Báo cáo khoa học: The propeptide of cruzipain ) a potent selective inhibitor of the trypanosomal enzymes cruzipain and brucipain, and of the human enzyme cathepsin F ppt

The propeptide of cruzipain
)
a potent selective inhibitor
of the trypanosomal enzymes cruzipain and brucipain, and
of the human enzyme cathepsin F
5
Flavia C. G. Reis
1
, Tatiana F. R. Costa
1
, Traian Sulea
2
, Alessandra Mezzetti
2
, Julio Scharfstein
1
,
Dieter Bro
¨
mme
3
, Robert Me
´
nard
2
and Ana Paula C. A. Lima
1
1 Instituto de Biofı
´
sica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, RJ, Brazil
2 Biotechnology Research Institute, National Research Council of Canada, Montreal, Canada

3 Department of Oral and Biological Sciences, University of British Columbia, Vancouver, Canada
Pathogenic trypanosomes are the cause of major para-
sitic diseases, which pose a threat to public health in
many developing countries [1]. Trypanosoma cruzi is
the etiologic agent of Chagas’ disease, a chronic illness
endemic in Central and South America, associated
with heart failure and⁄ or denervation of the digestive
system. Trypanosoma brucei causes sleeping sickness, a
widespread disease in sub-Saharan Africa that is char-
acterized by neurologic degeneration, psychiatric dis-
orders, and inevitable death if left untreated. Present
chemotherapy relies on compounds presenting low
efficacy and ⁄ or high toxicity, and there is an urgent
need for the development of alternative drugs.
In many pathogenic parasites, the activity of papain-
like cysteine proteases (CPs) seems to be crucial for
growth, development and tissue ⁄ host cell penetration
[2]. The use of synthetic irreversible CP inhibitors in
animals experimentally infected with T. cruzi [3] or
with T. brucei [4] has significantly reduced parasitemia
and mortality, validating these enzymes as promising
targets for the development of new drugs. The main
targets of these compounds are the well-characterized
major CPs of T. cruzi, cruzipain (or cruzain) [5–7], and
the CP of T. brucei, brucipain (or rhodesain) [8,9]. The
resolution of the X-ray structure of cruzain in com-
plex with diazomethane peptidyl inhibitors provided
the basis for structure-based inhibitor design [10].
Although several studies have explored the inhibition
of cruzipain by peptidyl epoxy ketones [11], aldehydes

[12], vinyl sulfones [13], thiosemicarbazones [14] and
alpha-keto-based compounds [15], there are still ongo-
ing efforts to find new mechanism-based small-molecule
Keywords
brucipain; cathepsin F; cruzipain; inhibition;
propeptide
Correspondence
A. P. C. A. Lima
3
, Instituto de Biofı
´
sica
Carlos Chagas Filho, Bloco G, Centro de
Cie
ˆ
ncias da Sau
´
de, Universidade Federal do
Rio de Janeiro, Cidade Universita
´
ria,
21949-900, Rio de Janeiro, RJ, Brazil
Fax: +55 21 2280 8193
Tel: +55 21 2209 6591
E-mail:
(Received 25 September 2006, revised 30
November 2006, accepted 22 December
2006)
doi:10.1111/j.1742-4658.2007.05666.x
Papain-like cysteine proteases of pathogenic protozoa play important roles

in parasite growth, differentiation and host cell invasion. The main cysteine
proteases of Trypanosoma cruzi (cruzipain) and of Trypanosoma brucei
(brucipain) are validated targets for the development of new chemothera-
pies. These proteases are synthesized as precursors and activated upon
removal of the N-terminal prodomain. Here we report potent and selective
inhibition of cruzipain and brucipain by the recombinant full-length prodo-
main of cruzipain. The propeptide did not inhibit human cathepsins S, K
or B or papain at the tested concentrations, and moderately inhibited
human cathepsin V. Human cathepsin F was very efficiently inhibited (K
i
of 32 pm), an interesting finding indicating that cruzipain propeptide is able
to discriminate cathepsin F from other cathepsin L-like enzymes. Compar-
ative structural modeling and analysis identified the interaction between the
b1p–a3p loop of the propeptide and the propeptide-binding loop of mature
enzymes as a plausible cause of the observed inhibitory selectivity.
Abbreviations
CP, cysteine protease
4
; PBL, proregion-binding loop; PCZ, recombinant propeptide of cruzipain.
1224 FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS
inhibitors and structural information that could lead
to the development of efficient and specific inhibitors
for both cruzipain and brucipain.
CPs are synthesized as inactive precursors that pos-
sess a signal peptide, a prodomain, and a mature
domain that retains catalytic activity. In trypanosomal
CPs, the mature enzyme is composed of a central
domain that corresponds to the mature domain of
mammalian CPs and an additional 130-residue C-ter-
minal extension with unknown function [2]. The C-ter-

minal extension is not essential for enzyme activity.
Activation of papain-like CPs is achieved by proteo-
lytic excision of the prodomain, an event believed to
occur in vivo by a multistep process that may involve
multiple endosomal ⁄ lysosomal peptidases or even pro-
teases present in the extracellular environment [16,17].
Diverse functions have been attributed to the prodo-
main present in CP precursors (proenzyme): (a) inhibi-
tion of enzyme activity through its interaction with the
active site; (b) folding assistance [18]; and (c) targeting
of the precursors to the endosomal–lysosomal system
[19]. Determination of the three-dimensional structure
of zymogens revealed that enzyme inhibition by the
propeptide is accomplished by its binding to the
enzyme’s active site with the backbone in the reverse
direction, thus protecting it from hydrolysis [20]. The
discovery that the propeptides of cathepsins B [21], L
[22], K and S [23–25] are potent inhibitors that display
a certain degree of selectivity for their parent enzymes
has paved the way for the design of new selective
inhibitory compounds. Indeed, this approach was suc-
cessful in the production of short peptidyl noncovalent
inhibitors based on the mode of inhibition of cathepsin
L by its prodomain. These synthetic inhibitors present
300-fold selectivity for cathepsin L over cathepsin K,
whereas the full-length propeptide of cathepsin L pre-
sented only two-fold selectivity for the parental enzyme
[26].
Although CPs are widely expressed in parasitic
organisms, very little information has been reported

