Stimulation with a monoclonal antibody (mAb4E4) of scavenger receptor-mediated uptake of chemically modified low density lipoproteins by THP-1-derived macrophages enhances foam cell generation.

mAb4E4, a murine monoclonal antibody that is specific for acetylated LDL and malondialdehyde-treated LDL, binds specifically to modified LDL present in human atherosclerotic lesions. It is directed against an epitope that is poorly exposed in delipidated and solubilized apolipoprotein B-100 from modified LDL. mAb4E4, as well as its F(ab')2 and Fab fragments, enhanced the uptake of both acetylated LDL and malondialdehyde-treated LDL by THP-1-derived macrophages resulting in a sixfold increase of cytoplasmic cholesteryl ester levels. The increased uptake of modified LDL/mAb4E4 complexes did not occur via the Fc receptor and did not depend on aggregation of modified LDL particles. However, their uptake was inhibited by blocking the scavenger receptors with fucoidin or by downregulation of receptor expression with endotoxins or interferon-gamma, indicating that their uptake is mediated via these receptors. Thus, generation of autoimmune antibodies against modified LDL and subsequent endocytosis of soluble modified LDL/antibody complexes via scavenger receptors may enhance foam cell generation. This mechanism may contribute to the progression of atherosclerotic lesions.

Structural organization of lipid phase and protein-lipid interface in apolipoprotein-phospholipid recombinants: influence of cholesterol.

The complexes of individual human plasma apolipoproteins (apo) A-I, E and A-II with dipalmitoylphosphatidylcholine (DPPC) in the absence or in the presence of cholesterol (Chol) were prepared with initial DPPC/Chol/protein weight ratio as 3:0.15:1. ApoA-I/DPPC/Chol complexes with different protein content (initial DPPC/apoA-I weight ratios were changed from 10.5:1 to 2.6:1) but with a fixed initial DPPC/Chol weight ratio of 20:1 were also prepared. The complexes were isolated by gel-filtration and characterized by size and composition. ApoA-I- and apoA-II-complexes had the same size (80-84 A) and the complexes became more heterogeneous upon Chol inclusion; apoE-complexes were larger (97-100 A) and more homogeneous and Chol addition had no effect on their hydrodynamic properties. Chol seems to be excluded partially in the following manner for isolated complexes with different apo's: A-II > E > A-I. The possible existence of two lipid regions in the complexes differing in lipid dynamics - the lipid shell in the vicinity of apolipoprotein (boundary lipid) opposite to the remaining part of the lipid bilayer - has been studied by absorbance and fluorescence spectroscopy with cis-parinaric acid (cis-PA) and trans-parinaric acid (trans-PA) embedded into the complexes. Their application is based on a strong preference of trans-PA for solid lipid while cis-PA distributes more equally between co-existing fluid and solid lipid regions (Sklar et al. (1979) Biochemistry 18, 1707-1716). (1) For apoA-I-complexes, the partition of cis-PA between water and lipid phase at temperatures below and above the transition temperature of DPPC (T(t)) was insensitive to Chol and temperature, while partition of trans-PA into the lipid phase of Chol-containing complex was increased at high temperature and decreased at low temperature. These results seem to be related to trans-PA redistribution between Chol-rich and protein-rich lipid domains, the latter being more disordered at T < T(t) and more immobilized at T > T(t) compared to the bulk bilayer; cis-PA localizes preferentially in boundary lipid. This hypothesis was directly confirmed by measurements of energy transfer between apoA-I tryptophanyls and probe molecules. (2) The relative response of trans-PA fluorescence intensity to temperature-induced phase transition of DPPC in apoA-I/DPPC/Chol complexes was decreased as a function of apolipoprotein content in a non-monotonic fashion with a transition midpoint at a mol ratio DPPC/A-I of 250:1, probably indicating two different modes of apolipoprotein/DPPC interaction in different sized complexes. (3) The comparative study of lipid dynamics in apoA-I-, apoE- and apoA-II-containing complexes with temperature response to phospholipid phase transition with fluorescence parameters such as intensity and anisotropy of cis-PA and trans-PA revealed the presence of boundary lipid in all three complexes without Chol. In contrast to apoA-I-containing complexes, in apoA-II/DPPC/Chol complexes, trans-PA seems to move preferentially into boundary lipid and cis-PA to distribute between two different regions probably as a result of more ordering action induced by apoA-II compared to apoA-I on the nearest phospholipid molecules in Chol-containing complexes; the apoE action on trans-PA and cis-PA distribution could be intermediate. Based on these results, the degree of Chol exclusion from the boundary lipid region for complexes with different apo's increasing in the order A-II > E > A-I can be suggested. Different Chol distributions between two lipid regions in the complexes seems not to be a function of complex size, but rather is an inherent property of the particular apolipoprotein molecule.

