Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

Abstract

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

Proteases from Trypanosoma brucei brucei

“…Trypanopain-Tb was shown to be a fairly typical cysteine proteinase, similar to trypanopain-Tc from 7: congolense (Mbawa et al, 1992) and cruzipain from 7: cruzi (Cazzulo et al, 1989). Interestingly, the aspartic proteinase inhibitor pepstatin A inhibits trypanopain-Tb slightly (Table 2 ; Pamer et al, 1989).…”

“…Interestingly, the aspartic proteinase inhibitor pepstatin A inhibits trypanopain-Tb slightly (Table 2 ; Pamer et al, 1989). In contrast, pepstatin A inhibits trypanopain-Tc by only 9.7% (Mbawa et al, 1992) and does not affect cruzipain (Murta et al, 1990;Cazzulo et al, 1989). Pepstatin A appears to be a competitive inhibitor of trypanopain-Tb (data not shown), and may act as a substrate analogue.…”

“…A cysteine proteinase has previously been isolated from 7: congolense (Mbawa et al, 1992) but we have now purified one from 7: b. brucei. OPTb from 7: b. brucei has been partially purified and characterised (Kornblatt et al, 1992), but here we report the purification of the enzyme to homogeneity as well as a more complete characterisation of certain aspects of its activity.…”

“…Trypanopain is lysosomally located (Mbawa et al, 1991) and, as such, is likely to be centrally involved in intracellular digestive and catabolic proteolysis. Additionally, the enzyme is proposed to help the parasite escape opsonisation by degrading internalised antibody-variant surface glycoprotein complexes (Russo et al, 1994).…”

Prolyl oligopeptidase-like deficient Trypanosoma evansi parasites are associated with reduced interleukin-10 concentrations in vivo and in vitro

“…All the mutant and wild-type parasites were able to hydrolyse Z-Phe-Arg-AMC, a substrate routinely used for measuring the activity of parasite cysteine peptidases and used as a control in this experiment (Mbawa et al 1992;Caffrey et al 2001). Wildtype T. evansi RoTat 1.2 parasites readily hydrolysed Z-ArgArg-AMC (expressed as 100% activity), while the Δpop and T. evansi RoTat 1.2 wild-type (L1) and Δpop null mutant (L2) genomic DNA incubated with probe 1 at slightly >3000 bp for TevPOP; (A2) incubated with probe 2 at slightly >3000 bp for TevPOP (L1), and at slightly >2100 bp and slightly >1700 bp for NEO and BLA respectively (L2).…”