on the inhibitory properties of their prodomains. The
propeptide of the main cathepsin L-like CP from the
worm Fasciola hepatica was described as highly select-
ive for the parasite’s enzymes, being practically inca-
pable of inactivating mammalian cathepsins L, K and
B and papain [27]. In another study, the inhibitory
potential of 23 overlapping synthetic peptides span-
ning the propeptide of a CP from the cattle parasite
Trypanosoma congolense
6
(congopain) has also been
described [28]. This study identified a few peptides
presenting a certain degree of selectivity for the inhi-
bition of congopain and cruzipain relative to cathep-
sins B and L [28].
In this study, the propeptide of cruzipain was pro-
duced and tested as an inhibitor of trypanosomal CPs.
Selectivity with regard to human lysosomal CPs was
also investigated. We show that the recombinant pro-
peptide of cruzipain is a potent inhibitor of the mature
enzyme, as well as brucipain, and further demonstrate
that the propeptide of cruzipain selectively inactivates
human cathepsin F with high potency. The molecular
basis for selective inhibition and the implications of
these findings are discussed.
Results and Discussion
Expression and purification of the propeptide
of cruzipain
The predicted full-length propeptide of cruzipain
(Cys19–Gly122) was cloned by PCR and expressed in

Escherichia coli as a fusion protein with a 6xHis tag at
the N-terminus. This type of N-terminal tag was previ-
ously used for the expression of the propeptide of
human cathepsin K, and it did not interfere with the
inhibitory activity of the protein [24]. Although some of
the recombinant propeptide of cruzipain (PCZ)
7
was pro-
duced in its soluble form, a great amount was found in
inclusion bodies. In order to maximize production and
to optimize purification, the fusion protein was dena-
tured in a buffer containing urea, and was subsequently
refolded and used in the inhibition assays. The purity of
refolded PCZ was checked by SDS ⁄ PAGE (Fig. 1A),
and its chemical integrity was verified by MS. Analysis
by MALDI-TOF MS showed a major single peak with a
molecular mass of 14 019 Da, and a minor peak with a
molecular mass of 14 161 Da (Fig. 1B). The major peak
corresponded to the full-length construct containing:
(a) residues MRGS (introduced by the plasmid multiple
cloning site immediately before the His-tag); (b) the six
histidines of the tag; (c) the full-length propeptide of cru-
zipain (Cys19–Gly122); and (d) residues RGVDLQPS-
LIS at the C-terminus, which resulted from the fusion of
the propeptide with a small stretch of coding sequence
within the multiple cloning site of the pQE30 plasmid
before the stop codon. The predicted molecular mass of
the fusion protein is 14 075 Da (14 kDa), which is
within 1% error of the mass corresponding to the major
peak. This indicates that the great majority of the puri-

fied propeptide was homogeneous and had no detectable
truncations.
Inhibitory activity of the propeptide
Next, we tested the inhibitory properties of PCZ
towards the parent enzyme, cruzipain, purified from
1
F. C. G. Reis et al.
2
Cathepsin F inhibition by propeptide of cruzipain
FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS 1225
T. cruzi epimastigotes. We observed time-dependent
inhibition of cruzipain (Fig. 2A) by PCZ, as noted pre-
viously for the inhibition of mammalian CPs by their
propeptides, which follows slow-binding kinetics, in the
pH 5–6 range [21–25]. The linear portions of the pro-
gress curves where steady state was reached were used to
calculate the rate constant of substrate hydrolysis in the
presence of the inhibitor (v
i
) and to determine K
i
. The
Dixon plots (1 ⁄ v versus [I]) [29] resulted in intersecting
lines, showing competitive inhibition (Fig. 2B). At
pH 6.5, PCZ inactivated cruzipain competitively, with
an inhibition constant (K
i
) of 0.018 nm. Remarkably,
this was the lowest K
i

value described so far for the inhi-
bition of a papain-like CP by its cognate propeptide.
The propeptide was also found to be a very good inhib-
itor of brucipain, with a K
i
value of 0.0163 nm at
pH 6.5. This finding is not surprising, as cruzipain and
brucipain share a high degree of sequence similarity,
both in the mature part and in their propeptides. Along
these lines, a previous study using 15-mer synthetic
14.3
20
26.6
1 2 MW KDa3
Mass (m/z)
% Intensity
0
878.2
0
12400
16600 20800 25000
10
20
30
40
50
60
70
80
90

100
14015.00
14063.56
A
B
Fig. 1. Expression of the full-length propeptide of cruzipain.
(A) A DNA fragment encoding the full-length propeptide of cruzipain
(Cys19–Gly122) was cloned in fusion with a polyhistidine tag in
pQE30 and expressed in E. coli. Denatured fusion protein (PCZ)
was purified in an Ni–nitrilotriacetic acid column and subsequently
renaturated, filtered and dialyzed. SDS ⁄ PAGE of the purification of
PCZ: bacterial homogenate after induction (lane 1), denaturated
PCZ eluted from Ni–nitrilotriacetic acid (lane 2), PCZ after renatura-
tion and dialysis (lane 3). (B) MS analysis by MALDI-TOF MS.
Approximately 5 nmol of the purified propeptide was precipitated
with trichloroacetic acid and analyzed by MS. The measured mass
for His-tagged PCZ was 14 019.01 Da, which is within 56 Da of the
average calculated mass.
0 200
400 600 800
1000
0
200
400
600
Control
0.02 n
M
0.03 nM
0.06 nM

