Abstract-The members of the serine protease inhibitor (serpin) family, which share a common tertiary structure and a role as serin protease inhibitors, are involved in a variety of newly discovered functions. For example, antithrombin III exerts a strong antiangiogenic activity. Angiotensinogen, the renin substrate, has a folded structure and is a member of the noninhibitory serpin subfamily. Two other noninhibitory serpins, maspin and pigment epithelium-derived factor, have antiangiogenic properties. We investigated the antiangiogenic effect of angiotensinogen and 2 related compounds: (1) des(angiotensin I)angiotensinogen, the product of angiotensinogen cleavage by renin, and (2) the reactive center loop-cleaved angiotensinogen, which is produced after selective and limited proteolysis by the protease V8. We used well-established in vitro (endothelial cell proliferation and migration, and capillary-like tube formation on Matrigel) and in vivo (the chick chorioallantoic membrane assay) models of angiogenesis to evaluate the antiangiogenic activities of these 3 related molecules. Our data demonstrated that these compounds exerted a clear and equipotent antiangiogenic effect, thus attributing a novel function to angiotensinogen and des(angiotensin I)angiotensinogen, for which no function was previously known. , an inactive decapeptide that is converted into Ang II, the main effector of the renin-angiotensin system (RAS). Its only role known is as a substrate for renin, a highly specific aspartyl protease. Renin cleaves the N-terminal end of AGT to generate Ang I. This leaves a much larger fragment intact (97.8% of the whole amino acid sequence), called des(angiotensin I)angiotensinogen (des[Ang I]AGT), which until now did not have any known function (Figure 1 for a schematic representation of AGT and its derivatives).The biochemical, enzymological, and structural characteristics of AGT have been thoroughly investigated as it is the rate-limiting step in the first reaction of the RAS cascade: its concentration in human plasma (1 mol/L) is close to its affinity (Km) for renin. 1 The liver is the main site of AGT synthesis, but other sites include the glial cells, adipocytes, kidney, and the walls of large vessels. 2 The concentration of AGT in the plasma is regulated by several endocrine factors 3 and also depends on the genotype of the AGT gene. An AGT gene variant at position 235 (235T) is associated with a 10% to 20% increase in plasma AGT concentration and high blood pressure. 4 Des(Ang I)AGT was long considered to be a degradation product and thus has not been studied extensively. What has been suggested is that des(Ang I)AGT may inhibit the renin AGT reaction. 5,6 AGT shares amino acid sequence and structural homologies with the serine protease inhibitor (serpin) family of proteins, but it has no inhibitor activity. Indeed, like 3 other noninhibitory serpins (ovalbumin, pigment epithelial-derived factor [PEDF], and maspin 7 ), AGT does not undergo the classical stressed-relaxed pathway of the inhibitory serpin...

Aminopeptidase A (EC 3.4.11.7, APA) is a 130 kDa membrane-bound protease that contains the HEXXH consensus sequence found in the zinc metalloprotease family, the zincins. In addition to the catalytic zinc atom, APA contains a Ca2+ ion that increases its enzymatic activity. Aligning the sequences of the mouse APA, APN, and other monozinc aminopeptidases led to the identification of a conserved histidine (His 450 in mouse APA). Replacing this residue with a phenylalanine (Phe 450) by site-directed mutagenesis resulted in markedly lower levels of APA activity and in a change in the sensitivity of APA to Ca2+ (the EC50 for Ca2+ was 25 microM in the wild type and only 279 microM in the mutant). Kinetic studies, with a supramaximal Ca2+ concentration (4 mM), showed that the Km of the mutant enzyme for the substrate alpha-L-glutamyl-beta-naphthylamide was 25 times higher than that of the wild type, whereas the kcat value was much lower (factor of 22). Thus, overall, the wild-type enzyme had a cleavage efficiency that was 571 times higher than that of the mutant. The inhibitory potencies of two different classes of inhibitors, a glutamate thiol and a glutamate phosphonate compound, were significantly lower (factors of 19 and 22, respectively) for the mutated enzyme than for the wild-type enzyme. In contrast, inhibition by lysine thiol was unaffected. These data strongly suggest that His 450 is critical for catalytic activity and is involved in substrate binding via interaction with the P1 carboxylate side chain of the substrate. Furthermore, His 450, together with Ca2+, may contribute to the substrate specificity of APA for N-terminal acidic amino acid residues.

The expression and function in growth and apoptosis of the renin -angiotensin system (RAS) was evaluated in human glioblastoma. Renin and angiotensinogen (AGT) mRNAs and proteins were found by in situ hybridisation and immunohistochemistry in glioblastoma cells. Angiotensinogen was present in glioblastoma cystic fluids. Thus, human glioblastoma cells produce renin and AGT and secrete AGT. Human glioblastoma and glioblastoma cells expressed renin, AGT, renin receptor, AT 2 and/or AT 1 mRNAs and proteins determined by RT -PCR and/or Western blotting, respectively. The function of the RAS in glioblastoma was studied using human glioblastoma cells in culture. Angiotensinogen, des(Ang I)AGT, tetradecapaptide renin substrate (AGT1 -14), Ang I, Ang II or Ang III, added to glioblastoma cells in culture, did not modulate their proliferation, survival or death. Angiotensin-converting enzyme inhibitors did not diminish glioblastoma cell proliferation. However, the addition of selective synthetic renin inhibitors to glioblastoma cells decreased DNA synthesis and viable tumour cell number, and induced apoptosis. This effect was not counterbalanced by concomitant addition of Ang II. In conclusion, the complete RAS is expressed by human glioblastomas and glioblastoma cells in culture. Inhibition of renin in glioblastoma cells may be a potential approach to control glioblastoma cell proliferation and survival, and glioblastoma progression in combination therapy.