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To target tumors overexpressing low-density lipoprotein receptors (LDLr), a pyropheophorbide cholesterol oleate conjugate was synthesized and successfully reconstituted into the low-density lipoprotein (LDL) lipid core. Laser scanning confocal microscopy studies demonstrated that this photosensitizer-reconstituted LDL can be internalized via LDLr by human hepatoblastoma G2 (HepG2) tumor cells.

We have recently shown that a class A amphipathic peptide 5F with increased amphipathicity protected mice from diet-induced atherosclerosis (Garber et al. J. Lipid Res. 2001. 42: 545-552). We have now examined the effects of increasing the hydrophobicity of a series of homologous class A amphipathic peptides, including 5F, on physical and functional properties related to atherosclerosis inhibition by systematically replacing existing nonpolar amino acids with phenylalanine. The peptides, based on the sequence Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH(2) (Ac-18A-NH(2) or 2F) were: 3F(3)(Ac-F(3)18A-NH(2)), 3F(14)(Ac-F(14)18A-NH(2)), 4F(Ac-F(3,14)18A-NH(2)), 5F(Ac-F(11,14,17) 18A-NH(2)), 6F(Ac-F(10,11,14,17)18A-NH(2)), and 7F(Ac-F(3,10,11,14,17) 18A-NH(2)). Measurements of aqueous solubility, HPLC retention time, exclusion pressure for penetration into an egg phosphatidylcholine (EPC) monolayer, and rates of EPC solubilization revealed an abrupt increase in the hydrophobicity between peptides 4F and 5F; this was accompanied by increased ability to associate with phospholipids. The peptides 6F and 7F were less effective, indicating a limit to increased hydrophobicity for promoting lipid interaction in these peptides. Despite this marked increase in lipid affinity, these peptides were less effective than apoA-I in activating the plasma enzyme, lecithin:cholesterol acyltransferase, with 5F activating LCAT the best (80% of apoA-I). Peptides 4F, 5F, and 6F were equally potent in inhibiting LDL-induced monocyte chemotactic activity. These studies suggest that an appropriate balance between peptide-peptide and peptide-lipid interactions is required for optimal biological activity of amphipathic peptides. These studies provide a rationale for the design of small apoA-I-mimetics with increased potency for atherosclerosis inhibition.

To understand the molecular basis for the differences in receptor-binding activity of the three common human apolipoprotein E (apoE) isoforms, we characterized the microenvironments of their LDL receptor (LDLR)-binding regions (residues 136;-150). When present in dimyristoyl phosphatidylcholine (DMPC) complexes, the 22-kDa amino-terminal fragments (residues 1;-191) of apoE3 and apoE4 bound to the LDLR with approximately 100-fold greater affinity than the 22-kDa fragment of apoE2. The pK(a) values of lysines (K) at positions 143 and 146 in the LDLR-binding region in DMPC-associated 22-kDa apoE fragments were 9.4 and 9.9 in apoE2, 9.5 and 9.2 in apoE3, and 9.9 and 9.4 in apoE4, respectively. The increased pK(a) of K146 in apoE2 relative to apoE3 arises from a reduction in the positive electrostatic potential in its microenvironment. This effect occurs because C158 in apoE2, unlike R158 in apoE3, rearranges the intrahelical salt bridges along the polar face of the amphipathic alpha-helix spanning the LDLR-binding region, reducing the effect of the R150 positive charge on K146 and concomitantly decreasing LDLR-binding affinity.The C112R mutation in apoE4 that differentiates it from apoE3 did not perturb the pK(a) of K146 significantly, but it increased the pK(a) of K143 in apoE4 by 0.4 pH unit. This change did not alter LDLR-binding affinity. Therefore, maintaining the appropriate positive charge at the C-terminal end of the receptor-binding region is particularly critical for effective interaction with acidic residues on the LDLR.

Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.

Despite extensive studies and characterizations of the high density lipoprotein-cholesteryl ester (HDL-CE)-selective uptake pathway, the mechanisms by which the hydrophobic CE molecules are transferred from the HDL particle to the plasma membrane have remained elusive, until the discovery that scavenger receptor BI (SR-BI) plays an important role. To elucidate the molecular mechanism, we examined the quantitative relationships between the binding of HDL and the selective uptake of its CE in the murine adrenal Y1-BS1 cell line. A comparison of concentration dependences shows that half-maximal high affinity cell association of HDL occurs at 8.7 +/- 4.7 micrograms/ml and the Km of HDL-CE-selective uptake is 4.5 +/- 1.5 micrograms/ml. These values are similar, and there is a very high correlation between these two processes (r2 = 0.98), suggesting that they are linked. An examination of lipid uptake from reconstituted HDL particles of defined composition and size shows that there is a non-stoichiometric uptake of HDL lipid components, with CE being preferred over the major HDL phospholipids, phosphatidylcholine and sphingomyelin. Comparison of the rates of selective uptake of different classes of phospholipid in this system gives the ranking: phosphatidylserine > phosphatidylcholine approximately phosphatidylinositol > sphingomyelin. The rate of CE-selective uptake from donor particles is proportional to the amount of CE initially present in the particles, suggesting a mechanism in which CE moves down its concentration gradient from HDL particles docked on SR-BI into the cell plasma membrane. The activation energy for CE uptake from either HDL3 or reconstituted HDL is about 9 kcal/mol, indicating that HDL-CE uptake occurs via a non-aqueous pathway. HDL binding to SR-BI allows access of CE molecules to a "channel" formed by the receptor from which water is excluded and along which HDL-CE molecules move down their concentration gradient into the cell plasma membrane.