Fluorescence units
seconds
1.5 µ
M
3.0 µM
[I] nM
1/v (M
–11
.s
–1
)
0.10.080.060.040.020–0.02
1
0.8
0.6
0.4
0.2
A
B
Fig. 2. Inhibition of cruzipain by its propeptide. (A) Progress curves
for the inhibition of cruzipain by its full-length recombinant propep-
tide. The figure shows the generation of product with time in the
absence or in the presence of PCZ at the concentrations indicated
for each curve. (B) Plots of 1 ⁄ v versus [I] showing competitive inhi-
bition. The substrate concentrations used in the kinetic measure-
ments are indicated in the graph.
2
Cathepsin F inhibition by propeptide of cruzipain
1
F. C. G. Reis et al.

1226 FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS
peptides based on the prodomain of the CP congopain
from the cattle trypanosome T. congolense showed
cross-inhibition of congopain and cruzipain [28]. How-
ever, the K
i
values of the inhibitory peptides were in the
micromolar range.
The potency of PCZ as an inhibitor of cruzipain
and brucipain decreased moderately at the more acidic
pH value of 5 (Table 1), but remained in the subnano-
molar range. The very low K
i
values at pH values ran-
ging from 5 to 7 suggest that if full-length propeptide
was generated upon procruzipain maturation, it would
probably inhibit the mature enzyme very efficiently.
Notably, it was suggested that in T. cruzi, the autocat-
alytic processing of cruzipain zymogens occurs at the
Golgi complex [30], a compartment that usually pre-
sents only a mildly acidic environment. In this context,
it is possible that the liberated full-length propeptide
exerts a crucial regulatory function in keeping cruzi-
pain inactive until it reaches lysosome-like organelles
(reservosomes). The propeptide of cruzipain is a potent
inhibitor of the cognate enzyme, and it could be expec-
ted that PCZ would likewise efficiently inactivate the
CPs from other trypanosomatids that share high
sequence similarity with cruzipain. Indeed, a prelimin-
ary test using the homogenates of trypanosomatids

such as T. rangeli, T. brucei and Leishmania donovani
revealed that PCZ inhibited approximately 90% of the
overall peptidase activity detected in L. donovani and
50–70% of the activity detected in the homogenates
from the other protozoa (Fig. 3). These results suggest
that PCZ would be a good candidate as a lead for the
design of potent peptidyl inhibitors aimed at the inacti-
vation of CPs from several pathogenic protozoa.
The propeptide of cruzipain is a potent inhibitor
of cathepsin F
To evaluate the selectivity of PCZ as an inhibitor of
papain-like enzymes, the propeptide was incubated for
30 min with papain or human cathepsins B, L, S, K, V
and F (in the subnanomolar range), and the residual
activity of the peptidases was measured using a syn-
thetic fluorogenic substrate. This analysis revealed rel-
atively weak inhibition of cathepsins L and V, whereas
neither papain nor cathepsins B, S and K were inacti-
vated by PCZ when incubated with up to 25 nm of
the propeptide (Table 2). We observed degradation of
PCZ by cathepsins S, L and V, but not by cathepsins
B or K after 60 min of incubation (data not shown).
During the invasion of mammalian cells by T. cruzi,
the host cell lysosomes are recruited to the point of
contact between host cell and parasites, and this is
followed by the delivery of the lysosomal content to
Table 1. Dissociation constants for the inhibition of cruzipain by its
propeptide. Cruzipain purified from T. cruzi epimastigotes was
assayed for inhibition by the propeptide as described in Experimen-
tal procedures. The steady-state velocities were calculated by linear

regression, and the results were used to plot 1 ⁄ v versus [I] to cal-
culate the K
i
values [36].
pH K
i
(nM)
5.0 0.2637 ± 0.0001
5.5 0.0565 ± 0.0005
6.0 0.027 ± 0.007
6.5 0.018 ± 0.002
7.0 0.034 ± 0.007
0,1
1
10
100
T. rangeli
T. brucei
L. donovani
T. cruzi
V
o
x 10
–11
(M/S)
Fig. 3. The propeptide of cruzipain inactivates endogenous CPs of
trypanosomatids. Parasites were cultivated as described in Experi-
mental procedures, and lysates were incubated with control buffer
(clear bars), with 5 n
M propeptide (gray bars), or with 30 lM E-64

(dark bars). The peptidase activity was measured by addition
of 10 l
M Z-Phe-Arg-MCA and monitored continuously. The graphs
represent initial velocities calculated by linear regression of the
progress curves.
Table 2. Inhibition of papain-like enzymes by the propeptide of cru-
zipain. The enzymes were assayed for inhibition by the propeptide
of cruzipain as described in Experimental procedures. The steady-
state velocities were calculated by linear regression and the results
were used to plot 1 ⁄ v versus [I] to calculate the K
i
values [29]. ND,
not detected.
Enzyme K
i
(nM) at pH 6.5
19
Cruzipain 0.018 ± 0.002
Brucipain 0.0163 ± 0.0001
Cathepsin B ND
Cathepsin F 0.032 ± 0.003
Cathepsin K ND
Cathepsin L
a
2.05 ± 0.25
Cathepsin S ND
Cathepsin V 5.18 ± 0.54
a
K
i

