Proteases from Trypanosoma brucei brucei

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Abstract
African trypanosomes contain proteases that may be released into the bloodstream of their infected hosts. This paper describes a novel, combined isolation of a cysteine proteinase (called trypanopain‐Tb) and a serine oligopeptidase (which we call oligopeptidase‐Tb) from Trypanosoma brucei brucei, as well as a comparison of the activities of these two enzymes against several host regulatory molecules. The enzymes differed in various respects. Firstly, purified trypanopain‐Tb was shown to readily cleave proteins such as gelatin maximally at acidic pH. In contrast, oligopeptidase‐Tb, which is optimally active at alkaline pH, did not hydrolyse proteins larger than 4 kDa. However, it readily hydrolysed various polypeptides, including neurotensin and atrial natriuretic factor. The interaction of the two enzymes with mammalian protease inhibitors also differed. Cystatins and α2‐macroglobulin effectively inhibited trypanopain‐Tb, with the Ki values for cystatin C and low‐molecular‐mass kininogen (≈10−11 M) predicting that trypanopain‐Tb is likely to be effectively controlled by these inhibitors if released into the host bloodstream. In contrast, oligopeptidase‐Tb was not inhibited by serpins or α2‐macroglobulin, suggesting that it may remain active if released into the host bloodstream. In support of these in vitro results, the blood of trypanosome‐infected rats displayed no trypanopain‐Tb‐like activity, but exhibited high oligopeptidase‐Tb‐like activity. Thus, while trypanopain‐Tb seems likely to be confined to an intracellular role within the parasite, oligopeptidase‐Tb has the potential to remain active in the host bloodstream and so contribute directly to pathogenesis.