Functional characterisation of Schistosoma japonicum acetylcholinesterase
You et al. Parasites & Vectors
Functional characterisation of Schistosoma japonicum acetylcholinesterase
Hong You 0 1
Geoffrey N. Gobert 0 1 3
Xiaofeng Du 0 1
Gabor Pali 0 1
Pengfei Cai 0 1
Malcolm K. Jones 1 2
Donald P. McManus 0 1
0 Molecular Parasitology Laboratory, Infectious Diseases Division, QIMR Berghofer Medical Research Institute , Brisbane, Queensland , Australia
2 School of Veterinary Sciences, The University of Queensland , Brisbane, Queensland , Australia
3 School of Biological Sciences, Queen's University Belfast , Belfast , UK
Background: Acetylcholinesterase (AChE) is an important metabolic enzyme of schistosomes present in the musculature and on the surface of the blood stage where it has been implicated in the modulation of glucose scavenging from mammalian host blood. As both a target for the antischistosomal drug metrifonate and as a potential vaccine candidate, AChE has been characterised in the schistosome species Schistosoma mansoni, S. haematobium and S. bovis, but not in S. japonicum. Recently, using a schistosome protein microarray, a predicted S. japonicum acetylcholinesterase precursor was significantly targeted by protective IgG1 immune responses in S. haematobium-exposed individuals that had acquired drug-induced resistance to schistosomiasis after praziquantel treatment. Results: We report the full-length cDNA sequence and describe phylogenetic and molecular structural analysis to facilitate understanding of the biological function of AChE (SjAChE) in S. japonicum. The protein has high sequence identity (88 %) with the AChEs in S. mansoni, S. haematobium and S. bovis and has 25 % sequence similarity with human AChE, suggestive of a highly specialised role for the enzyme in both parasite and host. We immunolocalized SjAChE and demonstrated its presence on the surface of adult worms and schistosomula, as well as its lower expression in parenchymal regions. The relatively abundance of AChE activity (90 %) present on the surface of adult S. japonicum when compared with that reported in other schistosomes suggests SjAChE may be a more effective drug or immunological target against this species. We also demonstrate that the classical inhibitor of AChE, BW285c51, inhibited AChE activity in tegumental extracts of paired worms, single males and single females by 59, 22 and 50 %, respectively, after 24 h incubation with 200 μM BW284c51. Conclusions: These results build on previous studies in other schistosome species indicating major differences in the enzyme between parasite and mammalian host, and provide further support for the design of an anti-schistosome intervention targeting AChE.
Schistosoma japonicum; Acetylcholinesterase; Drug or vaccine target
Schistosomiasis remains one of the most insidious and
serious of the tropical parasitic diseases of clinical and
public health significance. Currently, there is no effective
vaccine to prevent schistosomiasis [
] and treatment is
dependent on praziquantel chemotherapy. Previous
reports showed that human schistosomiasis could be
treated using the drug metrifonate [
], which can disrupt
the cholinergic system and neuromuscular signalling by
targeting acetylcholinesterase (AChE). Metrifonate was,
however, withdrawn from the market because of
unacceptable toxicity to the host and variable efficacy
against different schistosome species [
During the blood dwelling stages of schistosomes,
acetylcholinesterase (AChE) is present on the parasite
tegument membrane [
] and in the musculature [
both in adults and schistosomula. A previous study
implicated schistosome AChE in regulating glucose
scavenging from the host [
]. It has been shown that the basal
rate of glucose uptake in adult Schistosoma
haematobium and S. bovis is about twice that in S. mansoni [
Indicative of the higher metabolic requirements for
glucose in S. haematobium and S. bovis, relatively higher
amounts of AChE activity are present on their
teguments compared with S. mansoni [
]. These higher
levels of AChE activity result in the recorded higher
susceptibility to metrifonate [
]. It has also been shown that
S. mansoni AChE antibodies can lead to efficient
complement-mediated killing of schistosomula in vitro
]. Importantly, the absence of cross-reactivity with
human AChE further supports schistosome AChE as a
suitable target for immunological attack [
AChE has been characterised from S. mansoni, S.
haematobium and S. bovis [
], but not in S. japonicum.
Recently, using a schistosome protein microarray, a predicted
S. japonicum acetylcholinesterase precursor (AY810792)
was significantly targeted by protective IgG1 immune
responses in S. haematobium-exposed individuals that had
acquired drug-induced resistance to schistosomiasis after
praziquantel treatment . This observation further
supports consideration of S. japonicum AChE (SjAChE) as a
suitable vaccine candidate against schistosomiasis.
