A nonexchangeable apolipoprotein E peptide that mediates binding to the low density lipoprotein receptor.

ApoE is a 34-kDa apoprotein that mediates lipoprotein binding to the low density lipoprotein (LDL) receptor and to the LDL receptor-related protein. Receptor binding is mediated by a highly basic, alpha-helical sequence of approximately 15 amino acids that interacts with cysteine-rich repeat regions of the receptor. To determine the relationship between the receptor binding and lipid associating properties of apoE, we have synthesized a series of apoE peptides containing all (residues 129-169) or part (residues 139-169, 144-169, and 148-169) of the receptor-binding domain. The lipophilicity of these peptides was increased by modification of their N termini by acylation with either palmitic acid (C16-apoE peptide) or the N,N-distearyl derivative of glycine (diC18-Gly-apoE peptide). The unmodified peptides demonstrated low affinity for lipid surfaces (Kd > 10(-5) M) and moderate alpha-helicity in the presence of lipid (40%) and had no effect on LDL uptake by fibroblasts. N-Palmitoyl peptides had increased affinity for lipid (Kd approximately 10(-6) M) and increased alpha-helicity (55%) in the presence of lipid. The addition of the C16-apoE-(129-169)-peptide to 125I-LDL enhanced its uptake and degradation by fibroblasts 8-10-fold; however, < 50% of the degradation was mediated by the LDL receptor. By contrast, the diC18-Gly-apoE-(129-169)-peptide was essentially nonexchangeable (Kd < or = 10(-9) M) and highly helical (78%) in the presence of lipid. The addition of the diC18-Gly-apoE-(129-169)-peptide to 125I-LDL enhanced the specific uptake and degradation of LDL by both LDL receptor-mediated and non-LDL receptor-mediated mechanisms. Uptake and degradation of methylated LDL containing diC18-Gly-apoE-(129-169) revealed that the lipoprotein-bound peptide is the active agent. In agreement with this finding, a mutant diC18-Gly-apoE peptide (Arg142-->Gln) was much less effective than the wild-type peptide in potentiating binding, uptake, and degradation of 125I-LDL. Complexes of diC18-Gly-apoE-(129-169), apoA-I, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine containing four to six copies of the peptide/particle displayed an affinity for the LDL receptor similar to that of apoE-L-alpha-dimyristoylphosphatidylcholine discs containing four copies of apoE.(ABSTRACT TRUNCATED AT 400 WORDS)

Apolipoprotein E: phospholipid binding studies with synthetic peptides from the carboxyl terminus.

We have previously shown that the synthetic peptide apoE(129-169) forms lipid-peptide complexes with dimyristoylphosphatidylcholine (DMPC) with an L:P molar ratio of 125:1; the peptide in the isolated complex contains approximately 56% alpha-helicity. These results verify the presence of an amphipathic alpha-helix in this region of apoE as predicted by Chou-Fasman analysis and hydrophobicity calculations. To further define the lipid binding regions of apoE, we have synthesized four peptides, apoE(211-243), -(202-243), -(267-286), and -(263-286), from the carboxyl terminus of apoE and studied their lipid binding properties; apoE(202-243) contains two potential amphipathic helices. Although all four peptides formed alpha-helices in the helix-forming solvent 30% hexafluoropropanol, we found that only apoE(263-286) formed a stable complex with DMPC. The peptide contained approximately 80% alpha-helicity, and its Trp fluorescence spectrum was blue-shifted by 20 nm in the complex which had an L:P ratio of 163:1. We conclude that this sequence is a newly identified lipid binding region of apoE and that the amphipathic helices 203-221 and 226-243 are too hydrophilic to bind phospholipid.

Kinetics and mechanism of transfer of synthetic model apolipoproteins.

The effect of hydrophobicity on the rate and mechanism of transfer of a synthetic amphiphilic peptide between phosphatidylcholine single bilayer vesicles has been evaluated. These peptides, which had the sequence Cn-SSLKEYWSSLKESFS (where Cn represents a saturated acyl chain of n carbons that is attached to the amino terminus of the peptide and n = 8, 12, or 16), were distinguished by the length of the saturated acyl chain of n carbons that was covalently bonded to the amino terminus. The transfer of the peptides was monitored by following the rate of change of the intrinsic tryptophan fluorescence that followed mixing of donor vesicles, which contained peptide, phosphatidylcholine, and a fluorescence quencher, with acceptors composed only of phosphatidylcholine. The transfer rates were independent of the structure and concentration of the acceptor. The kinetics were biexponential with the contribution of the fast and slow components being nearly equal. The rates of both components decreased with increasing acyl chain length; the respective free energies of activation were linear with respect to the acyl chain length. These results showed that, unlike lipid transfer, peptide transfer is not always a simple unimolecular process. However, like lipid transfer, the transfer rates are a predictable function of hydrophobicity. It is proposed that the peptides exist as dimers on the phospholipid surface and that the two components of transfer are due to sequential transfer of each molecule in a dimer.

Apolipoprotein E: phospholipid binding studies with synthetic peptides containing the putative receptor binding region.

To define the lipid and receptor binding regions of apolipoprotein E (apoE), we have synthesized four peptides beginning at residue 169 and continuing through the putative receptor binding region and ending at residue 129 so as to include a proposed lipid binding domain. The peptides were synthesized by solid-phase techniques, cleaved with anhydrous HF, and purified by ion-exchange and semipreparative reversed-phase high-performance liquid chromatography (HPLC). The peptides had the correct amino acid composition and were greater than 99% pure by analytical reversed-phase HPLC. The circular dichroic spectrum of each peptide was recorded before and after mixing with dimyristoylphosphatidylcholine. With apoE (148-169), apoE (144-169), and apoE (139-169), no changes were observed in the ellipticity at 222 nm. However, with apoE (129-169), an increase in alpha-helicity to approximately 42% was observed. Density gradient ultracentrifugation of the lipid-peptide mixture permitted isolation of a complex with apoE (129-169) with a molar ratio of lipid to peptide of 125:1, which was stable to recentrifugation. The alpha-helicity of the peptide in the complex was estimated to be 56%. No complexes were isolated from the gradients of the shorter peptides. Therefore, we conclude that the amphipathic helix formed by residues 130-150 contains one of the lipid binding regions of apoE.