Lipid-free apolipoprotein (apo) A-I contributes to the reverse transport of cholesterol from the periphery to the liver by solubilizing plasma membrane phospholipid and cholesterol. The features of the apolipoprotein required for this process are not understood and are addressed in the current study. Membrane microsolubilization of human fibroblasts is not specific for apo A-I; unlipidated apos A-II, C, and E incubated with the fibroblast monolayers at a saturating concentration of 50 micrograms/ml are all able to release cholesterol and phospholipid similarly. To determine the properties of the apolipoprotein that drive the process, apo A-I peptides spanning the entire sequence of the protein were utilized; the peptides correspond to the 11- and 22-residue amphipathic alpha-helical segments, as well as adjacent combinations of the helices. Of the 20 helical peptides examined, only peptides representing the N-and C-terminal portions of the protein had the ability to solubilize phospholipid and cholesterol. Cholesterol efflux to the most effective peptides, 44-65 and 209-241, was approximately 50 and 70%, respectively, of that to intact apo A-I. Deletion mutants of apo E and apo A-I were constructed that have reduced lipid binding affinities as compared with the intact molecule. The proteins, apo A-I (Delta222-243), apo A-I (Delta190-243), apo E3 (Delta192-299) and apo E4 (Delta192-299) all exhibited a decreased ability to remove cellular cholesterol and phospholipid. These decreases correlated with the reduced ability of these proteins to penetrate into a phospholipid monomolecular film. Overall, the results indicate that insertion of amphipathic alpha-helices between the plasma membrane phospholipid molecules is a required step in the mechanism of apolipoprotein-mediated cellular lipid efflux. Therefore the lipid binding ability of the apolipoprotein is critical for efficient membrane microsolubilization.

NMR spectroscopy of 13C-labeled human low density lipoprotein (LDL) has been employed to characterize the lysine (Lys) residues in apo B-100. Reductive methylation with [13C]formaldehyde converts up to two-thirds of the Lys to the dimethylamino derivative; this pool of Lys is exposed at the surface of the LDL particle. The [13C]dimethyl-Lys which are visualized exhibit resonances at chemical shifts of 42.8 and 43.2 ppm (pH 7.6) indicating that they exist in two different microenvironments; this is a reflection of the native conformation of apo B associated with lipid, because the labeled, reduced, and alkylated protein gives a single resonance when dissolved in 7 M guanidine hydrochloride. The pH dependences of the Lys chemical shifts indicate that the two types of Lys titrate with different pK values; “active” Lys have a pK of 8.9, while “normal” Lys have a pK of 10.5. About 53 active Lys and 172 normal Lys are exposed on the surface of LDL with the remaining 132 Lys which are present in the human apo B-100 molecule being buried and unavailable for methylation. Addition of paramagnetic ions indicates that the active and normal Lys have different exposures to the aqueous phase; apparently this is a reflection of folding of the apo B molecule. The relative involvement of active and normal Lys in binding of apo B-100 to the LDL receptor on fibroblasts was explored by measuring the decrease in receptor binding as a function of the degree of methylation of the two types of Lys. Upper limits of 21 active and 31 normal Lys in the entire apo B-100 molecule are involved in the binding of LDL to the receptor. It is likely that these Lys are located in domains of apo B which contain clusters of basic amino acid residues and also bind heparin. If the sequence corresponding to apo B-48 (residues 1-2151) which does not bind to the receptor is excluded, then the above limits are halved; an upper limit of 10 active Lys may be particularly involved in receptor binding.

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.

High density lipoprotein (HDL) is able to remove unesterified cholesterol from peripheral cells in the process of reverse cholesterol transport by an aqueous diffusion mechanism as well as by an apolipoprotein (apo)-mediated process. The aqueous diffusion mechanism is understood but the molecular mechanism of lipid-poor pre-beta-HDL-(apo-) mediated cholesterol removal is not known. Measurements of the initial rates of efflux of unesterified cholesterol and phospholipid from human fibroblasts to lipid-free, human apoA-I showed that both lipids are released from the cells during a 10-min incubation with apoA-I. The concentration-dependence of efflux of the lipids is the same (Km = 0.4 and 0.6 microg apoA-I/ml for cholesterol and phospholipid flux, respectively), suggesting a membrane microsolubilization process. A finite pool of about 1% of the plasma membrane cholesterol is accessible for release by solubilization; the limited size of this cholesterol pool is not due to a lack of availability of apoA-I, but rather to the restricted amount of phospholipid that is removed from the plasma membrane. Plasma membrane domains may be involved in membrane microsolubilization, but caveolar cholesterol seems not to be specifically accessed in this process. Membrane microsolubilization is the process by which pre-beta1-HDL removes cell cholesterol in the first step of reverse cholesterol transport. When apoA-I is present in the extracellular space, the relative contributions of cholesterol efflux by membrane microsolubilization and by aqueous diffusion are determined by the degree of lipidation of the apoA-I molecules.

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.

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.

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.

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.

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)

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.