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

Article abstract of DOI:10.1016/j.molbiopara.2013.01.002

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 P ₁, 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 P ₃, 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 Ile³⁹³ 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.

Full text of DOI:10.1016/j.molbiopara.2013.01.002

Molecular & Biochemical Parasitology 187 (2013) 111–116
Contents lists available at SciVerse ScienceDirect
Molecular & Biochemical Parasitology
Substrate specificity studies of the cysteine peptidases falcipain-2 and falcipain-3
from Plasmodium falciparum and demonstration of their kininogenase activity
a
a
a
a
a
Simone S. Cotrin , Iuri E. Gouvêa , Pollyana M.S. Melo , Piero Bagnaresi , Diego M. Assis ,
b
a
c
d
Mariana S. Araújo , Maria Aparecida Juliano , Marcos L. Gazarini , Philip J. Rosenthal ,
Luiz Juliano , Adriana K. Carmona
Departamento de Biofísica, Universidade Federal de São Paulo, Rua 3 de maio 100, 04044-020 São Paulo, SP, Brazil
Departamento de Bioquímica, Universidade Federal de São Paulo, Rua 3 de maio 100, 04044-020 São Paulo, SP, Brazil
Departamento de Biociências, Universidade Federal de São Paulo, Av. Ana Costa, 95, 11060-001 Santos, São Paulo, Brazil
Department of Medicine, San Francisco General Hospital, University of California, 1001 Potrero Avenue, 94110 San Francisco, CA, USA
a
a,∗
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2012
Received in revised form 20 October 2012
Accepted 16 January 2013
Available online 24 January 2013
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 subsite specificity. Despite high sequence identity and structural similarity
2
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
Keywords:
Plasmodium falciparum
Falcipain
FRET substrates
Kinin-releasing
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 , while FP-3 accommodates
1
ꢀ
well peptides containing Arg in this position. The S subsite of both enzymes demonstrated broad speci-
2
ficity. 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
3
75
393
corresponds to the Met
to Ile
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.
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
(FP) and comprise four peptidases, namely falcipain-1 [5],
falcipain-2 [6], falcipain-2 [7] and falcipain-3 [3]. Compared to
ꢀ
Malaria is a disease caused by Plasmodium parasites and
papain, the prototypical cysteine protease of the family that
encompasses these enzymes, the falcipains display some unique
motifs. These sequences, found within the N-terminal part of the
prodomain, are crucial for protein targeting to the food vacuole
[4]. Also in the prodomain, it was found an inhibitory C-terminal
portion containing two motifs (ERFNIN and GNFD) that are con-
served in the cathepsin L [8]. The C-terminal part of the prodomain
is related to inhibition, and the N-terminal part of the mature
domain is required for the proper refolding of the protease. These
extensions are not found in other proteases of the papain family
remains one of the most prevalent and persistent diseases,
affecting hundreds of millions of people and causing approx-
imately 1–2 million deaths each year [1,2]. Several proteolytic
enzymes have been identified in Plasmodium species and
appear to be involved in important aspects of the para-
site biology. Among them, the papain-like cysteine peptidases
derived from Plasmodium falciparum, the most prolific human
malaria parasite, have
a crucial role in the physiology of
malaria parasites [3,4]. These enzymes are termed falcipains
[
9]. In addition, falcipains possess a unique motif that mediates
haemoglobin binding [8].
Falcipain-1 (FP-1) shares 38–40% sequence identity with
falcipain-2 (FP-2) and falcipain-3 (FP-3) and has been implicated in
erythrocyte invasion by merozoites [10]. Gene disruption studies
∗ Corresponding author. Tel.: +55 11 5576 4455; fax: +55 11 5575 9617.
E-mail addresses: ak.carmona@unifesp.br, adriana@biofis.epm.br
A.K. Carmona).