Structure of the apolipoprotein A-IV/lipid discoidal complexes: an attenuated total reflection polarized Fourier transform infrared spectroscopy study.

Discoidal lipid particles were prepared from a reaction mixture containing apo A-IV and dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) in the molar ratio of 185:1 (lipid/protein). The complexes were isolated by gel filtration and characterized in terms of composition and size. Infrared attenuated total reflection spectroscopy was used to estimate the secondary structure of apolipoprotein A-IV and the orientation of its amphipathic alpha-helices with respect to the lipid hydrocarbon chains. In addition, infrared spectra were analyzed in terms of the conformation and organization of different regions of the lipid molecules in the particles. This approach has been applied successfully to reconstituted HDL particles prepared from a reaction mixture containing DPPC and apo A-I in the molar ratio of 150:1 (Wald, J.H., Goormaghtigh, E., De Meutter, J., Ruysschaert, J.M. and Jonas, A. (1990) J. Biol. Chem. 265, 20044-20050). Apo A-IV helicity increased for the protein bound to DMPC or DPPC but the increase was more pronounced for the apo A-IV/DMPC particles. In both complexes, the alpha helical amphipathic segments of the protein were parallel to the lipid acyl chains and no significant modification of the overall organization of the lipid molecules in the lipid bilayer was observed. The presence of apo A-IV seems only to affect the conformation of the lipid hydrocarbon chains in close contact with the protein in the discoidal particles.

LCAT activation properties of apo A-I CNBr fragments and conversion of discoidal complexes into spherical particles.

We studied the substrate properties of the phospholipid-cholesterol-apolipoprotein complexes generated with apo A-I, apo A-I-CNBr fragments, apo A-II and apo A-IV for cholesterol esterification by the enzyme lecithin-cholesterol acyltransferase (LCAT). The kinetic parameters determined with the different complexes as substrates, showed that the complexes containing apo A-I and apo A-IV were about 40-times more efficient than those generated with the apo A-I fragments. In this system, the substrates containing apo A-II had the lowest efficiency. In spite of the differences in the kinetic parameters observed with the various apolipoprotein-lipid complexes, the cholesterol inserted in the complexes was esterified for more than 90% after 24 h in all systems studied. Based upon the results of the kinetic experiments, we followed the transformation of the discoidal complexes into spherical particles, due to the formation of a cholesteryl esters core, in the presence of low-density lipoproteins as an external source of cholesterol. We observed the formation of spherical particles by electron microscopy, after incubation of the discoidal complexes with LCAT for 24 h. The average percentage of cholesteryl esters in the converted particles was around 60% of the total cholesterol, varying between 40% for the apo A-I-CNBr-1-DPPC-cholesterol complex and up to 86% for the apo A-I-DPPC-cholesterol complex. The secondary structure of protein in the complexes was not significantly modified. However, the phospholipid phase transition disappeared, together with the parallel orientation of the phospholipid acyl chains with the helical segments of the apolipoproteins, as the phospholipids are organized in a monolayer at the surface of the spheres.

A novel bacteriocin with a YGNGV motif from vegetable-associated Enterococcus mundtii: full characterization and interaction with target organisms.