determination was performed at pH 5, due to enzyme instabil-
ity at higher pH values. Degradation of PCZ was observed when it
was incubated with cathepsins S, K, L and V at [E] > 500 n
M for
over 60 min.
1
F. C. G. Reis et al.
2
Cathepsin F inhibition by propeptide of cruzipain
FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS 1227
the extracellular environment [31]. In view of our
observations, if cathepsins S or L are present at high
concentrations at the parasite–host cell point of con-
tact, they could mediate degradation of the propeptide
present therein.
Interestingly, significant inhibition of cathepsin F by
PCZ was observed. The inactivation constants for inhi-
bition of cathepsin F were subsequently determined
(Table 2), and showed that PCZ exhibits very high
potency towards cathepsin F, and is 150-fold less
effective in inhibiting cathepsin V. Interestingly, the K
i
for cathepsin F was in the same range as that observed
with the cognate enzyme (cruzipain) and with bruci-
pain, although mature cathepsin F shares considerably
less sequence identity with mature cruzipain (49%)
than does mature brucipain (71%). The activity of
cathepsin L was minimally inhibited at the maximal
concentration of PCZ used (25 nm). The K
i

could not
be determined at pH 6.5, because cathepsin L inacti-
vates over time at this pH. However, when determined
at pH 5.0, the K
i
for inhibition of cathepsin L by PCZ
was in the same range as observed for the inactivation
of cathepsin V. This was expected, as cathepsin L is
very similar to cathepsin V, and its sequence identity
to cruzipain (43%) is also comparable to that of cath-
epsin V (41%).
In order to be able to inhibit endosomal ⁄ lysosomal
CPs in living cells, PCZ would have to be, at least to
some extent, resistant to degradation by peptidases
that are present in these acidic compartments. We
determined whether PCZ would be able to inhibit CPs
present in the pool of endogenous peptidases of mam-
malian cell homogenates. The tissue distribution of
cathepsin L is quite broad, whereas cathepsins F and
V present a much more restricted expression pattern.
It has been reported that both cathepsin V and cathep-
sin F are expressed in human monocyte-derived
macrophages [32,33] and that the latter is also
expressed in smooth muscle cells. Therefore, we tested
the inactivation of endogenous CPs of human cells
with diverse origins by PCZ (Fig. 4). The incubation
of whole cell homogenates with PCZ led to a 20–30%
drop in the total peptidase activity of mono-
cytes ⁄ macrophages (U937 cell line), smooth muscle
cells and Hela cells, whereas it reduced the peptidase

activity of prostate epithelial cells (DU145) by 4%.
Substrate hydrolysis by the cell homogenates was fully
inhibited by E-64 (data not shown), confirming that
we were measuring mainly the activity of papain-like
CP
8
s in our assays.
Cathepsin F is a key enzyme responsible for the deg-
radation of the invariant chain (Ii) in macrophages,
playing a fundamental role in antigen presentation
[34]. Cathepsin F also modifies low-density lipoprotein
particles in vitro, is present in human atherosclerotic
lesions [35], and partially degrades lipid free apoA-1
and high-density lipoprotein (HDL3) in macrophages,
thus blocking cholesterol efflux from these cells [32]. It
would be interesting to know whether T. cruzi can
secrete the cruzipain propeptide in its intact form. If
so, it is possible that antigen-presenting cells such as
macrophages could internalize the propeptide, which,
once in the endosomal ⁄ lysosomal compartments,
would be able to inhibit cathepsin F. As cathepsin F
participates in the degradation of Ii, indirectly modula-
ting the levels of class II MHC molecules available for
binding of antigenic peptides, one can speculate that
inhibition of this peptidase by the propeptide could
affect antigen presentation by such cells.
Comparative structural analysis of papain-like
CPs
Given the remarkable selectivity of PCZ as an inhib-
itor of cathepsin F and of the two trypanosomal CPs

tested, we set out to investigate the possible molecular
basis of selectivity. The sequence alignment of the
propeptide domains of several papain-like enzymes
(Fig. 5A) revealed that the prodomains of cruzipain
and of brucipain are highly similar. The high sequence
identity observed between the prodomains of the
trypanosomal enzymes (60%) could account for the
% inhibition of peptidase activity
Prostate
epithelium
Hela Smooth
muscle
Monocytes/
macrophages
0
5
10
15
20
25
30
Fig. 4. The propeptide of cruzipain inactivates endogenous CPs of
mammalian cells. Human cell lines were cultivated as described in
Experimental procedures, and cell lysates (10 lgÆmL
)1
) were incu-
bated with 5 n
M propeptide or with control buffer, for 15 min at
room temperature. The residual peptidase activity was measured
by the hydrolysis of 5 l

M Z-Phe-Arg-MCA. The peptidase activity of
all lysates was fully inhibited by 10 l
M E-64 (not shown). The initial
velocities were calculated by linear regression of the substrate
hydrolysis curves, and the graph represents the percentage of inhi-
bition, considering the peptidase activity of untreated lysates as
100%. The graph is representative of three independent experi-
ments.
2
Cathepsin F inhibition by propeptide of cruzipain
1
F. C. G. Reis et al.
1228 FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS
potent cross-inhibition of brucipain by PCZ. The pro-
domain of cruzipain shares the highest identity with
the C-terminal portion (residues Pro147–Leu251) of
the prodomain of cathepsin F (32%) and that of cath-
epsin W (33%) among the mammalian CPs, albeit at a
significantly lower level than with brucipain. The PCZ
similarity to the prodomains of human capthepsins L,
V, S and K is significantly lower (18–25%), showing
that sequence conservation qualitatively correlates to a
certain degree with the overall inhibition pattern
shown in Table 2. PCZ is significantly different from
the prodomain of cathepsin B (not shown) and is not
expected to inhibit this carboxypeptidase, due to the
occluding loop in the primed side of the binding site
groove. Although the prodomains of cathepsins F and
W share the same degree of similarity to the prodo-
main of cruzipain, the similarity of the mature domain