(
0
166-6851/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molbiopara.2013.01.002

1
12
S.S. Cotrin et al. / Molecular & Biochemical Parasitology 187 (2013) 111–116
suggest that FP-1 is not essential to the erythrocytic stage of
mass and purity of synthesised peptides were checked by
amino acid analysis and MALDI-TOF mass spectrometry using
a TofSpec-E from Micromass (Manchester, UK). Stock solutions
of the peptides were prepared in DMSO, and the concentra-
tions were measured spectrophotometrically using the molar
P. falciparum [5,11]. Gene silencing studies have alluded to a
ꢀ
functional biological role for FP-1 [10,12]. FP-2, FP-2 and FP-3
are food vacuole hemoglobinases, and their joint expression,
in concert with aspartic peptidases, seems to be essential for
efficient haemoglobin digestion [3,7,6]. Although the parasites can
survive when FP-2 expression is knocked down, the silencing of
FP-3 is lethal to P. falciparum [13], demonstrating the importance
of these proteases in the parasite’s life cycle. In the late stages
of its intraerythrocytic development, the parasite expresses an
endogenous cysteine protease inhibitor, falstatin [14].
−
1
−1
at
extinction coefficient of the EDDnp group, 17,300 M cm
365 nm.
2.3. Kinetic measurements
The hydrolysis of the FRET peptides was monitored in a
Hitachi F 2000 spectrofluorometer by continuously measuring the
fluorescence at ꢀex = 320 ± 10 nm and ꢀem = 420 ± 20 nm, and pho-
tomultiplier voltage set to 700 V. The assays were performed at
In the present work, we performed a comparative analysis of the
ꢀ
S − S subsite (according to the Schechter and Berger nomencla-
3
2
ture [15]) specificity of recombinant FP-2 and FP-3 using fluores-
cence resonance energy transfer (FRET) peptides derived from the
lead sequence Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic
acid; EDDnp = N-[2,4-dinitrophenyl] ethylenediamine). This pep-
tide was designed based in the results of a solid-phase substrate
library for cysteine proteases [16,17] and was cleaved only at the
Arg-Ser bond by FP-2 and FP-3. Five series of peptides were gen-
erated and tested: Abz-XLRSSKQ-EDDnp, Abz-KXRSSKQ-EDDnp,
Abz-KLXSSKQ-EDDnp, Abz-KLRXSKQ-EDDnp and Abz-KLRSXKQ-
EDDnp (X = different amino acids). Marked differences were
observed in all positions analysed, suggesting a very distinct speci-
ficity between the two enzymes.
◦
37 C in 100 mM sodium acetate buffer, pH 5.5, containing 2.5 mM
dithioerythritol (DTE). The 10-mm path length quartz cuvette
containing the buffer was placed in the thermostated cell compart-
◦
ment (37 C) of the fluorimeter until temperature equilibrium was
attained. Then, FP-2 or FP-3 were added to the cuvette and left for
5 min to pre-activate the enzymes. The assays were initiated by
the addition of the substrate in concentrations bellow to 1 ␮M, and
the increase in the fluorescence with the time was continuously
recorded in a final volume of 1 ml. The substrate hydrolyzed per
minute was converted into micromoles based on a calibration curve
obtained from the complete hydrolysis of each peptide. The exper-
iments were carried out at low substrate concentration where the
reactions followed first-order kinetics with the rate constant kobs
determined by the non-linear regression data analysis Grafit Ver-
sion 5 program (Erithacus Software Ltd., Horley, UK). The apparent
second-order rate constant kcat/Km was calculated by dividing kobs
by the enzyme concentration.
The recent description that plasmatic kininogen can be hydrol-
ysed by P. falciparum intracellularly generating active peptides [18]
led us to investigate whether kinins could be generated by falci-
pains, suggesting a real participation of these enzymes in malaria
infection. Therefore, we explored the capability of recombinant
FP-2 and FP-3 to release vasoactive peptides (kinins) from a synthe-
3
75
sised fluorogenic fragment spanning the sequence from Met
Ile
to
393
of human kininogen, Abz-MISLMKRPPGFSPFRSSRI-NH . This
2.4. Hydrolysis of the synthetic kininogen-related fluorogenic
peptide
2
peptide containing the bradykinin sequence (RPPGFSPFR) extended
at its C- and N-terminal sides, allowed us to evaluate the kinins
that the recombinant enzymes could release. In addition, we show
by radioimmunoassay that kinins can be generated by recombi-
nant FP-2- and FP-3-mediated proteolysis of high molecular weight
kininogen (HK).
