Expression Profile of the Schistosoma japonicum Degradome Reveals Differential Protease Expression Patterns and Potential Anti-schistosomal Intervention Targets
et al. (2014) Expression Profile of the Schistosoma japonicum Degradome Reveals Differential Protease Expression
Patterns and Potential Anti-schistosomal Intervention Targets. PLoS Comput Biol 10(10): e1003856. doi:10.1371/journal.pcbi.1003856
Expression Profile of the Schistosoma japonicum Degradome Reveals Differential Protease Expression Patterns and Potential Anti-schistosomal Intervention Targets
Shuai Liu 0
Pengfei Cai 0
Xianyu Piao 0
Nan Hou 0
Xiaosu Zhou 0
Chuang Wu 0
Heng Wang 0
Qijun Chen 0
Cynthia Gibas, University of North Carolina at Charlotte, United States of America
0 1 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China , 2 Key Laboratory of Zoonosis, Jilin University , Changchun , China , 3 Department of Microbiology and Parasitology, Institute of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China
Blood fluke proteases play pivotal roles in the processes of invasion, nutrition acquisition, immune evasion, and other hostparasite interactions. Hundreds of genes encoding putative proteases have been identified in the recently published schistosome genomes. However, the expression profiles of these proteases in Schistosoma species have not yet been systematically analyzed. We retrieved and culled the redundant protease sequences of Schistosoma japonicum, Schistosoma mansoni, Echinococcus multilocularis, and Clonorchis sinensis from public databases utilizing bioinformatic approaches. The degradomes of the four parasitic organisms and Homo sapiens were then comparatively analyzed. A total of 262 S. japonicum protease sequences were obtained and the expression profiles generated using whole-genome microarray. Four main clusters of protease genes with different expression patterns were identified: proteases up-regulated in hepatic schistosomula and adult worms, egg-specific or predominantly expressed proteases, cercaria-specific or predominantly expressed proteases, and constantly expressed proteases. A subset of protease genes with different expression patterns were further validated using real-time quantitative PCR. The present study represents the most comprehensive analysis of a degradome in Schistosoma species to date. These results provide a firm foundation for future research on the specific function(s) of individual proteases and may help to refine anti-proteolytic strategies in blood flukes.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All files are available from the Gene Expression
Omnibus database with accession numbers GPL18617, and series GSE57143.
Funding: This study was supported by the National Natural Science Foundation of China (#81270026), the National S & T Major Program (Grant
No. 2012ZX10004-220), the Special Fund for Health Research in the Public Interest (Grant No 201202019), and the Program for Changjiang Scholars and
Innovative Research Team in University (IRT13007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
Schistosomiasis is one of the most socioeconomic parasitic
diseases, afflicting millions of people in many tropical and
subtropical countries . Praziquantel is the only specific remedy
for the disease and no effective vaccine is available . However,
long-term, repeated mass chemotherapy in endemic regions may
give rise to praziquantel resistance, and Schistosoma mansoni
strains resistant or insensitive to the drug have been found in some
endemic areas . Therefore, additional chemotherapeutic
agents against blood flukes are desperately needed.
Proteases are considered druggable targets from the medical
and chemical viewpoints because of their known enzymatic
mechanism and regulatory roles in many pathologies . A
number of protease inhibitors have been developed and approved
for the treatment of various human diseases . Parasite proteases
contribute to pathogenesis in a variety of ways, including invasion,
nutrition acquisition, immune evasion, and other host-parasite
interactions . Although most parasite-derived proteases have
counterparts in their mammalian hosts, the mammalian proteases
are often sequestered in different cell organelles or play distinct
biological roles . For example, cysteine proteases perform
critical functions in extracellular proteolysis in parasites, but their
mammalian counterparts are found predominantly in intracellular
organelles. These discoveries make proteases promising targets for
the development of novel immunological or chemotherapeutic
anti-parasite agents . Indeed, cysteine protease inhibitor
K11777 has been tested in the murine model of schistosomiasis,
and the remarkable reduction in worm burden and pathology
validated schistosome cysteine proteases as novel potential drug
targets for chemotherapy [10,11].
Recently, draft genome sequences were published for the three
major pathogens of human schistosomiasis: Schistosoma japonicum,
Parasite proteases play critical roles in host-parasite
interactions and thus are considered to be potential
antischistosomal targets. Although numerous schistosome
proteases have been predicted based on recently
published genomes, no systematic analysis of their expression
in Schistosoma species has been performed. Thus, we
comparatively analyzed the degradomes of four parasitic
organisms and human host, and performed
wholegenome microarray analysis to analyze the expression
profile of the Schistosoma japonicum degradome at four
developmental stages. The expression profile generated
for the S. japonicum degradome was divided into four
main clusters with different expression patterns, and a
subset of selected proteases were further validated using
real-time quantitative PCR. Our work is the most
comprehensive analysis of a degradome in Schistosoma species to
date. Many protease genes were first characterized in
blood flukes, and some could be treated as potential
antischistosomal targets for intensive research in the future.
The results provide a firm foundation for deep study on
the specific function(s) of individual proteases or protease
families in schistosomes.
S. mansoni, and Schistosoma haematobium . These genomic
resources have provided insight into the molecular basis of
schistosome biology, host-parasite interactions, and the
pathogenesis of schistosomiasis, which will facilitate innovations in
schistosomiasis control . The degradome is defined as the
complete set of proteases present in an organism . The recent
availability of whole genomic sequences from blood flukes has led
us to predict the contents of the degradomes in Schistosoma
species. In S. japonicum, 314 putative proteases have been
identified that can be divided into five major classes of proteases:
aspartic, cysteine, metallo-, serine, and threonine . The vast
majority of schistosome proteases have been identified using
predicted proteomes, and only a few have been functionally
characterized. Choosing a putative protease gene for further
investigation will be difficult without transcript information.
Because schistosome parasites have a complicated developmental
lifecycle comprising seven morphologically discrete stages, and
the protease genes may be expressed in different lifecycle stages.
