Structural and functional identification of GP57/51 antigen of Trypanosoma cruzi as a cysteine proteinase

Purified GP57/51, a Trypanosoma cruzi glycoprotein earlier identified as a major antigen in infected humans, was subjected to N-terminal sequence analysis. Alignment of the first 30 amino acids revealed that its N-terminal region is virtually identical to that reported for a cysteine-proteinase isolated from the Tulahuen strain, including the presence of active site cysteine at position 25. The finding of serine at position 24 of GP57/51 (Y strain) has further increased the homology between this protozoan antigen with other members of the eukaryotic family of cysteine proteases, including human cathepsin L. Functional analysis of GP57/51 indicated that the antigen is indeed an active thiol proteinase, which is active across a wide pH range (5-7.5). This was shown using either human IgG or gelatin substrates co-polymerized into polyacrylamide gels prepared for electrophoresis, and also by enzyme assays peformed with the synthetic substrate Z-phe-arg-NMec. The enzyme was activated by thiol containing reagents, and was strongly inhibited by low concentrations of E-64 (IC50 0.1 microM), cystatin (IC50 1 microM), leupeptin (IC50 0.1 microM) and antipain (IC50 0.1 microM). Monoclonal antibodies directed against distinct epitopes of GP57/51 absorbed the hydrolytic activity from purified preparations, demonstrating that the antigenic and enzymatic activities were indeed expressed by the same molecular entities. The subcellular localization of immunoreactive molecules was investigated by electron microscopy; immunogold staining was conspicuously found in vesicles belonging to the endosomal-lysosomal system, in tissue culture trypomastigotes as well as in epimastigotes. The possibility that this highly antigenic protease is actively secreted and/or leaked out of damaged parasites is under investigation; its release to tissues and to the circulation may contribute to pathology, considering that it (i) can degrade proteins across a wide pH range and (ii) stimulates immune T cells from chronic chagasic patients.