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

Martha P. Mims, Asha T. Darnule, Reuben W. Tovar, Henry J. Pownall, Doris A. Sparrow, James T. Sparrow, David P. Via, Louis C. Smith

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)20539-20547
Number of pages9
JournalJournal of Biological Chemistry
Volume269
Issue number32
StatePublished - Aug 12 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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