Apolipoprotein A1 Forms 5/5 and 5/4 Antiparallel Dimers in Human High-density Lipoprotein.

Apolipoprotein A1 (APOA1), the major protein of high-density lipoprotein (HDL), contains 10 helical repeats that play key roles in protein-protein and protein-lipid interactions. The current structural model for HDL proposes that APOA1 forms an antiparallel dimer in which helix 5 in monomer 1 associates with helix 5 in monomer 2 along a left-left (LL5/5) interface, forming a protein complex with a 2-fold axis of symmetry centered on helix 5. However, computational studies suggest that other orientations are possible. To test this idea, we used a zero-length chemical cross-linking reagent that forms covalent bonds between closely apposed basic and acidic residues. Using proteolytic digestion and tandem mass spectrometry, we identified amino acids in the central region of the antiparallel APOA1 dimer of HDL that were in close contact. As predicted by the current model, we found six intermolecular cross-links that were consistent with the antiparallel LL5/5 registry. However, we also identified three intermolecular cross-links that were consistent with the antiparallel LL5/4 registry. The LL5/5 is the major structural conformation of the two complexes in both reconstituted discoidal HDL particles and in spherical HDL from human plasma. Molecular dynamic simulations suggest that that LL5/5 and LL5/4 APOA1 dimers possess similar free energies of dimerization, with LL5/5 having the lowest free energy. Our observations indicate that phospholipidated APOA1 in HDL forms different antiparallel dimers that could play distinct roles in enzyme regulation, assembly of specific protein complexes, and the functional properties of HDL in humans.

Apolipoprotein A-I mimetic 4F alters the function of human monocyte-derived macrophages.

HDL and its major protein component apolipoprotein A-I (apoA-I) exert anti-inflammatory effects, inhibit monocyte chemotaxis/adhesion, and reduce vascular macrophage content in inflammatory conditions. In this study, we tested the hypothesis that the apoA-I mimetic 4F modulates the function of monocyte-derived macrophages (MDMs) by regulating the expression of key cell surface receptors on MDMs. Primary human monocytes and THP-1 cells were treated with 4F, apoA-I, or vehicle for 7 days and analyzed for expression of cell surface markers, adhesion to human endothelial cells, phagocytic function, cholesterol efflux capacity, and lipid raft organization. 4F and apoA-I treatment decreased the expression of HLA-DR, CD86, CD11b, CD11c, CD14, and Toll-like receptor-4 (TLR-4) compared with control cells, suggesting the induction of monocyte differentiation. Both treatments abolished LPS-induced mRNA for monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), regulated on activation, normal T-expressed and presumably secreted (RANTES), IL-6, and TNF-alpha but significantly upregulated LPS-induced IL-10 expression. Moreover, 4F and apoA-I induced a 90% reduction in the expression of CD49d, a ligand for the VCAM-1 receptor, with a concurrent decrease in monocyte adhesion (55% reduction) to human endothelial cells and transendothelial migration (34 and 27% for 4F and apoA-I treatments) compared with vehicle treatment. In addition, phagocytosis of dextran-FITC beads was inhibited by 4F and apoA-I, a response associated with reduced expression of CD32. Finally, 4F and apoA-I stimulated cholesterol efflux from MDMs, leading to cholesterol depletion and disruption of lipid rafts. These data provide evidence that 4F, similar to apoA-I, induces profound functional changes in MDMs, possibly due to differentiation to an anti-inflammatory phenotype.

Solution NMR structure of a model class A (apolipoprotein) amphipathic alpha helical peptide.

To better understand the structural determinants of the physical-chemical and the biological properties of Ac-18A-NH(2) (acetyl-AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe-amide), we have determined its structure in 50% (v/v) trifluroethanol (TFE-d(3))/water mixture (5 mM potassium phosphate, pH 5.5, 310K) using two-dimensional proton NMR spectroscopy. Stereospecific assignments have been made for C(beta)H protons (all the residues except Ala and Val) and gammaCH(3) (Val) groups. Nuclear Overhauser effects are observed between the nonpolar side chains spaced at (i) and (i + 4) position in the primary sequence, e.g., Trp2 and Phe6, and Phe6 and Val10. This suggests that in addition to N-terminal acetyl and C-terminal amide groups, the amphipathic alpha helical structure of Ac-18A-NH(2) is further stabilized by interactions between the hydrophobic residues on the nonpolar face of the helix.

Interaction of model class A1, class A2, and class Y amphipathic helical peptides with membranes.