of cathepsin F to that of cruzipain is higher (48%)
than that between the mature domains of cruzipain
and cathepsin W (32%).
In order to understand the structural basis for the
PCZ inhibitory selectivity among endopeptidases, we
modeled the three-dimensional structure of cruzipain
propeptide in complex with the crystal structure of
mature cruzipain. The modeled PCZ–cruzipain com-
plex was then compared with the experimental struc-
tural data available for the other enzymes, particularly
surveying the residues and structural features at the
PCZ–cruzipain contact interface that are conserved in
brucipain and cathepsin F but not in cathepsins V, L,
S and K. The most significant difference is located in
the loop connecting strand b1p to helix a3p of the
proregion, and occurs concertedly with a structural
change in the interacting portion of the mature enzyme
known as the proregion-binding loop (PBL). As seen
in Fig. 5A, the b1p–a3p loop of the cruzipain prore-
gion, which is two residues shorter than those of the
cathepsin L, K, V or S proregions (see also Fig. 5A),
would result in a steric conflict with the PBL side
chain Phe145 of mature cathepsin L, as well as with
the corresponding side chains at this position in
mature cathepsins K (Phe144), V (Phe146) and S
(Leu147). The interfering side chain appears to be con-
formationally restricted by the PBL main chain in all
these enzymes. The collision with PCZ is, however, cir-
cumvented in the case of mature cruzipain and cathep-
sin F, due to a two-residue deletion in the PBL

sequence, which changes the main chain local confor-
mation relative to the other cathepsins, and in turn
moves the corresponding interfering side chain (the
smaller Thr147 of cruzipain, and Phe141 of cathep-
sin F) out of the way. On the basis of homology-based
sequence alignments of mature enzymes, the same
would hold true for PCZ binding to brucipain and
cathepsin W, which are, however, omitted from
Fig. 5B, due to the unavailability of experimental
structures for these enzymes. It remains to be tested
whether PCZ is indeed a potent inhibitor of cathep-
sin W, as predicted by this analysis. However, active
cathepsin W has not been successfully expressed to
date.
Conversely, the two-residue insertion Asn55p-(Ala ⁄
His)56p in the proregion loop b1p–a3p of cathepsins
L, K, V and S relative to the cruzipain, brucipain and
cathepsin F and W proregions (Fig. 5A), allows good
steric complementarity with the PBL side chain at
position 145 (cathepsin L numbering) of the corres-
ponding mature enzymes (Fig. 5B), in addition to a
hydrogen bond observed between the conserved
Asn55p side chain and the PBL main chain at position
144 (not shown). The predicted structural determinant
of PCZ specificity for cathepsin F can be verified
experimentally by mutagenesis in several ways, either
on the b1–a3 loops of propeptides or in the interacting
PBL segments of the mature enzymes. For example, a
recombinant PCZ variant with a two-residue-longer
b1–a3 loop, as in the prosegments of cathepsins L, K,

V and S, should exhibit a tendency to level off or even
reverse the cathepsin inhibition pattern of the wild-
type recombinant PCZ.
Other structural features may be invoked to fur-
ther explain the observed inhibitory selectivity of
cruzipain propeptide against cathepsins V, L, K and
S (Table 2). In general, it is expected that the fine
modulation of binding affinity and specificity
between mature enzymes and the  100-residue pro-
peptides results from the cumulative effect of small
contributions scattered along the extensive binding
interface. For example, the shorter b1p–a3p loop of
PCZ appears to force a small displacement of its
helix a3p and to allow the accommodation of an
additional residue (His81p) at the end of this helix,
close to the active site, in comparison to the struc-
tures of procathepsins L and K. These more subtle
structural changes may have a differential impact on
the affinity of binding to mature cathepsins V, L, K
and S. A movement of helix a3p perpendicular to its
axis has been documented between the proregions of
cathepsins L and K, and is speculated to affect the
proregion inhibitory selectivity [36]. Also, residues
Asn82p and Gly83p from the putative active site-
spanning segment YHNGAA of PCZ are conserved
in the proregions of cathepsins L and K, where they
bind in the reverse substrate-binding mode into the
S
1
¢ and S

1
subsites, respectively [20]. Their flanking
residues, however, are more conserved in brucipain
1
F. C. G. Reis et al.
2
Cathepsin F inhibition by propeptide of cruzipain
FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS 1229
and cathepsin F than in the cathepsin V, L, K and
S proregions, and thus they may represent another
source of selectivity among these enzymes, also
depending on the physicochemical properties of their
substrate-binding site groove. Given that CP prore-
gions adopt folded structures upon binding to the
mature enzymes, we cannot exclude that even the
PCZ regions located remotely from the interface and
active site could be implicated in the fine-tuning of
inhibitory selectivity.
The selectivity of PCZ for cathepsin F over other
mammalian cathepsins is a remarkable observation
indicating that it could serve as a lead for the develop-
ment of potent selective inhibitors of cathepsin F. The
knowledge generated by our study could contribute to
the design and production of potent small synthetic
inhibitors selective for either cruzipain or cathepsin F.
Such selective inhibitors would be of great interest for
the study of the biological functions and pathologic
states in which these enzymes are implicated.
A
B

