Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity

Abstract

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III.

Keywords: Apolipophorin III; Apolipoprotein; Lipid binding; Lipoprotein.

Figure 1

Panel A: Schematic representation of the apoLp-III_cys/CT-apoA-I chimera. Residues 179-243 from apoA-I were attached to apoLp-III from L. migratoria creating a 26 kDa chimeric apolipoprotein. Residues Thr-20 and Ala-149 were substituted with Cys to prevent opening of the apoLp-III helix bundle. Panels B and C: Identification of apoLp-III_cys/CT-apoA-I by SDS-PAGE (B, 20 μg of protein) and Western blot (C, 0.5 μg of protein using goat anti-apoA-I conjugated HRP). Lane 1: apoA-I; lane 2: apoLp-III_cys/CT-apoA-I; lane 3: apoLp-III.

Figure 2

Panel A: Non-denaturing PAGE analysis showing a much greater mobility of apoLp-III compared to apoA-I and the chimera. Twenty μg of protein was electrophoresed in the absence of SDS on a 4–20% Tris-glycine gel. Lane 1: apoA-I, lane 2: apoLp-III_cys/CT-apoA-I, lane 3: apoLp-III. Panel B: Size-exclusion chromatographic analysis. Protein (0.5 mg at a 1 mg/mL concentration) was applied to a Superdex-200 column. Elution of the proteins was monitored at 210 nm using a flow rate of 0.5 mL/min. ApoLp-III eluted as a single peak at 17 mL (dash-dotted line), while apoA-I (solid line) and the chimera (dotted line) elute much earlier at 11 mL.

Figure 3

Cross-linking analysis. Twenty μg of reduced protein in the presence (+) or absence (-) of DMS crosslinker was analyzed by SDS-PAGE. Shown are apoA-I (lane 1–2), apoLp-III_cys/CT-apoA-I (lane 3–4), and apoLp-III (lane 5–6). The molecular mass of marker proteins are shown at the right (M). In the presence of DMS apoA-I and apoLp-III_cys/CT-apoA-I form oligomers (as evident by multiple protein bands in the range of 50 to 170 kDa) while apoLp-III remains monomeric.

Figure 4

Secondary structure and protein stability analysis. Panel A: Far-UV spectra of the apolipoproteins at 0.2 mg/mL in 20 mM NaP pH 7.2. The average of 4 scans recorded from 185 to 260 nm at 50 nm/min speed is shown. Shown are apoLp-III_Cys (dashed-dotted line), apoLp-III_cys/CT-apoA-I (dotted line) and apoA-I (solid line). Panels B and C show the denaturation profile in the absence of DTT (B) and in the presence of DTT (C). Protein samples (0.2 mg/mL) were incubated with increasing concentrations of guanidine-HCl. Protein unfolding is plotted as % maximum change in which 100% is a completely unfolded protein. Shown are apoLp-III_cys/CT-apoA-I (○), apoLp-III_Cys (▼), and apoA-I (●), n = 3, ± standard deviation.

Figure 5

Phospholipid vesicle solubilization. Apolipoprotein was incubated with 0.5 mg/mL DMPC LUVs in a 1:1 mass ratio at 24.1 °C. The conversion of vesicles into the small discoidal complexes was monitored by the decrease in sample turbidity at 325 nm as a function of time. Shown are the average of 3 time scans of: DMPC only (a), apoLp-III_Cys (b), apoLp-III_Cys with DTT (c), apoLp-III_cys/CT-apoA-I with DTT (d), apoLp-III_cys/CT-apoA-I (e), and apoA-I (f).

Figure 6

Inhibition of LDL aggregation. LDL (50 μg protein) was treated with phospholipase-C (160 mU) and simultaneously incubated at 37 °C in the presence of apolipoproteins (150 μg) in the absence of DTT (A) or in the presence of DTT (B). Aggregation of LDL was monitored by the absorbance at 340 nm. Shown are the average of 3 measurements (± standard deviation) of apoLp-III_cys/CT-apoA-I (○), apoLp-III_cys(▼), apoA-I(●), and no protein (Δ).

Figure 7

Release of calcein from PG vesicles. Calcein-containing PG vesicles were equilibrated for 2 min, after which apolipoprotein was added triggering release of calcein measured by the increase in fluorescence intensity at 520 nm. At 10 min, detergent was added to release the remaining calcein. Solid line: apoA-I; dotted line: chimera; dash-dotted line: apoLp-III (n=3, ± standard deviation).

Figure 8

Native PAGE of apolipoprotein-LPS incubations. Apolipoproteins (20 μg) were incubated with increasing amounts of LPS for 1 h and analyzed by PAGE under non-denaturing conditions. Shown are apoA-I (lane 1), incubated with 80 μg (lane 2), 130 μg (lane 3) and 180 μg LPS (lane 4). Lanes 5–8 contain chimera only (lane 5) with 80 μg (lane 6), 130 μg (lane 7) and 180 μg LPS (lane 8); lanes 9–12 contain apoLp-III (lane 9) with 80 μg (lane 10), 130 μg (lane 11) and 180 μg LPS (lane 12).

Figure 9

TNF-α secretion by macrophages. Apolipoproteins were pre-incubated with LPS (10:1 ratio) after which macrophages were exposed to these mixtures; TNF-α concentrations were measured by ELISA (n = 3, ± standard deviation).