Apolipoprotein A-I configuration and cell cholesterol efflux activity of discoidal lipoproteins depend on the reconstitution process |
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Authors: | Luz Ángela Cuellar Eduardo Daniel Prieto Laura Virginia Cabaleiro Horacio Alberto Garda |
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Institution: | Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)/Universidad Nacional de La Plata (UNLP), Facultad de Ciencias Médicas, Calles 60 y 120, 1900 La Plata, Argentina |
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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. |
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Keywords: | ABCA1 ATP binding cassette A1 ABCG1 ATP binding cassette G1 apoA-I apolipoprotein A-I CD cholate-dialysis D-HDL discoidal high density lipoproteins DM direct microsolubilization DMPC 1 2-dimyristoyl phosphatidyl choline DPPC 1 2-dipalmitoyl phosphatidyl choline FRET fluorescence resonance energy transfer GdnHCl guanidine hydrochloride HDL high density lipoproteins IPTG isopropyl-1-thio-β-d-galactopyranoside LCAT lecithin-cholesterol acyl transferase MLV multilamellar vesicles PAGE polyacrylamide gel electrophoresis PAGGE polyacrylamide gradient gel electrophoresis POPC 1-palmitoyl 2-oleoyl phosphatidyl choline RCT reverse cholesterol transport SR-BI scavenger receptor BI Tt gel to liquid-crystalline phase transition temperature |
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