Probing the Function of Apolipoprotein A‐I Using Chimera Proteins

Abstract

Human apolipoprotein A‐I (apoA‐I) is a 243 residue, 28 kDa protein that consists of an N‐terminal (NT) helix bundle domain and a less structured C‐terminal (CT) domain. ApoA‐I is the major protein component in high‐density lipoprotein (HDL) and is known to play an important role in reverse cholesterol transport. HDL promotes the removal of excess cholesterol from peripheral tissues to the liver. The CT domain of apoA‐I has a high affinity for phospholipids and initiates lipid binding, whereas the NT domain harbors the site of lecithin‐cholesterol acyltransferase activity. A recent study showed that removal of residues 231–243 led to a decrease in lipid binding affinity. Other studies have suggested that segments in the NT domain, residues 1–43 and 44–65, initiate lipid binding. Thus, conflicting data exists regarding the location of high‐affinity lipid binding in apoA‐I. A chimera protein was recently designed in which the CT domain of apoA‐I was attached to insect apolipophorin (apoLp‐III). The insect apolipoprotein acquired apoA‐I like properties, including high affinity lipid binding. Thus, this system allows for the identification of apoA‐I segments that have a high affinity for lipid surfaces. To identify potential lipid binding regions within apoA‐I, three chimeric constructs were designed using L. migratoria apoLp‐III. Residues 1–43 or 44–65 of apoA‐I were attached to the NT end of apoLp‐III. In addition, residues 231–243 of apoA‐I were attached to the CT end of apoLp‐III. The three DNA constructs were inserted into the pET‐20b (+) expression vector through restriction enzymes HindIII and NdeI. Site‐directed mutagenesis was conducted to introduce two cysteines into apoLp‐III at positions Thr‐20 and Ala‐149. The two cysteines lock the protein in an inactive state under oxidizing conditions to prevent lipid binding. This enables to study the lipid binding properties of the attached apoA‐I segments in the chimera proteins. Proteins are currently being expressed in Escherichia coli BL21(DE3) pLysS cells and will be purified through nickel‐affinity and size‐exclusion chromatography. The lipid binding properties will be assessed by the ability to solubilize phospholipid vesicles.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.