Characterization of a leucine aminopeptidase from Toxoplasma gondii

Article abstract of DOI:10.1016/j.molbiopara.2009.11.005

The M17 family leucine aminopeptidase (LAP) hydrolyzes amino acids from the N-terminus of peptides. Many LAPs from parasitic protozoa, including Plasmodium, Trypanosoma, and Leishmania, have been intensely investigated because of their crucial roles in parasite biology. In this study, the functional recombinant Toxoplasma gondii LAP (rTgLAP) was expressed in Escherichia coli, and its enzymatic activity against synthetic substrates for aminopeptidase, as well as cellular localization, was determined. The activity was strongly dependent on metal divalent cations, and was inhibited by bestatin, which is an inhibitor for metalloprotease. Our results indicated that TgLAP is a functional aminopeptidase in the cytoplasm of T. gondii.

Full text of DOI:10.1016/j.molbiopara.2009.11.005

Molecular & Biochemical Parasitology 170 (2010) 1–6
Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
Characterization of a leucine aminopeptidase from Toxoplasma gondii
Honglin Jiaa,b, Yoshifumi Nishikawaa, Yuzi Luoa, Junya Yamagishia, Chihiro Sugimotob, Xuenan Xuana,∗
a National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
b Department of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Hokkaido 001-0020, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The M17 family leucine aminopeptidase (LAP) hydrolyzes amino acids from the N-terminus of pep-
tides. Many LAPs from parasitic protozoa, including Plasmodium, Trypanosoma, and Leishmania, have
been intensely investigated because of their crucial roles in parasite biology. In this study, the functional
recombinant Toxoplasma gondii LAP (rTgLAP) was expressed in Escherichia coli, and its enzymatic activity
against synthetic substrates for aminopeptidase, as well as cellular localization, was determined. The
activity was strongly dependent on metal divalent cations, and was inhibited by bestatin, which is an
inhibitor for metalloprotease. Our results indicated that TgLAP is a functional aminopeptidase in the
cytoplasm of T. gondii.
Received 18 August 2009
Received in revised form
11 November 2009
Accepted 11 November 2009
Available online 18 November 2009
Keywords:
Toxoplasma gondii
Leucine aminopeptidase
Enzymatic activity
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
However, no aminopeptidases in T. gondii have received much
attention to date. In this study, we cloned and expressed a leucine
Toxoplasma gondii is an obligate intracellular protozoan parasite
belonging to the phylum Apicomplexa. The parasite infects most
species of domestic animals, birds, and humans in both developed
and developing countries. It is estimated that up to one-third of the
population in the United States is infected with toxoplasmosis, and
up to 90% of the populations in other countries are infected with
T. gondii [1]. In immunocompromised individuals and pregnant
women, infection with the parasite can cause severe complications
[2].
Leucine aminopeptidases are exopeptidases that catalyze the
hydrolysis of leucine residues from the amino termini of pro-
tein or peptide substrates [3]. LAPs are one of the major cytosolic
aminopeptidases and have been identified in numerous microor-
ganisms, plants, vertebrates and invertebrates [4–7]. They play
diverse biological and physiological roles by either degrading
peptides or interacting with peptide-dependent signaling [8,9].
Changes in LAP expression pattern or catalytic function resulted
in altered peptide activation, leading to changes in tumor cell pro-
liferation, invasion, and angiogenesis.
aminopeptidase of T. gondii and determined its cellular localization
and enzymatic activity. The data we present here will considerably
expand existing knowledge of the protein metabolism of T. gondii.
2. Material and methods
2.1. Parasite culture and purification
T. gondii RH strain tachyzoites were maintained in vero cells
cultured in a minimum essential medium (MEM, Sigma, USA)
supplemented with 8% heat-inactivated fetal bovine serum and
50 ␮g/ml kanamycin at 37 ◦C in a 5% CO2 air environment. For
purification of T. gondii tachyzoites, parasites and host cells were
washed in cold PBS, and the final pellet was resuspended in cold
PBS and syringed three times with a 27-gauge needle. The parasites
were then filtered through a 5.0 ␮m pore filter (Millipore, USA),
washed twice with PBS, and store at −80 ◦C until use.
