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

Angiotensinogen, the renin (E.C. 3.4.23.15) substrate, belongs to the serpi ns superfamily and has been classified as a noninhibitory serpin, Using mas s spectroscopy, angiotensinogen purified from Chinese hamster ovary cell su pernatant shows a broad spectrum. The absence of protease inhibitors throug hout the purification leads to an angiotensinogen cleaved within the reacti ve center loop, This cleavage does not affect the Ang I generation because kinetic parameters are similar to the values of the full-length angiotensin ogen. Although cleavage is complete, the cleaved angiotensinogen migrates a fter deglycosylation on SDS-polyacrylamide gel electrophoresis as a doublet differing by 4 kDa. To test whether the circulating angiotensinogen is cle aved in the reactive center loop, it was purified from a pool of human plas ma and was shown to be uncleaved. Its migration was obviously slower than o f cleaved angiotensinogen but also consisted of two bands pointing to a so far unexplained residual heterogeneity. We then compared the heat-induced p olymerization of full-length-and reactive center loop-cleaved angiotensinog ens. Both monomers were able to aggregate, revealing a particular behavior of angiotensinogen distinct from that of reactive center loop-cleaved serpi ns, Lacking the three-dimensional structure of angiotensinogen, we propose and discuss a structural model of the serpin fold within the renin substrat e.