have a low CE/TG ratio would be promoted in the hypertriglyceridemic state in vivo.

Apolipoprotein A-I↗

PubMed1995

Effects of acceptor particle size on the efflux of cellular free cholesterol.

Several subspecies of human high density lipoprotein (HDL) have been shown to exist, and particle size is one parameter that can be used to distinguish them. Recently, a small HDL subspecies has been described that may be a particularly efficient acceptor of peripheral cell unesterified (free) cholesterol (FC). To address the effects of particle size on the ability of HDL to remove FC from cells, homogeneous, well defined HDL particles were reconstituted (rHDL) that varied in particle diameter within the size range of human HDL particles (7-13 nm). The abilities of each of these particles to remove cellular FC from mouse L-cells and rat Fu5AH hepatoma cells were compared on the basis of their phospholipid (PL) content as well as on a per particle basis. The effect of particle size was also examined using small unilamellar vesicles (SUV) of 25 nm in diameter and large unilamellar vesicles (LUVs) of 70-180 nm in diameter. The SUV were prepared by sonication, and the LUVs were prepared by extrusion techniques. The FC efflux efficiency of these particles (in order of decreasing efficiency) was: rHDL > SUV > LUV when compared on the basis of acceptor PL content across a range of concentrations (i.e. at a given PL concentration for these three acceptor classes, smaller particles were more efficient). The FC efflux differences between the rHDL and the vesicles were not due to the absence of apolipoprotein in the vesicles. No difference was detected among the rHDL of varying size, nor was a difference detected among the LUVs of varying size when compared on the basis of PL content. When the FC efflux data for rHDL and LUVs were normalized on the basis of the number of acceptor particles present at a given PL concentration, a correlation was found between acceptor particle radius and the ability to accept cellular FC with larger particles being the most efficient. However, the dependence of the rate of FC efflux on acceptor particle size was not quantitatively the same within the rHDL and LUV classes of acceptor particles. The dependence of FC efflux on acceptor particle size may reflect differing abilities of the variously sized acceptor particles to access the region very close to the cell plasma membrane where most of the FC removal is expected to occur.

Animals↗

PubMed1995

Microenvironments of basic amino acids in amphipathic alpha-helices bound to phospholipid: 13C NMR studies using selectively labeled peptides.

The lipid-binding properties of serum apolipoproteins are mediated by class A amphipathic alpha-helices in which basic and acidic amino acid residues tend to be localized at the helix polar-nonpolar boundary and the center of the polar face, respectively. To better understand the role of the interfacial basic residues, the microenvironments and titration characteristics of lysine (Lys) residues have been examined using two 18-residue peptides. The parent 18A molecule (AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe) binds phospholipid relatively well because of its class A amphipathic helix, whereas 18R, which has the positions of basic and acidic residues reversed, binds to phospholipid relatively weakly. Lys residues were 13C-dimethylated, and NMR spectra were obtained of the peptides complexed with dimyristoylphosphatidylcholine (1/1 (w/w) DMPC/peptide). The four [13C]-epsilon-dimethyl-Lys in 18A gave four resonances at chemical shifts (delta) of 42-43 ppm, whereas only a single resonance at delta 42.70 ppm was observed from the four Lys in 18R. Measurements of delta as a function of pH gave pKa values. The four Lys in 18R had a common pKa of 10.3, indicating that the Lys in the polar face of the amphipathic helix have the pKa expected for noninteracting, fully hydrated groups. In contrast, the four Lys in Ac-18A-NH2 had pKa's in the range 9.4-11.0, indicating that they exist in various microenvironments at the polar-nonpolar boundary of the 18A helix. To examine individual Lys, 18A was synthesized with various Lys selectively labeled. Lys-4, -9, -13, and -15 in Ac-18A-NH2 associated with DMPC had pKa values of 11.0, 9.4, 9.4, and 10.3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Efflux of cellular cholesterol and phospholipid to lipid-free apolipoproteins and class A amphipathic peptides.

