Kininogenase Activity by the Major Cysteinyl Proteinase (Cruzipain) from Trypanosoma cruzi (original) (raw)
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Host Cell Invasion by Trypanosoma cruzi Is Potentiated by Activation of Bradykinin B 2 Receptors
The parasitic protozoan Trypanosoma cruzi employs multiple molecular strategies to invade a broad range of nonphagocytic cells. Here we demonstrate that the invasion of human primary umbilical vein endothelial cells (HUVECs) or Chinese hamster ovary (CHO) cells overexpressing the B 2 type of bradykinin receptor (CHO-B 2 R) by tissue culture trypomastigotes is subtly modulated by the combined activities of kininogens, kininogenases, and kinin-degrading peptidases. The presence of captopril, an inhibitor of bradykinin degradation by kininase II, drastically potentiated parasitic invasion of HUVECs and CHO-B 2 R, but not of mock-transfected CHO cells, whereas the B 2 R antagonist HOE 140 or monoclonal antibody MBK3 to bradykinin blocked these effects. Invasion competence correlated with the parasites' ability to liberate the short-lived kinins from cell-bound kininogen and to elicit vigorous intracellular free calcium ([Ca 2 ϩ ] i ) transients through B 2 R. Invasion was impaired by membrane-permeable cysteine proteinase inhibitors such as Z-(SBz)Cys-Phe-CHN 2 but not by the hydrophilic inhibitor 1-trans-epoxysuccinyl-l -leucyl-amido-(4-guanidino) butane or cystatin C, suggesting that kinin release is confined to secluded spaces formed by juxtaposition of host cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas' disease.
Heparan Sulfate Modulates Kinin Release by Trypanosoma cruzi through the Activity of Cruzipain
Journal of Biological Chemistry, 2002
Trypanosoma cruzi activates the kinin pathway through the activity of its major cysteine proteinase, cruzipain. Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS). Kinetic assays show that HS reduces the cysteine proteinase inhibitory activity (K i app ) of HK about 10-fold. Conversely, the catalytic efficiency of cruzipain on kinin-related synthetic fluorogenic substrates is enhanced up to 6-fold in the presence of HS. Analysis of the HK breakdown products generated by cruzipain indicated that HS changes the pattern of HK cleavage products. Direct measurements of bradykinin demonstrated an up to 35fold increase in cruzipain-mediated kinin liberation in the presence of HS. Similarly, kinin release by living trypomastigotes increased up to 10-fold in the presence of HS. These studies suggest that the efficiency of T. cruzi to initiate kinin release is potently enhanced by the mutual interactions between cruzipain, HK, and heparan sulfate proteoglycans.
European Journal of Biochemistry, 1999
The substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, was investigated using a series of dansyl-peptides based on the putative autoproteolytic sequence of the proteinase (VVG-GP) located at the hinge region between the catalytic domain and the C-terminal extension. Replacing Val with Pro at P2 in this sequence greatly improved the rate of cleavage by cruzipain. Tyr and Val residues are preferred at P3 by all cysteine proteinases whatever their origin, whereas only cruzipain and cathepsin L cleaved substrate with a His at that position. The combination of a Pro at P2 and His at P3 abolished cleavage by cathepsin L, so that only cruzipain was able to cleave the HPGGP peptide at the GG bond. A substrate with intramolecularly quenched fluorescence was raised on this sequence (Abz-HPGGPQ-EDDnp) which was also specifically cleaved by cruzipain (k cat /K m of 157 000 m ±1´s±1) and by a homologous proteinase from Trypanosoma congolense. The pH activity profile of cruzipain on Abz-HPGGPQ-EDDnp showed a narrow peak with a maximum at pH 5.5 and no cleavage above pH 6.8, although trypanosomal cysteine proteinases remain active at basic pH. The lack of activity at neutral and basic pH was due to a decrease in k cat , while the K m remained essentially unchanged, demonstrating that the substrate still binds to the enzyme and therefore behaves as an inhibitor. Changing the substrate into an inhibitor depended on the deprotonation of the His residue in the substrate, as deduced from a comparison of the pH activity profile with that of a related, but uncharged, substrate. Abz-HPGGPQ-EDDnp also inhibited mammalian cathepsins B and L but was not cleaved by these proteinases at any pH. The importance of the His residue at P3 for cleavage by cruzipain was confirmed by substituting Lys for His at that position. The resulting peptide was not cleaved by cruzipain in spite of the presence of a positively charged group at P3, but still interacted with the enzyme. It was concluded that the presence of an imidazolium group at P3 was essential to endow the HPGGPQ sequence with the properties of a cruzipain substrate.
The substrate specificity of cruzipain 2, a cysteine protease isoform from Trypanosoma cruzi
FEMS Microbiology Letters, 2006
Papain-like cysteine proteases are important for the survival of the flagellated protozoa Trypanosoma cruzi, the causative agent of Chagas' Disease. The lysosomal cysteine protease designated as cruzipain or cruzain, is the archetype of a multigene family of related isoforms. We investigated the substrate specificity of the cruzipain 2 isoform using internally quenched fluorogenic substrates. We found that cruzipain 2 and cruzain differ substantially regarding the specificity in the S 2 , S 0 1 and S 0 2 pockets. Our study indicates that cruzipain 2 has a more restricted specificity than cruzain, suggesting that these isoforms might act on distinct natural substrates.
Current Medicinal Chemistry, 2009
This review aims to present different aspects related to cruzipain, one of the most important proteins of the etiological agent of Chagas disease that has been extensively studied in the last two decades, including all the particularities of the molecule as well as to highlight its participation in multiple relevant functions of the parasite to favour the cell invasion phenomena, to facilitate host tissues proteolytic degradation and to trigger the evasion mechanism from host immune response. Cruzipain has been related with parasite metabolism and identified as both an important candidate for vaccine development and for trypanocidal drug design. We have reported for the first time that this enzyme is a sulfated glycoprotein. Indeed, the sulfated oligosaccharides are main targets for immune responses and are involved in tissue damage in mice immunized in absence of infection contributing to get deeper into the knowledge of the molecule composition and helping to elucidate its role in the infection and/or pathogenesis of the disease. A whole view including all the aspects related to the major cysteine proteinase of Trypanosoma cruzi studied so far including recent advances as proteinase, antigen and glycoprotein will be discussed.
Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum
The Biochemical journal, 1995
African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in v...
Trypanosoma cruzi: Isolation and characterization of a proteinase
Experimental Parasitology, 1981
Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO 2 )-Phe-Val-Leu-O 4 MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (P68%) and the aspartic active site labeling agent, 1,2epoxy-3-(phenyl-nitrophenoxy) propane (P80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation.
Pharmacological Activities and Hydrolysis by Peptidases of [Phospho-Ser6]-Bradykinin (pS6-BK)
Biochemical Pharmacology, 2015
Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS 6 ]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS 6 ]-Bk or Lys-[pS 6 ]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF [pS 386 ]PFRSSRI-NH2. The pharmacological assays of [pS 6 ]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS 6 ]Bk, both if injected through femoral vein or aorta. [pS 6 ]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. 1 H-NMR experiments indicated that [pS 6 ]-Bk has lower flexibility, with the pS 6-P 7 bond restricted to the trans conformation, and can explain [pS 6 ]-Bk resistance to hydrolysis. In conclusion, [pS 6 ]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS 386 ]PFRSSRI-NH 2 , suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator.