Study on a proteolytic enzyme from Trypanosoma congolense

Rautenberg, Peter; Schädler, Regina; Reinwald, E.; Risse, H. J.

doi:10.1007/bf00229598

Cited by 21 publications

(9 citation statements)

References 34 publications

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“…This appears to be a characteristic of cysteine proteases of protozoan parasites in general (North et al, 1990). An earlier report (Rautenberg et al, 1982) described cysteine protease activity in T. congolense that probably corresponds to the minor activity we obscrve in the PI region around PI 5.0.…”

Section: Discussionmentioning

confidence: 48%

“…The assays were performed as described (Rautenberg et al, 1982). Brielly, 30 pl purified enzyme was added to 90 pl each of the protein substrates (0.8 mglml).…”

Section: Determination Of the Proteolytic Nature Of The Purified Cystmentioning

confidence: 99%

“…Activity was also tested against a variety of protein substrates (ovalbumin, fibrinogen, casein and bovine serum albumin). The assays were performed as described (Rautenberg et al, 1982). Brielly, 30 pl purified enzyme was added to 90 pl each of the protein substrates (0.8 mglml).…”

Section: Determination Of the Proteolytic Nature Of The Purified Cystmentioning

confidence: 99%

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Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

Mbawa¹,

Gumm²,

Shaw

et al. 1992

European Journal of Biochemistry

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A cysteine protease (trypanopain-Tc) with cathepsin-L-like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31 -3 2 kDa by SDS/ PAGE and 66 kDa by gel chromatography. It has a PI 7.4 and a high affinity for concanavalin A, Trypanopain-Tc catalyses the limited proteolysis of a variety of protein substrates such as fibrinogen, serum albumin and trypanosome variant-surface glycoprotein. It has minimal or no activity against casein or elastin.A variety of peptidyl amidomethylcoumarins and peptidyl diazomethanes were used to test the specificity of trypanopain-Tc. The better substrates had Arg or Lys in P1 and hydrophobic amino acids in P2 and P3. The best substrate found for trypanopain-Tc was Z-Phe-Arg-NHMec (Z, benzyloxycarbonyl ; NHMec, 7-amido-4-methylcoumarin). The kinetic constants for the hydrolysis, of ZPhe-Arg-NHMec were k,,, = 17.4 SKI, K, = 4.4 pM, kcal/Km = 4.0 pM-' ' s-', which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z-ArgArg-NHMec) and cathepsin H (Arg-NHMec) were not hydrolysed by trypanopain-Tc under the conditions tested. The pH optimum of trypanopain-Tc against Z-Phe-Arg-NHMec was pH 6.0 but it showed a broad peak of activity extending well into the alkaline region. The enzyme was activated by low-molecular-mass thiol compounds and inhibited by cystatin, ~-trans-epoxysucciny1-4-guanidinobutane (E-64) and a variety of peptidyl diazomethanes. The most effective diazomethane inhibitors (Z-Leu-Leu-Met-CHN2, Z-Leu-Met-CHN2 and Z-Leu-Lys-CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vituo after 24 -48 h incubation in 2 20 pM [inhibitor]. However, it is not clear whether the trypanocidal activity of these inhibitors is a consequence of the inhibition of trypanopains or of some other essential proteolytic activities within the parasites.Cathepsins L and B play important roles in intracellular protein turnover (for review see Ballard, 1987;Marzella and Glaumann, 1987). They are also implicated in the degradation of extracellular matrix in diseases such as muscular dystrophy, emphysema and arthritis, and in association with tumour metastasis (Rochefort ct al., 1987;Kominami et al., 1987; Burnett and Stockley, 1985;Etherington et al., 1988). Recently, interest has been shown in the role of cysteine proteases in the pathology of parasitic infections. These proteases can be released from the parasites (Blum, 1976;Keene, 1986; Lockwood et al., 1988; Garber and Lemchuk-Favel, 1989;North et al., 1989; Boutignon et al., 1990) andmay beinvolved in the pathology of the diseases and the survival of the parasites within the host (Bremmer et al

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Section: Discussionmentioning

confidence: 48%

“…The assays were performed as described (Rautenberg et al, 1982). Brielly, 30 pl purified enzyme was added to 90 pl each of the protein substrates (0.8 mglml).…”

Section: Determination Of the Proteolytic Nature Of The Purified Cystmentioning

confidence: 99%

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Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

Mbawa¹,

Gumm²,

Shaw

et al. 1992

European Journal of Biochemistry

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“…In the present investigation, T. evansi isolate of camel origin had eight proteinase bands apparently visible at 28, 32, 35, 41 and 69 kDa, and three bands in between the Mr 95-170 kDa and only two proteinase bands-of the eight proteinase bands-of 28 and 41 kDa were common in both Indian and African isolates of T. evansi. Rautenberg et al (1982) believed that higher MW forms represent the intracellular forms of the enzymes and demonstrated a progressive breakdown of the high MW forms of T. congolense to 31 kDa form. GS-PAGE revealed a strong proteolytic activity distinct at eight regions apparently in the MW 28-170 kDa-after the gel was incubated in incubation buffer containing DTT (pH range 3.0-5.5)-in the form of multiple zones of clearance, which may be attributed to all the three major acid proteinases viz., cysteine, serine and aspartic proteinases reported to be active only in acidic pH.…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of cysteine proteinases of Trypanosoma evansi

Yadav

Kumar

et al. 2011

Parasitol Res

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Trypanosoma evansi is a causative agent of 'surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28-41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.

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“…The possible modifications in uivo that could be responsible for the charge heterogeneity are presently under investigation. In African trypanosomes, charge heterogeneity in the surface glycoproteins has been attributed to phosphorylation [25] and sialylation [26].…”

Section: Discussionmentioning

confidence: 99%

Mapping of surface glycoproteins of Trypanosoma cruzi by two-dimensional electrophoresis. A correlation with the cell invasion capacity

Andrews¹,

Katzin²,

Colli³

1984

Eur J Biochem

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The cell-surface iodinatable proteins of Trypanosoma cruzi have been analyzed by two-dimensional polyacrylamide gel electrophoresis under equilibrium conditions. Antigenic polypeptides were characterized after immunoprecipitation and glycoproteins were identified by means of lectin-affinity chromatography.Two glycoproteins, with affinity for concanavalin A, were found to be common to both infective (trypomastigote) and non-infective (epimastigote) forms: protein 1 (90 kDa, PI 5.5-6.5) and protein 2 (80 kDa, PI 5.3 -6.3).In epimastigotes a specific concanavalin-A-binding surface glycoprotein (70 kDa, pl 5.5) was identified. Trypomastigote forms, on the other hand, presented several specific iodinatable surface components : glycoproteins 3 (85 kDa, PI 5.9, 4 (85 kDa, PI 5.0), 6 (100 kDa, PI 6.5), 7 (120 kDa, PI 6.3), 8 (68 kDa, PI 6.7) and several minor high-molecular-mass acid proteins, all containing glucose and/or mannose, and glycoprotein 5 (85 kDa, PI 6.3 -7.9, containing N-acetyl-D-glucosamine (Tc-85).Proteins 1, 2 and 5 were the only ones which gave clear evidence of charge heterogeneity. Most of the surface proteins of trypomastigote forms, the exception being proteins 3 , 4 and 8, were removed by treatment with trypsin. This proteolytic treatment results in 90 % inhibition of the in vitro vertebrate-cell-invasion capacity of the parasites. Upon reincubation in culture medium for 4 h, the trypsin-removed glycoproteins are again detected, an observation that correlates well with the recovery of the cell-penetration capacity observed in the same period.Trypanosoma cruzi, the causative agent of Chagas' disease, is a parasite with at least three well-defined differentiation stages : the amastigote, which lives and reproduces inside vertebrate cells; the epimastigote, a dividing form that inhabits the midgut of a reduviid insect; and the trypomastigote, anondividing cell which, in nature, is the link between the vertebrate and the invertebrate phases of the protozoan biological cycle. These forms are distinct not only in terms of their host environments but also with respect to their morphologies and biological behavior [I -31. method, although useful, does not discriminate between different proteins of similar molecular weight. For this reason we have made a comparative study of the differences in surface protein composition between the non-infective and infective forms of T. cruzi using two-dimensional gel electrophoresis under equilibrium conditions. This method, which provides better resolution and a more complete pattern, provides evidence for a correlation between trypsin-induced surface protein degradation and parasite infectivity. MATERIALS AND METHODS ParasitesEpimastigotes of the Y strain were grown in liver infusion/tryptose medium [16,17] under constant agitation at 28 "C. The parasites were collected on the third day after inoculation by centrifugation at 800 x g for 10min and washed six times with medium 199. Trypomastigotes of the Y strain were maintained by weekly transfers in A/Snell inbred ...

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Study on a proteolytic enzyme from Trypanosoma congolense

Cited by 21 publications

References 34 publications

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

Identification and characterization of cysteine proteinases of Trypanosoma evansi

Mapping of surface glycoproteins of Trypanosoma cruzi by two-dimensional electrophoresis. A correlation with the cell invasion capacity

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