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

Abstract：

: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.

journal_name

Mol Biochem Parasitol

journal_title

Molecular and biochemical parasitology

authors

Cotrin SS,Gouvêa IE,Melo PM,Bagnaresi P,Assis DM,Araújo MS,Juliano MA,Gazarini ML,Rosenthal PJ,Juliano L,Carmona AK

doi

10.1016/j.molbiopara.2013.01.002

subject

Has Abstract

pub_date

2013-02-01 00:00:00

pages

111-6

issue

eissn

0166-6851

issn

1872-9428

pii

S0166-6851(13)00004-2

journal_volume

187

pub_type

杂志文章

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