2.2. Cloning of the TgLAP gene
The LAPs of parasitic organisms such as Plasmodium, Try-
panosoma, and Leishmania have been proved to be involved in free
amino acid regulation [10–12]. Due to their important roles in par-
asite biology, many LAPs have recently been investigated as drug
targets [12] and vaccine candidates [13,14] in parasitic infections.
Total RNA of T. gondii RH strain was extracted with Trizol reagent
(Sigma, USA). Specific primers (forward primer, 5ꢀ-ata gaa ttc tat
gtc gag ggt tcc tgcg-3ꢀ; and reverse primer, 5ꢀ-ata gaa ttc cta gtt
ctc ttt cgt ttg tgt gc-3ꢀ) were designed according to the TgLAP
sequence in ToxoDB (accession number, TGME49 090670). The
full length of TgLAP cDNA was amplified by using the one-step
RT-RNA kit (Takara, Tokyo, Japan) and cloned into the PGEM-T
vector (Promega, USA). Subsequently, the gene was sequenced by
∗
Corresponding author. Tel.: +81 155 49 5648; fax: +81 155 49 5643.
E-mail address: gen@obihiro.ac.jp (X. Xuan).
0166-6851/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molbiopara.2009.11.005

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H. Jia et al. / Molecular & Biochemical Parasitology 170 (2010) 1–6
using an automated sequencer (ABI PRISM 3100 Genetic Analyzer,
Foster, USA) with amplification primers and additional internal
sequencing primers. A computer program, GENETYX version 7.0
(Software Development, Tokyo, Japan), was used for preliminary
sequence assembly and analysis. The protein sequence was then
sent for analysis with the NCBI/BLAST program. A phylogenetic tree
was generated from homologues of the full-length LAP amino acid
sequences by the neighbor-joining method, and the confidence of
the branching order was verified by making 1000 bootstrap repli-
cates.
2.8. IFAT and confocal laser microscopic observation
Wild-type and the transgenic parasites were plated on HFF
grown on 12-mm coverslips (approximately 2 × 105/well) in 24-
well plates. After 16–24 h incubation, coverslips were fixed with
3% paraformaldehyde in PBS for 15 min and were then permeabi-
lized with 0.3% Triton X-100 in PBS for 5 min. Mouse anti-rTgLAP
polyclonal antibody and anti-HA monoclonal antibody (Covance
Research Products, Berkeley, USA) diluted in PBS containing 3%
BSA were used as primary antibodies. Goat anti-mouse Alexa
488 antibodies (Molecular Probes) and goat anti-mouse Alexa
594 antibodies (Molecular Probes) were used as secondary anti-
bodies. Coverslips and glass slides were mounted with Mowiol
(Calbiochem) and observed under a confocal laser microscope (TCS
NT, Leica, Wetzlar, Germany).
2.3. Expression of the recombinant TgLAP in Escherichia coli and
production of anti-rTgLAP sera
The cDNA of the TgLAP gene was cloned into the prokary-
otic expression vector pGEX-4T-3, and the resulting plasmid
was transformed into an E. coli BL21 strain (Amersham Phar-
macia Biotech). Purification of the rTgLAP was performed with
Glutathione Sepharose 4B beads (Amersham Pharmacia Biotech)
according to the manufacturer’s instructions. The rTgLAP fused
with GST was eluted by 20 mM reduced glutathione (GE Health-
care, Piscataway, USA) and dialyzed against 50 mM Tris–HCl (pH
8.0). The protein concentrations were measured with the Bio-Rad
Protein Assay (Bio-RAD Laboratories, Hercules, USA).
