Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells.
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.
Célérier
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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
Schmid
G
G
Le Caer
J P
JP
Gimenez-Roqueplo
A P
AP
Bur
D
D
Friedlein
A
A
Langen
H
H
Corvol
P
P
Jeunemaitre
X
X
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
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000808:Angiotensinogen; D012083:Renin