Substrate specificity studies of the cysteine peptidases falcipain-2 and falcipain-3 from Plasmodium falciparum and demonstration of their kininogenase activity

We studied the substrate specificity requirements of recombinant cysteine peptidases from Plasmodium falciparum, falcipain-2 (FP-2) and falcipain-3 (FP-3), using fluorescence resonance energy transfer (FRET) peptides as substrates. Systematic modifications were introduced in the lead sequence Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = N-[2,4-dinitrophenyl]ethylenediamine) resulting in five series assayed to map S-3 - S-2' subsite specificity. Despite high sequence identity and structural similarity between FP-2 and FP-3, noteworthy differences in substrate specificity were observed. The S-1 subsite of FP-2 preferentially accommodates peptides containing the positively charged residue Arg in P-1, while FP-3 has a clear preference for the hydrophobic residue Leu in this position. The S-2 subsite of FP-2 and FP-3 presents a strict specificity for hydrophobic residues, with Leu being the residue preferred by both enzymes. FP-2 did not show preference for the residues present at P-3, while FP-3 hydrolysed the peptide Abz-ALRSSRQ-EDDnp, containing Ala at P-3, with the highest catalytic efficiency of all series studied. FP-2 has high susceptibility for substrates containing hydrophobic residues in P-1', while FP-3 accommodates well peptides containing Arg in this position. The S-2' subsite of both enzymes demonstrated broad specificity. In addition, radioimmunoassay experiments indicated that kinins can be generated by FP-2 and FP-3 proteolysis of high molecular weight kininogen (HK). Both enzymes excised Met-Lys-bradykinin, Lys-bradykinin and bradykinin from the fluorogenic peptide Abz-MISLMKRPPGFSPFRSSRI-NH2, which corresponds to the Met(375) to Ile(393) sequence of HK. The capability of FP-2 and FP-3 to release kinins suggests the involvement of these enzymes in the modulation of malaria pathophysiology. (c) 2013 Elsevier B.V. All rights reserved.