2.9. Enzyme assay
The aminopeptidase activity was determined by measuring the
rate of liberation of l-leucine from a fluorogenic substrate, l-
leucine-4-methyl-coumaryl-7-amide (Leu-MCA, Peptide Institute,
Osaka, Japan). Thereleased AMC was measuredusingafluorescence
micro-plate reader, Fluoroskan Ascent FL (Thermo Electron Cor-
poration, Waltham, USA), with a wavelength pair of 355–460 nm
for both emission and excitation. To determine the pH depen-
dent activity, acetate/Tris buffers (50 mM acetic acid and 100 mM
Tris–HCl, pH 4–11) containing 1 mM MnCl2 and rTgLAP and 0.1 mM
Leu-MCA at 37 ◦C were used. Cation sensitivity was investigated
by assaying the rTgLAP activity after pre-incubating the enzyme
at 37 ◦C for 30 min in 50 mM Tris–HCl (pH 8.0) containing a metal
chloride (Sigma–Aldrich, St Louis, USA). To determine the inhibitory
efficacy of bestatin to the enzymatic activity of rTgLAP, the enzyme
was pre-incubated with bestatin for 30 min at 37 ◦C before the
substrate was added to measure the residual activity. The rela-
tive inhibition levels of the rTgLAP were assessed using bestatin
at various concentrations.
2.4. Electrophoresis
SDS-PAGE was carried out on a 12% (w/v) polyacrylamide gel
as described by Liao et al. [15]. PAGE under native conditions was
performed by using NativePAGETM Novex® Bis-Tris Gel System and
following the instructions of the manufacturer (Invitrogen, Carls-
bad, USA). Proteins were stained with Coomassie Brilliant Blue
R-250.
2.5. Preparation of mouse sera against TgLAP
2.10. Enzyme kinetics
Female Jcl:ICR mice (6 weeks old; CLEA Japan Inc., Tokyo, Japan)
were immunized intraperitoneally with 100 ␮g of purified rTgLAP
emulsified with an equal volume of Freund’s complete adjuvant
(Difco Laboratories, Detroit, USA) for the first injection. Fifty micro-
gram of the same antigen in Freund’s incomplete adjuvant (Difco)
was intraperitoneally injected into the mice on days 14 and 28. Sera
were collected 14 days after the last immunization.
The Km (Michaelis constant) and Vmax (maximum velocity) val-
ues of rTgLAP were determined by incubating the enzyme in the
reaction mixture in the presence of increasing concentrations of
various fluorogenic substrates (Peptide Institute) at 37 ◦C. The ini-
tial velocity was calculated from the slope of the linear range of
fluorescence versus the time curve. The Km and Vmax values were
recorded with their standard errors derived from three indepen-
dent experiments.
2.6. Western blotting
3. Results and discussion
The T. gondii lysates were separated on 12% SDS-PAGE under
reducing conditions, and Western blotting was performed as
described by Liao et al. [15].
3.1. Identification of the TgLAP gene
The full-length TgLAP cDNA was intermediate in length among
the LAPs of apicomplexan parasites. Two functional domains,
a less conserved N-terminal domain (residues 48–208) and a
more conserved catalytic C-terminal domain (residues 245–551),
were identified using the Pfam protein search algorithm of the
SMART program (http://smart.embl-heidelberg.de/). Zn-binding
sites (residues 327, 332, 351, 411, 413, and 415) and substrate-
binding/catalytic sites (residues 327, 332, 339, 351, 411, 413,
and 440) were identified in TgLAP by the BLAST program. M17
aminopeptidases are reported to be homohexameric enzymes
[12]. In the amino acid sequence of TgLAP, the interface sites
between two trimers were also identified, which indicated that the
native TgLAP could exist as homohexamers in the parasite as well.
Although the identity of these sites was relatively low, their posi-
tions are very similar, especially in the conserved C-terminal. We
2.7. Construction of transgenic T. gondii tachyzoites
overexpressing TgLAP
A transgenic T. gondii RH parasite strain overexpressing the
homogeneous TgLAP was generated to further confirm this point.
The transfer vector was constructed as follows: the open reading
frame of TgLAP was first used to replace the fragment between the
NcoI and NheI restrictions of the pHXNTPHA [16]. Subsequently,
a sequence housing full-length TgLAP fused with the HA fragment
at C-terminal was amplified with two primers (5ꢀ-atg tcg agg gtt
cct gcg cc-3ꢀ and 5ꢀ-gaa gag gct att atc cgc tgt-3ꢀ) and then inserted
into the EcoRV site of the pDMG vector [17]. The transfection was
performed as described previously [17].

