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 S3-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 S1 subsite of FP-2 preferentially accommodates peptides containing the positively charged residue Arg in P1, while FP-3 has a clear preference for the hydrophobic residue Leu in this position. The S2 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 P3, while FP-3 hydrolysed the peptide Abz-ALRSSRQ-EDDnp, containing Ala at P3, 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.
Departamento de Biofísica, Universidade Federal de São Paulo, Rua 3 de maio 100, 04044-020 São Paulo, SP, Brazil.
Simone S
SS
Cotrin
Iuri E
IE
Gouvêa
Pollyana M S
PM
Melo
Piero
P
Bagnaresi
Diego M
DM
Assis
Mariana S
MS
Araújo
Maria Aparecida
MA
Juliano
Marcos L
ML
Gazarini
Philip J
PJ
Rosenthal
Luiz
L
Juliano
Adriana K
AK
Carmona
D003546:Cysteine Endopeptidases / Q000378:metabolism*; D007610:Kallikreins / Q000378:metabolism*; D007704:Kininogens / Q000378:metabolism; D007705:Kinins / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D013379:Substrate Specificity
D007704:Kininogens; D007705:Kinins; D007610:Kallikreins; D003546:Cysteine Endopeptidases; C416224:falcipain 2; C443101:falcipain 3