Four developmental stages are closely associated with
mammalian hosts: cercariae, by which mammalian hosts are infected;
juvenile schistosomula, which enter mammalian hosts capillaries
and lymphatic vessels en route to the lungs and liver; adult
worms, which migrate to the veins of the intestines or bladder
and produce eggs; and eggs, which cause serious granulomatous
reactions and fibrosis in the affected organs . Elucidation of
the expression of the proteases in these four important stages of
the parasite will contribute to the future function dissection of the
enzymes, which will facilitate discovery of anti-schistosomal
However, no systematic analysis of degradome profiles has been
performed in Schistosoma species to date. Therefore, we used
whole-genome microarray analysis to profile the expression of the
majority of protease genes in these four developmental stages of S.
japonicum. The gene expression patterns of a subset of proteases
were further validated using real-time quantitative PCR
(qRTPCR). The results obtained from this work provide a foundation
for the further functional characterization of protease genes in
Materials and Methods
All procedures performed on animals within this study were
conducted following animal husbandry guidelines of the Chinese
Academy of Medical Sciences and with permission from the
Experimental Animal Committee with the Ethical Clearance
Protease sequence retrieval and analysis
The degradomes of four parasitic organisms with known
genome sequences were analyzed in this study. A total of 314 S.
japonicum protease sequences predicted in genome-wide , and
253 sequences obtained from the MEROPS database  were
integrated to generate the degradome of S. japonicum. The
degradome of S. mansoni was composed of protease sequences
predicted according to the putative proteome . The
degradome of Clonorchis sinensis comprised protease sequences from
the MEROPS database. CD-HIT v4.5.4 software (http://www.
bioinformatics.org/cd-hit/) was used to remove redundant
sequences, with the standard of 90% identity and 80% coverage
between two sequences (the shorter one was eliminated). If the
identity was 100% between two sequences with more than 100
aligned consecutive amino acid residues, one sequence was
Next, the degradome of S. japonicum was analyzed using
several bioinformatic approaches. Protease sequences were
functionally annotated using Blast2GO , and the output provided
as combined graphics in three categories of gene ontology (GO)
terms: biological processes, molecular functions, and cellular
components. The KEGG automated annotation server (KAAS)
was used to assign pathway-based functional orthology to protease
sequences . Signal peptides were predicted using the SignalP
4.1 server , and transmembrane helices were predicted using
TMHMM 2.0 .
Phylogenetic analysis of the schistosome cathepsin gene
The S. japonicum degradome sequences were searched to
identify cathepsin proteins using BLASTp program with the
published schistosome cathepsin protein sequences and Homo
sapiens cathepsin Aprotein sequence (4CI9_A) as query sequences.
All obtained protein sequences were further examined for the
presence of cathepsin conserved motif and domains by searching
the Conserved Domain Database (v. 3. 11) on NCBI [23,24]. The
amino acid sequences of cathepsins from S. japonicum and S.
mansoni were first aligned using ClustalX , and then refined
manually. Finally, phylogenetic tree was constructed using MEGA
5.0 software by the neighbor-joining (NJ) method, and the
bootstrap test was replicated 1000 times .
The freshly released cercariae were harvested from S.
japonicum-infected Oncomelania hupensis provided by Hunan
Institute of Parasitic Diseases, Yueyang, China. Hepatic
schistosomula were isolated from infected New Zealand rabbits at 2
weeks post-infection. Mixed adult worms were isolated from
infected rabbits at 6 weeks post-infection. Male and female worms
were manually separated with the aid of a light microscope. Eggs
were purified from liver tissues of infected rabbits by enzyme
digestion method . All parasites were soaked in RNAlater
solution (Ambion, CA, USA), and stored at 280uC until total
RNA was isolated.
Total RNA isolation
Total RNAs were isolated from parasites at different
developmental stages (eggs, cercariae, hepatic schistosomula, and adult
worms) using RNeasy Mini kit (QIAGEN), and the contaminating
genomic DNA were removed from RNA samples with Tubro
DNA-free kit (Ambion, CA, USA). The quantity and quality of the
RNA samples were assessed by NanoDropND-1000
spectrophotometer (NanoDrop Technologies, Wilmington, DE) and
denaturing agarose gel electrophoresis.
Microarray analysis of S. japonicum degradome
Schistosome genome-wide microarray was used to analysis the
expression profile of the S. japonicum degradome. The design and
construction of the microarray, and the methods used in
microarray hybridization and feature extraction have been
previously reported . Microarray hybridization was performed
in three biological replicates for all samples. Raw data and
normalized gene level data from the array have been deposited at
the public database Gene Expression Omnibus (http://www.ncbi.
nlm.nih.gov/geo) under accession numbers for the platform
GPL18617, and series GSE57143. Finally, local BLAST searches
were performed to identify the microarray sequences
corresponding to S. japonicum protease sequences which were used as query
sequences. For protease sequences with more than one microarray
sequences, the highest expression value was considered. Protease
genes were considered as statistical differentially expressed by
expression fold-change $2 between any two compared
developmental stages, and p-value ,0.05 (one tailed Students t-test). The
coefficient of variation (CV) was employed to extract the
constantly expressed protease genes among the four
developmental stages by cut-off value of 0.15. Hierarchical clustering analysis
of selected genes was performed to generated heat maps using
Cluster 3.0 software , and Heatmap Builder 1.0 software .
Real-time quantitative PCR
A subset of protease genes with different expression patterns
were selected for further validation using qRT-PCR as previously
described . Reactions were carried out in technological
triplicate on the 7300 Real-Time PCR system (Applied
Biosystems) using Brilliant II SYBR Green QPCR Master Mix (Agilent
Technologies, USA) according to the manufacturers instructions.
26S proteasome non-ATPase regulatory subunit 4 (PSMD4),
which has been validated as a reliable reference gene in
transcriptomic analysis of S. japonicum , was employed as a
control gene in the qRT-PCR analysis. The qRT-PCR primers
were designed using Primer Express 3.0 software (Applied
Biosystems, Foster City, USA) (Table S1). The relative expression
Results and Discussion
Overview of the S. japonicum degradome
After culling the redundant sequences, we identified a total of
262 genes encoding known or putative proteases among 64
families (Table 1 and S2), comprising approximately 2% of the S.
japonicum predicted proteome. Depending on the key residue for
protease catalytic mechanism, all of these proteases can be
grouped into five catalytic classes: aspartyl, cysteine, metallo-,
serine, and threonine. In this study, we culled the redundant
protease sequences according to a strict standard. For instance,
short sequences were removed under conditions of 90% identity
and 80% coverage between two sequences. Indeed, we found that
some short predicted protein sequences aligned to the same long
protein sequence with a complete coding sequence on NCBI.
Therefore, the number of proteases we identified is less than the
number of proteases (314 protease sequences) predicted from the
recently published genome sequence of S. japonicum. Similarly, a
total of 255 putative protease sequences (269 proteases in our
result) were identified using different data mining methodologies,
compared to 335 protease sequences identified from proteins
predicted for the S. mansoni genome . Most protease families
have shrunk, and the greatest reduction in number was the C01
family, which is now at half of previously published numbers (16
sequences vs. 31 sequences). However, our data added several new
protease families for S. japonicum, including C83, C85, C86, and
Comparative analysis of the degradomes in parasitic
flatworms and human host
Genome sequencing has paved the way for a systematic
dissection of parasite biology, and an increasing number of
parasite genomes have been decoded recently [15,3235]. Here,
we chose three more parasitic worms for comparative analysis of
their degradomes: S. mansoni (Platyhelminthes, Trematoda), C.
