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, α-helical sequence of ~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 (K(d) > 10^-5 M) and moderate α-helicity in the presence of lipid (40%) and had no effect on LDL uptake by fibroblasts. N-Palmitoyl peptides had increased affinity for lipid (K(d) ~ 10^-6 M) and increased α-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 (K(d) ≤ 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-α-dimyristoylphosphatidylcholine discs containing four copies of apoE. In summary, the receptor binding properties of apoE depend on its association with phospholipid; transfer of peptide fragments of the receptor-binding domain of apoE from the aqueous phase to a lipid surface converts them from a random coil to an α-helical conformation that is recognized by the LDL receptor. Moreover, since the N,N- distearylglycyl peptide is nonexchangeable, the data indicate that it can be used to probe the structural requirements for ligand binding and the processing of apoE-containing lipoproteins.