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A 33-kDa protein of Trypanosoma congolense is a major antigen in infected cattle and the production of antibody to this antigen appeared to correlate with enhanced resistance to trypanosomiasis [4]. Immunoelectron microscopy using a monoclonal antibody (mAb 4C5) raised against the 33-kDa antigen showed a lysosomal localisation, similar to that of a previously described 32-kDa cysteine protease of T. congolense. Both mAb 4C5 and anti-33 kDa antibody from infected cattle bound on Western blots to the cysteine protease that had been purified by affinity chromatography on cystatin-Sepharose. Sepharose-coupled mAb 4C5 was used to affinity purify the antigen from bloodstream forms of T. congolense. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the affinity-purified antigen had a molecular mass of 33 kDa under non-reducing conditions, and 40 kDa under reducing conditions. Anti-33-kDa antibody from infected cattle bound to both non-reduced and reduced affinity-purified antigen on Western blots. Serum from a rabbit immunised with the biochemically purified enzyme also bound the affinity-purified antigen. The affinity-purified antigen displayed proteolytic activity in fibrinogen-containing SDS-PAGE and against Azocoll. It hydrolysed benzyloxycarbonyl-Phe-Arg-7-amino-methyl coumarin (Z-Phe-Arg-NHMec) with a Km similar to that of the biochemically purified enzyme. Proteolytic and peptidolytic activities of the antigen were inhibited by the inhibitors of cysteine proteases, cystatin and trans-epoxysuccinyl-L-leucyl-amido (4-guanidino)butane (E-64). On two-dimensional gel electrophoresis, the antigen displayed similar characteristics to those of the biochemically purified enzyme. We conclude that the 33-kDa antigen of T. congolense and the cysteine protease are the same molecule.

A cysteine protease (trypanopain-Tc) with cathepsin-L-like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31-32 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 Z-Phe-Arg-NHMec were kcat = 17.4 s-1, Km = 4.4 microM, kcat/Km = 4.0 microM-1.s-1, which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z-Arg-Arg-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, L-trans-epoxysuccinyl-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 vitro after 24-48 h incubation in greater than or equal to 20 microM [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.

A cysteine protease has been purified from bloodstream forms of Trypanosoma congolense by affinity chromatography on cystatin-Sepharose. A polyclonal antibody was raised against the purified enzyme and used for immunocytochemical localization of the enzyme by electron microscopy. Antibody labeling of the cysteine protease, using colloidal gold-labeled protein A (PrA-Au), was observed over amorphous material within subcellular organelles which have the appearance of lysosome-like bodies. This intracellular labeling colocalized in organelles containing bovine serum albumin-gold (BSA-Au) that had been endocytosed by the living parasites. The PrA-Au/antibody also labeled the flagellar pocket and parasite cell surface, albeit less consistently. Volume density analysis showed that the organelles containing endocytosed BSA-Au, after 30 min incubation at 37 degrees C in BSA-Au, comprised approximately 22% of the total parasite cell volume. Under similar conditions, but employing horseradish peroxidase (HRP) as a fluid-phase marker of the lysosomal system, only 5.7% of the cell contained HRP. This value dropped to 3.6% after 60 min incubation. Volume density analysis showed that the amorphous material which was labeled by the antibody to the cysteine protease occupied 6.9% of the cell volume. This amorphous material was contained within a membrane-bound lysosome-like organelle that occupied 11.5% of the cell. Thus, the cysteine protease appears to be present in half, or less, of the lysosomal system of T. congolense.

Lysates of different life-cycle stages of Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei were analysed for endopeptidase activity, using reaction conditions which permitted a distinction to be made between lysosomal and non-lysosomal activity [Lonsdale-Eccles, J. D. & Grab, D. J. (1987) Eur. J. Biochem. 169, 467-475]. Hydrolysis of Z-Arg-Arg-NHMec (Z = benzyloxycarbonyl, NHMec = 7-amino-4-methylcoumaryl) and Z-Gly-Gly-Arg-NHMec occurred predominantly at alkaline pH and was observed in lysates of both insect and mammalian infective forms of T. brucei and T. congolense. Compared to their other life-cycle stages, procyclic forms of T. brucei and epimastigote forms of T. congolense exhibited enhanced hydrolysis of these substrates. Low levels of hydrolysis of Z-Arg-Arg-NHMec were observed in the bloodstream and epimastigote forms of T. vivax. The hydrolysis of Z-Gly-Gly-Arg-NHMec in each of the life-cycle stages of T. vivax was generally below detectable levels. In lysates of T. congolense, proteolytic and Z-Phe-Arg-NHMec-hydrolytic activity in bloodstream forms greater than metacyclic greater than epimastigote greater than procyclic forms. In T. vivax Z-Phe-Arg-NHMec-hydrolytic activity differed slightly according to the origin of the parasite but, in general, followed the same pattern (i.e. bloodstream forms greater than epimastigote forms, with metacyclic forms usually intermediate between these two). In T. brucei, Z-Phe-Arg-NHMec-hydrolytic activity in bloodstream forms greater than procyclic forms. Upon differentiation of the long, slender bloodstream forms into short, stumpy forms the Z-Phe-Arg-NHMec-hydrolytic activity was elevated even further. Thus, during their life cycle, each of these African trypanosomes exhibits complex changes of endopeptidase activity, suggestive of an induction of lysosomal activity between the insect and mammalian forms.