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.
D000596:Amino Acids / Q000032:analysis; D001057:Apolipoproteins E / Q000378:metabolism*; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism*; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010455:Peptides / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*
D000596:Amino Acids; D001057:Apolipoproteins E; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010455:Peptides; D011956:Receptors, Cell Surface; D004134:Dimyristoylphosphatidylcholine