A novel broad-spectrum antimicrobial peptide produced by vegetable-associated Enterococcus mundtii was purified and characterized, and designated mundticin. To our knowledge, this is the first report on bacteriocin production by this organism. The elucidation of the full primary amino acid sequence of mundticin (KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATGGAAGWSK) revealed that this antimicrobial peptide belongs to the class IIa bacteriocins of lactic acid bacteria which share a highly conserved N-terminal 'YGNGV' motif. Data obtained by computer modelling indicated an oblique orientation of the alpha-helical regions of mundticin and homologous class IIa bacteriocins at a hydrophobic-hydrophilic interface, which may play a role in the destabilization of phospholipid bilayers. The average mass of mundticin, as determined by electron spray mass spectrometry, was found to be 4287.21+/-0.59 Da. With respect to its biological activity, mundticin was shown to inhibit the growth of Listeria monocytogenes, Clostridium botulinum and a variety of lactic acid bacteria. Moreover, it was demonstrated to have a bactericidal effect on L. monocytogenes as a result of the dissipation of the membrane potential, and a loss of intracellular ATP in absence of ATP leakage. Its good solubility in water, and its stability over a wide pH and temperature range indicate the potential of this broad spectrum bacteriocin as a natural preservation agent for foods.

Evaluation of the secondary structure of apo B-100 in low-density lipoprotein (LDL) by infrared spectroscopy.

The secondary structure of the apo B-100 protein present in human low density lipoprotein has been investigated by transmission and attenuated total reflection infrared spectroscopy. The amount of beta-sheet (41%) is significantly higher than that determined by CD spectroscopy in the present study (12%) and elsewhere (15-16%). The high percentage of beta-sheet structure in apo B-100 supports the importance of such segments in maintaining the lipid-protein assembly in LDL. Polarized infrared spectroscopy indicates that the beta-sheet component of apo B-100 adopts a preferential orientation with respect to the phospholipid monolayer surrounding the LDL, whereas no such orientation is observed for the other secondary structure components.

Mode of assembly of amphipathic helical segments in model high-density lipoproteins.

The structure of discoidal apo A-I-phospholipid complexes, representing the metabolic precursors of mature high-density lipoprotein particles, was studied by a combination of both a theoretical and an experimental approach. The secondary structure of the complex was determined by circular dichroic measurements, while the relative orientation of the apo A-I helical segments and of the phospholipid acyl chains was determined by ATR infrared measurements. Fluorescence energy transfer between the tryptophan residues of apo A-I and fluorescent phospholipid probes yielded an estimation of the relative topography of the lipid and apolipoprotein components in discoidal and spherical particles. The theoretical approach consisted of the identification of the helical segments in various apo A-I species. These segments were then oriented at a lipid/water interface by minimization of their hydrophobic and hydrophilic transfer energies. The calculation of the hydrophobicity profiles along the axis of the helices leads to the identification of specific interactions between pairs of helices. The helices were further assembled together with the phospholipids by computer modelling, enabling an estimation of the dimensions of the complex. The combination of the experimental and theoretical results yielded a model for discoidal apolipoprotein-phospholipid complexes, in which the amphipathic helical segments are oriented along the edges of the discs. Such a model can be extended to the conversion of these complexes into mature spherical HDL, through the formation of a cholesteryl ester core.

Synthetic model peptides for apolipoproteins. I. Design and properties of synthetic model peptides for the amphipathic helices of the plasma apolipoproteins.

Amphipathic helical peptides are the lipid-binding motives of the plasma apolipoproteins, and synthetic peptide analogs have been used to unravel the mechanism of lipid association within this class of proteins. Hydrophobic interactions between the apolar amino acid residues belonging to the hydrophobic face of the amphipathic helices and the lipids are the major driving forces in the peptide-lipid association to form discoidal complexes. Ionic interactions and salt bridge formation between contiguous peptide chains in the complex can, however, contribute to the overall stability of the lipid-protein particle. This was studied by designing peptide analogs to the helical repeats of the apolipoproteins with variable degrees of salt bridge formation between adjacent peptide chains. The most stable conformation for pairs of synthetic peptides was calculated by energy minimisation together with the energy of interaction between peptides. The sequence of the peptides was derived from that of the 18A peptide synthesized by Segrest et al., and the theoretical calculations confirmed that ionic interactions between residues close to each other, along the edge of two adjacent anti-parallel peptides, can significantly contribute towards the stability of a peptide-phospholipid complex.

