Binding Interaction of Lipid-Bound Apoa-I with Lipopolysaccharides and Phosphatidylglycerol

Apolipoprotein A‐I (apoA‐I) is a major component of high‐density lipoprotein (HDL), an anti‐antherogenic complex responsible for reverse cholesterol transport from peripheral tissues to liver, and mediates several essential metabolic functions related to heart disease. Human apoA‐I is 28 kDa protein found in lipid‐free and lipid‐bound forms. Previous studies have shown that lipid‐free apoA‐I binds and neutralizes lipopolysaccharides (LPS), which are major constituents of the outer bacterial membrane of gram‐negative bacteria, therefore reducing endotoxic effects. In addition, the protein destabilizes bilayers of negatively charged phosphatidylglycerol (PG), a phospholipid found in the inner bacterial membrane, which may result in cell lysis. However, the majority of apoA‐I is found in the lipid‐bound form as HDL; therefore binding interactions with LPS and PG were studied. To this end reconstituted HDL (rHDL) was produced with 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and apoA‐I, which was isolated by KBr density ultracentrifugation. rHDL was analyzed by native PAGE and showed three distinct lipid‐protein complexes of 246, 203, and 149 kDa. To compare the binding of lipid‐free and lipid bound apoA‐I to LPS, non‐denaturing PAGE was employed. This showed that LPS binding was decreased for rHDL in comparison to lipid‐free apoA‐I. Both forms of apoA‐I were able to disaggregate LPS micelles, as shown by LPS carbohydrate staining of native PAGE. To investigate binding to PG, unilamellar PG vesicles with encapsulated calcein were prepared by extrusion. Calcein is a fluorescent dye and used as an indicator of lipid vesicle leakage, and is self‐quenched at a concentration above 70 mM. Binding to the vesicles was measured by the increase in calcein fluorescence, which was released upon protein‐vesicle binding. Lipid‐free apoA‐I induced the release of 60 to 70% calcein from PG vesicles; however, calcein release was reduced to 20 to 30% for rHDL. These results show that when apoA‐I is in the lipid‐bound state, binding interaction with LPS and PG bilayer vesicles was decreased significantly. Thus in order to be most effective as an antimicrobial protein, apoA‐I needs to be in the lipid‐free conformation. Support or Funding Information This research was supported by a grant from the National Institutes of Health (NIGMS #GM089564).