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Binding of lysine residues of apolipophorin III to phosphatidylglycerol membranes
Author(s) -
Shah Kriti,
Weers Paul M.M.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.661.5
Subject(s) - biochemistry , phosphatidylglycerol , lysine , bacterial outer membrane , antimicrobial peptides , membrane , atp binding cassette transporter , biology , chemistry , phospholipid , peptide , amino acid , escherichia coli , transporter , gene , phosphatidylcholine
Apolipoproteins are the protein components of lipoproteins, which are responsible for transport of lipids to target tissues through the aqueous environment of the body. Apolipophorin III (apoLp‐III) is an abundant apolipoprotein of the hemolymph of insects. The 18 kDa protein is made of a bundle of five amphipathic α‐helices, which are the structural elements responsible for lipid binding. ApoLp‐III facilitates the lipoprotein mediated transport of diacylglycerols, which are released from fat body tissue for transport to the flight muscles during flight. Another novel function performed by apoLp‐III is modulating the cellular and humoral immune response. The protein up‐regulates expression of antimicrobial proteins, neutralizes lipopolysaccharides, and binds and deforms bacterial membranes. Lipopolysaccharides and phosphatidylglycerol (PG) are abundant components of the bacterial membrane, and our studies aim to better understand the antimicrobial properties of apoLp‐III of Locusta migratoria. This eventually may lead to improved strategies to treat bacterial infection and sepsis. ApoLp‐III contains 8 lysine residues, and the objective of this study was to characterize the ionic interactions between lysine residues of apoLp‐III and the negatively charged membrane components of gram‐negative bacteria, lipopolysaccharides and PG. To mimic bacterial membranes, PG vesicles with encapsulated calcein were prepared by extrusion. Protein‐induced membrane perturbation causes release of calcein which fluoresces intensively. When lysine side‐chains were neutralized by acetylation, binding to PG membranes was greatly reduced as indicated by the large reduction in calcein release from 80 ± 3.3% (unmodified apoLp‐III) to 21 ± 0.2% (acetylated apoLp‐III). In contrast, no effect on lipopolysaccharide binding was observed. To identify the lysine residues responsible for PG binding, site‐directed mutagenesis was used to change lysine into glutamine at position 52, 54, 63, 68 and 121. The proteins were expressed in bacteria and purified to homogeneity. The lysine variants were all able to release calcein, with values for most mutants around 70 to 80%. Since this is similar to the wild‐type protein, this indicates that these residues are not critical for PG binding. The remaining lysine residues at positions 143,145 and 161 were also changed into glutamine; the mutant proteins are currently expressed and purified. In addition, studies are underway to specifically acetylate the N‐terminal amino group by lowering the pH since the pK a for α amino groups is lower than the lysine ɛ‐amino group and hence making it a better nucleophile.