To test the hypothesis that differences in the lipid affinity of exchangeable apolipoproteins are due to the presence of different classes of amphipathic alpha-helical motifs which differ primarily in the distribution of charged amino acid residues, we designed and synthesized model peptides mimicking class A1, class A2, and class Y amphipathic helices present in these apolipoproteins. Both class A1 and class A2 helices have positive residues at the polar-nonpolar interface and negative residues at the center of the polar face. However, clustering of positive and negative residues is less exact in class A1 compared to class A2 helices. The class Y helices have two negative residue clusters on the polar face separating the two arms and the base of the Y motif formed by three positive residue clusters. The lipid affinities of three 18 residue model peptides representing these classes, Ac-18A1-NH2 (Ac-ELLEKWAEKLAALKEALK-NH2), Ac-18A2-NH2 (Ac-ELLEKWKEALAALAEKLK-NH2), and Ac-18Y-NH2 (Ac-ELLKAWKEALEALKEKLA-NH2), were determined by right-angle light scattering, circular dichroism spectroscopy, differential scanning calorimetry, and fluorescence spectroscopy. The observed rank order of lipid affinity of these three peptides is: Ac-18A2-NH2 > Ac-18Y-NH2 > Ac-18A1-NH2. This order is consistent with the known lipid affinity of exchangeable apolipoproteins containing class A1, class A2, and class Y helices (class A2 > class Y > class A1). Results of this study illustrate the important role of interfacial lysine residues in modulating the lipid affinity of amphipathic helices and suggest that the effect of interfacial lysine residues in increasing lipid affinity is additive. We propose that interfacial lysine residues, in addition to widening the hydrophobic face because of snorkeling, also help anchor the amphipathic helix in the lipid bilayer.

Interactions of synthetic peptide analogs of the class A amphipathic helix with lipids. Evidence for the snorkel hypothesis.

Class A amphipathic helixes present in exchangeable plasma apolipoproteins are characterized by the location of positively charged amino acid residues at the non-polar-polar interface and negatively charged amino acid residues at the center of the polar face. The objectives of the present study were: (i) to investigate the role of hydrocarbon side chain length of the interfacial positively charged amino acid residues in the lipid affinity of class A amphipathic helixes, and (ii) to investigate the importance of the nature of interfacial charge in the lipid affinity of class A amphipathic helixes. Toward this end, lipid interactions of the following two analogs of the class A amphipathic helix, Ac-18A-NH2 (acetyl-Asp-Trp-Leu-Lys-Ala-Phe-Tyr- Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe-NH2), and Ac-18A(Lys > Haa)-NH2 (acetyl-Asp-Trp-Leu-Haa-Ala-Phe-Tyr-Asp-Haa-Val-Ala-Glu-Haa-Leu-Haa-Glu- Ala-Phe-NH2) (Haa = homoaminoalanine), were studied. The side chain of Haa has two CH2 groups less than that of lysine. The lipid affinities of these two peptide analogs were compared with that of Ac-18R-NH2, an analog of Ac-18A-NH2 with positions of the charged amino acid residues reversed. The techniques used in these studies were circular dichroism, fluorescence spectroscopy, right-angle light scattering measurements, and differential scanning calorimetry. The results of these studies indicated the following rank order of lipid affinity: Ac-18A-NH2 > Ac-18A(Lys > Haa)-NH2 > Ac-18R-NH2. These results are in agreement with the "snorkel" model proposed earlier to explain the higher lipid affinity of class A amphipathic helixes (Segrest, J. P., Loof, H. D., Dohlman, J. G., Brouillette, C. G., and Anantharamaiah, G. M. (1990) Proteins Struct. Funct. Genetics 8, 103-117). In addition, it was observed from the differential scanning calorimetry studies that Ac-18A-NH2 and Ac-18A(Lys > Haa)-NH2 interact more strongly than Ac-18R-NH2 with negatively charged dimyristoyl phosphatidylglycerol. The weaker interaction of Ac-18R-NH2 with dimyristoyl phosphatidylglycerol is suggested to be due to electrostatic repulsion between the negatively charged lipid and the interfacial negative charges of the peptide.

Molecular basis for prokaryotic specificity of magainin-induced lysis.

Magainins and mastoparans are examples of peptide antibiotics and peptide venoms, respectively. They have been grouped together as class L amphipathic helixes [Segrest, J.P., et al. (1990) Proteins 8, 103-117] because of similarities in the distribution of Lys residues along the polar face of the helix. Class L venoms lyse both eukaryotic and prokaryotic cells whereas class L antibiotics specifically lyse bacteria. The structural basis for the specificity of class L antibiotics is not well understood. Sequence analysis showed that class L antibiotics have a Glu residue on the nonpolar face of the amphipathic helix; this is absent from class L venoms. We synthesized three model class L peptides with or without Glu on the nonpolar face: 18LMG (LGSIWKFIKAFVGGIKKF), [E14]18LMG and [G5,E14]18LMG. Hemolysis, bacteriolysis, and bacteriostasis studies using these peptides showed that the specificity of lysis is due to both the presence of a Glu residue on the nonpolar face of the helix and the bulk of the nonpolar face. Studies using large unilamellar phospholipid vesicles showed that the inclusion of cholesterol greatly inhibited leakage by the two Glu-containing peptides. These results cannot be attributed to changes in the phase behavior of the lipids caused by the inclusion of cholesterol or to differences in the secondary structure of the peptides. These results suggest that eukaryotic cells are resistant to lysis by magainins because of peptide-cholesterol interactions in their membranes that inhibit the formation of peptide structures capable of lysis, perhaps by hydrogen bonding between Glu and cholesterol. Bacterial membranes, lacking cholesterol, are susceptible to lysis by magainins.