The hydrolysis of the fluorogenic kininogen peptide Abz-
MISLMKRPPGFSPFRSSRI-NH2 by FP-2 and FP-3 was analysed by
liquid chromatography/electrospray ionisation mass spectrometry
(LC/ESI-MS) using a Shimadzu 2010 apparatus (Shimadzu Corpo-
ration, Tokyo, Japan) with an SPD-20A UV/vis detector and an
RF-10AXL fluorescence detector coupled with an Ultrasphere C-
2
. Materials and methods
1
8 column (5 ␮m, 4.6 mm × 250 mm). FP-2 (200 nM) and FP-3
(200 nM) were incubated in 0.1 M sodium acetate, pH 5.5, for 5 h at
2.1. Enzymes
◦
3
7 C, allowing the complete hydrolysis of the substrate (30 ␮M).
The enzymes were pre-activated with 2.5 mM DTE for 5 min at
The enzymes falcipain-2 (FP-2) and falcipain-3 (FP-3) were
expressed in Escherichia coli following a previously described pro-
◦
3
7 C, before the addition of the substrate. A sample of the reaction
mixture was applied in the Ultrasphere C-18 column and eluted
with two solvent systems: (A) trifluoroacetic acid/water (1:1000,
v/v) and (B) trifluoroacetic acid/acetonitrile/water (1:900:100, v/v)
at a flow rate of 1.7 ml/min with a 10–80% gradient over 20 min
after 8 min of isocratic flow. The percentage of the formation of
each fragment was calculated by estimating the peak area of the
generated fragment, taking the totally hydrolysed substrate to be
tocol [3,19]. The recombinant enzymes were stored in 50% glycerol
◦
at −80 C. The molar concentrations of the enzymes were deter-
mined by active site titration with E-64 according to Barrett et al.
[
20].
2
.2. Peptides
1
00%.
FRET peptides containing the fluorescent group ortho-
aminobenzoic acid (Abz) and the fluorescence acceptor
N-[2,4-dinitrophenyl]ethylenediamine (EDDnp) attached to
2.5. Radioimmunoassay determination of kininogenase activity
a
glutamine residue were synthesised by the solid-phase syn-
thesis method as described previously [21]. The fluorogenic
The ability of recombinant FP-2 (200 nM) and FP-3 (200 nM) to
generate kinins from 0.52 ␮g of HK (Enzyme Research Co., USA)
was evaluated incubating the enzymes in 0.1 M sodium acetate, pH
kininogenase fragment Abz-MISLMKRPPGFSPFRSSRI-NH , which
2
◦
is devoid of the quenching effect, was synthesised following
the same methodology. An automated bench-top simultane-
ous multiple solid-phase peptide synthesiser (PSSM 8 system,
Shimadzu, Japan) was used to synthesise the peptides using
the Fmoc-procedure. All peptides were purified by semi-
preparative HPLC on an econosil C-18 column. The molecular
5.5, at 37 C for 1 h in a final volume of 0.08 ml. The enzymes were
◦
pre-activated with 0.5 mM DTE for 5 min, at 37 C, before the addi-
tion of the HK. The assays in the presence of 5.0 ␮M of E-64 were
performed under the same conditions, after 30 min pre-incubation
with the inhibitor. Ethanol (3:1, v/v) was added, and the mixture
was centrifuged at 1000 × g for 15 min. The kinin content in the

S.S. Cotrin et al. / Molecular & Biochemical Parasitology 187 (2013) 111–116
113
followed by Abz-KFRSSKQ-EDDnp (kcat/K = 133 mM 1
−
s
−1
) and
supernatant was measured by radioimmunoassay as previously
described [22].
m
−1
−
1
Abz-KMRSSKQ-EDDnp (kcat/Km = 70 mM
s ).
The subsite S3 of FP-2 shows a broad specificity with a slight
preference for the lead peptide, Abz-KLRSSKQ-EDDnp, which con-
3
. Results
−
1
−1
). In contrast, the
tains Lys in P3 position (kcat/Km = 1850 mM
s
same peptide was hydrolysed by FP-3 with the lowest catalytic
efficiency in the series (kcat/Km = 55 mM
3.1. Substrate specificity studies
−1
−1
s ). Surprisingly, the
peptide Abz-ALRSSRQ-EDDnp, containing the non-polar aliphatic
residue Ala in P3, was hydrolysed by FP-3 with the highest catalytic
The FRET peptides derived from Abz-KLRSSKQ-EDDnp were
ꢀ
used to perform a comparative analysis of the S − S sub-
3
2
−
1
−1
efficiency of all series studied (kcat/Km = 2333 mM
s ).
