A pyrene based fluorescence approach to study conformation of apolipoprotein E3 in macrophage-generated nascent high density lipoprotein

作者：Kim Sea H; Kothari Shweta; Patel Arti B; Bielicki John K; Narayanaswami Vasanthy^*

来源：Biochemical and Biophysical Research Communications, 2014, 450(1): 124-128.

DOI：10.1016/j.bbrc.2014.05.071

• Macrophage
• Apolipoprotein E3
• Nascent HDL
• Pyrene fluorescence
• Cross-linking
• Reverse cholesterol transport

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 similar to 12 to similar to 19 nm in diameter, corresponding to molecular mass of similar to 450 to similar to 700 kDa. The lipid: apoE3 ratio varied from similar to 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.

• 出版日期2014-7-18