Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells

J Biol Chem. 2000 Apr 7;275(14):10648-54. doi: 10.1074/jbc.275.14.10648.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Angiotensinogen / chemistry*
  • Angiotensinogen / metabolism*
  • Animals
  • Binding Sites
  • CHO Cells
  • Callithrix
  • Cricetinae
  • Humans
  • Kinetics
  • Mice
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Protein Conformation
  • Recombinant Proteins / metabolism
  • Renin / metabolism*
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Sheep
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Transfection

Substances

  • Peptide Fragments
  • Recombinant Proteins
  • Angiotensinogen
  • Renin