Fig. 5. Structural basis of PCZ inhibitory selectivity. (A) Sequence alignment of propeptides from papain-like CPs. Residues identical in at
least half of the sequences are highlighted on a black background; those similar in at least half of the sequences are highlighted on a gray
background. Secondary structure elements, as observed in the crystal structure of procathepsin L (Protein Data Bank accession number:
1CS8), are indicated below the alignment. Amino acid identities (%) of the cruzipain proregion with other CP proregions are given on the
right. (B) Interactions with the PBLs of the mature enzymes as a basis for the PCZ inhibitory selectivity. The PBL segments of mature cruzi-
pain (Protein Data Bank accession number: 1ME3, modeled in complex with its propeptide) and mature cathepsins F, L, K, V and S (Protein
Data Bank accession numbers: 1MD6, 1CS8, 1BY8, 1FH0 and 1NQC, respectively), displayed as Ca traces, correspond to the sequences
aligned in the upper right corner. The PBL segments of brucipain and cathepsin W, whose propeptides are aligned in (A), are not shown in
(B), due to the unavailability of experimental structures for these mature enzymes. The PBL-interacting propeptide segments of cruzipain
(model) and procathepsins L and K (Protein Data Bank accession numbers: 1CS8 and 1BY8, respectively), rendered by secondary structure,
correspond to the sequences aligned in the lower left corner. The last four displayed residues (underlined) of these propeptide segments
extend into subsites S1¢ to S3 of the mature enzymes. Cruzipain is shown in green, cathepsin F in magenta, and the other cathepsins in
white. An arrow indicates the two-residue insertions (highlighted) in the b1p–a3p loops of cathepsin L and K propeptides relative to the cruzi-
pain propeptide. This length variability in the propeptide sequences correlates with the steric requirements for interaction imposed by the
conformation adopted by a PBL residue, displayed as a ball-and-stick model and highlighted in the sequence alignment.
2
Cathepsin F inhibition by propeptide of cruzipain
1
F. C. G. Reis et al.
1230 FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS
Experimental procedures
Expression of the propeptide of cruzipain
Complementary oligonucleotides directed to residues
Cys19 (5¢-GCGGATCCTGCCTCGTCCCCGCGGCG-3¢)
and Gly122 (5¢-CGCCCGGGGCCAACAACCTCCACG-
TC-3¢), which span the full-length predicted prodomain of
the cruzipain gene of T. cruzi Dm28c, were used as primers in
a PCR using the cruzipain gene as a template and Pfx
polymerase (Invitrogen, Grand Island, NY, USA). The
amplification conditions were as follows: 30 cycles of dena-

turation (94 °C, 1 min), annealing (55 °C, 1 min), and exten-
sion (68 °C, 1 min). The resulting fragment was excised from
a 0.8% agarose gel, digested with BamHI and SmaI, cloned
into the respective sites of pQE30 (Qiagen, Valencia, CA,
USA), and sequenced in an automated Applied Biosystems
(Foster City, CA, USA)
9
sequencer. E. coli M15 (pREP4)
(Qiagen) was used as a host for the expression of the fusion
protein containing a 6xHis N-terminal tag and the full-length
predicted propeptide of cruzipain. The cells were grown in
LB medium supplemented with 100 lgÆmL
)1
ampicillin and
25 lgÆmL
)1
kanamycin overnight at 37 °C, diluted 20 times
in fresh medium, and cultivated until a D
600 nm
of 0.8
10
was
reached. Recombinant protein expression was induced by
addition of 1 mm isopropyl thio-b-d-galactoside at 30 °C, for
4 h. The cells were harvested by centrifugation
11
at 5000 g
using a Sorvall Centrifuge RC28S (Wilmington, DE, USA)
with GSA rotor, washed in 20 mm phosphate buffer (pH 7.2)
and 150 m m NaCl (NaCl ⁄ P

i
), and lysed in 100 mm
NaH
2
PO
4
,10mm Tris, and 8 m urea (pH 8), under agitation,
for 1 h at room temperature. The soluble material was recov-
ered by centrifugation
12
at 10 000 g for 20 min using a Sorvall
Centrifuge RC28S with F28 ⁄ 36 rotor, and the recombinant
propeptide was purified in an Ni–nitrilotriacetic acid–agarose
resin according to the manufacturer’s instructions. The eluted
samples (1 mgÆmL
)1
) were pooled and submitted to in vitro
refolding by incubation with 5 mm dithiothreitol at 37 °C for
45 min, followed by 100-fold dilution in ice-cold 100 mm
Tris ⁄ HCl (pH 8), 1 mm EDTA, 1 m KCl, and 20% glycerol,
and further incubation at 4 °C for 24 h. The solution was
filtered through 0.2 lm cellulose acetate filters (Millipore,
Bedford, MA, USA), and centrifuged
13
at 10 000 g for 20 min
using a Sorvall Centrifuge RC28S with F28 ⁄ 36 rotor, and the
soluble fraction was concentrated and dialyzed by successive
dilutions in ice-cold Milli-Q water by ultrafiltration in 3 kDa
cut-off DIAFLO membranes (Amicon, Bedford, MA, USA).
The titer of the propeptide was determined by active site titra-

tion of cruzipain. The activated enzyme (previously titrated
with E-64) was incubated with different volumes of the pro-
peptide in 50 mm Na
2
HPO
4
, 200 mm NaCl, 5 mm EDTA
(pH 6.5), and 2.5 mm dithiothreitol, for 20 min at room tem-
perature. The residual activity of the enzyme was determined
by the hydrolysis of 5 lm Z-Phe-Arg-AMC (Sigma, St Louis,
MO, USA) in 50 mm Na
2
HPO
4
, 200 mm NaCl, 5 mm EDTA
(pH 6.5), 2.5 mm dithiothreitol, and 5% dimethylsulfoxide.
Substrate hydrolysis was monitored in a Hitachi
14
F-4500
spectrofluorimeter (Tokyo, Japan) at 380 nm excitation and
440 nm emission, in continuous assays. The initial velocities
were calculated by linear regression of the hydrolysis curves.
The propeptide titer was calculated by linear regression of
the titration curves as being equal to the X-value at which
V
0
¼ 0. The titrations were performed with the propeptide at
concentrations at least five-fold above the K
i
.

