A Novel Pyrene Based Fluorescence Approach to Study Conformation of Apolipoprotein E3 in Macrophage-generated Nascent High Density Lipoprotein

摘要

Apolipoprotein E3 (apoE3) is an anti-atherogenic apolipoprotein with the ability to exist in lipid-free and lipoprotein-associated states. During atherosclerosis, its function in promoting cholesterol efflux from macrophages via the ATP-binding cassette transporter A1 (ABCA1) takes a prominent role, leading to generation of nascent high density lipoprotein (nHDL) particles. The objective of this study is to understand the conformation adopted by apoE3 in macrophage-generated nHDL using a fluorescence spectroscopic approach involving pyrene. Pyrene-labeled recombinant human apoE3 displayed a robust ability to stimulate ABCA1-mediated cholesterol efflux from cholesterol-loaded J774 macrophages (which do not express apoE), comparable to that elicited by unlabeled apoE3. The nHDL recovered from the conditioned medium revealed the presence of apoE3 by immunoblot analysis. A heterogeneous population of nHDL bearing exogenously added apoE3 was generated with particle size varying from ~ 12 to ~19 nm in diameter, corresponding to molecular mass of ~ 450 to ~700 kDa. The lipid: apoE3 ratio varied from ~60:1 to 10:1. A significant extent of pyrene excimer emission was noted in nHDL, indicative of spatial proximity between Cys112 on neighboring apoE3 molecules similar to that noted in reconstituted HDL. Cross-linking analysis using Cys-specific cross-linkers revealed the predominant presence of dimers. Taken together the data indicate a double belt arrangement of apoE molecules on nHDL. A similar organization of the C-terminal tail of apoE on nHDL was noted when pyrene-apoEA277C(201-299) was used as the cholesterol acceptor. These studies open up the possibility of using exogenously labeled apoE3 to generate nHDL for structural and conformational analysis.