sinensis (Platyhelminthes, Trematoda), and Echinococcus
multilocularis (Platyhelminthes, Cestoda). After eliminating the
redundant sequences, the total numbers of known or putative proteases
in the four parasite species were as follows: 262 (S. japonicum), 269
(S. mansoni), 212 (E. multilocularis, the lists were abstracted from
the genome, supplementary table 13.9 ) and 244 (C. sinensis)
(See more details in Table S3). Except C. sinensis, the proportion
of protease classes in each of the parasites was largely in harmony
with each other (Figure 1). A significant expansion was observed in
the relative proportion of aspartic proteases in C. sinensis
Proteases with predicted
Proteases with predicted
Figure 1. Proportions of each protease class in the degradomes of four parasites. S. japonicum (262 proteases), S. mansoni (269 proteases),
E. multilocularis (212 proteases), and C. sinensis (244 proteases).
compared to that of the other three species, which was mainly due
to A01 family (41 members of the A01 family in C. sinensis,
compared to five genes in S. japonicum, seven in S. mansoni, and
only one in E. multilocularis). On the contrary, the number of
serine proteases and metalloproteases was obviously reduced in C.
sinensis (the first was S09 family of serine proteases and M08
family of metalloproteases). Although the proportion of protease
classes was largely similar between Schistosoma species and E.
multilocularis, there were several protease families exhibiting
significant difference, such as C01, M08 and M13 family (See
more details in Table S3).
We further performed comparative analysis of the degradomes
between parasitic flatworms and human host (the degradome of
human was abstracted from the mammalian degradome database
). There are 54 common protease families among the four
flatworms, of which 53 families are shared between flatworm and
human. Notably, one protease family (C83) is obviously exclusive
to these four parasitic flatworms and may be ideal anti-parasite
targets for the future investigation. Moreover, we performed
BLASTp searches against the non-redundant protein sequences
(nr) database of S. mansoni (taxid: 6183) and H. sapiens (taxid:
9606) on NCBI using the S. japonicum protease sequences as
query sequences. The results indicated that the majority of
schistosome proteases shared relatively low sequence identity with
their homologous counterparts in H. sapiens. More importantly,
some crystal structures of these human proteases have been
resolved, which lays good foundation for the future screening of
compounds selectively inhibiting the parasite proteases based on
structural disparity (Table S2). Meanwhile, there were several
schistosome proteases sharing relatively high sequence identity
with their counterparts in H. sapiens, such as O-sialoglycoprotein
endopeptidase (Sjp_0083850, M22 family), methionine
aminopeptidase 2 (ACU78097.1, M24 family) and 20S proteasome
subunits (T01 family) (Table S2). It might suggest that these
proteases evolve slowly at the sequence level, and still retain their
functions. In addition, we found that a few long S. japonicum
Figure 2. Gene ontology (GO) distributions for the S. japonicum degradome. The Blast2Go program defined the GO terms using three
categories: (A) biological processes, (B) molecular functions, and (C) cellular component.
protease sequences aligned with more than one short
nonredundant S. mansoni protease sequence in different sequence
positions, and vice versa. For example, the S. japonicum protease
sequence (AAW26282.1) aligned with the S. mansoni protease
sequence (Smp_061510.1) from the first amino acid to amino acid
570 (identity 83%), and the protease sequence (Smp_155220) from
amino acid 579 to the last amino acid (identity 84%) (Figure S1).
Thus, some short schistosome protease sequences may belong to
the same protease sequence, considering the majority of proteases
were predicted protein sequences based on schistosome genome
sequences. The comparative analysis of sequences in the two
Schistosoma species will further improve the quality of genomes
and proteomes in the future .
Gene ontology analysis of the S. japonicum degradome
Gene ontology (GO) analysis was performed to summarize and
explore the functional categories of the S. japonicum degradome in
this study. A total of 240 of the 262 S. japonicum protease
sequences were annotated with GO terms in three independent
categories: biological processes (210 protease sequences),
molecular functions (231 protease sequences), and cellular components
(78 protease sequences) (Table S4). The biological processes
analysis (level 2) showed that the predominant proteases (203,
56%) were involved in the response to metabolic processes
(Figure 2A). In the case of molecular functions (level 3), the
majority of proteases were annotated with hydrolase activity (208,
60%), which is in consistent with the molecular role of proteases in
proteolysis (Figure 2B). Notably, proteases with the ion binding
term (63, 18%) may be metal ion-dependent enzymes.
Interestingly, only 78 proteases were annotated with GO terms in the
cellular components analysis (level 3). Twenty-one proteases (22%)
were components of protein complexes, including 14 proteases
that were subunits of the proteasome core complex (Figure 2C,
Global expression profiles of the S. japonicum
degradome at four developmental stages
We used an oligonucleotide microarray to measure the patterns
of expression for the obtained proteases at four developmental
stages in S. japonicum. After BLAST searching, 257 of 262
protease sequences were aligned with sequences used for our
microarray design. Using a fluorescence signal value of 100 as the
cut-off value, we ultimately extracted 247 protease sequences with
probes that produced detectable signals in microarray analysis; the
cathepsin B, Antigen Sj31
lysosomal Pro-Xaa carboxypeptidase
lysosomal Pro-Xaa carboxypeptidase
transmembrane protease, serine 6
lysosomal aspartic protease precursor
ubiquitin carboxyl-terminal hydrolase 2
ubiquitin-specific peptidase 24
other 10 sequences maybe pseudogenes or expressed in other
developmental stages or conditions (See supplementary Table S5
for all the protease gene expression data extracted from our
microarray). Using fold change cut-off value $2 between any two
developmental stages and p-value ,0.05 (one tailed Students
ttest), a total of 136 differentially expressed protease genes were
identified in the four developmental stages and 47 proteases in
sexual distinction (Table S6). Hierarchal clustering was used to
investigate the transcriptional patterns of proteases in the four
developmental stages. The heat map showed that three major
transcriptional patterns were closely related to the developmental
stages: pattern I, genes were significantly up-regulated in the
schistosomula and adult stages (the mammalian host dwelling
schistosome life-stages); pattern II, genes were predominantly
expressed in the egg stage; and pattern III, genes were highly
expressed in the cercaria stage (Figure 3 and S2). Examples of
protease genes with different expression patterns are presented in
Table 2. Proteases with different expression patterns may be
closely bound up with the host-parasite interaction. For instance,
the S. japonicum elastase gene (SjCE, ACR27083.1/
Sjp_0028090), which has been validated to be important for
cercariae in host skin invasion , was clustered into expression
pattern III (Table 2). After invading the mammalian host, the
blood flukes growth, development, and reproduction were
dependent on the acquisition of nutrients from the host
bloodstream . Over the past few decades, an increasing
number of proteases of different classes have been ascribed roles in
host protein digestion in schistosomes (reviewed in references
[9,37]), including cysteine protease legumain , cathepsin B, C
and L, aspartic protease cathepsin D, and metalloprotease leucine
aminopeptidase. The majority of proteases mentioned above were
clustered into expression pattern I (Table 2, Figure S2). The
pairwise comparative analysis of gene expression provided detailed
information for the identification of proteases related to
schistosome developmental biology and host-parasite interactions. In
addition, a set of stably expressed proteases genes was extracted
from the analyses. By setting 1.5% as the cut-off value for the
coefficient of variation, 33 constantly expressed protease genes
were identified, and the majority were abundantly transcribed
among the four developmental stages or between adult males and
females (Figure 4). These constantly expressed protease genes may
play fundamental roles in the life cycle of blood flukes, but only the
Figure 4. Constantlyexpressed S. japonicum protease genes in four developmental stages were identified by microarray analysis.
