title: A pyrene based fluorescence approach to study conformation of apolipoprotein E3 in macrophage-generated nascent high density lipoprotein.
abstract: 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.
authors.affiliation: Department of Chemistry & Biochemistry, California State University Long Beach, Long Beach, CA 90840, USA.
authors.forename: Sea H
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Chemistry & Biochemistry, California State University Long Beach, Long Beach, CA 90840, USA.
authors.forename: Shweta
authors.initials: S
authors.lastname: Kothari
authors.affiliation: Department of Chemistry & Biochemistry, California State University Long Beach, Long Beach, CA 90840, USA.
authors.forename: Arti B
authors.initials: AB
authors.lastname: Patel
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: John K
authors.initials: JK
authors.lastname: Bielicki
authors.affiliation: Department of Chemistry & Biochemistry, California State University Long Beach, Long Beach, CA 90840, USA. Electronic address: vas.narayanaswami@csulb.edu.
authors.forename: Vasanthy
authors.initials: V
authors.lastname: Narayanaswami
mesh_terms: D000818:Animals; D053318:Apolipoprotein E3 / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D053442:High-Density Lipoproteins, Pre-beta / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D011487:Protein Conformation; D011721:Pyrenes / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D053318:Apolipoprotein E3; D053442:High-Density Lipoproteins, Pre-beta; D011721:Pyrenes; C030984:pyrene
keywords: Apolipoprotein E3; Cross-linking; Macrophage; Nascent HDL; Pyrene fluorescence; Reverse cholesterol transport