The mechanism(s) by which lipid-free apolipoprotein (apo) AI is able to stimulate efflux of cholesterol and phospholipid from cells in cultures has (have) been examined. This process was found to be enhanced when macrophages were enriched with cholesterol. There were 12- and 4-fold increases in cholesterol and phospholipid efflux, respectively, from cholesterol-enriched mouse macrophages when compared to cells not loaded with cholesterol. This enhancement in cholesterol efflux to lipid-free apo AI from macrophages enriched with cholesterol was found to be controlled by the level of free cholesterol in the cells. When cholesterol-enriched mouse macrophages were exposed to lipid-free apo AI at 20 micrograms/mL (706 nM), there was significant efflux of [14C]cholesterol and [3H]phospholipid (20% +/- 0.5%/24 h and 6% +/- 0.3%/24 h, respectively). In comparison, HDL at equivalent protein concentrations only stimulated 11% and 4% efflux of cholesterol and phospholipid, respectively. Synthetic peptides containing amphipathic helical segments that mimic those present in apo AI were used to examine the structural features of the apoprotein which stimulate lipid efflux. Peptides containing only one (18A) or two (37pA) amphipathic helical segments stimulated as much cholesterol efflux from both mouse macrophages and L-cells as apo AI. The order of efficiency, as assessed by the mass concentration at which half-maximal efflux was reached (EC50), was apo AI > 37pA > 18A, indicating that acceptor efficiency was dependent on the number of amphipathic helical segments per molecule. When the helical content of 18A was increased by neutralizing the charges at the ends of the peptide (Ac-18A-NH2), there was a substantial increase in the efficiency for cholesterol efflux (EC50 18A = 17 micrograms/mL vs Ac-18A-NH2 = 6 micrograms/mL). In contrast, when the amphipathicity of the helix in 18A was decreased by scrambling the amino acid sequence, thereby reducing its lipid affinity, cholesterol and phospholipid efflux were not stimulated. The efficiency with which the peptides stimulated cholesterol efflux was in order of their lipid affinity (37pA > Ac-18A-NH2 > 18A), and this order was similar for phospholipid efflux. The time course of lipid release from mouse macrophages and L-cells indicated that phospholipid appeared in the extracellular medium before cholesterol. These results suggest that the apo AI or peptides first interacted with the cell to form protein/phospholipid complexes, that could then accept cholesterol.

Amino Acid Sequence↗

PubMed1995

Molecular determinants of plasma cholesteryl ester transfer protein binding to high density lipoproteins.

The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and is associated with high density lipoproteins (HDL). To understand the mechanism of interaction of CETP with HDL, we studied the binding of pure recombinant CETP to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/apoA-I discoidal particles. Separating bound from free CETP using native gradient gel electrophoresis, complexes of CETP with 10-nm hydrodynamic diameter discoidal particles migrated with a diameter of 12-16 nm, compared with approximately 7.5 nm for CETP. At lower ratios of CETP to discs, CETP bound to discs without displacement of apoA-I. CETP alone was unable to generate discoidal complexes. Cross-linking and fluorescence resonance energy transfer experiments indicated that CETP bound to discs as monomers. Cross-linking of CETP to apoA-I in discs suggested proximity of apoA-I and CETP. By negative-stain electron microscopy, discoidal complexes containing CETP and CETP monoclonal antibody showed localization of antibody molecules to the disc edge, suggesting that CETP was bound to the disc edge. The binding of CETP to discs of different composition or size was studied. Discs (10-nm Stokes diameter) prepared with either apoA-I or apoA-II had a similar Kd (120 nM). Inclusion of 1 mol % cholesteryl oleate, 5 mol % cholesterol, or 6 mol % phosphatidylinositol increased the binding affinity of CETP 3-10 times (20-30 nM). In comparison, plasma HDL3 had a Kd of approximately 450 nM. For POPC/apoA-I discs, 10-nm discs bound CETP with much higher affinity than smaller 7.8-nm discs (Kd = 1-2 microM). 7.7-nm hydrodynamic diameter POPC/apoA-I spherical particles containing either triolein or cholesteryl oleate in their core bound CETP with higher affinity (Kd = 50-100 nM) than 7.8-nm POPC/apoA-I discs. Thus, CETP appears to bind to the perimeter of discoidal particles, possibly in a process in which flexible segments in apoA-I or apoA-II accommodate CETP at the disc edge. The binding of CETP to HDL is markedly influenced by overall particle size and shape and by lipid composition, and the increased binding affinity for cholesterol- and cholesteryl ester-containing discs suggests a higher affinity of CETP for nascent than mature HDL.

Animals↗

PubMed1995

The effect of high density lipoprotein phospholipid acyl chain composition on the efflux of cellular free cholesterol.

