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 l ow 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-ri ch repeat regions of the receptor. To determine the relationship betwe en the receptor binding and lipid associating properties of apoE, we h ave 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(1 8)-Gly-apoE peptide). The unmodified peptides demonstrated low affinit y for lipid surfaces (K-d > 10(-5) M) and moderate alpha-helicity in t he presence of lipid (40%) and had no effect on LDL uptake by fibrobla sts. N-Palmitoyl peptides had increased affinity for lipid (K-d simila r to 10(-6) M) and increased alpha-helicity (55%) in the presence of l ipid. The addition of the C-16-apoE-(129-169)-peptide to I-125-LDL enh anced its uptake and degradation by fibroblasts 8-10-fold; however, <5 0% of the degradation was mediated by the LDL receptor. By contrast, t he 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 p resence of lipid. The addition of the diC(18)-Gly-apoE-(129-169)-pepti de 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 age nt. In agreement with this finding a mutant diC(18)-Gly-apoE peptide ( Arg(142) --> Gln) was much less effective than the wild-type peptide i n potentiating binding, uptake, and degradation of I-125-LDL. Complexe s 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 r eceptor, Moreover, since the N,N-distearylglycyl peptide is nonexchang eable, the data indicate that it can be used to probe the structural r equirements for ligand binding and the processing of apoE-containing l ipoproteins. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine containi ng four to six copies of the peptide/particle displayed an affinity fo r the LDL receptor similar to that of apoE-L-alpha-dimyristoylphosphat idyl-choline discs containing four copies of apoE.