Carboxydipeptidase activities of recombinant cysteine peptidases

Abstract

The recombinant cysteine peptidases, cruzain from Trypanosoma cruzi and CPB2.8DCTE from Leishmania mexicana, are cathepsin L-like and characteristically endopeptidases. In this study, we characterized the carboxydipeptidase activities of these enzymes and compared them with those of human recombinant cathepsin B and cathepsin L. The analysis used the internally quenched fluorescent peptide Abz-FRFK*-OH and some of its analogues, where Abz is ortho-aminobenzoic acid and K* is (2,4-dinitrophenyl)-e-NH 2 -lysine. These peptides were demonstrated to be very sensitive substrates, due to the strong quenching effect of K* on the fluorescence of the Abz group. The carboxydipeptidase activity of cruzain was shown to be very similar to that of cathepsin B, while that of CPB2.8DCTE is closer to the carboxydipeptidase activity of cathepsin L. The S 2 subsite architecture of cruzain and the nature of the amino acid at the P 2 position of the substrates determine its carboxydipeptidase activity and gives further and direct support to the notion that the carboxydipeptidase activity of the papain family cysteine peptidases rely on the S 2 -P 2 interaction [Na¨gler D. K., Tam, W., Storer, A.C., Krupa, J.C., Mort, J.S. & Menard, R. (1999) Biochemistry 38, 4868-4874]. Cruzain and CPB2.8DCTE presented a broad pH-range for both the endo-and exo-peptidase activities, although the later is approximately one order of magnitude lower. This feature, that is not common in related mammalian cysteine peptidases, is consistent with the enzymes being exposed to different environmental conditions and having different locations during parasite development.

Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment

“…A similar cleavage pattern was noted for processing of proenkephalin by cathepsin L at particular mono or dibasic amino acids where an aromatic acid is located at P2 or P3 of the cleavage site (21 , and Tyr 146 may be regarded as the C terminus of peptides generated by endocleavage at an adjacent upstream typical motif (Fig. 7), which is subsequently blunted by the very weak carboxypeptidase activity of cathepsin L itself (40), in a stepwise manner. Thus, the 8-kDa subunit and four other peptides derived from the linker (e.g.…”

Proteinases of Trypanosoma cruzi: Potential Targets for the Chemotherapy of Chagas Disease

“…The peculiar substrate specificity of cruzipain, somehow intermediate between those of cathepsins L and B, since the enzyme is able to accomodate either a hydrophobic or a positively charged residue at P2, is consistent with the presence of Glu at position 205, as first pointed out by Lima and co-workers [24]. Recently, recombinant cruzain has been shown to have carboxydipeptidase activity, very similar to that of cathepsin B [25].…”

Repurposing of 5‐nitrofuran‐3,5‐disubstituted isoxazoles: A thriving scaffold to antitrypanosomal agents

“…Cruzain, truncated in the C-terminal extension, is a recombinant enzyme obtained from Escherichia coli (strain DH5α containing the expression plasmid, Sigma-Aldrich ® ), that was expressed, purified, and activated following the previously reported procedure [37,38] Cruzain was assayed in a reaction buffer of 100 mM sodium acetate (Sigma-Aldrich ® ), containing 100 mM NaCl (Sigma-Aldrich ® ), 0.01% Triton-X 100 (Sigma-Aldrich ® ), 5 mM EDTA (Sigma-Aldrich ® ), and 20% glycerol (Sigma-Aldrich ® ) at pH 5.5. The enzyme was pre-incubated in the presence of 3 mM DTT (Sigma-Aldrich ® ) for 5 min at 37°C in a 1 ml final volume with constant stirring.…”