title: A specific binding site for the prorenin propart peptide Arg10-Arg20 does not occur on human endothelial cells.
abstract: BACKGROUND
  We looked for novel binding sites for the human prorenin 'decoy peptide' sometimes called 'handle region peptide' on human endothelial cells.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Vascular Medicine Group, Department of Cardiovascular Sciences, University of Leicester, Leicester, UK. bjl2@le.ac.uk
authors.forename: Brenda J
authors.initials: BJ
authors.lastname: Leckie
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Bottrill
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D019921:Biotinylation; D015153:Blotting, Western; D002853:Chromatography, Liquid; D012085:Chymosin / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014471:Umbilical Veins / Q000166:cytology; D000093342:Prorenin Receptor
chemical_list: D004792:Enzyme Precursors; D010455:Peptides; D011956:Receptors, Cell Surface; D001120:Arginine; C032443:prorennin; D012085:Chymosin; D000093342:Prorenin Receptor