site specificity of FP-2 and FP-3. Five series of peptides with
general sequences Abz-XLRSSKQ-EDDnp, Abz-KXRSSKQ-EDDnp,
Abz-KLXSSKQ-EDDnp, Abz-KLRXSKQ-EDDnp and Abz-KLRSXKQ-
EDDnp (X = different amino acids) were tested, and the relative
value of the kcat/Km parameters are presented in Fig. 1. Due
to the low catalytic efficiency presented by FP-3 towards some
sequences, the studies with this enzyme were performed with
fewer peptides than that the number used in FP-2 characterisa-
tion. Thus, for FP-3, analogues containing representative residues
of each class of amino acid (hydrophobic, positively and negatively
charged and non-polar) were selected. The substrates assayed were
cleaved by FP-2 and FP-3 at the Arg-Ser bond or at the X-Ser
ꢀ
The S of FP-2 shows clear preference for peptides containing
1
ꢀ
the aromatic hydrophobic residue Phe in P with the peptide Abz-
KLRFSKQ-EDDnp (kcat/Km = 2440 mM
1
−
1
−1
s
) being hydrolysed with
the highest catalytic efficiency of all the series studied. Related to
FP-3, the results show that the enzyme accommodates well the
ꢀ
peptide containing the positively charged residue Arg in P1 with
the peptide Abz-KLRRSKQ-EDDnp being hydrolysed with the high-
−
1
−1
est catalytic efficiency in this series (kcat/Km = 577 mM
s ). It is
ꢀ
of note that a Pro residue is not accepted by the S1 of FP-1 or FP-2.
Thereby, the peptide Abz-KLRPSKQ-EDDnp is cleaved at the Leu-
Arg bond by both enzymes, positioning the Pro residue at P . The
ꢀ
2
ꢀ
ꢀ
S subsite studies of FP-2 and FP-3 indicated a broad specificity, well
and Arg-X when the modifications were introduced at P − P
2
1
1
ꢀ
accommodating the different class of amino acids at P . However,
positions, respectively. The only exception was the peptide Abz-
KLRPSKQ-EDDnp, which was cleaved at the Leu-Arg bond by both
falcipains.
2
this subsite has a marked influence on the FP-3 catalytic activ-
−
1
−1
s )
ity. The peptides Abz-KLRSIKQ-EDDnp (kcat/Km = 1217 mM
and Abz-KLRSVKQ-EDDnp (kcat/Km = 1111 mM
−
1
−1
s ), which con-
The kinetic data show that FP-2 preferentially accommo-
dates at the S1 subsite the positively charged residue Arg
tain the aliphatic hydrophobic residues Val and Ile, respectively, in
ꢀ
−
−
1
1
−1
−1
P , were hydrolysed by FP-3 with approximately a 20-fold higher
(
(
kcat/Km = 1850 mM
kcat/Km = 1042 mM
s
s
) followed by the non-polar residue Gln
) and all other peptides were poorly
2
value of catalytic efficiency than the one presented by the lead
−
1
−1
s ).
peptide, Abz-KLRSSKQ-EDDnp (kcat/Km = 55 mM
hydrolysed. In contrast, the S1 subsite of FP-3 accommodates very
well the hydrophobic aliphatic residue Leu, with the substrate
Abz-KLLSSKQ-EDDnp being hydrolysed with the highest catalytic
3.2. Hydrolysis of kininogen-related peptide and HK by FP-2 and
FP-3
−1
−1
), while the lead
efficiency in the series (kcat/Km = 660 mM
s
peptide-Abz-KLRSSKQ-EDDnp was hydrolysed by FP-3 with the
−
1
−1
s ).
