Analysis of peptidase activities of a cathepsin B-like (TcoCBc1) from Trypanosoma congolense

• Carboxydipeptidase activity of TcoCBc1 depends on the substrate sequences.
• TcoCBc1 prefers acidic residues at P2 and P1 for carboxydipeptidase activity.
• TcoCBc1 endopeptidase activity is better with substrates with E at P1 position.
• TcoCBc1 has positively charged catalytic site while it is negative in cathepsin B.
• Could these peculiarities involve TcoCBc1 in T. congolense apoptotic processes?

The substrate specificity of TcoCBc1 was evaluated using two internally quenched fluorescent peptide libraries with randomized sequences designed to detect carboxydipeptidase (Abz-GXXZXK(Dnp)-OH) and endopeptidase (Abz-GXXZXXQ-EDDnp) activities at acidic and neutral pHs, respectively. All the data obtained with TcoCBc1 were compared with those of human cathepsin B, including the pH profiles of the hydrolytic reactions. The most relevant observation is the preference of TcoCBc1 for substrates with a pair of acidic amino acids at positions P2 and P1 for its carboxydipeptidase activity and the well acceptance for E and D at P1 position for endopeptidase activity. These peculiar preferences for negatively charged groups of TcoCBc1 and its requirements for carboxydipeptidase activity were also observed on Abz labeled analogues of bradykinin (Abz-RPPG↓FSAFR-OH, Abz-RPPG↓FS↓AF-OH, Abz-RPPG↓DE↓AF-OH) and angiotensin I (Abz-DR↓VYIHAFHL-OH), where ↓ indicates the cleavage site. TcoCBc1 was modeled based on the atomic coordinates of the cathepsin B from Trypanosoma brucei and the positively charged environment in TcoCBc1 catalytic site contrasts with the negatively charged environment in human cathepsin B. The preferences of S1 and S2 subsites of TcoCBc1 for acidic amino acids have to be taken into consideration for future studies of physiological roles of TcoCBc1 as for instance in apoptotic processes of Trypanosoma congolense.