Proteolytic enzymes
Cruzipain was purified from T. cruzi Dm28c epimastigotes
as previously described [37]. Human cathepsin B and
human cathepsin S were purchased from Sigma and Calbio-
chem (La Jolla, CA, USA), respectively. Human cathepsins
L, V, K and F were expressed in Pichia pastoris as previ-
ously described [38,39]. The detailed cloning and produc-
tion of recombinant brucipain will be described elsewhere
(T. F. R. Costa, F. C. G. Reis, L. Juliano, J. Scharfstein, &
A. P C. A. Lima, unpublished results). Briefly, a DNA
fragment spanning the predicted first residue of the prodo-
main (Cys22) and the last residue of the mature domain
(Val339) of brucipain was cloned in the BamHI and KpnI
sites of pQE30 for expression in E. coli M15 (pREP4). The
purification of His-tagged recombinant probrucipain and
in vitro refolding were performed as described above for the
propeptide of cruzipain, with the exception of a 6-day incu-
bation at 4 °C for complete refolding of the proenzyme.
The proenzyme was activated by diluting in 50 mm NaOAc
(pH 5.5), 200 mm NaCl, 5 mm EDTA, and 1 mm dithio-
threitol. Mature enzyme was subsequently affinity-purified
in thiopropyl sepharose 6B (Pharmacia-Amersham, Upsala,
Sweden), according to the manufacturer’s instructions. The
purified enzyme was stable at 4 °C for 1 month. The
enzyme concentration was determined by active site titra-
tion with E-64, as previously described [40].
MS analysis
The refolded propeptide (5.4 nmol) was precipitated with
trichloroacetic acid and resupended in de-ionized water.
The protein was mixed at 1 : 1 v ⁄ v with the matrix solution

(a-cyano-4-hydroxycinnamic acid), applied on the steel
plate appropriate for the introduction of samples in the
Voyager DE Pro equipment, and allowed to dry at room
temperature. The mass profile was subsequently obtained
with MALDI-TOF MS (Voyager DE-Pro ⁄ Applied Biosys-
tem, Foster City, CA, USA).
Kinetic measurements
Kinetic measurements were carried out in continuous assays
using a Hitachi
15
F-4500 spectrofluorimeter at 28 °C. All the
1
F. C. G. Reis et al.
2
Cathepsin F inhibition by propeptide of cruzipain
FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS 1231
enzymes were stable at the different pH values during
the assays. The kinetics of inhibition of cruzipain were
measured using the Z-Arg-Arg-MCA substrate at 1.5 lm
and 3 lm ([S] << K
m
)(K
m
¼ 14 lm). All reactions were
started with the addition of enzyme to the cuvette. The
steady-state velocities in the presence of 0–0.1 nm propep-
tide, at the different [S] values, were used for 1 ⁄ v versus [I]
plots [29]. The K
i
value was calculated independently from

each curve for different [S] values (intercept ⁄ slope), and the
average of both values was considered as the final value. All
measurements were performed in 100 mm sodium phosphate
(pH 6–7) or 100 mm sodium acetate (pH 5–5.5) buffers con-
taining 5 mm EDTA, 200 mm NaCl, 2.5 mm dithiothreitol
and 5% dimethylsulfoxide. The kinetics of inhibition of
cathepsin V and cathepsin L were determined using the
Z-Phe-Arg-MCA substrate at 0.77 lm and 1.5 lm (K
m
¼
7.6 lm) or at 0.2 lm and 0.4 lm (K
m
¼ 2 lm), respectively.
The kinetics of inhibition of cathepsin F was determined
using the Z-Phe-Arg-MCA substrate at 0.4 lm and 0.8 lm
(K
m
¼ 4 lm). The kinetics of inhibition of brucipain were
measured using e-NH
2
-(Cap)Leu-(SBzl)Cys-MCA as sub-
strate at 1.5 lm and 3 lm (K
m
¼ 9.3 lm). Under the experi-
mental conditions used, progress curves for the inhibition of
cruzipain and cathepsin F were linear, whereas the progress
curves for brucipain showed curvature and fitted a model of
inhibition through slow binding kinetics.
Cell culture and homogenates
The human monocyte cell line U937 (ATCC) was cultivated

