title: The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts.
abstract: Human apolipoprotein E (apo E) consists of two distinct domains, the lipid-associating domain (residues 192-299) and the globular domain (residues 1-191) 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 lipid-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApoE[141-150]-18A (hE18A) and its end-protected analogue, Ac-hE18A-NH(2). The importance of positively charged residues and the role of the hydrophobic residues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH(2) [Ac-hE(R)18A-NH(2)] and Ac-LRKMRKRLMR-18A-NH(2) (Ac-mE18A-NH(2)) contained an extended hydrophobic face, including the receptor binding region. Control peptides, Ac-LRLLRKLKRR-18A-NH(2) [Ac-hE(Sc)18A-NH(2)], had the amino acid residues of the apo E receptor binding domain scrambled to disrupt the extended hydrophobic face, and Ac-RRRRRRRRRR-18A-NH(2) (Ac-R(10)18A-NH(2)) had only positively charged Arg residues as the receptor binding domain. 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-NH(2), and Ac-hE(R)18A-NH(2), respectively, whereas the control peptides had no significant biological activity. All three active peptides increased the level of degradation of LDL by 100%. The LDL binding and internalization to MEF1 cells in the presence of these peptides was not saturable over the LDL concentration range that was studied (1-10 microgram/mL). Furthermore, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor-related protein (LRP), LDLR, or both. Pretreatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We conclude that the dual-domain peptides enhanced LDL uptake and degradation by fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.
authors.affiliation: Department of Medicine, Atherosclerosis Research Unit, University of Alabama at Birmingham Medical Center 35294, USA.
authors.forename: G
authors.initials: G
authors.lastname: Datta
authors.forename: M
authors.initials: M
authors.lastname: Chaddha
authors.forename: D W
authors.initials: DW
authors.lastname: Garber
authors.forename: B H
authors.initials: BH
authors.lastname: Chung
authors.forename: E M
authors.initials: EM
authors.lastname: Tytler
authors.forename: N
authors.initials: N
authors.lastname: Dashti
authors.forename: W A
authors.initials: WA
authors.lastname: Bradley
authors.forename: S H
authors.initials: SH
authors.lastname: Gianturco
authors.forename: G M
authors.initials: GM
authors.lastname: Anantharamaiah
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001057:Apolipoproteins E / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000378:metabolism; D018110:Receptors, Lipoprotein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001057:Apolipoproteins E; D008077:Lipoproteins, LDL; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010455:Peptides; D011973:Receptors, LDL; D018110:Receptors, Lipoprotein