The interaction between acetylcholine (ACh) and its
receptor, the nicotinic acetylcholine receptor (nAChR),
results in the opening of the ion channel in mammalian
]. Schistosome AChE plays an important role in
limiting this interaction as the inhibition of AChE mimics
ligand excess and causes receptor desensitisation [
has been shown that circulating concentrations of ACh
can result in an increase in glucose uptake in schistosomes
in vitro, and this effect is ablated in the presence of
antiacetylcholinesterase antibodies [
]. Furthermore, the
influence of acetylcholine on glucose uptake in these worms
can be modulated through inhibition of either tegumental
AChE or nAChR [
]. nAChRs are ligand-gated ion
channels within the nervous system that mediate the excitatory
responses to acetylcholine. Three types of acetylcholine
receptors have been identified in S. haematobium:
ShAR1α (AY392150) [
], ShAR1β (AY392151) 
and ShAR2β [
]. It has been demonstrated that ShAR1α
is located on the parasite surface and may contribute to
the potentiation of the uptake of glucose from the host
blood in response to circulating concentrations of ACh.
As the first step in determining the functional
characteristics of AChE from S. japonicum, we present the
isolated full-length sequence of the protein from this
schistosome species, describe the distribution of the
enzyme in schistosomula and adult worms, and show that
the classic inhibitor of BW284c51 effectively suppresses
AChE activity in adult worms in vitro.
Schistosoma japonicum adult worms were collected by
perfusion of female ARC Swiss mice infected
percutaneously with 60 cercariae of S. japonicum (Anhui
population, mainland China) shed from Oncomelania
hupensis hupensis snails as described [
]. In order to
obtain schistosomula, cercariae were passed through a
22gauge emulsifying needle 25 times to mechanically shear
the cercarial tails from the bodies. The resulting larvae
were separated from the free tails by centrifugation,
washed three times with a modified Basch’s medium [
and incubated at 37 °C under a 5 % CO2 atmosphere
Cloning S. japonicum AChE
A Qiagen RNeasy kit (Qiagen, Hilden, Germany) was
used to purify total RNA from adult S. japonicum. A one
step RT-PCR (Qiagen) kit was employed to amplify
specific cDNA. Based on the conserved sequences of AChE
in S. mansoni (AF279461), S. haematobium (AF279462)
and S. bovis (AF279463), and partial S. japonicum
sequences available at http://www.genedb.org/Homepage/
Sjaponicum, four pairs of primers for SjAChE were
designed (Table 1) to obtain the full-length cDNA
sequence to PCR amplify the full-length sequence of
SjAChE using an overlap strategy.
Sequence and phylogenetic analysis
Searches for homologous acetylcholinesterase protein
sequences were performed using BLAST on the NCBI web
site (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the
WormBase ParaSite web site (http://parasite.wormbase.org/
Multi/Tools/Blast). Phylogenetic analysis was performed
using online resources
] by uploading the set of available AChE
sequences from the different species presented. Molecular
weight and isoelectric point determinations were
performed using the ExPASy-Compute pI/Mw tool (http://
web.expasy.org/compute_pi/). The PHYRE2 protein fold
recognition server (http://www.sbg.bio.ic.ac.uk/phyre2/)
was used to generate the three-dimensional (3D) model of
] and binding site predictions were carried out
using the 3DLigandSite
(Bio-Rad, Mississauga, Canada). Data are presented as
antibody endpoint titres, defined as the highest dilution
of test serum that yielded an average O.D. two standard
deviations (SDs) greater than that obtained in the
absence of primary antibody.
Protein expression, purification and antibody generation
A C-terminal fragment of SjAChE (from Q465 to V680,
named SjAChEC) was amplified and cloned into the
pET28b vector (Novagen, Madison, USA), by using
forward (5′-CGG GAT CCT CAG TTG CCG ACA CTT
GAA AGT TGG A-3′ with BamHI restriction site
underlined) and reverse (5′-CGC TCG AGC ACG CCT
AAA CAA TGC TGA CGA TTA CG-3′ with XhoI
restriction site underlined) primers. The reconstructed
vector was then transformed into Escherichia coli (BL21
strain) for expression induced with 1 mM IPTG
(isopropyl thio-b-D-galactoside) at 37 °C for 3 h. Recombinant
protein was purified from inclusion bodies by
chromatography using a Ni-NTA His-tag affinity kit (Novagen)
under denaturing conditions using 6 M guanidine
according to the manufacturer’s instructions.