Enzymatic hydrolysis of reconstituted dimyristoylphosphatidylcholine-apo A-I complexes.

Apolipoproteins share a common structural feature, their interaction with phospholipids. It is believed that amphipathic helical sequences enable apolipoproteins to bind to lipid bilayer and to form discoidal particles of defined dimensions. While the knowledge of the apo A-I sequence and secondary structure has been used to make predictions about its mode of association with lipids, the available experimental data necessary to propose a precise model of these discoidal structures are still limited. An important step in our understanding of these structures would be to identify the apolipoprotein lipid-associated domains. Proteolysis of apo A-I-DMPC reconstituted HDL (rHDL) and free apo A-I is used here to identify lipid-protected domains of apo A-I. Free cleaved peptides were separated from rHDL associated peptides by density gradient centrifugation. The lipid-associated peptides were further analyzed by SDS-PAGE and transferred by Western blot to a ProBlott membrane for sequencing. Cleavage occurred at residue 43 with proteinase K, 46 with trypsin and residue 47 or 48 with pronase. A large domain from about residue 45 to the C-terminal remains highly protected against hydrolysis eventhough it contains several bonds susceptible to proteolytic cleavage. No protected fragments were detected by SDS-PAGE after enzymatic cleavage of free apo A-I in identical experimental conditions.

Headgroup specificity of lecithin cholesterol acyltransferase for monomeric and vesicular phospholipids.

In this study, we investigated how the nature of the phospholipid head group and the macromolecular structure of the phospholipid, either as a monomer or incorporated into a lipid matrix, influence the activity of lecithin cholesterol acyltransferase (LCAT). As substrates we used 1,2-bis-(1-pyrenebutanoyl)-phosphatidylcholine, 1, 2-bis-(1-pyrenebutanoyl)-phosphatidylethanolamine and 1, 2-bis-(1-pyrenebutanoyl)-phosphatidyl-alcohols, either as monomers or incorporated into small unilamellar vesicles consisting of dipalmitoylphosphatidylcholine ether. The rate of hydrolysis of the pyrene-labeled phospholipids was determined both by fluorescence and by high performance liquid chromatography. V(max) and K(m) were calculated for the different substrates. The data show that V(max) is 10- to 30-fold higher for the hydrolysis of monomeric phosphatidylcholine (PC) compared to phosphatidylethanolamine (PE) and the phosphatidylalcohols, while K(m) values are comparable. When the fluorescent substrates were incorporated into dipalmitoylphosphatidylcholine ether vesicles, we observed a 4- to 10-fold increase of V(max) for PE and the phosphatidylalcohols, and no significant change for K(m). V(max) for PC remained the same. Natural LCAT mutants causing Fish-Eye Disease (FED) and analogues of these mutants expressed in Cos-1 cells, had similar activity on monomeric PC and PE. These data suggest that the activity of LCAT is determined both by the molecular structure of the phospholipid and by its macromolecular properties. The LCAT activity on monomeric substrates decreases as: phosphatidylcholine&z. Gt;phosphatidylethanolamine congruent withphosphatidylpropanol congruent withphosphatidylethanol congruent withphosphatidylethyleneglycol. The incorporation of PE and the phosphatidylalcohols into a matrix of dipalmitoylphosphatidylcholine decreases the specificity of the phospholipid head group.

Helix-helix interactions in reconstituted high-density lipoproteins.