H. Jia et al. / Molecular & Biochemical Parasitology 170 (2010) 1–6
3
Fig. 1. Phylogenetic relationship of TgLAP with other LAPs of apicomplexan parasites. (A) Phylogenetic tree of the TgLAP with other LAPs. The scale at the bottom measures
the distance between sequences. Sequences used in this study are as follows: Cryptospridum parvum LAP (accession number XP 626197), C. hominis LAP (accession number
XP 667960), T. gondii RH LAP (accession number EEB01321), P. yoelli LAP (accession number XP 729735), P. falciparum LAP (accession number XP 001348613), Theileria
parva LAP (accession number XP 764196), T. annulata LAP (accession number CAI76586), Babesia gibsoni LAP (accession number AB490782), and B. bovis LAP (accession
number XP 001609968). Homo sapiens LAP (accession number AAD17527) was used as an outgroup. T. gondii ME49 LAP (accession number TGME49 090670) was from
ToxoDB (http://toxodb.org/toxo/) and Neospora caninum LAP (accession number NC LIV 113250) was from GeneDB (http://www.genedb.org/). (B) Alignment of the amino
acid sequences of the LAPs of apicomplexan parasites. Conservation between amino acids is indicated by asterisk and dots in the alignment. The conserved C-terminal region
is presented in bold. The substrate-binding site residues are marked in red, the interface sites between two trimers are marked in green, and the essential Zn-binding sites
are underlined. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
assessed the phylogenetic relationship between TgLAP and its clos-
est homologues of apicomplexan protozoan parasites (Fig. 1). The
overall sequence identity between the TgLAP and the LAP of P. falci-
parum was 36.1%, and that within the C-terminal domain was 55.5%.
transformed into an E. coli BL21 strain. An rTgLAP fused with
GST at the N-terminal was expressed as expected. The molecular
weight of the monomer of rTgLAP was around 85 kDa as observed
in SDS-PAGE (Fig. 2A). The polymer of rTgLAP with molecular
weight of around 500 kDa was observed in native PAGE analy-
sis (Fig. 2B). The use of G-250 charge-shift in NativePAGETM gels
results in protein resolution based upon protein size allowing
accurate size estimation of native proteins and protein com-
plexes [18]. Although the size estimation may have an expected
size estimation error of ∼15%, this result indicated that the
3.2. Polyacrylamide gel electrophoresis of rTgLAP expressed in E.
coli
The cDNA of the TgLAP gene was cloned into the prokary-
otic expression vector pGEX-4T-3, and the resulting plasmid was

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H. Jia et al. / Molecular & Biochemical Parasitology 170 (2010) 1–6
As shown in Fig. 3A, an anti-HA monoclonal antibody specifically
recognized HA-tagged TgLAP in the transgenic parasites, although
an unexpected band could be observed in the blot. Since there was
not signal was detected in the control at all, the unexpected band
should be degraded product.
3.4. Localization of TgLAP
The specific fluorescence stained by anti-TgLAP polyclonal
mouse antibodies under confocal laser scanning microscopy indi-
cated the localization of TgLAP in the cytoplasm of T. gondii (Fig. 3B).
The cytosolic localization was also confirmed by staining the trans-
genic parasites with an anti-HA monoclonal antibody (Fig. 3B). Que
et al. [19] suggested that cytosolic neutral aminopeptidases were
likely involved in the hydrolysis of the dipeptides digested by the
TgCPCs in parasitophorous vacuoles (PV). It requires the dipeptides
be sent to the cytoplasm where TgLAP localized. However there
was no evidence that indicated T. gondii tachyzoites could uptake
them. Alternatively, another source of the substrates of TgLAP could
be the peptides derived from the proteasomal protein degradation
pathways.