lowest kcat/Km value (55 mM
To verify the kinin-releasing activity of FP-2 and FP-3, a fluoro-
genic fragment spanning the sequence from Met3 to Ile
75
393
of the
The S2 subsite of FP-2 and FP-3 is restricted to hydrophobic
residues following the typical and well-defined characteris-
tic of the cysteine peptidases from the papain superfamily
23]. At the P2 position, FP-2 shows a clear preference for
the hydrophobic aliphatic residue Leu (kcat/Km = 1850 mM
over the aromatic residue Phe (kcat/Km = 333 mM
S2 subsite of FP-3 also preferred the Leu containing peptide
Abz-KLRSSKQ-EDDnp, as this substrate is hydrolysed with the
human kininogen, Abz-MISLMKRPPGFSPFRSSRI-NH , was synthe-
2
sised and tested with the recombinant enzymes. The hydrolysis
this peptide, which is devoid of the quenching effect, was ana-
lysed by LC/ESI-MS, and the products were characterised according
to their calculated molecular mass (Table 1). FP-2 and FP-3
presented a very similar pattern of cleavage, generating Met-Lys-
bradykinin (MKRPPGFSPFR), Lys-bradykinin (KRPPGFSPFR) and
bradykinin (RPPGFSPFR) as shown in Fig. 2. However, both enzymes
[
−
1
−1
s )
−
1
−1
s ). The
−
1
−1
s ),
highest catalytic efficiency in the series (kcat/Km = 174 mM
ꢀ
Fig. 1. Analysis of S3–S2 subsite specificity of recombinant FP-2 and FP-3 using fluorescence resonance energy transfer (FRET) peptides derived from the sequence Abz-
KLRSSKQ-EDDnp. The enzymes were incubated as described in Section 2. The assays were performed in low substrate concentration where the reactions followed first-order
ꢀ
conditions. The x axis shows the different amino acid residues at P3–P2 and the y axis shows the catalytic efficiencies (kcat/Km) values.

1
14
S.S. Cotrin et al. / Molecular & Biochemical Parasitology 187 (2013) 111–116
Table 1
Hydrolysis of the fluorescent-labelled kininogen related peptide Abz-MISLMKRPPGFSPFRSSRI-NH2 by FP-2 and FP-3 and identification of the generated fragments.
Enzyme
Abz-MISL↓M↓K↓RPPGFSPFR↓SSRI-NH2
Molecular weight
Calculated
Observed ion (m/z)
MKRPPGFSPFR (7%)
KRPPGFSPFR (63%)
RPPGFSPFR (30%)
1319.59
1188.39
1060.22
1319.31
1188.29
1060.39
FP-2
FP-3
MKRPPGFSPFR (20%)
KRPPGFSPFR (68%)
RPPGFSPFR (12%)
1319.59
1188.39
1060.22
1319.22
1188.29
1060.49
◦
Hydrolysis conditions: The assays were performed at 37 C in 0.1 M sodium acetate buffer, pH 5.5. The enzymes were preactivated with 2.5 mM DTE for 5 min before the
addition of the substrate. Hydrolysis was followed as described in Section 2 and the generated fragments were identified by LC/ESI-MS mass spectrometry. ↓ cleavage sites.
preferentially released Lys-bradykinin, as indicated by the relative
percentage of the generated kinins (Table 1).
The capability of FP-2 and FP-3 to release kinins from HK was
also tested and quantified by radioimmunoassay (Table 2). We
could detect that FP-3 presents a 8-fold higher catalytic activ-
ity on HK compared to FP-2. The irreversible cysteine proteinase
inhibitor E-64 blocked the activity of both enzymes, demonstrat-
ing the specificity of the assay. Control of kininogenase activity with
human plasma kallikrein was performed in the same experimen-
tal conditions, except for the buffer (0.05 M Tris–HCl, pH 8.0). The
amount of released kinins by FP-2 and FP-3 represented 5% and 40%
of the kallikrein activity, respectively (data not shown).
Fig. 2. Mass spectrometry analyses of Abz-MISLMKRPPGFSPFRSSRI-NH2 (A) hydrolysis by FP-2 (B) and FP-3 (C) and the identification of the generated kinins. Assay conditions
are described in Section 2.

S.S. Cotrin et al. / Molecular & Biochemical Parasitology 187 (2013) 111–116
115
Table 2
did not detect the hydrolysis of Boc-LL-MCA by FP-3. The expla-
nation for this discrepancy may be the fact that depending on the
peptide sequence and interaction of the side chains of amino acid
residues with subsites S and S , variations can be observed in the
susceptibility to hydrolysis.
FP-2 and FP-3 kininogenase activity on HK measured by radioimmunoassay.
