Reconstituted high-density lipoproteins with a disulfide-linked apolipoprotein A-I dimer: Evidence for restricted particle size heterogeneity

The apolipoprotein A-I-Milano (apoA-I-M) is a molecular variant of apoA-I characterized by the Arg(173)-->Cys substitution, resulting in the formation of homodimers (A-I-M/A-I-M) 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-I-M/A-I-M and apoA-I with dimyristoylphosphatidylcholine (DMPC), and the structure and properties of reconstituted HDL containing palmitoyloleoylphosphatidylcholine (POPC) and either A-I-M/A-I-M 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-I-M/A-I-M 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-I-M/A-I-M molecules and have a diameter of 7.8 nm and 12.5 nm. Investigations of the A-I-M/A-I-M structure in these two rHDL, by circular dichroism, fluorescence, and second-derivative UV spectroscopy, suggest that the secondary and tertiary structures of A-I-M/A-I-M 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.