In this work we calculated the ionic interactions between adjacent amphipathic helices of apo A-I and apo A-IV. The calculation of the electrostatic potential around the helices helps identify the charged residues susceptible to form salt bridges between adjacent helices. An estimation of the stability of the different pairs of helices is derived from the calculation of the energy of interaction between contiguous helices at a water/lipid interface after energy minimization. The most stable energetic conformation corresponds to the 17-residue helices oriented anti-parallel and separated by a stretch of 5 residues in an extended beta-strand conformation, as calculated through the 'stereo alphabet' calculation procedure. In a pair of helices, the hydrophobic faces are directed towards the lipid core of the discoidal phospholipid-apolipoprotein complex and the hydrophobic lipid-protein interactions are major determinants for the stability of the complex. Interactions between polar residues located on the opposite face of the helix and water molecules can also contribute to the overall energy of the system. Finally, salt bridge formation between residues of opposite charge along the edge of the helical segments contribute to the cooperativity of the phospholipid-apolipoprotein complex formation. The mode of assembly of the amphipathic helical repeats of the apolipoproteins around the edge of a discoidal complex is therefore determined both by the hydrophobic character of the residues and by the charge complementarity along the edge of the helices which increases the structural stability and determines the relative orientation of the helices.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic model peptides for apolipoproteins. II. Characterization of the discoidal complexes generated between phospholipids and synthetic model peptides for apolipoproteins.

The structure, composition and physico-chemical properties of complexes generated between phospholipids and synthetic model peptides for the amphipathic helices of the plasma apolipoproteins were studied. The sequences of the peptides were derived from that of the 18A peptide and designed to either enhance or decrease ionic interactions between pairs of peptides, as described in the accompanying paper. Complexes were prepared with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or with DPPC and cholesterol, and isolated on a Superose 6HR column. Association kinetics for the DMPC-peptides complexes were followed by measuring the turbidity as a function of the temperature. The diameters of the DPPC-peptide complexes, measured by gradient gel electrophoresis (GGE), were about 120 A. Fluorescence polarization measurements after labeling with diphenyl hexatriene (DPH) yielded transition temperatures of, respectively, 40.6, 41.5 and 41.8 degrees C for the DPPC/18AM1-, DPPC/18AM4- and DPPC/18A-peptide complexes. These values were confirmed by differential scanning calorimetry. Circular dichroism and infrared spectroscopy revealed that the peptides adopt an alpha-helical structure in solution and this percentage increased from 30-40% in the free peptides up to 50-60% in the complexes. Attenuated total reflection (ATR) infrared measurements of the complexes indicated that the peptides are oriented parallel to the acyl chains of the phospholipid bilayer. Denaturation of the peptides and of the peptide-lipid complexes was monitored by Trp fluorescence under addition of increasing amounts of GdmCl. The mid-points of the denaturation curves lie at, respectively, 0.05, 0.25 and 0.35 M GdmCl for the 18AM4, 18A and 18AM1 peptide and are shifted towards higher GdmCl concentrations after peptide-lipid binding. GdmCl denaturation decreased the alpha-helical content of the peptides and of the complexes, as monitored by circular dichroism measurement. The helix to random coil structure transition occurred at, respectively, 2.1, 2.2, and 2.0 M GdmCl for 18A, 18AM1 and 18AM4, compared to 5.1, 5.0, and 5.3 M in the corresponding complexes. These data suggest altogether that the structural properties, the mode of lipid-protein association and the stability of the phospholipid-peptide complexes are similar to those of native plasma apolipoproteins. The 18A and 18AM4 peptides which contain charged residues along the edge of the helix, leading to salt bridge formation between peptides were shown to mimic the amphipathic helices of the plasma apolipoproteins.

The 118-135 peptide of the human prion protein forms amyloid fibrils and induces liposome fusion.

