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

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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 16.4 nm diameter, a molecular mass of 460 kDa, 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.

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Acknowledgements

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number GM089564. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding

This study was funded by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number GM089564.

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  1. Department of Chemistry and Biochemistry, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA

    Tilini U. Wijeratne & Paul M. M. Weers

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Correspondence to Paul M. M. Weers.

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Wijeratne, T.U., Weers, P.M.M. Lipid-bound apoLp-III is less effective in binding to lipopolysaccharides and phosphatidylglycerol vesicles compared to the lipid-free protein. Mol Cell Biochem 458, 61–70 (2019). https://doi.org/10.1007/s11010-019-03530-x

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