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.

Probing structure and function of VLDL by synthetic amphipathic helical peptides.

Class A amphipathic helical peptides have been shown to mimic many properties of exchangeable apolipoproteins. The three analogs of the class A amphipathic peptides were used to probe the structure and function of human very low density lipoproteins (VLDL): 1) 18 residue peptide possessing a single helical domain (18A) with the sequence Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe; 2) two domains of 18A separated by a Pro (37pA); and 3) and 18A analog with the end groups protected to increase helicity (Ac-18A-NH2). Upon incubation of the peptides with VLDL at a peptide to VLDL, (protein) ratio of 1:1, the 37pA and Ac-18A-NH2 were able to displace most of apolipoprotein (apo) Cs and E from VLDL without alteration in its lipid composition and morphology while 18A had minimal effect. The extent of displacement was a function of the peptide to VLDL ratio. The rank order of displaceability of apolipoproteins on VLDL was apoE > C-III > C-II. The displacement of apoE and/or Cs from VLDL by peptides variably affected the ability of VLDL to interact with purified bovine milk lipoprotein lipase (LpL) and cultured macrophages. Treatment of VLDL with Ac-18A-NH2 markedly lowered its reactivity to LpL and its ability to induce lipid accumulation in cultured macrophages: however, treatment of VLDL with 37pA or 18A only minimally lowered their abilities. Ac-18A-NH2 treatment of VLDL resulted in the increase of apparent K(m) and a decrease of Vmax for lipoprotein lipase (LpL)-catalyzed hydrolysis of VLDL triglycerides. When an artificial triglyceride emulsion was used as a substrate of LpL, 37pA, but not Ac-18A-NH2, activated LpL. The above data indicate that 1) amphipathic helical peptides can alter the metabolic and functional properties of VLDL by dissociating the functionally important exchangeable apolipoproteins from VLDL as well as by acting as a functional element of VLDL after their incorporation; and 2) the class A amphipathic peptides having different lipid-associating properties exert significantly different effect on VLDL function.

Human placental tissue expresses a novel 22.7 kDa apolipoprotein A-I-like protein.

Since apolipoprotein A-I (apo A-I) and HDL stimulate the expression of the placental hormone human placental lactogen (hPL), experiments were performed to determine whether the human placenta synthesizes apo A-I. Western blot analysis of a partially purified extract of human term placenta with an antiserum to human apo A-I yielded an immunoreactive band with an apparent mass of approximately 23.5 kDa, which is smaller than human plasma apo A-I (28 kDa). HPLC chromatography of the partially purified placental extract on a preparative reverse-phase C-18 column yielded two fractions that reacted to the apo A-I antiserum. The mass of both fractions by mass spectral analysis was 22 721 daltons, and N-terminal amino acid sequences were identical to the first four amino acids of apo A-I (Asp, Glu, Pro, Pro). The apo A-I-like protein was not a proteolytic product of apo A-I since Northern analysis of placental RNA with a 641 bp apo A-I cDNA fragment encoding most of the 5' region of the apo A-I mRNA detected a single band of 850 nt, which is smaller than the size of apo A-I mRNA (1100 nt). Placental mRNA, however, did not hybridize with a 3' apo A-I riboprobe, indicating that the 3' region of the apo A-I-like mRNA is different from that of apo A-I mRNA. Differences in the mRNAs were confirmed by S1 nuclease analysis of placental RNA with a cDNA probe that included the 3' end of the apo A-I cDNA and by RT-PCR analysis with a series of oligonucleotide primers that span the entire cDNA for apo A-I. Since there is only a single apo A-I gene in the human genome, these findings strongly suggest that human placental tissue expresses a novel 22.7 kDa apo A-I-like protein (ALP) that results from alternative splicing of the apo A-I primary transcript.

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.

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)

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.

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.

Studies of synthetic peptides of human apolipoprotein A-I containing tandem amphipathic alpha-helixes.

