On the post-translational modifications at the C-terminal domain of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, has a 130 amino acid-long C-terminal domain, which, although microheterogeneous in SDS-PAGE, has a single N-terminal amino acid sequence. Most of the Thr residues present at the beginning of this sequence are modified; the nature of this modification is still unknown, but O-glycosylation and phosphorylation seem both to be absent. The only potential site for N-glycosylation (Asn 254) is glycosylated in vivo. Most of the eight Cys residues are involved in disulfide bridges. The results are consistent with cruzipain being made of two well-defined domains, a catalytic one with high homology to cathepsin L, and a C-terminal domain, linked to the former by a 'hinge' corresponding to the Pro- and Thr-rich region at its N-terminus.     

Polymorphisms of the genes encoding cruzipain, the major cysteine proteinase of Trypanosoma cruzi, in the region encoding the C-terminal domain

Forty-eight cDNA clones obtained from different developmental stages of Trypanosoma cruzi and all encoding the C-terminal domain of the major cysteine proteinase (cruzipain) have been sequenced. A number of polymorphisms were detected, seven of them resulting in amino acid replacements. The predicted pI values of the corresponding gene products varied between 7.05 and 8.12. These changes in amino acid sequence, together with previously reported variations in carbohydrate composition at the only N-glycosylation site in the C-terminal domain, may account for most of the heterogeneities found in the mature enzyme.     

Digestion of human immunoglobulin G by the major cysteine proteinase (cruzipain) from Trypanosoma cruzi

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi was able to digest human IgG, as shown by polyacrylamide gel electrophoresis in the presence of SDS, and by gel filtration on a Superose 12 column, in a FPLC system. The Fab fragment of IgG was only slightly degraded, but Fc was extensively hydrolyzed to small peptides. The results suggest that cruzipain might be involved in the defense mechanisms of the parasite against the immune response of the host.     

A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi

Epimastigotes of different stocks of Trypanosoma cruzi contain similar levels of proteinase activity on azocasein; amastigotes and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.     

Purification and partial characterization of a cysteine proteinase from Trypanosoma rangeli

Abstract Epimastigotes of the American Trypanosome Trypanosoma rangeli contain a very low cysteine proteinase (CP) activity. The enzyme was purified to homogeneity by affinity chromatography on ConA-Sepharose and Cystatin-Sepharose. This CP had a similar apparent molecular mass and an identical N-terminal sequence (15 amino acids) as compared with cruzipain from Trypanosoma cruzi ; cross-reacted immunologically with the latter enzyme, it was inhibited by E-64 and TLCK, but not by PMSF, o-phenanthroline or Pepstatin, and was able to use the same substrates, although with different order of effectiveness and optimum pH.     

Nitric oxide inhibits cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi

Nitric oxide (NO) is a pluripotent regulatory molecule showing, among others, an antiparasitic activity. Moreover, NO inhibits cysteine proteinase action by nitrosylating the Cys catalytic residue. In the present study, the inhibitory effect of the substrate N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methyl coumarin) and of NO on the catalytic activity of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi (the hemoflagellate protozoan parasite which causes the American trypanosomiasis), is reported. In particular, NO-donors S-nitroso-glutathione (GSNO), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), S-nitroso-acetyl-penicillamine (SNAP), and sodium nitroprusside (SNP) dose-dependently inhibited cruzipain, this effect being likely attributable to the S-nitrosylation of the Cys25 catalytic residue. These results were analyzed in parallel with those concerning the inhibitory effect of the substrate and of NO on the catalytic activity of falcipain, the cruzipain-homologous cysteine proteinase from Plasmodium falciparum. The modulation of the cruzipain and falcipain activity by NO may be relevant in developing new strategies against T. cruzi and P. falciparum in human host. As a whole, the NO-mediated S-nitrosylation of pathogenic viral, bacterial, fungal, and parasitic cysteine proteinases may represent a general mechanism of antimicrobial and antiparasitic host defences.     

Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi

A cysteine proteinase, purified to homogeneity from epimastigotes of Trypanosoma cruzi, was strongly inhibited by L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64). The second-order rate constant was 20,800 M-1.s-1, and the reagent could be used for active site titration. The enzyme hydrolysed chromogenic peptides at the carboxyl Arg or Lys; it required at least one more amino acid, preferably Arg, Phe, Val or Leu, between the terminal Arg or Lys and the amino-blocking group. Enzyme activity on azocasein at pH 5.0 was increased by urea, maximal activity being attained at 2 M, and was still as active at 5 M urea as in its absence. Guanidine hydrochloride and KSCN also activated at low concentrations, but caused a strong inhibition above 2 M and 1 M, respectively. When azocasein was tested as a substrate at pH 7.0, there was no activation, and when synthetic substrates were used all chaotropic agents tested were inhibitory. The results suggest that the enzyme, for which we propose the trivial name 'cruzipain', differs in some aspects from all other cysteine proteinases described so far, although it shares several of the properties of mammalian cathepsin L.     

High resolution localization of cruzipain and Ssp4 in Trypanosoma cruzi by replica staining label fracture

Summary— The replica staining label fracture technique was used to analyse the distribution of cruzipain and Ssp4 in Trypanosoma cruzi. Intense labeling for the two proteins was seen on the E fracture face of amastigote forms. Gold particles did not co-localize with the intramembranous particles. Labeling was abolished by previous treatment of the parasites with phospholipase C from Trypanosoma brucei, which removes glycosylphosphatidyl inositol (GPI) anchored proteins. These observations suggest that cruzipain and Ssp4 are attached to the parasite surface via a GPI anchor.     

Effects of proteinase inhibitors on the growth and differentiation of Trypanosoma cruzi

Abstract Three proteinase inhibitors, one peptidyl acyloxymethyl ketone (AMK), Z-Phe-Lys-CH₂-OCO-(2,4,6-Me₃)Ph.HCl, and two diazomethyl ketones (DMKs), Z-Phe-Phe-DMK and Z-Phe-Ala-DMK, have been studied for their effects in vitro on the four developmental stages of Trypanosoma cruzi . The three inhibitors penetrated living parasites and inhibited the major cysteine proteinase, cruzipain. The AMK was the most potent inhibitor of cruzipain itself and at 20 μM caused lysis of epimastigotes and trypomastigotes. When at lower concentrations, however, it had little effect on epimastigote growth but reduced metacyclogenesis. The DMKs had no effect against epimastigotes but inhibited differentiation to metacyclics. All three inhibitors markedly reduced infection of Vero cells by the parasite and the multiplication of the intracellular amastigotes, whereas release of trypomastigotes was almost entirely prevented. The results confirm the importance of cysteine proteinases in the life cycle of T. cruzi , and suggest that the differentiation steps are the most susceptible to cysteine proteinase inhibitors.     

NMR spectroscopic and molecular modeling investigations of the trans-sialidase from Trypanosoma cruzi

Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalyzed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalyzed by TcTS were also investigated using a range of N-acetylneuraminosyl-based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl alpha-d-N-acetylneuraminide (MUN) is hydrolyzed by the enzyme approximately 3-5 times faster than either the disaccharide Neu5Acalpha(2,3)Galbeta1Me or the trisaccharide Neu5Acalpha(2,3)Lacbeta1Me. In the transfer reaction, we show that Neu5Acalpha(2,3)Lacbeta1Me is the most favorable substrate for TcTS and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor alpha1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesize that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312. The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments, taken together with other published data, have clearly demonstrated that lactose in the absence of other coligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.     