Fig. 2. Electrophoresis of purified rTgLAP. (A) SDS-PAGE of purified rTgLAP. (Lane
1) 10 ␮g of rTgLAP and (lane 2) 5 ␮g of rTgLAP. (B) Native PAGE analysis of purified
rTgLAP. (Lanes 1 and 2) 10 ␮g of rTgLAP and (lanes 3 and 4) 5 ␮g of rTgLAP.
functional form (homohexamer) was present in the purified
rTgLAP.
3.5. Enzymatic activity of rTgLAP
3.3. Western blot analysis of native and HA-tagged TgLAP
The enzymatic activity of rTgLAP was analyzed against synthe-
sized substrates for aminopeptidase. The protein concentration was
chosen in order to obtain the linearity of the reactions (Fig. 4A).
Data were fit to the Michaelis–Menten equation using GraphPad
Prism version 4.0c (GraphPad Software, San Diego, USA) (Fig. 4B).
Activity was noted at around a neutral to slightly alkaline pH range
A polyclonal anti-rTgLAP serum raised in an ICR mouse was
used to identify the native TgLAP in the lysate of T. gondii para-
sites. A specific band with a size of around 60 kDa was detected by
Western blotting in the T. gondii lysates (Fig. 3A). A transgenic T.
gondii expressing HA-tagged TgLAP was successfully constructed.
Fig. 3. Western blotting and IFAT of native and HA-tagged TgLAP. (A) Western blot analysis of native and HA-tagged TgLAP. OE, lysates of T. gondii RH strain overexpressing
TgLAP; GFP, lysates of T. gondii RH strain expressing GFP alone; W, lysates of wild-type T. gondii RH strain; HFF, lysate of HFF cells. (B) Indirect immunofluorescence detection
of the stained TgLAP in cytoplasm. (a) Wild-type RH T. gondii stained with anti-rTgLAP polyclonal antibodies; (b) phase contrast of Panel a; (c) an overlay of Panel a on Panel
b; (d) transgenic T. gondii stained with an anti-HA monoclonal antibody; (e) fluorescence of GFP in transgenic T. gondii and (f) an overlay of Panel d on Panel e.

H. Jia et al. / Molecular & Biochemical Parasitology 170 (2010) 1–6
5
Fig. 4. Enzymatic activity of the rTgLAP. (A) An example of linear reaction of the rTgLAP against synthetic substrates; (B) enzyme assays with the fluorogenic peptide substrate
Leu-MCA demonstrate that the rTgLAP exhibits Michaelis–Menten enzymatic kinetics. One unit was defined as picomoles of AMC released per milligram of recombinant
protein; (C) pH-dependence of rTgLAP was analyzed against Leu-MCA from pH 4 to 11; (D) inhibition of rTgLAP activity against Leu-MCA by bestatin. Inhibition of the rTgLAP
activity was studied using bestatin (Sigma–Aldrich) at final concentrations of 0, 0.125, 0.25, and 1 ␮M in the reaction mixture. Data points indicate the mean activity SD
(n = 3).
(pH 7–10), and optimum activity was achieved at pH 8.0 and 37 ◦C
(Fig. 4C). The metal cation sensitivity was investigated by assay-
ing the rTgLAP activity after pre-incubating the enzyme at 37 ◦C
for 30 min in 50 mM Tris–HCl (pH 8.0) containing a metal chloride
(Sigma–Aldrich, St. Louis, USA). In the absence of metal ion from the
reaction buffer, the rTgLAP activity was almost undetectable. How-
ever, the activity was markedly activated by the addition of Mn2+
or Co2+ (Table 1). Therefore, the rTgLAP seems to be purified as an
apoenzyme, although the metal contents in the rTgLAP still need
further investigations. Similar results were also observed in other
reports [4,6]. The in vitro inhibition assay of rTgLAP activity using
bestatin, which is known as the inhibitor of M1 and M17 cytoso-
lic aminopeptidases [20], is shown in Fig. 4D. The rTgLAP activity
was inhibited by bestatin in a dose-dependent manner. Bestatin is
a dipeptide analog first discovered as an antibiotic of the bacterium
Streptomyces olivoreticuli. It has very low toxicity in experimental
animals and humans [21,22] and, consequently, has been formu-
lated for the safe therapeutic treatment of certain cancers such as
squamous cell carcinoma in humans [23]. These dipeptide analogs
will be useful scaffolds on which novel small molecule inhibitors
could be designed to potently and selectively inhibit the T. gondii
M17 family aminopeptidases.