The heat map shows the fluorescent intensity values for the 33protease genes with the lowest coefficient of variation (1.5%) among the four
developmental stages (E, eggs; C, cercariae; S, hepatic schistosomula; A, adult worm pairs; M, adult male worms; F, adult female worms). Each of the
stages contained three biological replicates.
enzymes that are unique to the parasite may serve as potential
targets for anti-schistosomal drugs. For example, the proteasome,
multisubunit enzyme complex, plays a key role in non-lysosomal
protein degradation. The S. mansoni proteasome subunit beta
type 6 (Smp_034490) has been predicted to be a possible drug
target using an in silico approach . Several proteasome
inhibitors have been developed and evaluated in clinical trials as
anticancer drugs , which may be used to study on anti-parasite
in future. In mammals, the 26S proteasome contains a
barrelshaped proteolytic core complex (the 20S proteasome) and two
19S regulatory cap subunits. The 20S proteasome is composed of
a four-ring arrangement of alpha and beta subunits. Mammals
have seven alpha and seven beta proteasome subunits, and all of
the counterparts were identified in S. japonicum (Table S2) as the
main part of threonine proteases. With the exception of
proteasome subunit alpha 4, the subunits were found to be stably
transcribed (Figure 5A). Using S. japonicum PSMD4 (one of 26S
proteasome non-ATPase regulatory subunits) as a reference gene
for qRT-PCR validation, the relative expression levels of two
selected subunit genes correlated well with the microarray results
(Figure 5B). The COP9 signalosome (CSN) is a conserved
multiprotein complex typically consisting of eight subunits
(CSN1CSN8) that plays a crucial role in the
ubiquitinproteasome-mediated protein degradation pathway by regulating
the activity of E3-cullin RING ubiquitin ligases (CRLs) . As a
multifunctional subunit in the CSN complex, CSN5 not only
functions as the catalytic center for CSN isopeptidase activity, but
also independently participates in important biological functions
. Two CSN subunit genes with metalloprotease motifs (CSN5,
Sjp_0052560 and CSN6, Sjp_0109020) were also among the
constantly expressed protease genes in S. japonicum. All of the
results suggest that the schistosome proteases involved in the
ubiquitin-proteasome pathway may be considered potential
antischistosomal intervention targets in future research.
Phylogenetic analysis and global expression profiling of
the schistosome cathepsin gene families
Proteases frequently function not only as individual enzymes,
but also in cascades or networks. A notable evolutionary switch
occurred in one such protease network involved in protein
digestion in the intestine . In vertebrates, serine proteases of
the trypsin family are mainly responsible for the work, whereas
cysteine proteases of the papain family and aspartic proteases
assume the role in invertebrates . The cathepsins of blood flukes
are thought to be the main proteases involved in the digestion of
host blood proteins [37,43]. The members of the cathepsin
families characterized in Schistosoma species currently include
cathepsin B (SjCB1, SmCB1, SjCB2, and SmCB2) ,
cathepsin C (SjCC and SmCC), cathepsin L (SjCL1/SjCF,
SmCL1/SmCF, SmCL2, and SmCL3) , and cathepsin D
(SjCD and SmCD). Although such a multienzyme network in
invertebrate intestinal protein digestion has been validated using a
combination of protease class-specific inhibitors and RNA
interference in S. mansoni, the precise proteolytic cascade or
network involving multiple proteases has not yet been determined
definitively . To identify the potential members of the
schistosome cathepsin family, we used the above published
schistosome cathepsin genes and H. sapiens cathepsin A gene
(4CI9_A) as query sequences to perform a BLASTp search against
our non-redundant degradome database. A total of 21 cathepsin
genes were identified in the S. japonicum degradome and 20 genes
in S. mansoni. The presence of cathepsin conserved domains
(cd02620, cd02248, cd05490, cd05485, pfam00450 and
pfam08773) was confirmed in all of the sequences using the
conserved domain database on NCBI, and 18 S. japonicum
cathepsins were predicted with signal peptides using the SignalP
4.1 server (Table S2). To examine the phylogenetic relationships
among the cathepsin genes in the two Schistosoma species, we
constructed a phylogenetic tree by aligning the 41 full-length
schistosome cathepsin protein sequences using the
neighborjoining method in MEGA 5.0. The phylogenetic analysis showed
that the schistosome cathepsin gene family can be divided into
three classes of proteases (C01 family of cysteine proteases, A01
family of aspartic proteases, and S10 family of serine proteases)
and five main kinds of cathepsins (cathepsin A, B, C, D, and L)
Next, we systematically analyzed the expression profile of the S.
japonicum cathepsin family in the four developmental stages. As
expected, the majority of these proteases were expressed primarily
in schistosomula and adult worms, which is consistent with their
roles in the digestion of host blood proteins (Figure 7A). The gene
expression patterns detected by qRT-PCR for 16 selected S.
japonicum cathepsin genes were generally consistent with the
microarray results and could be further classified into several
different expression patterns (Figure 7B). Eight of the 16 cathepsin
genes were developmentally expressed from egg to adult worm
(higher in adult female worms than adult male worms; Figure 7B).
Among the eight cathepsin genes, schistosome cathepsin B1 (SjCB1,
P43157.1 and SmCB1, P25792.1/Smp_103610), schistosome
cathepsin C (SjCC, AAC32040.1 and SmCC, Q26563.1/
Smp_019030), schistosome cathepsin L (SjCL2, AAW25326.1
and SmCL2, CAA83538.1/Smp_193000), and schistosome
cathepsin D (SjCD, AAC37302/CAX72323.1 and SmCD,
AAB63442/Smp_013040.2) have been shown to be important for
host hemoglobin digestion [9,37]. Notably, SjCD (CAX72323.1)
was also highly expressed in eggs, in contrast to SjCB1, SjCC, and
SjCL2, which were expressed at very low levels in eggs, if at all
(Figure 7B, Table S5). Interestingly, we found that one cathepsin
gene (Sjp_0049310) was highly expressed in the cercaria stage
compared to the other three stages, two genes (Sjp_0027020 and
CAX70514.1) were predominantly expressed in female worms, and
three genes (AAW25775.1, AAO59414.2, and AAW24518.1) were
strongly expressed in male worms (Figure 7B).
