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A fluorescence based assay of apolipoprotein E ‐ low density lipoprotein receptor interactions
Author(s) -
Yamamoto Taichi,
Ryan Robert O.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a94
Subject(s) - ldl receptor , chemistry , apolipoprotein b , fluorescence , ligand (biochemistry) , apolipoprotein e , lipoprotein , receptor , cysteine , tryptophan , mutant , biochemistry , biophysics , microbiology and biotechnology , cholesterol , biology , medicine , enzyme , amino acid , physics , disease , quantum mechanics , gene
Members of the low density lipoprotein receptor (LDLR) family function to maintain plasma cholesterol homeostasis. One of the best characterized LDLR ligands is apolipoprotein E (apoE). Whereas lipid‐free apoE does not serve as a ligand, lipid associated apoE binds to LDLR. The NT domain of apoE is responsible for recognition by LDLR. A soluble fragment of LDLR (N‐terminal residues 1‐699) was expressed in HEK 293 cells and isolated from the culture medium by affinity chromatography. A tryptophan deficient apoE3 N terminal (NT) domain (Trp null apoE3‐NT) mutant was expressed in E. coli, isolated and covalently labeled on cysteine 112 with the extrinsic fluorescent probe, N‐iodoacety‐N’‐(5‐sulfo‐1‐naphthyl) ethyeylenediamine (AEDANS). AEDANS Trp null apoE3‐NT was complexed with dimyristoylphosphatidylcholine (DMPC) and incubated with LDLR. Enhancement in AEDANS fluorescence emission via energy transfer from LDLR Trp residues (excitation 280 nm) was observed. The enhancement in AEDANS fluorescence was saturable and was abolished by incubation with EDTA or pre‐incubation with a non‐fluorescent competitor ligand (Trp null apoE3‐NT DMPC). The present assay provides a sensitive, convenient and quantitative means to evaluate ligand binding to LDLR.