The prion protein (PrPC) is a glycoprotein of unknown function normally found at the surface of neurons and of glial cells. It is involved in diseases such as bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease in the human, where PrPC is converted into an altered form (termed PrPSc). PrPSc is highly resistant towards proteolytic degradation and accumulates in the central nervous system of affected individuals. By analogy with the pathological events occuring during the development of Alzheimer's disease, controverses still exist regarding the relationship between amyloidogenesis, prion aggregation and neuronal loss. To unravel the mechanism of PrP neurotoxicity and understand the interaction of PrP with cellular membranes, a series of natural and variant peptides spanning residues 118 to 135 of PrP was synthesized. The potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. According to computer modeling calculations, the 120 to 133 domain of PrP is predicted to be a tilted lipid-associating peptide, and to insert in a oblique way into a lipid bilayer through its N-terminal end. In addition to amyloidogenic properties exhibited in vitro by these peptides, peptide-induced vesicle fusion was demonstrated by several techniques, including lipid- and core-mixing assays. Elongation of the 120 to 133 peptide towards the N- and C-terminal ends of the PrP sequence showed that the 118 to 135 PrP peptide has maximal fusogenic properties, while the variant peptides had no effect. Due to their high hydrophobicity, all peptides tested were able to interact with liposomes to induce leakage of encapsulated calcein. We demonstrate also that the propensity of the peptides to fold as an alpha-helix increases their fusogenic activity, thus accounting for the maximal fusogenic activity of the most stable helix at residues 118 to 135. These data suggest that, by analogy with the C-terminal domain of the beta-amyloid peptide, the fusogenic properties exhibited by the prion peptides might contribute to the neurotoxicity of these peptides by destabilizing cellular membranes.

The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.

The assembly of atherogenic lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein (apo)B, a microsomal triglyceride transfer protein (MTP) and protein disulphide isomerase (PDI). Here we show by molecular modelling and mutagenesis that the globular amino-terminal regions of apoB and MTP are closely related in structure to the ancient egg yolk storage protein, vitellogenin (VTG). In the MTP complex, conserved structural motifs that form the reciprocal homodimerization interfaces in VTG are re-utilized by MTP to form a stable heterodimer with PDI, which anchors MTP at the site of apoB translocation, and to associate with apoB and initiate lipid transfer. The structural and functional evolution of the VTGs provides a unifying scheme for the invertebrate origins of the major vertebrate lipid transport system.

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling.

The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp-Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three-dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the alpha/beta hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta-strands connected by four alpha-helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site-directed mutagenesis for the localization of the active site residues. The wild-type enzyme and mutants were expressed in Cos-1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomeric substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential "lid" domain at residues 50-74 of LCAT might be involved in the enzyme-substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three-dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish-eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.

Structural and functional properties of apolipoprotein B in chemically modified low density lipoproteins.

The structural and compositional changes occurring during in vitro chemical modification of apolipoprotein B-100 (apo B), the apolipoprotein component of low density lipoproteins (LDL), were investigated in this study. The functional properties of chemically modified apo B and especially its potential to induce accumulation of cholesterol esters in macrophages were related to the structural changes of apo B. Acetylation, maleylation or malondialdehyde conjugation did not significantly affect the lipid composition of LDL. However, the unsaturated cholesteryl esters content, especially that of cholesteryl arachidonate was significantly decreased through Cu-oxidation. The number of reactive lysine residues in apo B was decreased by Cu-catalyzed LDL oxidation, acetylation, maleylation and by malondialdehyde conjugation. The number of free cysteines decreased from six in native apo B-100 to three in Cu-oxidized LDL. The tryptophan fluorescence intensity decreased most in malondialdehyde-conjugated LDL and in Cu-oxidized LDL, compared with acetylated and maleylated LDL. The secondary structure of native and chemically modified LDL was measured by attenuated total reflection infrared spectroscopy and by circular dichroism. No significant changes were observed in the secondary structure of any of the modified LDL. These data suggest that neither acetylation, malondialdehyde treatment or even Cu-oxidation substantially altered the secondary structure of apo B, in spite of significant modifications in the primary structure. Incubation of chemically modified LDL with J774 macrophages induced an accumulation of cellular cholesteryl esters and foam cell formation. The highest cholesterol accumulation was induced after malondialdehyde treatment of LDL. These data suggest that the cellular uptake and accumulation of modified LDL is not modulated by changes in the apo B structure. Rather it seems dependent upon the net charge of the apo B protein and probably involves the modification of critical lysine residues.

Displacement of apo A-I from HDL by apo A-II or its C-terminal helix promotes the formation of pre-beta1 migrating particles and decreases LCAT activation.

