title: Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells.
abstract: Angiotensinogen, the renin (E.C. 3.4.23.15) substrate, belongs to the serpins superfamily and has been classified as a noninhibitory serpin. Using mass spectroscopy, angiotensinogen purified from Chinese hamster ovary cell supernatant shows a broad spectrum. The absence of protease inhibitors throughout the purification leads to an angiotensinogen cleaved within the reactive center loop. This cleavage does not affect the Ang I generation because kinetic parameters are similar to the values of the full-length angiotensinogen. Although cleavage is complete, the cleaved angiotensinogen migrates after deglycosylation on SDS-polyacrylamide gel electrophoresis as a doublet differing by 4 kDa. To test whether the circulating angiotensinogen is cleaved in the reactive center loop, it was purified from a pool of human plasma and was shown to be uncleaved. Its migration was obviously slower than of cleaved angiotensinogen but also consisted of two bands pointing to a so far unexplained residual heterogeneity. We then compared the heat-induced polymerization of full-length- and reactive center loop-cleaved angiotensinogens. Both monomers were able to aggregate, revealing a particular behavior of angiotensinogen distinct from that of reactive center loop-cleaved serpins. Lacking the three-dimensional structure of angiotensinogen, we propose and discuss a structural model of the serpin fold within the renin substrate.
authors.affiliation: INSERM U36-Pathologie Vasculaire et Endocrinologie Rénale, Collège de France-Chaire de Médecine Expérimentale et d'Endocrinologie Rénale 3, rue d'Ulm 75005 Paris, France. jerome.celerier@college-de-france.fr
authors.forename: J
authors.initials: J
authors.lastname: Célérier
authors.forename: G
authors.initials: G
authors.lastname: Schmid
authors.forename: J P
authors.initials: JP
authors.lastname: Le Caer
authors.forename: A P
authors.initials: AP
authors.lastname: Gimenez-Roqueplo
authors.forename: D
authors.initials: D
authors.lastname: Bur
authors.forename: A
authors.initials: A
authors.lastname: Friedlein
authors.forename: H
authors.initials: H
authors.lastname: Langen
authors.forename: P
authors.initials: P
authors.lastname: Corvol
authors.forename: X
authors.initials: X
authors.lastname: Jeunemaitre
mesh_terms: D000595:Amino Acid Sequence; D000808:Angiotensinogen / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002144:Callithrix; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012083:Renin / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D000808:Angiotensinogen; D012083:Renin