There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact Ox-LDL with high affinity. This binding was very effectively inhibited (ϳ50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.

It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4°C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognize both the lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and͞or covalently linked to the apoB of OxLDL, likely play a role in that recognition.

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 g/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 ␣-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 (⌬222-243), apo A-I (⌬190 -243), apo E3 (⌬192-299) and apo E4 (⌬192-299) 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 ␣-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.

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)

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Maltose transport across the cytoplasmic membrane of Escherichia coli is catalyzed by a periplasmic binding protein-dependent transport system and energized by ATP. The maltose system, a member of the ATP-binding cassette or ABC transport family, contains two copies of an ATP-binding protein in a complex with two integral membrane proteins. ATP hydrolysis by the transport complex can be assayed following reconstitution into proteoliposomes in the presence of maltose binding protein and maltose. Mutations in the transport complex that permit binding protein-independent transport render ATP hydrolysis constitutive so that hydrolysis can also be assayed with the transport complex in detergent solution. We have used both of these systems to study the role of two ATP binding sites in ATP hydrolysis. We found that both the wild-type and the binding protein-independent systems hydrolyzed ATP with positive cooperativity, suggesting that the two ATP binding sites interact. Vanadate inhibited the ATPase activity of the transport complex with 50% inhibition occurring at 10 mum vanadate. In detergent solution, the degree of cooperativity in the binding protein-independent complex decreased with increasing pH. The loss of cooperativity was accompanied by a decrease in ATPase activity and a decrease in sensitivity to vanadate. Because reconstitution of the complex into a lipid bilayer prevented the loss of cooperativity, we expect that ATP hydrolysis is cooperative in vivo. The mutations leading to binding protein-independent transport do not significantly alter the affinity, cooperativity, vanadate sensitivity, or substrate specificity of the ATP binding sites during hydrolysis. These results justify the use of the binding protein-independent system to investigate the mechanism of transport and hydrolysis.

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

Previous studies have shown that macrophage receptors for oxidized LDL (OxLDL) recognize both the lipid and protein moieties, and that a monoclonal antibody against OxLDL, EO6, also recognizes both species. The present studies show directly that during LDL oxidation phospholipids become covalently attached to apolipoprotein B (apoB). After exhaustive extraction of lipids, apoB of native LDL contained 4 ؎ 3 moles of phosphorus/mole protein. In contrast, apoB of OxLDL contained approximately 75 moles of phosphorus/mole protein. Saponification of this apoB released phosphorus, choline, and saturated fatty acids in a molar ratio of 1.0:0.98:0.84. When LDL was reductively methylated prior to oxidation, the amount of phospholipid covalently bound was reduced by about 80%, indicating that the phospholipids attach at lysine epsilon amino groups. Progressive decreases in the phospholipid associated with apoB of OxLDL decreased the ability of the protein to compete for binding to macrophage scavenger receptors and decreased its reactivity with antibody EO6. We postulate that some oxidized phospholipids containing fatty acid aldehydes at the sn -2 position bind to lysine residues of apoB while others remain unreacted within the lipid phase. This would account for the interchangeability of lipid and apolipoprotein of OxLDL with respect to receptor binding and antibody recognition. -