The receptor binding domain of apolipoprotein E, linked to a model class Aamphipathic helix, enhances internalization and degradation of LDL by fibroblasts

Human apolipoprotein E (apo E) consists of two distinct domains, the lipid- associating domain (residues 192-299) and the globular domain (residues 1-1 91) which contains the LDL receptor (LDLR) binding site (residues 129-169), To test the hypothesis that an arginine-rich apo E receptor binding domain (residues 141-150) is sufficient to enhance low-density lipoprotein (LDL) uptake and clearance when covalently linked to a class A amphipathic helix, a peptide in which the receptor binding domain of human apo E, LRKLRKRLLR (hApoE[141-150]), is linked to 18A, a well-characterized high-affinity lipi d-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApo E[141-150]18A (hE18A) and its end-protected analogue, Ac-hE18A-NH2. The imp ortance of positively charged residues and the role of the hydrophobic resi dues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH2 [Ac-hE(R)18A-NH2] and Ac-LRKMRKRLMR-18A-NH2 (Ac-mE18 A-NH2) contained an extended hydrophobic face, including the receptor bindi ng region. Control peptides, Ac-LRLLRKLKRR-18A-NH2 [Ac-hE(Sc)18A-NH2], had the amino acid residues of the apo E receptor binding domain scrambled to d isrupt the extended hydrophobic face, and Ac-RRRRRRRRRR 18A-NH2 (Ac-R(10)(1 8)A-NH2) had only positively charged Arg residues as the receptor binding d omain. The effect of the dual-domain peptides on the uptake and degradation of human LDL by fibroblasts was determined in murine embryonic fibroblasts (MEF1). LDL internalization was enhanced 3-, 5-, and 7-fold by Ac-mE18A-NH 2, Ac-hE18A-NH2, and Ac-hE(R)18A-NH2 respectively, whereas the control pept ides had no significant biological activity. All three active peptides incr eased the level of degradation of LDL by 100%, The LDL binding and internal ization to MEF1 cells in the presence of these peptides was not saturable o ver the LDL concentration range that was studied (1-10 mu g/mL). Furthermor e, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor related protein (LRP), LDLR, or both. Pre treatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We c onclude that the dual-domain peptides enhanced LDL uptake and degradation b y fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.