Abstract
Modifying apolipoprotein (apo) A-I mimetic peptides to include a proline-punctuated alpha-helical repeat increases their anti-inflammatory properties as well as allows better mimicry of full-length apoA-I function. This study compares the following mimetics, either acetylated or biotinylated (b): 4F (18mer) and 4F-proline-4F (37mer, Pro). b4F interacts with both mouse HDL (moHDL) and LDL in vitro. b4F in vivo plasma clearance kinetics are not affected by mouse HDL level. Administration of biotinylated peptides to mice demonstrates that b4F does not associate with lipoproteins smaller than LDL in vivo, though it does associate with fractions containing free hemoglobin (Hb). In contrast, bPro specifically interacts with HDL. b4F and bPro show opposite binding responses to HDL by surface plasmon resonance. Administration of acetylated Pro to apoE(-/-) mice significantly decreases plasma serum amyloid A levels, while acetylated 4F does not have this ability. In contrast to previous reports that inferred that 4F associates with HDL in vivo, we systematically examined this potential interaction and demonstrated that b4F does not interact with HDL in vivo but rather elutes with Hb-containing plasma fractions. bPro, however, specifically binds to moHDL in vivo. In addition, the number of amphipathic alpha-helices and their linker influences the anti-inflammatory effects of apoA-I mimetic peptides in vivo.
Highlights
Modifying apolipoprotein A-I mimetic peptides to include a proline-punctuated ␣-helical repeat increases their anti-inflammatory properties as well as allows better mimicry of full-length apoA-I function
area under the curve (AUC) analysis of plasma clearance curves demonstrated no significant differences in total plasma biotinylated 4F (b4F) regardless of mouse HDL (moHDL) status (Fig. 1B)
To shed light on the basis of this unexpected finding that HDL levels do not influence b4F clearance, we investigated the lipoprotein-association behavior of b4F in vitro. b4F was equilibrated with isolated lipoprotein populations and the mixture was separated by ultracentrifugation on density gradients
Summary
Peptides were either synthesized from L-amino acids and purified as described [3] or purified peptides were purchased from BioSynthesis (Lewisville, TX). 244 mg unmodified (+)-biotin (Sigma) was solubilized in 2 ml dimethylsulfoxide to which was added 1 ml dimethylformamide (DMF), 2 ml 0.5M 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in DMF, and 452 μl diisopropylethylamine, sequentially This solution was added to the peptide-containing uncleaved resin serially for room temperature stirring until the coupling was complete by qualitative ninhydrin assay. Mice in peptide clearance or lipoprotein association studies were injected IP with 300 μg biotinylated 4F (b4F) or biotinylated Pro (bPro) solubilized in ~500 μl sterile PBS. For assays of plasma clearance of biotinylated peptide, the ECL signal from the 0 h bleed (before peptide injection) was subtracted from all time points to normalize for the preexisting biotin concentration in mouse plasma. For assays of FPLC fractions, each treated mouse was paired with a control PBS-treated mouse and the biotin peptide-specific signal was determined by subtraction. Data processing and statistical analyses were performed by Microsoft Excel using the Student’s t-test for significant differences
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