Analysis of the cysteine proteinases of Leishmania mexicana and Trypanosoma cruzi using specific antisera

Abstract Antisera raised against papain and cysteine proteinases (CPs) purified from Leishmania mexicana and Trypanosoma cruzi have been used to study the proteins in the two parasites. The antisera against the major CP of T. cruzi (cruzipain) not only cross-reacted with known CPs of L. mexicana but also detected stage-specific molecules that may represent previously unrecognised CPs. The binding of the same abtisera to extracts of different life cycle stages of T. cruzi suggested that the stages possess different isoforms of cruzipain. The lack of cross-reactivity of anti-papain antiserum against cruzipain suggests that the major immunogenic epitopes of these CPs are different, whereas the detection of the major CPs of L. mexicana with both heterologous antisera shows that the parasite's enzymes share epitopes with the other CPs.     

Proteolytic Activities in Cell Extracts of Trypanosoma cruzi*

SYNOPSIS. Cell extracts of culture forms of Trypanosoma cruzi are capable of hydrolysing substances belonging to 4 different groups of protease substrates: (a) substrates for trypsin-like enzymes: benzoyl-arginine-p-nitroanilide and benzoylarginine-naphtylamide: (b) substrates for aminopeptidases: leucyl. lysyl and glutamyl-β-naphtylamide; (c) a substrate for chymotrypsin-like enzymes: carbobenzoxy-L-tyrosine-p-nitrophenylester, and (d) a nonspecific substrate for a broad range of proteases: azocasein. Some physico-chemical characteristics of each enzymic reaction were studied. They were found to be distinct enough to allow attributing each hydrolytic activity to a separate enzyme.     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.     

Binding of Lectins to the Cell Surface of Trypanosoma cruzi*

SYNOPSIS. Differences in the composition and distribution of cell membrane carbohydrates were demonstrated in the 3 life cycle forms of 3 Trypanosoma cruzi strains by using lectins with different specificities. The results suggest that lectin binding may be useful in characterization of the parasite strains.     

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS–PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K_i = 3.29 nM) and human cathepsin L (K_i = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.     

Proteomic analysis of the human pathogen Trypanosoma cruzi

Trypanosoma cruzi, the protozoan that causes Chagas disease, possesses a complex life cycle involving different developmental stages. Experimental conditions for two-dimensional electrophoresis (2-DE) analysis of T. cruzi trypomastigote, amastigote and epimastigote proteomes were optimized. Comparative proteome analysis of the cell-cycle stages were carried out, revealing that few proteins included in the 2-DE maps displayed significant differential expression among the three developmental forms of the parasite. In order to identify landmark proteins, spots from the trypomastigote 2-DE map were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mass fingerprinting, resulting in 26 identifications that corresponded to 19 different proteins. Among the identified polypeptides, there were heat shock proteins (HSP; chaperones, HSP 60, HSP 70 and HSP 90), elongation factors, glycolytic pathway enzymes (enolase, pyruvate kinase and 2,3 bisphosphoglycerate mutase) and structural proteins (KMP 11, tubulin and paraflagellar rod components). The relative expression of the identified proteins in the 2-DE maps of the T. cruzi developmental stages is also presented.     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages

, , and 1986. Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages. International Journal for Parasitology 16: 629–632. Prelabelling of lysosomes with acridine orange has been performed in order to verify whether metacyclic forms of Trypanosoma cruzi are capable of inhibiting lysosomal fusion during the first moments of interiorization in non-sensitized mouse peritoneal macrophages. Thus, the degree of degranulation (lysosomal fusion) in metacyclic forms is low while epimastigote forms present higher levels. When epimastigote forms are made to interact with the macrophages in the presence of various concentrations of the medium used for transformations of epimastigotes to metacyclic forms or when interaction was performed in the presence of NH₄Cl, the degree of degranulation was similar to that obtained when interaction was carried out with metacyclic forms.

The present results suggest that during the first moments of the interaction of T. cruzi, only the infective forms may increase the cytoplasmic pH value of the host phagocytic cell, avoiding lysosomal fusion and the subsequent destruction of the parasite.