Sample
Kinin released (pg)
ꢀ
HK
FP-2
7
6
FP-3
6
In relation to the S3 subsite, we highlight the major influence of
this subsite on FP-3 activity, as the peptide Abz-ALRSSKQ-EDDnp
showed the highest value of catalytic efficiency of all series studied
HK + FP-2
HK + FP-3
HK + FP-2 + E-64
HK + FP-3 + E-64
240
1965
6.5
2.7
−
1
−1
(kcat/Km = 2333 mM
s ). This result may be explained by the nar-
rowing of the S2 subsite of FP-3 observed by Kerr et al. [30]. Thus, a
peptide containing a small residue at P3, such as Ala, may be better
accommodated at the active site of the enzyme than the peptides
containing residues with long side chains.
Assay conditions: FP-2 (200 nM) and FP-3 (200 nM) were incubated with 0.52 ␮g
of HK in 0.1 M sodium acetate, pH 5.5 for 1 h, at 37 C, in a final volume of 0.08 ml.
The assays in the presence of 5.0 ␮M of E-64 were performed under the same con-
ditions, after 10 min of pre-incubation. Experimental conditions were as describe
under Section 2.
◦
ꢀ
Our studies indicated that the S subsites have a notable influ-
ence on the specificity of FP-2 and FP-3. The presence of a Phe
ꢀ
residue in position P1 of the Abz-KLRFSKQ-EDDnp made the
4
. Discussion
peptide very suitable to hydrolysis by FP-2, being the substrate
cleaved with the highest catalytic efficiency of all the series tested
−
1
−1
). It is noteworthy that FP-2 hydrolyses
Despite the great efforts made by the scientific community,
(kcat/Km = 2440 mM
the substrate Z-LR-MCA with kcat/Km of 103 mM
3 with kcat/Km value of 0.590 mM
s
−
1
−1
s
malaria remains one of the most prevalent and persistent diseases
that affect people worldwide, with hundreds of millions of people
infected [24]. The disease is caused by Plasmodium parasites that
use several processes to complete the different stages of their life
cycle, and a series of peptidases are essential to the achievement of
one or more of these phases.
Proteins and peptides can reach and be processed by P. falcip-
arum proteases inside erythrocytes [18,25]. Among them, the P.
falciparum cysteine peptidases, especially falcipain-2 and falcipain-
[8] and FP-
−1
s [3], which confirmed the
−
1
favourable effect of the interaction of a more relaxed substrate
ꢀ
with the enzyme S and S subsites occupied. Thus, our compara-
tive analysis of the specificity FP-3 and FP-2 showed differences at
all analysed positions, demonstrating that the two enzymes have
very different substrate specificities. Whereas haemoglobin is the
natural substrate for both, the two enzymes cleave the protein at
different sites, generating fragments equally important for the par-
asite [4]. The results presented here may be useful for developing
selective inhibitors for each of the enzymes studied and for drug
development.
3
, stand out because they are involved in the hydrolysis of
haemoglobin and erythrocyte rupture [26]. In the present study,
we used FRET peptides to explore the S − S subsite specificity
ꢀ
3
2
requirements of recombinant FP-2 and FP-3. For this purpose,
we synthesised and tested analogues of the lead sequence Abz-
KLRSSKQ-EDDnp. Our results show some differences from those
obtained in previous studies with a library with general sequence
Z-peptidyl-MCA [27], making clear that the subsites are not
independent and are influenced by residues occupying “primed”
subsites. This is an advantage of our methodology because when a
The capability of FP-2 and FP-3 to generate active kinins was
also tested to verify the participation of these enzymes in infec-
tion caused by malaria. Kininogenase activity of some cysteine
peptidases has been described for enzymes from Porphyromonas
gengivalis [31], Streptococcus pyogenes [32], Trypanosoma cruzi [33]
and Fasciola hepatica [34]. In the late 1960s, some studies suggested
that kinins could be involved in the pathological manifestations
of malaria [35]. Recently, Bagnaresi et al. described the uptake
of kininogen by malarial parasites, followed by the generation of
kinins, which are products of the parasite’s proteolytic activity [18].
These generated peptides can elicit a calcium response in HUVEC
cells in culture, suggesting that these fragments have biological
activity and could be involved in the disease progression. Here, we
demonstrated that FP-2 and FP-3 are able to hydrolyse the fluo-
ꢀ
fixed residue occupies the position P , such as the MCA group in
1
ꢀ
the compound Z-peptidyl-MCA, it is not possible to analyse the S
subsites.
