Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The p resence of cystatin-like inhibitors in lower eukaryotes such its protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibito rs to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas' heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cystein e protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cyste ine protease inhibitor in T cruzi. We used high temperature induced denatur ation to isolate a heat-stable cruzipain-binding protein (apparent molecula r mass, 12 kDa) from epimastigote lysates. This protein was subsequently ch aracterized as a tight-binding and reversible inhibitor of papain-like cyst eine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cyt oplasmic vesicles of trypomastigotes and to the cell surface of amastigotes . Binding assays performed by probing living parasites with fluorescein (FI TC)cruzipain or FITC-chagasin revealed the presence of both inhibitor and p rotease at the cell surface of amastigotes. The intersection of chagasin an d cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.