Why is there such a discrepancy in expression profiles among
the schistosome cathepsins? The answer to this question will shed
new light on the functions of parasite cathepsins, which is crucial
for understanding parasite virulence and adaptation . The
different expression patterns of S. japonicum cathepsins among the
developmental stages implied that schistosome cathepsins may be
involved in diverse functions and biological processes. The
cathepsin L protease family, as one of important protease families
for parasites, has been extensively studied, especially in Fasciola
species whose cathepsin L proteases have undergone a great
expansion . Herein, we constructed a phylogenetic tree using
the protein sequences of schistosome cathepsin L and their
homologous sequences characterized in other flatworms. The
result showed that these proteases fell into two phylogenetic clades:
three schistosome cathepsin Ls (cathepsin L1, L2 and L3) were
allied closely in the first clade of the phylogenetic tree with other
flatworm cathepsin Ls, and the others formed the second clade
alone (Figure S3). The previous studies revealed that the cathepsin
L proteases in the first clade were mainly presented in the
excretory/secretory products of the adult worms, such as SmCL3
, FhCL1 and FhCL2 [50,51], CsCL1 , and EmCL1 
which may contribute to the network of proteases involved in
digestion of host proteins as nutrients. Notably, two S. japonicum
cathepsin Ls (Sjp_0027020 and CAX70514.1) in the second clade
were significantly up-regulated in adult females which may be
related to reproduction.
Skin penetration, facilitated by cercarial secretions, is the initial
event in infection of the mammalian hosts by Schistosoma species.
Understanding the molecular and biochemical mechanisms of
parasite invasion could provide a theoretical basis for rational
vaccine and drug development. Previous studies from other groups
revealed distinct invasion strategies among schistosome blood
flukes [9,54,55]. In S. mansoni, cercarial elastases play essential
roles in host skin invasion, and multiple elastases have been
identified in genome-wide analyses . However, S.
japonicum may mainly utilize a papain-like cysteine protease to
facilitate host invasion [58,59]. Schistosome cathepsin B2 has been
shown to degrade multiple host skin proteins, and S. japonicum
has 40-fold greater cathepsin B activity in cercarial secretions than
S. mansoni . We found that S. japonicum cathepsin B2
(SjCB2, AAO55414.2) exhibits a distinct expression pattern
compared to SjCB1 (P43157.1), which plays a key role in the
digestion of host hemoglobin (Figure 7B). Although the relative
expression of SjCB1 was the lowest in cercariae according to
qRTPCR analysis, the signal produced in the microarray analysis of
cercariae was very strong (Table S5). SjCB1 and cathepsin B
isoforms (AAW26625.1 and CAX70257.1) were also detected via
strong hybridization signals in the microarray analysis of the
cercaria stage (Table S5). These S. japonicum cathepsin B isoforms
may be also involved in host skin invasion, in addition to their roles
in host protein digestion. Defining the roles of these major
enzymes will not only provide a clearer understanding of the
functions of the complex parasite protease network, but also
provide insights into which of these proteases are logical targets for
the development of chemotherapy for parasitic diseases [49,60].
The schistosome stage- and gender-specific or
predominantly expressed proteases
Notably, most attention has been focused on the schistosome
aspartic and cysteine proteases that assist worms in obtaining
nutrients from the host. Except the cercarial elastase serine
proteases, which facilitate host invasion by infective schistosome
larvae, few serine proteases have been identified and characterized
in the past few years . Recently, two trypsin-like serine
proteases of the S01 family (Smp_030350 and Smp_103680) were
shown to be predominantly expressed in S. mansoni eggs , and
their counterparts (CAX73257.1 and Sjp_0012180) in S.
japonicum were found to have similar expression patterns in our research
(Table 2, Figure S2). Remarkably, we found that several serine
proteases have similar expression patterns as those involved in host
protein digestion. For instance, S. japonicum cathepsin A
(CAX69725.1, S10 family), also known as carboxypeptidase C,
had a similar expression pattern as SjCB1 (P43157.1), SjCC
(AAC32040.1), and SjCL2 (AAW25326.1) (Figure 7B). Two
lysosomal Pro-Xaa carboxypeptidases (CAX71062.1 and
Sjp_0085250, S28 family), which can hydrolyze carboxy-terminal
amino acids linked to proline in peptides, had a similar expression
pattern as SjCD (CAX72323.1 and AAW24549.1) (Table S5). It
will be engrossing to determine whether these serine proteases are
members of the multienzyme network involving in host protein
digestion by schistosome parasite.
In the complex lifecycle of schistosomes, the adult females
pairing with adult males finally reside in the mesenteric or bladder
circulation, where they produce infectious eggs. The majority of the
eggs trapped in the host tissues, resulting in serious granulomatous
reactions and fibrosis, are the major cause of pathology in
schistosomiasis; the others eliminated into the environment with
the host feces or urine are responsible for lifecycle progression .
As eggs play central roles in the pathology of schistosomiasis and
transmission of the blood fluke, understanding the female
reproductive biology and egg development could lead to novel strategies
Figure 8. Expression analysis of six stage- and gender-specific or predominantly expressed genes using qRT-PCR. The expression was
validated in the four developmental stages (E, eggs; C, cercariae; S, hepatic schistosomula; M, adult male worms; F, adult female worms) by qRT-PCR
analysis. The relative expression levels of genes were calculated using SDS v1.4 software (Applied Biosystems). The error bars represent the standard
deviation for three technical replicates.
for combating schistosomiasis. Two S. mansoni tyrosinases
specifically expressed in adult female worms have been shown to be
critical for egg formation and production . Three S. japonicum
serine proteases (CAX69683.1, AAW25748.1, and CAX73292.1)
were found to be specifically or abundantly expressed in adult
female worms in microarray and qRT-PCR analyses (Figure 8,
Table S5). The S. japonicum serine protease (CAX69683.1, S33
family) was annotated as putative lysosomal acid lipase (LAL) or
cholesterol esterase. LAL plays a critical role in the hydrolysis of
triglycerides and cholesterolesters, and LAL deficiency in humans
leads to two phenotypes, cholesterolester storage disease and
Wolman disease . Fatty acid oxidation (FAO) is essential for
schistosome egg production, which is consistent with the finding that
fecund female worms possess abundant fat reserves, whereas virgin
females have significantly lower lipid stores . Meanwhile,
genome-wide analysis of the metabolic pathway reveals that
schistosomes can not de novo synthesize fatty acids or sterols, and
the parasite genome certainly encodes multiple transporters and
lipases to exploit fatty acids and cholesterol from the hosts . As
S. japonicum LAL (SjLAL, CAX69683.1) was significantly
upregulated in fecund female worms, and the expression pattern
coincided with the previous FAO finding in schistosomes, SjLAL
may be critical for female reproduction and the biological function
of this protease needs to be investigated further.
