Reconstituted High-Density Lipoproteins with a Disulfide-Linked Apolipoprotein A-I Dimer:  Evidence for Restricted Particle Size Heterogeneity

Abstract

The apolipoprotein A-IMilano (apoA-IM) is a molecular variant of apoA-I characterized by the Arg173-->Cys substitution, resulting in the formation of homodimers (A-IM/A-IM) and heterodimers with apoA-II. In order to examine the effects of the introduction of an interchain disulfide bridge on the lipid-binding properties of apoA-I, the present studies compare the kinetics of association of A-IM/A-IM and apoA-I with dimyristoylphosphatidylcholine (DMPC), and the structure and properties of reconstituted HDL containing palmitoyloleoylphosphatidylcholine (POPC) and either A-IM/A-IM or apoA-I. The results show that apoA-I dimerization does not affect the rate of association with DMPC. Apolipoprotein-POPC complexes instead, when analyzed by nondenaturing gradient gel electrophoresis, demonstrate that, differently from apoA-I, A-IM/A-IM forms only two species of rHDL particles despite a wide range of initial lipid to protein ratios. These two rHDL species contain one or two A-IM/A-IM molecules and have a diameter of 7.8 nm and 12.5 nm. Investigations of the A-IM/A-IM structure in these two rHDL, by circular dichroism, fluorescence, and second-derivative UV spectroscopy, suggest that the secondary and tertiary structures of A-IM/A-IM are remarkably similar in both small and large particles. These results suggest that the introduction of an interchain disulfide bridge does not affect the association of apoA-I with lipids but restricts HDL particle size heterogeneity, thus possibly affecting HDL function in lipid metabolism and atherosclerosis protection.

Increased Cholesterol Efflux Potential of Sera From ApoA-I Milano Carriers and Transgenic Mice

“…The A-I M and A-I WT mice have low-normal serum ApoA-I levels, no lipid-free ApoA-I in serum, 18,31 and a similar HDL phospholipid/ApoA-I ratio. The distinct physicochemical properties of acceptor particles containing wild-type or mutant ApoA-I 18,28,34,35 may also contribute to the discrepancy between the present and previous findings, by affecting the interaction of HDL with scavenger receptor BI.…”

Apolipoprotein A-I Assumes a “Looped Belt” Conformation on Reconstituted High Density Lipoprotein

“…The variability of apoA-I structure in this region may also explain the effect of the apoA-I Milano (R173C) mutation on rHDL disc size (43,44), which is restricted in size to 7.8-nm particles and is a poor activator of LCAT (45). The disulfide bridge formed between paired apoA-I at position 173 may lock apoA-I monomers in an alternative alignment (25) wherein the disruption of the protein-protein contacts necessary for adaptive changes in the central loop would create an insurmountable barrier.…”

Enzymatically Active Paraoxonase-1 Is Located at the External Membrane of Producing Cells and Released by a High Affinity, Saturable, Desorption Mechanism

“…Reconstituted HDL (rHDL) were prepared as described (24). Discoidal rHDL containing apoA-I (rHDLAI) or apoA-II (rHDLAII) were prepared by the cholate dialysis technique using a palmitoyloleophosphatidyl choline (POPC)/protein ratio (w/w) of 2.5:1.0.…”

“…Cholesterol-containing rHDL were prepared using a POPC/cholesterol/protein ratio (w/w) of 2.5:0.12:1.0. The size of mixtures and homogeneous particles was estimated by non-denaturing gradient gel electrophoresis (24), using the Pharmacia Phast System (Amersham Biosciences, Inc.). rHDLAI consisted of a major population of particles with a diameter of 9.6 nm.…”