High density lipoprotein (HDL) phospholipid (PL) fatty acyl chain composition has been proposed to affect the ability of HDL to participate in the first step of reverse cholesterol transport. To examine the effects of PL fatty acid chain length and degree of unsaturation in this process, reconstituted HDL (rHDL) particles were made with human apolipoprotein (apo) A-I and PL containing fatty acid chains from 14 to 18 carbons in length, which were either fully saturated or unsaturated in one or both chains. These particles were characterized structurally and for their ability to promote free (unesterified) cholesterol (FC) efflux from cells growing in culture. The discoidal rHDL particles were homogeneous and exhibited similar hydrodynamic diameters (10.4 +/- 1.0 nm) indicating that apoA-I forms similarly sized discs with a variety of PL. Measurements of particle surface charge, apoA-I alpha-helix content, and conformational stability indicated that the conformation of apoA-I varies among the particles. These conformational effects on apoA-I are consistent with the PL fluidity influencing the interaction between the amphipathic alpha-helical segments and PL acyl chains. Differential scanning calorimetry demonstrated that the physical state of the rHDL PL at 37 degrees C varied according to acyl chain length and degree of unsaturation; the FC efflux efficiencies for particles with PL in either the gel or liquid crystal states were determined. The ability of the rHDL to accept cellular FC depended on the physical state of the PL in the rHDL. Liquid crystal PL formed the most efficient FC acceptor particles exhibiting a maximal efflux velocity (Vmax) of 12-14% release of total cellular FC per h. Gel-phase PL formed inefficient rHDL acceptors with a Vmax of about 3%/h. A similar hierarchy of FC efflux efficiency was noted when either mouse L-cells or rat Fu5AH hepatoma cells were used as the FC donors. Furthermore, this hierarchy was found to be due to the characteristics of the PL and not due to variable apoA-I conformation because protein-free, small unilamellar vesicles made with the same PL exhibited similar relative efflux capabilities. Generally, the ability of a given rHDL particle to accept cellular FC was related to rHDL PL acyl chain length and degree of unsaturation; decreases in PL acyl chain length and increases in chain unsaturation tended to result in more efficient FC acceptor particles.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Effect of the arrangement of tandem repeating units of class A amphipathic alpha-helixes on lipid interaction.

Exchangeable apolipoproteins possess tandem repeating units of class A amphipathic helical segments and many of them are linked together by proline residues. To understand the optimal arrangement of the amphipathic helixes for lipid association, we have studied the interactions of three model class A amphipathic helical peptides with lipids. The three peptides are: 37pA, a dimer of 18A (DWLKAFYDKVAEKLKEAF) linked together by a Pro (18A-Pro-18A); 37aA, a dimer of 18A linked together by an Ala (18A-Ala-18A); and 36A, a dimer of 18A without any linker residue (18A-18A). Circular dichroism (CD) spectra showed that the peptides are predominantly alpha-helical in aqueous and lipid environments. Temperature dependent CD studies indicated that in buffer helix stability decreases in the order 36A > 37aA > 37pA; however, in the presence of dimyristoyl phosphatidylcholine (DMPC), the above order is reversed. The retention times of the peptides on a C18 reversed-phase high performance liquid chromatography column decreased in the order 36A > 37aA > 37pA, consistent with the lengths of the nonpolar faces of the alpha-helixes being in the same order; the retention time of the parent 18A was shorter than 37pA. While 37pA adsorbed to egg phosphatidylcholine monolayers most strongly, the degree and rate of association of 36A were significantly lower. Differential scanning calorimetry indicated that, while 37pA was most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of DMPC multilamellar vesicles, 36A was least effective; 36A was even less effective than 18A. Fluorescence quenching experiments with iodide and acrylamide indicated that, in the presence of DMPC, Trp residues in 36A are most exposed to the quenchers while in 37pA they are least exposed. In the presence of DMPC, shielding of Trp in 18A from the quenchers was more than that observed with Trp residues in 36A. The results of this study suggest that the arrangement of tandem repeating amphipathic helical units which results in the formation of a class A amphipathic helix with a nonpolar face longer than five or six turns reduces the ability of the helix to associate with phospholipid.

Amino Acid Sequence↗

PubMed1995

Cell membrane structure and lipoprotein metabolism.

Knowledge of the effect of the plasma membrane structure on lipoprotein metabolism is relatively limited. Receptor activity and, thereby, the endocytic pathway of lipoprotein particle catabolism is reported to be affected. The reverse cholesterol transport pathway is clearly affected because altered rates of cholesterol efflux from different cellular plasma membranes have been observed. Changes in the structure and lipid organization of the membranes in arterial wall cells can also alter vessel relaxation.

