Sidedness of interfacial arginine residues and anti-atherogenicity of apolipoprotein A-I mimetic peptides

Abstract

To test the hypothesis that sidedness of interfacial arginine (Arg) in apoA-I mimetic peptides, similar to that observed in apoA-I (Bashtovyy, D. et al. 2011. Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function. J. Lipid Res. 52: 435-450.), may be important for biological activity, we compared properties of 4F and analogs, [K⁴,¹⁵>R]4F and [K⁹,¹³>R]4F, with Lys>Arg substitutions on the right and left side, respectively, of the 4F amphipathic helix. Intraperitoneal administration of these peptides into female apoE null mice (n = 13 in each group) reduced en face lesions significantly compared with controls; 4F and [K⁴,¹⁵>R]4F were equally effective whereas [K⁹,¹³>R]4F was less effective. Turnover experiments indicated that [K⁴,¹⁵>R]4F reached the highest, whereas [K⁹,¹³>R]4F had the lowest, plasma peak levels with a similar half life as the [K⁴,¹⁵>R]4F analog. The half life of 4F was two times longer than the other two peptides. The order in their abilities to associate with HDL in human plasma, generation of apoA-I particles with pre-β mobility from isolated HDL, lipid associating ability, and sensitivity of lipid complexes to trypsin digestion was: 4F>[K⁴,¹⁵,>R]4F>[K⁹,¹³>R]4F. These studies support our hypothesis that the sidedness of interfacial Arg residues in the polar face of apoA-I mimetics results in differential biological properties.

Fig. 1.

WHEEL/HELNET analysis of 4F, [K^4,15>R]4F, and [K^9,13>R]4F. A: Helical wheel diagrams of 4F and its two Arg containing analogs. Lys and Arg residues are colored light and dark blue, respectively. Notice that in [K^4,15>R]4F analog, Arg residues are located on the right hand side of the polar face (similar to that observed in human apoA-I helical wheels with the polar face pointing downwards), whereas in [K^9,13>R]4F, Arg residues are located on the left hand side of the polar face. Color schemes for other residues are as follows: negatively charged amino acids, red; hydrophobic amino acids, black; Ala, no color; Lys, light blue; Arg dark blue. Nonpolar and polar faces of the amphipathic helices are labeled. B: Helical net diagrams of 4F, [K^4,15>R]4F, and [K^9,13>R]4F. Contour lines drawn at 0, 5, and 10 Å into the lipid milieu are indicated. C: WHEEL/HELNET derived helix parameters for 4F, [K^4,15>R]4F, and [K^9,13>R]4F.

Fig. 2.

Perturbation of methyl and methylene protons of POPC LUV by 4F, [K^4,15>R]4F, and [K^9,13>R]4F. Partial ¹H NMR (500 MHz) spectra of POPC LUV in the absence and presence of 4F, [K^4,15>R]4F, and [K^9,13>R]4F. ¹H NMR spectra were obtained at 37°C using a lipid to peptide ratio of 20:1 (wt:wt). Chemical structure of POPC and methyl (labeled as 1) and methylene (labeled as 2) protons are shown.

Fig. 3.

Quantification of atherosclerotic lesions. A: Quantification of aortic sinus lesions. Oil Red O stained areas in the aortic sinus were measured in saline and 4F, [K^4,15>R]4F, and [K^9,13>R]4F treated groups at the end of treatment period. Data are expressed as mean ± SEM. Both 4F and [K^4,15>R]4F, but not [K^9,13>R]4F, reduced aortic sinus lesion significantly compared with control as determined by one-way ANOVA. B: En face preparations of the entire aorta from the aortic arch to the iliac bifurcation were done in mice treated with 4F, [K^4,15>R]4F, and [K^9,13>R]4F. The aorta was excised, cleaned, and opened longitudinally with extremely fine Vannas scissors then pinned flat on a black wax surface. The aorta was then stained with Oil Red O and lesions were quantified by video capture under a stereo dissecting microscope. Lesion and total areas were determined using SigmaScan (Systat) and lesion area was expressed as a percentage of total area.

Fig. 4.

Plasma turnover studies in female apoE null mice. (A) Bioavailability of 4F (■), [K^4,15>R]4F (○), and [K^9,13>R]4F (△). One hundred micrograms of ¹⁴C-labeled peptides in saline were administered ip to female apoE null mice. Blood was drawn at various time points as indicated. Radioactivity in plasma at different time points (n = 3 per time point) was expressed as percent of injected cpm. Data are expressed as mean ± SEM. B: Area under the curve was calculated for each peptide, and is expressed as % injected cpm × h.

Fig. 5.

Trypsin digestion of POPC complexes of 4F (■), [K^4,15>R]4F (○), and [K^9,13>R]4F (△). One hundred micrograms/milliliter of the complex was digested with trypin (enzyme:complex, 1:40, wt:wt) at 37°C. At the indicated time points, an aliquot was taken out and frozen at −20°C for RP-HPLC analysis. Area under the peak was used to calculate the amount of undigested peptide in the complex. Error bars represent SD of two independent experiments.

Fig. 6.

Distribution of ¹⁴C-labeled 4F (■), [K^4,15>R]4F (○), and [K^9,13>R]4F (△) in human plasma. One hundred micrograms of ¹⁴C-labeled peptide were incubated with 1 ml human plasma at 37°C overnight in the dark. The plasma was fractionated using a fast protein liquid chromatography system (BioLogic, DuoFlow; Bio-Rad) and Superdex 200 10/300 GL column (GE Healthcare) run at a flow rate of 0.4 ml/min in PBS containing 0.02% sodium azide (pH 7.4). Complex elution was monitored using absorbance at 280 nm. One milliliter fractions were collected. Total cholesterol and radioactivity in each fraction were measured. Cholesterol values are indicated by red-filled circles. In addition to cholesterol, apoA-I was determined in FPLC fractions 12-20 using Western blot analysis. Compared with other fractions, fractions 13-15 were enriched in apoA-I.

Fig. 7.

Generation of apoA-I containing particles with preβ mobility by incubation of peptides with isolated human HDL. HDL (d = 1.063-1.210 g/ml) was isolated from human plasma using sequential density gradient ultracentrifugation. Peptides were incubated with HDL at HDL protein to peptide ratio of 5:1 (wt:wt) at 37°C overnight in the dark. Top panel shows Coomassie Blue stained gel and bottom panel shows a Western blot using a biotin-labeled polyclonal antibody to human apoA-I. In the top panel, lanes 1-4 and 5-8 represent 5 µL and 10 µL, respectively, of the sample volume loaded. Top panel: 1, control; 2, 4F; 3, [K^4,15>R]4F; 4, [K^9,13>R]4F; 5, control; 6, 4F; 7, [K^4,15>R]4F; 8, [K^9,13>R]4F. Bottom panel: 1, control; 2, 4F; 3, [K^4,15>R]4F; 4, [K^9,13>R]4F.

Fig. 8.

Agarose gel electrophoresis of peptides in the absence and presence of Ox-PAPC. The lipid to peptide ratio was 10:1 (wt:wt). 1, 4F; 2, [K^4,15>R]4F; 3, [K^9,13>R]4F; 4, 4F:Ox-PAPC; 5, [K^4,15>R]4F:Ox-PAPC; 6, [K^9,13>R]4F:Ox-PAPC.