Antibodies were raised against the SjAChEC fusion
protein in a rabbit at the South Australian Health and
Medical Research Institute (SAHMRI). Briefly, the rabbit
was immunized three times each with 500 μg
recombinant protein at three week intervals. Based on the fact
that complete Freund’s adjuvant is the most effective
adjuvant available for consistently producing high titer
antibodies to diverse antigens, we used complete
Freund’s adjuvant in the initial injection, but in the
subsequent two used incomplete Freund’s adjuvant. The
injections were delivered subcutaneously at multiple sites
along the neck and spine. Blood was collected two weeks
after the final boost. The titre of the antibody was
determined using an enzyme-linked immunosorbent assay
(ELISA). Briefly, Maxisorb immunoplates (Nalge Nune
International, USA) were coated overnight at 4 °C with
rSjAChE protein (100 μl of 0.5 μg/ml) in coating buffer
(100 μl/well). After three washes with 0.05 % (v/v)
Tween in PBS (PBST), wells were blocked with 200 μl of
5 % (v/v) skim milk in PBS (SMP) and incubated for 1 h
at 37 °C. The rabbit anti-SjAChE serum was serially
diluted (from 1:200 to 1:102,400) in SMP and 100 μl in
duplicate of each dilution were added to individual wells.
After incubation at 37 °C for 1 h, the wells were washed
with PBST (3X) and 100 μl (1:2,000 dilution) of
horseradish peroxidise (HRP)-conjugated goat anti-rabbit IgG
(Invitrogen) was added. After incubation at 37 °C for
1 h, the wells were washed with PBST (5X), 100 μl of
substrate solution [2,2-azino-di-(ethyl-benzithiozolin
sulfonate)] (Sigma, Castle Hill, Australia) was added and
the wells were incubated at room temperature and read
on a plate reader by using Microplate manager software
Western blot analysis
The rabbit anti-SjAChEC serum was used in Western
blotting to probe to the electrophoresed purified
recombinant SjAChEC protein and the native SjAChE protein
in a separated crude S. japonicum antigen extract. The
crude antigen was prepared from adult worms of S.
japonicum freshly perfused from mice percutaneously
infected with 60 cercariae six weeks previously. After
three washes in perfusion buffer (8.5 g NaCl and 15 g
NaCitrate in 1 l of water), to minimise contamination of
the schistosome protein extract with host components,
an adult worm antigen preparation (SWAP) was made
as described [
]. The recombinant SjAChEC and
SWAP samples were separated on a 15 % (w/v)
SDSPAGE gel and transferred to an Immun-Blot® low
fluorescence-PVDF membrane. Overnight blocking was
performed with Odyssey buffer at 4 °C. Then, the
membrane was subjected to incubation with the rabbit
antiSjAChE anti-serum (1:100 dilution in Odyssey buffer
and 0.1 % Tween-20) for 1 h followed by incubation
with IRDye-labeled 680LT goat anti-rabbit IgG antibody
(Li-COR Biosciences) (1:15,000 diluted in Odyssey buffer
with 0.1 % Tween-20 and 0.01 % SDS) for 1 h on a
shaker in a dark chamber. After a final wash with
distilled water, the membrane was allowed to dry in the
dark and visualized using the Odyssey® CLx Infrared
Imaging System [
Adult S. japonicum
Horseradish peroxidise (HRP) labelling was used for the
immunolocalisation of SjAChE in adult S. japonicum.
Freshly perfused male and female worms were fixed in
100 % methanol, embedded in Tissue-Tek Optimal
Cutting Temperature (OCT) compound (ProSciTech,
Queensland, Australia), and 7.0 μm cryostat sections
produced. The HRP labelling was performed according
to standard procedures [
]. The primary antibody
solution was a 1:200 dilution of the rabbit anti-SjAChE
serum, and normal rabbit serum was used as control.
Non-specific antibody binding was inhibited by
incubating the section in 10 % (v/v) normal goat serum in PBS.
ImmPRESSTM HRP Anti-Rabbit IgG (Peroxidase)
Polymer (Vector Labs, California USA) was used as second
antibody for the immunolocalisation. Slides were
scanned and digitised using a ScanScope XT (Aperio,
Four-day old transformed larvae were cultured in Basch’s
] containing rabbit anti-SjAChEC serum
(1:100 dilution), or pre-immune rabbit serum (1:100
dilution) as negative control, at 4 °C overnight [
larvae were washed three times with Basch medium and
incubated with 1:300 donkey anti-rabbit IgG Alexa Fluor
555 (2 mg/ml, Invitrogen) for 1 h at room temperature,
followed by three further washes in the medium. The
larvae were fixed in 4 % paraformaldehyde in PBS for
10 min at room temperature, and then visualised under
fluorescence using a Zeiss 780 NLO confocal
microscope (Zeiss, Germany).
