Discrimination of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, and mammalian cathepsins B and L, by a pH-inducible fluorogenic substrate of trypanosomal cysteine proteinases

The substrate specificity of cruzipain, the major cysteine proteinase of Tr ypanosoma cruzi, was investigated using a series of dansyl-peptides based o n the putative autoproteolytic sequence of the proteinase (VVG-GP) located at the hinge region between the catalytic domain and the C-terminal extensi on. Replacing Val with Pro at P2 in this sequence greatly improved the rate of cleavage by cruzipain. Tyr and Val residues are preferred at P3 by all cysteine proteinases whatever their origin, whereas only cruzipain and cath epsin L cleaved substrate with a His at that position. The combination of a Pro at P2 and His at P3 abolished cleavage by cathepsin L, so that only cr uzipain was able to cleave the HPGGP peptide at the GG bond. A substrate wi th intramolecularly quenched fluorescence was raised on this sequence (Abz- HPGGPQ-EDDnp) which was also specifically cleaved by cruzipain (k(cat)/K-m of 157 000 (M-1.s-1)) and by a homologous proteinase from Trypanosoma congo lense. The pH activity profile of cruzipain on Abz-HPGGPQ-EDDnp showed a na rrow peak with a maximum at pH 5.5 and no cleavage above pH 6.8, although t rypanosomal cysteine proteinases remain active at basic pH. The lack of act ivity at neutral and basic pH was due to a decrease in k(cat), while the K- m remained essentially unchanged, demonstrating that the substrate still bi nds to the enzyme and therefore behaves as an inhibitor. Changing the subst rate into an inhibitor depended on the deprotonation of the His residue in the substrate, as deduced from a comparison of the pH activity profile with that of a related, but uncharged, substrate. Abz-HPGGPQ-EDDnp also inhibit ed mammalian cathepsins B and L but was not cleaved by these proteinases at any pH. The importance of the His residue at P3 for cleavage by cruzipain was confirmed by substituting Lys for His at that position. The resulting p eptide was not cleaved by cruzipain in spite of the presence of a positivel y charged group at P3, but still interacted with the enzyme. It was conclud ed that the presence of an imidazolium group at P3 was essential to endow t he HPGGPQ sequence with the properties of a cruzipain substrate.