High density lipoproteins (HDL) mediate cholesterol transport and protection from cardiovascular disease. Although synthetic HDLs have been studied for 30 years, the structure of human plasma-derived HDL, and its major protein apolipoprotein (apo)A-I, is unknown. We separated normal human HDL into 5 density subfractions and then further isolated those containing predominantly apoA-I (LpA-I). Using cross-linking chemistry and mass spectrometry, we found that apoA-I adopts a structural framework in these particles that closely mirrors that in synthetic HDL. We adapted established structural models for synthetic HDL to generate the first detailed models of authentic human plasma HDL in which apoA-I adopts a symmetrical cage-like structure. The models suggest that HDL particle size is modulated via a twisting motion of the resident apoA-I molecules. This understanding offers insights into how apoA-I structure modulates HDL function and its interactions with other apolipoproteins.

Human high density lipoproteins (HDL) are divided into two major subpopulations based on lipoprotein composition. The predominant subpopulation contains both major apolipoproteins apoA-I and apoA-II (termed LpA-I/A-II), while the minor subpopulation contains only apoA-I (termed LpA-I). It is speculated that LpA-I/A-II could demonstrate different anti-oxidative and anti-inflammatory properties compared to LpA-I. The goal of the current work was to dissect out any contributions from apoA-II on such beneficial functions of HDL. We generated a series of reconstituted (r)LpA-I/A-II in which major apolipoprotein ratios mimic native LpA-I/A-II (apoA-I: apoA-II; 2:1, 2:2 and 2:3) but lack the anti-oxidative enzymes in native HDL. First, we generated the precursor rLpA-I by reacting discoidal HDL with recombinant lecithin cholesterol acyl transferase (LCAT) in the presence of low density lipoproteins. The rLpA-I consist of a single population of ∼93 Å particles. Cross-linking followed by MALDI-MS indicated exactly three apoA-I per rLpA-I. Different rLpA-I/A-II complexes were generated by adding the required amount of apoA-II into rLpA-I followed by isolation of rLpA-I:A-II using size exclusion chromatography. All rLpA-I/A-II showed comparable particle diameter to rLpA-I. Electron microscopy clearly captured the spherical nature of these particles and agarose gel electrophoresis demonstrated the presence of a neutral lipid core in rLpA-I and rLpA-I/A-II compared to discoidal HDL. All particles contain comparable amounts of total protein 36-40%, phospholipids 40-45%, cholesteryl esters 17-22%, free cholesterol <1% and triglycerides <1%. In vitro anti-oxidative functional assays utilizing copper-mediated oxidation demonstrated ∼20% better anti-oxidative capacity for rLpA-I/A-II (apoA-I: apoA-II, 2:1) compared to rLpA-I. We are currently in the process of testing anti-oxidative properties of other rLpA-I/A-II complexes. Experiments are currently underway to assess differences of anti-inflammatory properties of these complexes by quantitative measurements of adhesion molecule VCAM, ICAM and E-selection using flow cytometry in cell culture.