Structure/function relationships of apolipoprotein a-I mimetic peptides: implications for antiatherogenic activities of high-density lipoprotein

Abstract

Rationale: Apolipoprotein (apoA)-I mimetic peptides are a promising type of anti-atherosclerosis therapy, but how the structural features of these peptides relate to the multiple antiatherogenic functions of HDL is poorly understood.

Objective: To establish structure/function relationships of apoA-I mimetic peptides with their antiatherogenic functions.

Methods and results: Twenty-two bihelical apoA-I mimetic peptides were investigated in vitro for the capacity and specificity of cholesterol efflux, inhibition of inflammatory response of monocytes and endothelial cells, and inhibition of low-density lipoprotein (LDL) oxidation. It was found that mean hydrophobicity, charge, size of hydrophobic face, and angle of the link between the helices are the major factors determining the efficiency and specificity of cholesterol efflux. The peptide with optimal parameters was more effective and specific toward cholesterol efflux than human apoA-I. Charge and size of hydrophobic face were also the major factors affecting antiinflammatory properties, and the presence of cysteine and histidine residues was the main factor determining antioxidant properties. There was no significant correlation between capacities of the peptides to support individual functions; each function had its own optimal set of features.

Conclusions: None of the peptides was equally effective in all the antiatherogenic functions tested, suggesting that different functions of HDL may have different mechanisms and different structural requirements. The results do suggest, however, that rationalizing the design of apoA-I mimetic peptides may improve their therapeutic value and may lead to a better understanding of mechanisms of various antiatherogenic functions of HDL.

Fig. 1. Dependence of the capacity of the peptides to support cholesterol efflux on mean hydrophobicity and charge of the peptides (a) and relationship between cholesterol efflux capacity and contribution of ABC transporters (b)

Data for cholesterol efflux capacity and specificity are taken from Table 2 and data for hydrophobicity and charge are taken from Table 1. Squares denote positively charged peptides; triangles denote negatively charged peptides.

Fig. 2. Specificity of cholesterol efflux from THP-1 cells

Cholesterol efflux was expressed as the proportion of [³H]cholesterol transferred from cells to medium; concentration of the peptides was 80 μg/ml. Fine cross-hatched bars denote peptides with charge ≥ +2; coarse cross-hatched bar denotes peptides with A for P substitution. a – Efflux from the fixed cells b - Contribution of the specific efflux (efflux from live cells to the total efflux (efflux from live cells – efflux from fixed cells/efflux from live cells x100%). Negative values are shown as “0”.

Fig. 3. Cholesterol efflux from BHK/ABCA1 cells

Cholesterol efflux was expressed as the proportion of [³H]cholesterol transferred from cells to medium; concentration of the peptides was 20 μMol/ml (or approximately 90 μg/ml). a – ABCA1-dependent efflux from BHK cells. Data presented are a difference between the efflux from BHK/ABCA1 cells and BHK/mock cells. Means ± SEM are presented. b - Correlation between the ABC-dependent efflux from THP-1 and ABCA1-dependent efflux from BHK cells. Two peptides, ELK-2A and 5A-CH2, shown as ◆, were excluded from the analysis of correlations.

Fig. 4. The effect of peptides on anti-inflammatory properties

a - CD11b expression in human monocytes. CD11b expression was measured by flow cytometry; results were expressed as percentage of the CD11b expression compared to cells stimulated with PMA in the presence of a vehicle; concentration of peptides was 40 μg/mL. Means ± SEM are presented; *p<0.01 (versus vehicle). Table shows peptide properties that are likely to influence CD11b expression. b - VCAM-1 expression in mouse endothelial cells, SVEC4/VCAM-1. Data were expressed per milligram of cellular protein and related to the luciferase activity in cells incubated with a vehicle instead of the peptides; concentration of peptides was 0.75 mg/mL. Means ± SEM are presented; *p<0.01 (versus vehicle). Table shows peptide properties that are likely to influence VCAM-1 expression.

Fig. 5. The effect of peptides on LDL oxidation

Concentration of peptides was 100 μg/mL. Rate of oxidation was calculated as maximum absorbance divided to the length of the lag period. *p<0.01 (calculated from comparing the time-dependence curves presented in the Supplementary Figure VI). Table shows peptide properties that are likely to influence anti-oxidant properties. G – G-helix, Y-Y-helix, AP-substitution of A for P.