The displacement of apolipoprotein (apo) A-I by apo A-II is a major event in the remodeling of high density lipoproteins (HDL). In the present study, we investigated the displacement of apo A-I both from native and reconstituted HDL (rHDL) by either apo A-II or by the C-terminal helical peptide (i.e. residues 53-70). We studied the remodeling process of the original particles, the changes in size and composition and in their lecithin:cholesterol acyltransferase (LCAT) activating properties. Using gel filtration, we show that, at low apo A-II/AI ratios, the initial lipid apolipoprotein complex containing 2 mol apo A-I is remodeled into a mixed complex containing apo A-I and apo A-II, involving the displacement of one apo A-I by apo A-II. Upon addition of a larger amount of apo A-II, the rHDL particles become more heterogeneous and of larger size. Immunoblotting of the particles separated by non denaturing gradient gel electrophoresis shows that most of the apo A-I remains associated with the largest particles. The LCAT activation properties of the remodeled complexes decrease upon addition of either apo A-II or its C-terminal helix. This decrease is more pronounced when rHDL are incubated with the apo A-II C-terminal helix than with native apo A-II, as VmaX decreases from 28 to 16 and 7 nmol cholesteryl ester/ml per h respectively, whereas Km remains unchanged. The displacement of apo A-I observed with rHDL also occurred with native HDL particles as demonstrated by two-dimensional gel electrophoresis, using pyrene-phospholipid labeled HDL. Displacement of apo A-I generates pre-beta1 migrating particles containing apo A-I and phospholipids. We therefore propose that apo A-II has a dual effect on the role of HDL in reverse cholesterol transport: displacement of apo A-I from rHDL results in a negative control of the LCAT activity, while generation of pre-beta1 migrating particles enhances the formation of potential acceptors of cellular cholesterol.

Branched synthetic peptide constructs mimic cellular binding and efflux of apolipoprotein AI in reconstituted high density lipoproteins.

This study investigates the suitability of the trimeric apolipoprotein (apo)AI(145-183) peptide that we recently described, to serve as a model to probe the relationship between apoAI structure and function. Three copies of the apoAI(145-183) unit, composed each of two amphipathic alpha-helical segments, were branched onto a covalent core matrix and the construct was recombined with phospholipids. A similar construct was made with the apoAI(102-140) peptide and used as a comparison with dimyristoylglycerophosphocholine (DMPC)-apoAI complexes. The DMPC-trimeric-apoAI(145-183) complexes had similar immunological reactivity with monoclonal antibodies directed against the 149-186 apoAI sequence (A44), suggesting that the A44 epitope is exposed similarly in both the synthetic peptide and the native apoAI complexes. The complexes generated with the trimeric-apoAI(145-183) bind specifically to HeLa cells with comparable affinity to the DMPC apoAI complexes; they are a good competitor for binding of apoAI to both HeLa cells and Fu5AH rat hepatoma cells; finally, these complexes promote cholesterol efflux from Fu5AH cells with an efficiency comparable with the apo AI/lipid complexes. To study LCAT activation by the trimeric apo AI(145-183) construct, complexes were prepared with dipalmitoylphosphatidylcholine (DPPC), cholesterol (C) and either the trimeric construct or apoAI. LCAT activation by the trimeric construct was much lower than by apo AI, possibly because the conformation of the trimeric 145-183 peptide in DPPC/C/peptide complexes does not mimic that of apoAI in the corresponding complexes. In comparison, the complexes generated with the multimeric apoAI(102-140) construct had a poor capacity to mimic the physico-chemical and biological properties of apoAI. The apoAI(102-140) construct had low affinity for lipid compared with the (145-183) construct. After association with lipids, it was a poor competitor of DMPC-apoAI complexes for cellular binding and had only limited capacity to promote cholesterol efflux. These results suggest trimeric constructs can serve as an appropriate models for apoAI, enabling further investigations and new experimental approaches to determine the structure-function relationship of apoAI.

Comparison of lipid-binding and lecithin:cholesterol acyltransferase activation of the amino- and carboxyl-terminal domains of human apolipoprotein E3.

