Characterization of the ApoLp-III/LPS Complex: Insight into the Mode of Binding Interaction

Apolipoproteins are able to associate with lipopolysaccharides (LPS), potentially providing protection against septic shock. To gain insight into the molecular details of this binding interaction, apolipophorin III (apoLp-III) from <i>Galleria mellonella</i> was used as a model. The binding of apoLp-III to LPS was optimal around 37–40 °C, close to the LPS phase transition temperature. ApoLp-III formed complexes with LPS from <i>E. coli</i> (serotype O55:B5) with a diameter of ∼20 nm and a molecular weight of ∼390 kDa, containing four molecules of apoLp-III and 24 molecules of LPS. The LPS-bound form of the protein was substantially more resistant to guanidine-induced denaturation compared to unbound protein. The denaturation profile displayed a multiphase character with a steep drop in secondary structure between 0 and 1 M guanidine-HCl and a slower decrease above 1 M guanidine-HCl. In contrast, apoLp-III bound to detoxified LPS was only slightly more resistant to guanidine-HCl induced denaturation compared to unbound protein. Analysis of size-exclusion FPLC elution profiles of mixtures of apoLp-III with LPS or detoxified LPS indicated a much weaker binding interaction with detoxified LPS compared to intact LPS. These results indicate that apoLp-III initially interacts with exposed carbohydrate regions, but that the lipid A region is required for a more stable LPS binding interaction

Phospholipid vesicle solubilization capability of chimeras.

<p>About 125 μg of DMPC MLVs were equilibrated in 400 μl of PBS in a cuvette at 23.7°C in a Peltier-controlled spectrophotometer. Vesicle solubilization was initiated by addition of 125 μg of apolipoprotein, mixed rapidly and the change in absorbance at 325 nm measured for 30 min. Data were normalized to initial absorbance immediately following addition of protein. ApoAI (_____); apoAI-NT/apoE-CT (·······); apoE3 (-------); apoE3-NT/apoAI-CT (··-··-··); and DMPC vesicles alone in the absence of apolipoproteins (__ __ __).</p

Chimeric apolipoproteins mediated cholesterol efflux from J774 macrophages.

<p><b>Panels A and B.</b> J774 macrophages were labeled with [³H]cholesterol, and treated in the absence (open bars) or presence (closed bars) of cAMP. Lipid-free chimeras or parent apolipoproteins (10 μg/ml) were added to cells in serum-free RPMI-1640 medium, and amount of cholesterol efflux determined at 4 h. Conditions of time and dose were within a linear response range to allow for potential differences (if any) to be observed with and without treatment of acceptors in the absence (<b>Panel A</b>) or presence (<b>Panel B</b>) of 5x molar excess of BME. <b>Panel C.</b> Dependence of cholesterol efflux on chimera concentration. The cells were treated with the indicated concentrations of the chimeras for 4 h. <b>Panel D.</b> Kinetics of chimera-mediated cholesterol efflux. Chimera-mediated cholesterol efflux was followed at the indicated time points using 32 μg/μl of each protein. Values are means ±SD from triplicate determinations within a single experiment representative of two. For <b>Panel C</b> and <b>Panel D</b>, apoE3-NT/apoAI-CT (open inverted triangles); apoAI-NT/apoE-CT (open circles).</p

Characterization of chimeric apolipoproteins.

<p><b>Panel A</b>. SDS-PAGE analysis of the chimeric apolipoproteins. Electrophoresis of chimeric and parent proteins (20 μg protein) was carried out using a 4–20% acrylamide gradient Tris-glycine gel under reducing conditions in the presence of BME. <b>Panel B</b>. Western blot analysis of chimeras (0.5 μg protein) using mouse HRP-conjugated apoE polyclonal antibody (<i>Left</i>) or apoAI antibody (<i>Right</i>). Lane assignments are as follows: Lane 1, apoAI; Lanes 2, apoAI-NT/apoE-CT; Lanes 3, apoE3; Lanes 4, apoE3-NT/apoAI-CT.</p

LDLr binding ability of chimeras.

<p>DMPC/chimera complexes (10 μg protein) were incubated with 10 μg of sLDLr, followed by co-IP with anti-c-Myc-agarose. sLDLr-bound apoE was detected by Western blot using HRP-conjugated polyclonal apoE antibody (<i>Top</i>); the lane assignments are: Lane 1, apoAI; lane 2, apoAI-NT/apoE-CT; lane 3, apoE3; lane 4, apoE3-NT/apoAI-CT; lane 5, apoAI added in the absence of sLDLr; lane 6, apoE3 added in the absence of LDLr; lane 7, LDLr alone in the absence of added proteins. A replica experiment was conducted wherein an anti-c-Myc antibody (9E10) was utilized to identify the presence of LDLr in each reaction (<i>Bottom</i>).</p

Unfolding and folding behavior of chimeras.

<p><b>Panels A and B</b>. GdnHCl-induced denaturation profiles of apoAI-NT/apoE-CT (<b>A</b>) and apoE3-NT/apoAI-CT (<b>B</b>). The samples (0.2 mg/ml) were incubated with increasing concentration of GdnHCl and 5x molar excess of TCEP for 16 h at 24°C. The ellipticity value at 222 nm was measured and protein unfolding plotted as % maximal change in which 100% represents completely unfolded protein. ApoAI (filled circles); apoAI-NT/apoE-CT (open circles); apoE3 (closed inverted triangles); and apoE3-NT/apoAI-CT (open inverted triangles). <b>Panel C</b>. ANS fluorescence emission spectra of chimeric and parent proteins. About 50 μg of each protein sample was excited at 395 nm and the emission spectra were recorded at 100 nm/min from 400 to 600 nm. ApoAI (_____); apoAI-NT/apoE-CT (·······); apoE3 (-------); apoE3-NT/apoAI-CT (··-··-··) and ANS (__ __ __).</p

Characterization of DMPC/chimera particle size and protein/lipid composition.

<p>Characterization of DMPC/chimera particle size and protein/lipid composition.</p