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 similar to 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 (C-16-apoE peptide) or the N,N-distearyl derivative of glycine (diC(18)-Gly-apoE peptide). The unmodified peptides demonstrated low affinity for lipid surfaces (K-d > 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 (K-d similar to 10(-6) M) and increased alpha-helicity (55%) in the presence of lipid. The addition of the C-16-apoE-(129-169)-peptide to I-125-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 diC(18)-Gly-apoE-(129-169)-peptide was essentially nonexchangeable (K-d less than or equal to 10(-9) M) and highly helical (78%) in the presence of lipid. The addition of the diC(18)-Gly-apoE-(129-169)-peptide to I-125-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 diC(18)-Gly-apoE-(129-169) revealed that the lipoprotein-bound peptide is the active agent. In agreement with this finding a mutant diC(18)-Gly-apoE peptide (Arg(142) --> Gln) was much less effective than the wild-type peptide in potentiating binding, uptake, and degradation of I-125-LDL. Complexes of diC(18)-Gly-apoE-(129-169), apoA-I, and 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 alpha-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. 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-dimyristoylphosphatidyl-choline discs containing four copies of apoE.