Animals↗

PubMed1994

The influence of apolipoprotein structure on the efflux of cellular free cholesterol to high density lipoprotein.

The influence of apolipoprotein conformation on the ability of high density lipoprotein (HDL) to remove cellular free cholesterol (FC) has not been studied in detail. To address the effects of amphipathic alpha-helix structure on cellular FC efflux, three class A helical peptides and apolipoprotein (apo) AI were complexed to dimyristoyl phosphatidylcholine (DMPC) to make discoidal complexes that were used as acceptors of cell cholesterol. The peptides consisted of an 18-amino acid, amphipathic, alpha-helical peptide with the sequence DWLKAFYDKVAEKLKEAF (18A), a dimer of 18A covalently linked by a proline residue (37pA), and acetyl-18A-amide (Ac-18A-NH2) that has a higher alpha-helix content than the unblocked 18A molecule. The three peptides strongly mimic the lipid-binding characteristics of the amphipathic segments of apolipoproteins and form discoidal complexes with DMPC that are similar in diameter (11-12 nm) to those formed by human apoAI when reconstituted at a 2.5:1 (w:w) phospholipid to protein ratio. The abilities of these complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts; efflux of FC from both the plasma membrane and the lysosomal pools was examined. For each of the acceptors, the removal of cholesterol from the plasma membrane and lysosomal pools was equally efficient. All four discoidal complexes were equally efficient cell membrane FC acceptors when compared at saturating acceptor concentrations of > 200 micrograms of DMPC/ml of medium. However, at the same lipid concentration, protein-free DMPC small unilamellar vesicles (SUV) were significantly less efficient. The initial rates of FC removal from cells at saturating concentrations of acceptor particles (Vmax) were 12, 10, 10, and 11% per h, respectively, for the complexes containing either 18A, Ac-18A-NH2, 37pA, or apoAI, but only 1% cellular FC per h for the DMPC SUV. The 10-fold higher Vmax for the apoprotein/peptide-containing acceptors was likely due to a reversible interaction of apoprotein or peptide with the plasma membrane that changed the lipid packing characteristics in such a way as to increase the rate of FC desorption from the cell surface. This interaction required amphipathic alpha-helical segments, but it was not affected by the length, number, or lipid-binding affinity of the helices. Furthermore, the efflux efficiency was not dependent on the amino acid sequence of the helical segments which suggests that this interaction is not mediated by a specific cell surface binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.

Several subspecies of human high density lipoprotein (HDL) exist, and particle surface charge is one parameter that can be used to distinguish them. For instance, pre-beta HDL has a lower negative surface charge than the bulk of plasma HDL resulting in slower migration when subjected to electrophoresis in an agarose gel at pH 8.6. The molecular basis for the variations in HDL particle surface charge have been established in this work. Quantitative analysis of electrophoretic mobilities in agarose gels indicates that the surface potentials of reconstituted pre-beta HDL and alpha-HDL3 are -7.6 and -11.4 mV, respectively. The difference of 3.8 mV corresponds to an apparent 1e more net negative charge on alpha-migrating HDL3. The contributions of the lipid and protein components to the charge of HDL3 were defined using reconstituted particles (rHDL) containing various combinations of HDL3 protein and lipid. The surface potentials of spherical and discoidal rHDL are not affected by the particle apoprotein composition. Thin layer chromatography of the HDL3 phospholipid fraction from five normolipidemic subjects revealed that it consists of approximately 84% phosphatidylcholine, 12% sphingomyelin, and 4% phosphatidylinositol (PI) by weight. PI significantly affects the charge of discoidal rHDL and is responsible for approximately 0.7 mV of the surface potential difference between pre-beta- and alpha-HDL. Reconstitution of spherical complexes containing apolipoprotein AI, palmitoyl-oleoyl phosphatidylcholine, and HDL3 neutral lipid esters showed that particles containing a neutral lipid core migrate to an alpha position in an agarose gel. The change in particle shape accounts for approximately 3 mV of the difference in surface potential between pre-beta and alpha-HDL. Overall, the charge of HDL particles is determined by 1) a direct contribution of negative charge from PI molecules in the surface of the lipoprotein and 2) the conformation of the resident apolipoprotein(s) which is influenced by the presence of neutral lipids and the particle shape. This quantitative understanding of surface charge provides a basis for understanding variations in function of HDL subspecies.

Apolipoproteins A↗