Apolipoprotein A-I configuration and cell cholesterol efflux activity of discoidal lipoproteins depend on the reconstitution process

Abstract

Discoidal high-density lipoproteins (D-HDL) are critical intermediates in reverse cholesterol transport. Most of the present knowledge of D-HDL is based on studies with reconstituted lipoprotein complexes of apolipoprotein A-I (apoA-I) obtained by cholate dialysis (CD). D-HDL can also be generated by the direct microsolubilization (DM) of phospholipid vesicles at the gel/fluid phase transition temperature, a process mechanistically similar to the "in vivo" apoAI lipidation via ABCA1. We compared the apoA-I configuration in D-HDL reconstituted with dimyristoylphosphatidylcholine by both procedures using fluorescence resonance energy transfer measurements with apoA-I tryptophan mutants and fluorescently labeled cysteine mutants. Results indicate that apoA-I configuration in D-HDL depends on the reconstitution process and are consistent with a "double belt" molecular arrangement with different helix registry. As reported by others, a configuration with juxtaposition of helices 5 of each apoAI monomer (5/5 registry) predominates in D-HDL obtained by CD. However, a configuration with helix 5 of one monomer juxtaposed with helix 2 of the other (5/2 registry) would predominate in D-HDL generated by DM. Moreover, we also show that the kinetics of cholesterol efflux from macrophage cultures depends on the reconstitution process, suggesting that apoAI configuration is important for this HDL function.

Keywords: 1,2-dimyristoyl phosphatidyl choline; 1,2-dipalmitoyl phosphatidyl choline; 1-palmitoyl, 2-oleoyl phosphatidyl choline; ABCA1; ABCG1; ATP binding cassette A1; ATP binding cassette G1; Apolipoproteins; CD; Cysteine mutants; D-HDL; DM; DMPC; DPPC; FRET; Fluorescence resonance energy transfer (FRET); GdnHCl; HDL; IPTG; LCAT; Lipoprotein structure; MLV; PAGE; PAGGE; POPC; RCT; SR-BI; Single tryptophan mutants; Site directed mutagenesis; Tt; apoA-I; apolipoprotein A-I; cholate-dialysis; direct microsolubilization; discoidal high density lipoproteins; fluorescence resonance energy transfer; gel to liquid-crystalline phase transition temperature; guanidine hydrochloride; high density lipoproteins; isopropyl-1-thio-β-d-galactopyranoside; lecithin-cholesterol acyl transferase; multilamellar vesicles; polyacrylamide gel electrophoresis; polyacrylamide gradient gel electrophoresis; reverse cholesterol transport; scavenger receptor BI.