A specific binding site for the prorenin propart peptide Arg 10 -Arg 20 does not occur on human endothelial cells

INTRODUCTION: We looked for novel binding sites for the human prorenin 'decoy peptide' sometimes called 'handle region peptide' on human endothelial cells. METHOD: The biotinylated peptide biotin-Acp-RIFLKRMPSIR (B-PR), an unlabelled peptide PR1 (RIFLKRMPSIR) and a scrambled peptide scPR1 (SRRMIFPIKLR) were synthesized. B-PR was added to human umbilical cord endothelial cells (HUVECs) maintained in serum-free medium, with or without excess unlabelled peptide or 'scrambled' peptide as blocker. Biotin-labelled HUVEC proteins were extracted, the amount of bound tracer was measured, and the identity of the binding proteins was analysed by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). RESULTS: Biotinylated peptide bound to the HUVEC proteins with a major labelled band at 68,600 ± 1503 kDa (mean ± SEM, n = 5 runs). Unlabelled peptide and scrambled peptide equally displaced the labelled peptide, indicating that the binding was non-specific for amino acid sequence. LC-MS/MS showed that binding was mainly to cytoskeletal proteins. CONCLUSION: The binding of the human prorenin peptide R¹⁰IFLKRMPSIR²⁰ to HUVEC proteins is not specific for amino acid sequence and probably involves a general peptide/protein uptake mechanism. We could not detect a specific prorenin propart binding site in these cells.