A specific binding site for the prorenin propart peptide Arg10-Arg20 does not occur on human endothelial cells

Abstract

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.