The other two female-specific or highly expressed proteases
(AAW25748.1 and CAX73292.1, S01 family) were annotated as
trypsin-like serine proteases in S. japonicum. In the genetic model
Drosophila melanogaster, female reproductive tract proteins play
essential roles in sexual reproduction, and five mating-induced
serine proteases expressed in the female reproductive tract have
been identified using EST collections and microarray analyses
; the two S.japonicum trypsin-like serine proteases share the
same conserved domain (cd00190) with these mating-induced
serine proteases. Thus, the function of the two schistosome
proteases may also be associated with sexual reproduction and
could serve as new potential anti-schistosomal intervention targets.
As one of the most expanded gene families in schistosomes
compared to their mammalian hosts, the M8 family of
metalloproteases may yield new and valuable insights about the
requirements for a parasitic lifestyle . This family is composed
of leishmanolysins (also called invadolysins), which were first
reported in the protozoan parasite Leishmania. Fourteen putative
M8 family members have been identified in the S. japonicum
genome. This family includes important surface proteases of
parasitic protozoa that play critical roles in the degradation of host
extracellular matrix proteins to facilitate tissue or cell invasion
. The majority of leishmanolysins identified in our microarray
were egg-enriched or cercaria-enriched genes (Table S5). Three
egg-specific or predominantly expressed leishmanolysin genes
(CAX73243.1, CAX75587.1, and CAX75591.1) were further
validated by qRT-PCR (Figure 8). Analysis of S. mansoni cercarial
secretions showed that leishmanolysin, now annotated in the
genome as invadolysin, ranked second only to cercarial elastase as
the most prominent component . All of the
cercaria-upregulated leishmanolysins (Sjp_0048370, Sjp_0067490, and
Sjp_0067500) (Table 2 and S5) have high homology (identity $
65%) with S. mansoni invadolysins (Smp_153930, Smp_090100
and Smp_090110), which Parker-Manuel et al found to be
significantly up-regulated in intramolluscan germ balls .
Numerous proteins utilized by the cercaria for host invasion have
been suggested to be expressed during the development of germ
balls in the snail . Thus, it is tempting to speculate that
leishmanolysin (invadolysin) may also contribute to tissue invasion
by schistosome cercariae, besides cercarial elastase and cathepsin
B. These egg-up-regulated leishmanolysins may also play vital
roles in the release of eggs from host tissues or the hatching of
miracidia from eggs. Therefore, leishmanolysin inhibition could
serve as a novel intervention strategy for schistosomiasis. All of the
suppositions need to be validated by experiments, which will
contribute to the determination of protease functions and further
improve the development of novel intervention strategies for
The present study presents the most comprehensive analysis of
degradomes in Schistosoma species to date. A total of 262 S.
japonicum proteases were identified and the global expression
profile at four developmental stages was obtained by microarray
analysis. The proteases can be divided into four clusters according
to the transcriptional pattern: proteases significantly up-regulated
in schistosomula and adult stages, proteases highly expressed in the
cercaria stage, proteases predominantly expressed in the egg stage,
and proteases constantly expressed among the four developmental
stages. Numerous potential anti-schistosomal targets were
identified with the expression profile information, including cathepsin A,
trypsin-like serine proteases, lysosomal Pro-Xaa
carboxypeptidases, lysosomal acid lipase, leishmanolysins, and the 20S proteasome.
Although the functions of schistosome proteases remain largely
unknown, and many experiments are needed to determine their
precise functions, our analysis of the S. japonicum degradome
establishes a firm foundation for future research on the specific
function(s) of individual proteases or protease families and may
help refine anti-proteolytic strategies in blood flukes.
Figure S2 Three clusters of protease genes with different
expression patterns among four developmental stages (E, eggs;
C, cercariae; S, hepatic schistosomula; A, adult worm pairs). I,
genes significantly up-regulated in the schistosomula and adult
stages; II, genes abundantly expressed in the egg stage; III, genes
highly expressed in the cercaria stage. The color scale represents
relative expression levels, with red as up-regulated, green as
downregulated, and black as unchanged.
Figure S3 Phylogenetic relationships between cathepsin L
proteases of Schistosoma species, Fasciola species, C. sinensis and
Echinococcus species. The unrooted phylogenetic tree was
constructed using MEGA 5.0 and the neighbor-joining method
with 1000 bootstrap replicates. The bootstrap values are shown at
the nodes. SjCL, S. japonicum cathepsin L; SmCL, S. mansoni
cathepsin L; FhCL, Fasciola hepatica cathepsin L; FgCL, Fasciola
gigantica cathepsin L; EgCL, Echinococcus granulosus cathepsin
L; EmCL, Echinococcus multilocularis cathepsin L; CsCL, C.
sinensis cathepsin L. The color bars represent the relative
expression levels of the S. japonicum cathepsin Ls in the four
developmental stages, with red as up-regulated and green as
downregulated. E, eggs; C, cercariae; S, hepatic schistosomula; M, adult
male worms; F, adult female worms.
List of primers used for qRT-PCR analysis.
Comprehensive information of the S. japonicum
Blast2Go annotation details of S. japonicum protease
We appreciate very much the efforts of technicians at CapitalBio for
microarray performance. We also thank the Schistosoma japonicum
Genome Sequencing and Functional Analysis Consortium for making
the S. japonicum genome available on the public domain.
Conceived and designed the experiments: SL QC. Performed the
experiments: SL PC XP NH XZ CW. Analyzed the data: SL QC.
Contributed reagents/materials/analysis tools: HW. Wrote the paper: SL
1. Gray DJ , McManus DP , Li Y , Williams GM , Bergquist R , et al. ( 2010 ) Schistosomiasis elimination: lessons from the past guide the future . Lancet Infect Dis 10 : 733 - 736 .
2. Doenhoff MJ , Cioli D , Utzinger J ( 2008 ) Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis . Curr Opin Infect Dis 21 : 659 - 667 .
3. Melman SD , Steinauer ML , Cunningham C , Kubatko LS , Mwangi IN , et al. ( 2009 ) Reduced susceptibility to praziquantel among naturally occurring Kenyan isolates of Schistosoma mansoni . PLoS Negl Trop Dis 3 : e504 .
4. Caffrey CR ( 2007 ) Chemotherapy of schistosomiasis: present and future . Curr Opin Chem Biol 11 : 433 - 439 .
5. Deu E , Verdoes M , Bogyo M ( 2012 ) New approaches for dissecting protease functions to improve probe development and drug discovery . Nat Struct Mol Biol 19 : 9 - 16 .
6. Turk B ( 2006 ) Targeting proteases: successes, failures and future prospects . Nat Rev Drug Discov 5 : 785 - 799 .
7. McKerrow JH , Caffrey C , Kelly B , Loke P , Sajid M ( 2006 ) Proteases in parasitic diseases . Annu Rev Pathol 1 : 497 - 536 .