In mature human apolipoprotein A-I (apo A-I), the amino acid residues 1-43 are encoded by exon 3, whereas residues 44-243 are encoded by exon 4 of the apo A-I gene. The region encoded by exon 4 of the apo A-I gene contains 10 tandem amphipathic alpha-helixes; their location and the class to which they belong are as follows: helix 1 (44-65, class A1), helix 2 (66-87, class A1), helix 3 (88-98, class Y), helix 4 (99-120, class Y), helix 5 (121-142, class A1), helix 6 (143-164, class A1), helix 7 (165-186, class A1), helix 8 (187-208, class A1), helix 9 (209-219, class Y), and helix 10 (220-241, class Y). To examine the effects of multiple tandem amphipathic helixes compared to individual helixes of apo A-I on lipid association, we have studied lipid-associating properties of the following peptides: Ac-44-87-NH2 (peptide 1-2), Ac-66-98-NH2 (peptide 2-3), Ac-66-120-NH2 (peptide 2-3-4), Ac-88-120-NH2 (peptide 3-4), Ac-99-142-NH2 (peptide 4-5), Ac-121-164-NH2 (peptide 5-6), Ac-143-186-NH2 (peptide 6-7), Ac-165-208-NH2 (peptide 7-8), Ac-187-219-NH2 (peptide 8-9), and Ac-209-241-NH2 (peptide 9-10). To study lipid-associating properties of the region encoded by exon 3 of the apo A-I gene, 1-33-NH2 (peptide G) has also been studied. The results of the present study indicate that, among the peptides studied, peptides 1-2 and 9-10 possess significantly higher lipid affinity than the other peptides, with peptide 9-10 having higher lipid affinity than peptide 1-2, as evidenced by (i) higher helical content in the presence of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), (ii) faster rate of association with DMPC multilamellar vesicles (MLV), (iii) greater reduction in the enthalpy of gel to liquid-crystalline phase transition of DMPC MLV, (iv) higher exclusion pressure from an egg yolk phosphatidylcholine monolayer, and (v) higher partitioning into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine MLV. A comparison of the free energies of lipid association (DeltaG) of the peptides studied here with those studied previously by us [Palgunachari, M. N. , et al. (1996) Arterioscler. Thromb. Vasc. Biol. 16, 328-338] indicates that, except for the peptides 4-5 and 5-6, other peptides possess higher lipid affinities compared to constituent helixes. However, the lipid affinities of the peptides studied here are neither higher than nor equal to the sum of the lipid affinities of the constituent helixes. This indicates the absence of cooperativity among the adjacent amphipathic helical domains of apo A-I for lipid association. As indicated by DeltaG, the lipid affinity of peptide 4-5 is higher than peptide 5 but lower than peptide 4; the lipid affinity of peptide 5-6 is lower than both peptides 5 and 6. Implications of these results for the structure and function of apo A-I are discussed.

A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis.

Several synthetic class A peptide analogues have been shown to mimic many of the properties of human apo A-I in vitro. A new peptide [acetyl-(AspTrpLeuLysAlaPheTyrAspLysValPheGluLysPheLysGluPhePhe)-NH2; 5F], with increased amphipathicity, was administered by intraperitoneal injection, 20 microg/day for 16 weeks, to C57BL/6J mice fed an atherogenic diet. Mouse apo A-I (MoA-I) (50 microg/day) or phosphate-buffered saline (PBS) injections were given to other mice as controls. Total plasma cholesterol levels and lipoprotein profiles were not significantly different between the treated and control groups, except that the mice receiving 5F or MoA-I had lower high density lipoprotein (HDL) cholesterol when calculated as a percentage of total cholesterol. No toxicity or production of antibodies to the injected materials was observed. When HDL was isolated from high fat diet-administered mice injected with 5F and presented to human artery wall cells in vitro together with human low density lipoprotein (LDL), there were substantially fewer lipid hydroperoxides formed and substantially less LDL-induced monocyte chemotactic activity than with HDL from PBS-injected animals. Injection of human apo A-I produced effects similar to 5F on lipid peroxide formation and LDL-induced monocyte chemotactic activity, but injection of MoA-I was significantly less effective in reducing lipid hydroperoxide formation or lowering LDL-induced monocyte chemotactic activity. Mice receiving peptide 5F had significantly less aortic atherosclerotic lesion area compared with mice receiving PBS, whereas lesion area in mice receiving MoA-I was similar to that of the PBS-injected animals. This is the first in vivo demonstration that a model class A amphipathic helical peptide has antiatherosclerotic properties. We conclude that 5F inhibits lesion formation in high fat diet-administered mice by a mechanism that does not involve changes in the lipoprotein profile, and may have potential in the prevention and treatment of atherosclerosis.

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.

Apolipoprotein-mediated plasma membrane microsolubilization. Role of lipid affinity and membrane penetration in the efflux of cellular cholesterol and phospholipid.

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.