title: Proteolytic processing of angiotensin-I in human blood plasma.
abstract: In mammalian species, except humans, N-terminal processing of the precursor peptide angiotensin I (ANG-1-10) into ANG-2-10 or ANG-3-10 was reported. Here we hypothesize that aminopeptidase-generated angiotensins bearing the same C-terminus as ANG-1-10 are also present in humans. We demonstrate the time dependent generation of ANG-2-10, ANG-3-10, ANG-4-10, ANG-5-10 and ANG-6-10 from the precursor ANG-1-10 by human plasma proteins. The endogenous presence of ANG-4-10, ANG-5-10 and ANG-6-10 in human plasma was confirmed by an immuno-fluorescence assay. Generation of ANG-2-10, ANG-3-10 and ANG-4-10 from ANG-1-10 by immobilized human plasma proteins was sensitive to the cysteine/serine protease inhibitor antipain. The metal ion chelator EDTA inhibited Ang-6-10-generation. Incubation of the substrates ANG-3-10, ANG-4-10 and ANG-5-10 with recombinant aminopeptidase N (APN) resulted in a successive N-terminal processing, finally releasing ANG-6-10 as a stable end product, demonstrating a high similarity concerning the processing pattern of the angiotensin peptides compared to the angiotensin generating activity in plasma. Recombinant ACE-1 hydrolyzed the peptides ANG-2-10, ANG-3-10, ANG-4-10 and ANG-5-10 into ANG-2-8, ANG-3-8, ANG-4-8 and ANG-5-8. Since ANG-2-10 was processed into ANG-2-8, ANG-4-8 and ANG-5-8 by plasma proteases the angiotensin peptides bearing the same C-terminus as ANG-1-10 likely have a precursor function in human plasma. Our results confirm the hypothesis of aminopeptidase mediated processing of ANG-1-10 in humans. We show the existence of an aminopeptidase mediated pathway in humans that bypasses the known ANG-1-8-carboxypeptidase pathway. This expands the knowledge about the known human renin angiotensin system, showing how efficiently the precursor ANG-1-10 is used by nature.
authors.affiliation: University Medical Centre Hamburg-Eppendorf, Institute of Clinical Chemistry, Mass Spectrometric Proteomics, Hamburg, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Hildebrand
authors.forename: Philipp
authors.initials: P
authors.lastname: Merkel
authors.forename: Lars Florian
authors.initials: LF
authors.lastname: Eggers
authors.forename: Hartmut
authors.initials: H
authors.lastname: Schlüter
mesh_terms: D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D000809:Angiotensins / Q000378:metabolism; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D018826:CD13 Antigens / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008875:Middle Aged; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D059748:Proteolysis / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000809:Angiotensins; D001798:Blood Proteins; D055767:Immobilized Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D000803:Angiotensin I; D018826:CD13 Antigens; C488034:ACE protein, human; D007703:Peptidyl-Dipeptidase A