in RPMI (Sigma), supplemented with 10% fetal bovine
serum, at 37 °C, in a 5% humidified CO
2
atmosphere, until
semiconfluence was reached. The cells were incubated with
10 nm 4b-phorbol 12-myristate 13-acetate (Sigma) for 3 days
to induce differentiation to macrophages. Hela cells and and-
rogen-independent DU145 prostate cancer cells derived from
brain metastasis of human prostate cancer were obtained
from the ATCC, and human primary cultures of smooth
muscle cells (Cell Bank of Rio de Janeiro) were cultivated in
DMEM supplemented with 10% fetal bovine serum, as des-
cribed above (<20 passages), until semiconfluence was
reached. The cells were washed twice with Hank’s balanced
salt saline before being lysed by addition of 50 mm NaOAc,
200 mm NaCl, 5 mm EDTA (pH 5.5), and 1% NP-40. The
soluble fraction was recovered by centrifugation
16
at 10 000 g
for 20 min using a Sorvall Centrifuge RC28S with F28 ⁄ 36
rotor and kept at ) 70 °C until assayed for peptidase activity.
T. cruzi epimastigotes (Dm28c) were cultivated in LIT med-
ium supplemented with 10% fetal bovine serum at 28 °C,
L. donovani was cultivated in M199 medium, 10% fetal
bovine serum at 27 °C, T. brucei bloodstream forms (427
strain) were cultivated in HMI9 medium supplemented with
10% fetal bovine serum and 10% serum plus (Invitrogen) at
37 °C in a 5% CO
2
humidified atmosphere, and T. rangeli

was a gift from G. Atella (Federal University of Rio de
Janeiro, Brazil). The parasites were collected at mid-log
phase, washed three times with Hank’s balanced salt saline,
and lysed as described above. The inhibition of peptidases by
the propeptide of cruzipain was evaluated by incubating the
cell homogenates (10 lgÆmL
)1
)in50mm Na
2
HPO
4
, 200 mm
NaCl, 5 mm EDTA (pH 6.0) and 2.5 mm dithiothreitol with
5nm recombinant propeptide for 15 min at room tempera-
ture. Controls were performed by incubating the lysates with
an equal volume of 20 mm Tris ⁄ HCl (pH 8.0), 0.2 mm
EDTA, 200 mm KCl, and 4% glycerol. The residual pepti-
dase activity was measured by the hydrolysis of 5 lm of Z-
Phe-Arg-MCA in 50 mm Na
2
HPO
4
, 200 mm NaCl, 5 mm
EDTA (pH 6.0), and 2.5 mm dithiothreitol. The activity
detected was fully inhibited by E-64, indicating that it was
due to papain-like CPs.
Sequence alignment and homology modeling
The sequences of cruzipain and human cathepsins L, S, F, V,
K and W were retrieved from GenBank (accession numbers:
M84342, NM_001912, NM_004079, BC036451, AB001928,

NM_000396 and NM_001335, respectively), and the
sequence of brucipain was determined in the laboratory after
the cloning of the brucipain gene. Propeptide sequences were
initially aligned with the clustalw program [41] and using
the structure-based sequence alignment between the prore-
gions of procathepsins L and K whose crystal structures are
available [42,43] (Protein Data Bank accession numbers:
1CS8 and 1BY8, respectively). The multiple sequence align-
ment was further refined manually to ensure: (a) positioning
of gaps and insertions between secondary structure elements;
and (b) suitability of the overall three-dimensional model of
the cruzipain proregion bound to mature cruzipain.
A structural model of the cruzipain propeptide bound to
cruzipain was derived from the crystal structure of the
mature enzyme [44] (Protein Data Bank accession number:
1ME3) and a homology model of the prodomain. Homo-
logy modeling was done with the composer program [45]
and the protein loops module implemented in the sybyl
6.9 software (Tripos, Inc., St Louis, MO), using as template
the crystal structure of procathepsin L [42] (Protein Data
Bank accession number: 1CS8) and following the sequence
alignment of the propeptides. Thirteen crystallographic
water molecules buried in the mature enzyme were retained.
Hydrogen atoms were added explicitly, and the protonation
state at pH 6.5 was adopted (i.e. ionized forms for the Asp,
Glu, Arg, Lys and His side chains, for the chain termini,
and for the catalytic residue Cys25). The model of the pro-
peptide–cruzipain complex was refined by conjugate gradi-
ent energy minimization with the AMBER molecular
mechanics force field [46], using a distance-dependent

dielectric (4R
ij
) and an 8 A
˚
nonbonded cutoff. Gradual
structural relaxation was accomplished in a four-stage step-
wise minimization protocol: (a) only the propeptide loops
corresponding to insertions and deletions relative to the
2
Cathepsin F inhibition by propeptide of cruzipain
1
F. C. G. Reis et al.
1232 FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS
template structure were relaxed; (b) the entire propeptide
was relaxed in the context of the rigid mature portion;
(c) the propeptide and the cruzipain side chains in contact
with the propeptide were free to relax while the remainder
of cruzipain was constrained to the crystallographic geom-
etry with a harmonic potential of 5 kcalÆmol
)1
ÆA
˚
)2
; (d) all
constraints were lifted, and the complex was minimized up
to an rms gradient of 0.05 kcalÆmol
)1
ÆA
˚
)1

.
Acknowledgements
We thank Angela Alves and Alda Fidelis for technical
assistance. We are grateful to Luiz Juliano (Federal
University of Sa
˜
o Paulo, Sa
˜
o Paulo, Brazil) for the
synthesis of e-NH
2
-(Cap)L-(SBzl)C-MCA, to Dr Amil-
car Tanuri (Federal University of Rio de Janeiro, Rio
de Janeiro, Brazil) for access to the Perkin-Elmer ABI
PRISM 3100 automated sequencer, and to Denis L. S.
Dutra (Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil) for assistance with MS. This work was
supported in part by grants from the Conselho Nac-
ional de Desenvolvimento Cientı
´
fico e Tecnolo
´
gico
(CNPq), the Fundac¸ a
˜
o Carlos Chagas Filho de
Amparo a
`
Pesquisa do Estado do Rio de Janeiro
(FAPERJ), the Howard Hughes Medical Institute

(Departmental sharing grant no. 55003669), NIH (AR
46182) (to DB) and the CIHR (to DB). This is NRCC
publication number 47553
17
.
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