Table 1
Effect of divalent metal ions on rTgLAP activity.
Metal ion
Fe(II)
Concentration (mM)
Activity (units/min)a
3.6. Substrate specificity of rTgLAP
0.1
1
0.07 0.1
0.46 0.09
The enzyme kinetics of rTgLAP against fluorogenic synthetic
substrates matched its classification as a member of the M17
leucine aminopeptidase family. Hydrophobic amino acid leucine
could be efficiently cleaved by the rTgLAP from the N-terminus of
synthetic substrates. The overall catalytic efficiency of rTgLAP to
hydrolyze this amino acid, kcat/Km, was 832 M−1 s−1 (Table 2). The
preference of rTgLAP for other synthesized substrates is slightly
different from that of the LAP of Babesia gibsoni [4] or P. falciprum
[12]. In the case of hydrophobic amino acids phenylalanine and
0.1
1
0.02 0.22
0.22 0.29
Mg(II)
Ca(II)
Ni(II)
Mn(II)
Cu(II)
Zn(II)
0.1
1
0.09 0.03
0.13 0.07
0.1
1
0.07 0.01
24.72 1.29
0.1
1
109.49 9.22
225.72 14.37
Table 2
0.1
1
0.01 0.08
0.16 0.04
Kinetic parameters for the hydrolysis of peptide substrates by rTgLAP.
a
Substrate
kcat (×10−2 s−1
)
Km (mM)a
kcat/Km (M−1 s−1
)
0.1
1
0.43 0.05
0.46 0.02
Leu-MCA
Ala-MCA
Arg-MCA
Pro-MCA
Phe-MCA
8.89 0.86
3.04 0.58
4.05 0.84
0.16 0.03
0.107 0.013
0.169 0.041
0.426 0.108
0.124 0.025
832
179.83
95.10
13.2
0.1
1
89.45 8.66
105.2 1.25
Co(II)
0.002 0.0003
0.010
0
2.30
a
One unit was defined as picomoles of AMC released per milligram of recombi-
nant protein. Data represent means SD from three independent experiments.
a
Data represent means SD from three independent experiments.

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H. Jia et al. / Molecular & Biochemical Parasitology 170 (2010) 1–6
proline, the enzyme’s efficiency values (kcat/Km) were only 2.30
and 13.2 M−1 s−1, respectively. For the neutral amino acid ala-
nine, the kcat/Km value was 179.83 M−1 s−1. Similar to the LAP
of B. gibsoni but not P. falciparum LAP, rTgLAP cleaved to the N-
terminal basic amino acid, arginine, with comparable efficiency
(kcat/Km = 95.10 M−1 s−1). These slight differences among the LAPs
of the parasites were probably caused by the requirement for dif-
ferent environments.
In summary, we report here the cloning, genetic analysis,
and biochemical characterization of a novel leucine aminopepti-
dase from T. gondii. The optimal amidolytic activity of TgLAP was
observed against a fluorogenic synthetic substrate of Leu-MCA at
pH 8.0 and 37 ◦C. In addition, consistent with its classification as
a member of the M17 leucyl aminopeptidase family, the activity
of TgLAP was enhanced by Mn2+ and Co2+ at millimolar con-
centrations and inhibited by the broad spectrum aminopeptidase
inhibitor bestatin.
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by endoproteases and sent to cytoplasma. Parasite proteases are
increasingly recognized as potential targets for chemotherapeutic
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Acknowledgements
This work was funded by a grant from the Ministry of Educa-
tion, Culture, Sports, Science, and Technology of Japan. We thank
Dr. K.A. Joiner (Yale University) for providing the plasmid vector
pHXNTPHA.
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