To extend the characterization of the functional domains of apolipoprotein E (apoE), the amino-(residues 1-191, 22-kDa) and carboxyl-terminal (residues 216-299, 10-kDa) fragments were tested for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. A disulfide bond linking helices 2 and 3 of the four-helix bundle amino-terminal domain was introduced by mutating threonine-57 to cysteine (Thr57-->Cys) in apoE3 (cysteine at position 112) to determine the influence of the disulfide bond on the properties of this domain. Lipid-binding properties were determined by the ability to form complexes with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine, assessed by measuring decreases in turbidity as a function of temperature. The results demonstrate that the relative lipid binding efficiencies were intact apoE3 approximately 10-kDa fragment > 22-kDa fragment > Thr57-->Cys variant. In addition, free, non-lipid-associated protein was observed with the two 22-kDa fragments but not with intact apoE3 or the 10-kDa fragment. The transition temperatures determined by fluorescence polarization were higher for the DMPC complexes with intact apoE3 and with 22- and 10-kDa fragments (25.5 degrees C) than with the 22-kDa Thr57-->Cys variant (23.5 degrees C), suggesting that the variant fragment possessed the lowest affinity for lipid. Attenuated total reflection infrared measurements of the complexes indicated that the long axes of the alpha-helices of the various apoE forms were parallel to the acyl chains of the phospholipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Design of a new class of amphipathic helical peptides for the plasma apolipoproteins that promote cellular cholesterol efflux but do not activate LCAT.

Amphipathic helical peptides represent the lipid-binding units of the soluble plasma apolipoproteins. Several synthetic peptide analogues have been designed to mimic such structures and have been used to unravel some of the mechanisms involved in the physiological function of the apolipoproteins, including lipid binding, LCAT activation, and enhancement of cholesterol efflux from lipid-laden cells. A series of novel synthetic peptides, named ID peptides, was modeled on the basis of the structural properties common to the amphipathic helices of apolipoprotein (apo) A-I. In these new peptides, however, the segregation between hydrophobic and hydrophilic faces of the helices is more pronounced than in apoA-I, so that the surface of the hydrophobic and hydrophilic faces of the amphipathic helices is equal. Moreover, there are fewer negatively charged residues in the center of the hydrophilic face of the helical peptides. Most charged amino acids are located along the edge of the helix and are susceptible to forming salt bridges with residues of an antiparallel helix, such as around a discoidal phospholipid/peptide complex. The physicochemical characteristics of these peptides and their complexes with phospholipids were compared with those of the 18A peptide and its lipid/peptide complex. All ID peptides bind dimyristoylphosphatidylcholine vesicles more rapidly than the 18A peptide to yield discoidal peptide/phospholipid complexes of comparable size. The alpha-helical content of the lipid-free ID peptides is close to that of the 18A peptide and increases slightly on lipid binding. The stability of the ID and 18A peptides and of the phospholipid/peptide complexes against guanidinium hydrochloride denaturation is higher than that of lipid-free and lipid-bound apoA-I. LCAT activation by the 18A/phospholipid/cholesterol complexes equals that of apoA-I/ phospholipid/cholesterol complexes, whereas none of the ID peptides tested is able to activate LCAT to a significant extent. Incubation of the peptide/phospholipid complexes with lipid-laden macrophages induces cellular cholesterol efflux and incorporation of cholesterol into the complexes. The cholesterol efflux capacity of the peptide/phospholipid complexes is comparable among the peptides and higher than that of apoprotein/phospholipid complexes. In conclusion, although the amphipathicity of the new peptides is higher than that of the 18A model peptide, the lack of LCAT activation by the ID peptides suggests that an enhanced segregation of the hydrophobic and hydrophilic residues, equal magnitude of hydrophobic and hydrophilic faces of the helix, and the absence of negatively charged residues in the central part of the hydrophilic face might account for the lack of LCAT activity of these peptides. These parameters do not affect the capacity of the peptide/phospholipid complexes to promote cellular cholesterol efflux.