Lipid-bound apoLp-III is less effective in binding to lipopolysaccharides and phosphatidylglycerol vesicles compared to the lipid-free protein.

Abstract

Apolipophorin III (apoLp-III) is an insect apolipoprotein that is predominantly present in a lipid-free state in the hemolymph. ApoLp-III from Galleria mellonella is able to interact with membrane components of Gram-negative bacteria, as part of an innate immune response to infection. The protein also exists in a lipoprotein-associated state when large amounts of lipids are mobilized. Therefore, lipid-bound apoLp-III was generated to analyze the binding interaction with lipopolysaccharides and phosphatidylglycerol, both abundantly present in membranes of Gram-negative bacteria. G. mellonella apoLp-III was lipidated with palmitoyl-2-oleoyl-glycero-3-phosphocholine to form lipid-protein complexes. The particle shape was discoidal with a 16.4nm diameter, a molecular mass of 460kDa, and contained 4 apoLp-III molecules. These discoidal lipoproteins were used to compare the lipopolysaccharide and phosphatidylglycerol binding activity with lipid-free apoLp-III. Lipopolysaccharide binding interaction was analyzed by non-denaturing PAGE, showing reduced ability of the lipid-bound protein to form lipopolysaccharide-protein complexes and to disaggregate lipopolysaccharide micelles. The apoLp-III-induced release of calcein from phosphatidylglycerol vesicles was decreased approximately fivefold when the protein was in the lipid-bound form, indicating reduced binding interaction with the phosphatidylglycerol membrane surface. These results show that when apoLp-III adopts a lipid-bound conformation, it is markedly less effective in interacting with lipopolysaccharides and phosphatidylglycerol vesicles. Thus, in order to be an effective antimicrobial protein, apoLp-III needs to be in a lipid-free state.