8. Renslo AR , McKerrow JH ( 2006 ) Drug discovery and development for neglected parasitic diseases . Nat Chem Biol 2 : 701 - 710 .
9. Kasny M , Mikes L , Hampl V , Dvorak J , Caffrey CR , et al. ( 2009 ) Chapter 4. Peptidases of trematodes . Adv Parasitol 69 : 205 - 297 .
10. Jilkova A , Rezacova P , Lepsik M , Horn M , Vachova J , et al. ( 2011 ) Structural basis for inhibition of cathepsin B drug target from the human blood fluke, Schistosoma mansoni . J Biol Chem 286 : 35770 - 35781 .
11. Abdulla MH , Lim KC , Sajid M , McKerrow JH , Caffrey CR ( 2007 ) Schistosomiasis mansoni: novel chemotherapy using a cysteine protease inhibitor . PLoS Med 4 : e14 .
12. Zhou Y , Zheng H , Chen Y , Zhang L , Wang K , et al. ( 2009 ) The Schistosoma japonicum genome reveals features of host-parasite interplay . Nature 460 : 345 - 351 .
13. Berriman M , Haas BJ , LoVerde PT , Wilson RA , Dillon GP , et al. ( 2009 ) The genome of the blood fluke Schistosoma mansoni . Nature 460 : 352 - 358 .
14. Young ND , Jex AR , Li B , Liu S , Yang L , et al. ( 2012 ) Whole-genome sequence of Schistosoma haematobium . Nat Genet 44 : 221 - 225 .
15. Webster JP , Oliviera G , Rollinson D , Gower CM ( 2010 ) Schistosome genomes: a wealth of information . Trends Parasitol 26 : 103 - 106 .
16. Quesada V , Ordonez GR , Sanchez LM , Puente XS , Lopez-Otin C ( 2009 ) The Degradome database: mammalian proteases and diseases of proteolysis . Nucleic Acids Res 37 : D239 - 243 .
17. Gryseels B , Polman K , Clerinx J , Kestens L ( 2006 ) Human schistosomiasis . Lancet 368 : 1106 - 1118 .
18. Rawlings ND , Barrett AJ , Bateman A ( 2012 ) MEROPS: the database of proteolytic enzymes, their substrates and inhibitors . Nucleic Acids Res 40 : D343 - 350 .
19. Gotz S , Garcia-Gomez JM , Terol J , Williams TD , Nagaraj SH , et al. ( 2008 ) High-throughput functional annotation and data mining with the Blast2GO suite . Nucleic Acids Res 36 : 3420 - 3435 .
20. Moriya Y , Itoh M , Okuda S , Yoshizawa AC , Kanehisa M ( 2007 ) KAAS: an automatic genome annotation and pathway reconstruction server . Nucleic Acids Res 35 : W182 - 185 .
21. Petersen TN , Brunak S , von Heijne G , Nielsen H ( 2011 ) SignalP 4.0: discriminating signal peptides from transmembrane regions . Nat Methods 8 : 785 - 786 .
22. Krogh A , Larsson B , von Heijne G , Sonnhammer EL ( 2001 ) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes . J Mol Biol 305 : 567 - 580 .
23. Marchler-Bauer A , Anderson JB , Chitsaz F , Derbyshire MK , DeWeese-Scott C , et al. ( 2009 ) CDD: specific functional annotation with the Conserved Domain Database . Nucleic Acids Res 37 : D205 - 210 .
24. Marchler-Bauer A , Lu S , Anderson JB , Chitsaz F , Derbyshire MK , et al. ( 2011 ) CDD: a Conserved Domain Database for the functional annotation of proteins . Nucleic Acids Res 39 : D225 - 229 .
25. Larkin MA , Blackshields G , Brown NP , Chenna R , McGettigan PA , et al. ( 2007 ) Clustal W and Clustal X version 2.0. Bioinformatics 23 : 2947 - 2948 .
26. Tamura K , Peterson D , Peterson N , Stecher G , Nei M , et al. ( 2011 ) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods . Mol Biol Evol 28 : 2731 - 2739 .
27. Dalton JP , Day SR , Drew AC , Brindley PJ ( 1997 ) A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues . Parasitology 115 (Pt 1): 29 - 32 .
28. Liu S , Cai P , Hou N , Piao X , Wang H , et al. ( 2012 ) Genome-wide identification and characterization of a panel of house-keeping genes in Schistosoma japonicum . Mol Biochem Parasitol 182 : 75 - 82 .
29. de Hoon MJ , Imoto S , Nolan J , Miyano S ( 2004 ) Open source clustering software . Bioinformatics 20 : 1453 - 1454 .
30. King JY , Ferrara R , Tabibiazar R , Spin JM , Chen MM , et al. ( 2005 ) Pathway analysis of coronary atherosclerosis . Physiol Genomics 23 : 103 - 118 .
31. Bos DH , Mayfield C , Minchella DJ ( 2009 ) Analysis of regulatory protease sequences identified through bioinformatic data mining of the Schistosoma mansoni genome . BMC Genomics 10 : 488 .
32. Wang X , Chen W , Huang Y , Sun J , Men J , et al. ( 2011 ) The draft genome of the carcinogenic human liver fluke Clonorchis sinensis . Genome Biol 12 : R107 .
33. Tsai IJ , Zarowiecki M , Holroyd N , Garciarrubio A , Sanchez-Flores A , et al. ( 2013 ) The genomes of four tapeworm species reveal adaptations to parasitism . Nature 496 : 57 - 63 .
34. Mitreva M , Jasmer DP , Zarlenga DS , Wang Z , Abubucker S , et al. ( 2011 ) The draft genome of the parasitic nematode Trichinella spiralis . Nat Genet 43 : 228 - 235 .
35. Zheng H , Zhang W , Zhang L , Zhang Z , Li J , et al. ( 2013 ) The genome of the hydatid tapeworm Echinococcus granulosus . Nat Genet 45 : 1168 - 1175 .
36. Swain MT , Larkin DM , Caffrey CR , Davies SJ , Loukas A , et al. ( 2011 ) Schistosoma comparative genomics: integrating genome structure, parasite biology and anthelmintic discovery . Trends Parasitol 27 : 555 - 564 .
37. Caffrey CR , McKerrow JH , Salter JP , Sajid M ( 2004 ) Blood 'n' guts: an update on schistosome digestive peptidases . Trends Parasitol 20 : 241 - 248 .
38. Ovat A , Muindi F , Fagan C , Brouner M , Hansell E , et al. ( 2009 ) Aza-peptidyl Michael acceptor and epoxide inhibitors-potent and selective inhibitors of Schistosoma mansoni and Ixodes ricinus legumains (asparaginyl endopeptidases) . J Med Chem 52 : 7192 - 7210 .