As expected for a cysteine peptidase [23], the highest selectivity
ofFP-2 and FP-3 is givenby the S subsite, whichclearlypreferspep-
2
tides containing aliphatic or aromatic hydrophobic residues at P2.
Ramjee et al. [27], using Z-peptidyl-MCA substrates, observed that
the S2 subsite of FP-2 preferentially accepts Leu and Phe residues,
while FP-3 hydrolyses only peptides containing Leu or Val in posi-
rogenic peptide Abz-MISLMKRRPGFSPFRSSRI-NH , which contains
2
the kinins sequence in kininogen. Lys-BK was the predominant
kinin generated by both enzymes, followed by BK and Met-Lys-
BK. Interestingly, the profile of the fragments generated from
tion P . Our studies indicated that Leu is the preferred residue by the
2
S2 subsite of FP-2 and FP-3; however, the latter also well accepts
well Phe and Met residues at the P2 position. Indeed, the struc-
the processing of the peptide Abz-MISLMKRRPGFSPFRSSRI-NH by
2
tural data showed the preference of a Leu at P , as this hydrophobic
recombinant falcipains differs from that reported for parasites by
Bagnaresi et al. [18]. These authors have shown the formation of
des-Arg -BK, but not Met-Lys-BK, after cell parasites incubation
2
aliphatic residue fits comfortably into the narrow S subsite of both
2
9
enzymes, thus allowing it to be involved in a number of favourable
interactions [28–30].
with these kininogen-related fluorogenic peptide, demonstrating
that besides FP-2 and FP-3 other proteases and cellular events
are also involved in kininogen processing during malaria devel-
opment, as already described for haemoglobin [37]. Enzymes such
as Plasmodium aminopeptidases, which X-ray structure and sub-
strate specificity was recently characterised [36–39], are probably
involved in kinin formation. In addition to aminopeptidases, P. fal-
ciparum carboxypeptidases may be involved in the processing of
With respect to the S1 subsite, we observed that FP-2 preferred
the peptides containing Arg and Gln at the P1 position, as Abz-
KLRSSKQ-EDDnp and Abz-KLQSSKQ-EDDnp were hydrolysed with
the highest values of kcat/Km of the series. In contrast, the compound
Abz-KLQSSKQ-EDDnp was one of the worst substrates in the series
for FP-3. This enzyme preferentially hydrolysed the peptide Abz-
KLLSSKQ-EDDnp, which contains the hydrophobic residue Leu at
P1. Reinforcing our findings, the crystal structure of FP-3 complexed
with vinyl sulphones showed that the nonpolar environment of the
S2 and S1 pockets favour the interaction with the hydrophobic P1
and P2 residues of the inhibitor, which allow favourable contacts
9
the generated kinins, thereby justifying the formation of des-Arg -
BK; however, these enzymes had not yet been described.
The hydrolysis of HK by FP-2 and FP-3 was also analysed
and quantified by radioimmunoassay, and the kininogenase activ-
ity presented by both enzymes towards the protein corroborates
[
30]. Ramjee et al. [27] obtained results similar to ours for FP-2 but

1
16
S.S. Cotrin et al. / Molecular & Biochemical Parasitology 187 (2013) 111–116
the results obtained with the mimetic-HK peptide. Our results
demonstrated that the well-known inhibitory effects of the two
cystatin-like domains of kininogen on falcipains [7] are not suffi-
cient to block FP-2 and FP-3 activity but can be responsible for the
high enzyme concentrations required to yield a sufficient amount
of kinins for detection, particularly in a very sensitive method
such as an radioimmunoassay. Bagnaresi et al. [18] verified that
HK fragments resulting from FP-2 and FP-3 cleavage were bio-
logically active and were able to promote the contraction of the
isolated guinea pig ileum, which was prevented by the B2 recep-
tor antagonist HOE-140. When HK was incubated with FP-2 and
FP-3 in the presence of the specific cysteine protease inhibitor E-
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6
4, the contraction response was ablated. The answer obtained in
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understanding of the role of these enzymes in parasite biology.
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This work was supported by the Brazilian Agencies Funda c¸ ão de
Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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