39. Crowther GJ , Shanmugam D , Carmona SJ , Doyle MA , Hertz-Fowler C , et al. ( 2010 ) Identification of attractive drug targets in neglected-disease pathogens using an in silico approach . PLoS Negl Trop Dis 4 : e804 .
40. Moreau P , Richardson PG , Cavo M , Orlowski RZ , San Miguel JF , et al. ( 2012 ) Proteasome inhibitors in multiple myeloma: 10 years later . Blood 120 : 947 - 959 .
41. Wei N , Deng XW ( 2003 ) The COP9 signalosome . Annu Rev Cell Dev Biol 19 : 261 - 286 .
42. Wei N , Serino G , Deng XW ( 2008 ) The COP9 signalosome: more than a protease . Trends Biochem Sci 33 : 592 - 600 .
43. Robinson MW , Dalton JP , Donnelly S ( 2008 ) Helminth pathogen cathepsin proteases: it's a family affair . Trends Biochem Sci 33 : 601 - 608 .
44. Tchoubrieva EB , Ong PC , Pike RN , Brindley PJ , Kalinna BH ( 2010 ) Vectorbased RNA interference of cathepsin B1 in Schistosoma mansoni . Cell Mol Life Sci 67 : 3739 - 3748
45. Caffrey CR , Salter JP , Lucas KD , Khiem D , Hsieh I , et al. ( 2002 ) SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni . Mol Biochem Parasitol 121 : 49 - 61 .
46. Skelly PJ , Da'dara A , Harn DA ( 2003 ) Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference . Int J Parasitol 33 : 363 - 369 .
47. Horn M , Jilkova A , Vondrasek J , Maresova L , Caffrey CR , et al. ( 2011 ) Mapping the Pro-Peptide of the Schistosoma mansoni Cathepsin B1 Drug Target: Modulation of Inhibition by Heparin and Design of Mimetic Inhibitors . ACS Chem Biol 6 : 609 - 617 .
48. Dvorak J , Mashiyama ST , Sajid M , Braschi S , Delcroix M , et al. ( 2009 ) SmCL3, a gastrodermal cysteine protease of the human blood fluke Schistosoma mansoni . PLoS Negl Trop Dis 3 : e449 .
49. Delcroix M , Sajid M , Caffrey CR , Lim KC , Dvorak J , et al. ( 2006 ) A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite . J Biol Chem 281 : 39316 - 39329 .
50. Robinson MW , Corvo I , Jones PM , George AM , Padula MP , et al. ( 2011 ) Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica . PLoS Negl Trop Dis 5 : e1012 .
51. Robinson MW , Tort JF , Lowther J , Donnelly SM , Wong E , et al. ( 2008 ) Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulenceassociated factors . Mol Cell Proteomics 7 : 1111 - 1123 .
52. Li Y , Hu X , Liu X , Xu J , Hu F , et al. ( 2009 ) Molecular cloning and analysis of stage and tissue-specific expression of Cathepsin L-like protease from Clonorchis sinensis . Parasitol Res 105 : 447 - 452 .
53. Sako Y , Yamasaki H , Nakaya K , Nakao M , Ito A ( 2007 ) Cloning and characterization of cathepsin L-like peptidases of Echinococcus multilocularis metacestodes . Mol Biochem Parasitol 154 : 181 - 189 .
54. Ruppel A , Chlichlia K , Bahgat M ( 2004 ) Invasion by schistosome cercariae: neglected aspects in Schistosoma japonicum . Trends Parasitol 20 : 397 - 400 .
55. Doleckova K , Kasny M , Mikes L , Cartwright J , Jedelsky P , et al. ( 2009 ) The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti . Int J Parasitol 39 : 201 - 211 .
56. Ingram JR , Rafi SB , Eroy-Reveles AA , Ray M , Lambeth L , et al. ( 2012 ) Investigation of the Proteolytic Functions of an Expanded Cercarial Elastase Gene Family in Schistosoma mansoni . PLoS Negl Trop Dis 6 : e1589 .
57. Lopez Quezada LA , Sajid M , Lim KC , McKerrow JH ( 2011 ) A blood fluke serine protease inhibitor regulates an endogenous larval elastase . J Biol Chem 287 : 7074 - 7083 .
58. Ingram J , Knudsen G , Lim KC , Hansell E , Sakanari J , et al. ( 2011 ) Proteomic analysis of human skin treated with larval schistosome peptidases reveals distinct invasion strategies among species of blood flukes . PLoS Negl Trop Dis 5 : e1337 .
59. Dvorak J , Mashiyama ST , Braschi S , Sajid M , Knudsen GM , et al. ( 2008 ) Differential use of protease families for invasion by schistosome cercariae . Biochimie 90 : 345 - 358 .
60. Horn M , Nussbaumerova M , Sanda M , Kovarova Z , Srba J , et al. ( 2009 ) Hemoglobin digestion in blood-feeding ticks: mapping a multipeptidase pathway by functional proteomics . Chem Biol 16 : 1053 - 1063 .
61. Horn M , Fajtova P , Rojo Arreola L , Ulrychova L , Bartosova-Sojkova P , et al. ( 2014 ) Trypsin- and Chymotrypsin-Like Serine Proteases in Schistosoma mansoni - 'The Undiscovered Country' . PLoS Negl Trop Dis 8 : e2766 .
62. Fitzpatrick JM , Hirai Y , Hirai H , Hoffmann KF ( 2007 ) Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases . FASEB J 21 : 823 - 835 .
63. Bernstein DL , Hulkova H , Bialer MG , Desnick RJ ( 2013 ) Cholesteryl ester storage disease: review of the findings in 135 reported patients with an underdiagnosed disease . J Hepatol 58 : 1230 - 1243 .
64. Huang SC , Freitas TC , Amiel E , Everts B , Pearce EL , et al. ( 2012 ) Fatty Acid Oxidation Is Essential for Egg Production by the Parasitic Flatworm Schistosoma mansoni . PLoS Pathog 8 : e1002996 .
65. Lawniczak MK , Begun DJ ( 2007 ) Molecular population genetics of femaleexpressed mating-induced serine proteases in Drosophila melanogaster . Mol Biol Evol 24 : 1944 - 1951 .
66. Pina-Vazquez C , Reyes-Lopez M , Ortiz-Estrada G , de la Garza M , SerranoLuna J ( 2012 ) Host-parasite interaction: parasite-derived and -induced proteases that degrade human extracellular matrix . J Parasitol Res 2012 : 748206 .
67. Curwen RS , Ashton PD , Sundaralingam S , Wilson RA ( 2006 ) Identification of novel proteases and immunomodulators in the secretions of schistosome cercariae that facilitate host entry . Mol Cell Proteomics 5 : 835 - 844 .
68. Parker-Manuel SJ , Ivens AC , Dillon GP , Wilson RA ( 2011 ) Gene expression patterns in larval Schistosoma mansoni associated with infection of the mammalian host . PLoS Negl Trop Dis 5 : e1274 .