Fluorescence-based enzyme assays
The enzymatic activity of AChE in S. japonicum was
determined using the Amplex Red
Acetylcholine/Acetylcholinesterase Assay Kit (Invitrogen) according to the
manufacturer’s instructions. The assay is
fluorescencebased and utilises the highly fluorescent end product
resorufin which is processed in black Costar 96-well
plates (Sigma) and measured using the POLARstar
OPTIMA (BMG Labtech, Ortenberg Germany) at an
absorption of 560 nm and an emission of 590 nm.
Negative and positive control samples are provided in the
assay kit and BW284c51
[1,5-bis(4allyldimethylammoniumphenyl)pentan-3-one dibromide] (Sigma), a specific
inhibitor of AChE, was also used in the enzyme assay.
Different protein extracts of S. japonicum used in the
(1) Tegument protein and residual carcass protein
extracted from adult S. japonicum freshly perfused from
mice. The tegument was removed from paired adult
worms by the freeze/thaw/vortex method [
freshly perfused paired adult S. japonicum (50 pairs)
were frozen in liquid nitrogen, thawed on ice and 400 μl
of ice-cold TBS (10 mM Tris-HCl, 0.84 % NaCl, pH 7.4)
was added to each tube. The supernatant was removed
after 1 min and then 400 μl of Tris-HCl, pH 7.4 was
added and the tube left to incubate on ice for 5 min.
Tubes were vortexed 8 times for 1 s to ensure tegument
release. The tegument-rich supernatant was transferred
to another tube where it was centrifuged for 30 min at
12,000 g, 4 °C, the supernatant was discarded and the
tegument-rich pellet re-suspended in 60 μl 10 mM
TrisHCl, pH 8.0. The remaining carcasses were homogenised
using the protocol for making SWAP essentially as
described in [
], and above. The protein concentrations of
the enriched tegument fraction and the residual carcass
preparation were measured using the Bio-Rad protein
assay dye reagent (Bio-Rad, California, USA). These
protein extracts (0.005 mg/ml) were pre-incubated at
room temperature for 30 min with BW284c51 at
concentrations of 0, 10, 100 and 1000 μM and then
used in the AChE activity assays.
(ii) Freshly perfused adult S. japonicum were cultured
in RPMI medium containing 10 % (v/v)
heatinactivated fetal calf serum overnight. The worms were
then divided into three groups: single males, single
females and paired male and female worms (40 single
worms or 20 pairs/group). Each group was then
treated with or without 200 μM BW284c51 for 24 h,
after which time the worms were rinsed 3 times in
RPMI medium, collected and used for tegumental
protein and carcass protein extraction as described above.
AChE activities of all the protein samples (0.005 mg/
ml) from the various worm samples were measured
using the Amplex Red
Acetylcholine/Acetylcholinesterase Assay Kit.
Worm collections, protein extractions and AChE
activity measurements in (i) and (ii) were performed
three times. T-test was employed to make the
comparison between samples by using GraphPad software
Full-length sequence of SjAChE
The complete SjAChE cDNA sequence was obtained,
comprising an open reading frame (ORF) of 2,040 bp
(submitted to GenBank under accession number
KX268651) encoding 680 amino acids. SjAChE shares
88 % amino acid sequence identity with the AChEs from
S. mansoni, S. haematobium and S. bovis, and 55 %
identity with the AChEs in Echinococcus granulosus [
E. multilocularis [
]. In contrast, SjAChE shares only
25 % amino acid identity to human AChE and 26 %
identity to the AChE from Torpedo californica (Pacific
electric ray). Phylogenetic analysis was performed
using AChE protein sequences from a variety of
species to produce a cladogram to infer evolutionary
relationships between taxa (Fig. 1). Of the schistosome
sequences, the AChE coding region for S. japonicum
is most similar to that of S. haematobium. The
Schistosoma spp. sequences are separated considerably
from those of Echinococcus species and, as expected,
have more sequence similarity with the AChEs of
other trematode species including Clonorchis sinensis,
Opisthorchis viverrini and Fasciola hepatica. Crucially,
all residues within the AChE protein sequence that
are currently known to be important for substrate
binding and catalytic activity, i.e. those comprising
the peripheral anionic site [
], the catalytic triad
substrate inhibition of acetylcholinesterase residues
involved in signal transduction from the surface to the
catalytic center [
], and those lining the catalytic
], are conserved across all Schistosoma
Using an in silico motif and domain search tool
(http://prosite.expasy.org/), we identified two conserved
sub-domains in the SjAChE protein sequence - a
carboxylesterase type-B signature 2 (E156-P166) region, and a
carboxylesterase type-B serine active site (F258-G273),
both of which are shown boxed in red in Fig. 2. Several
other motifs were also found in SjAChE (Fig. 2); these
After comparisons with other species and the
schistosome sequences published by Bentley et al [
demonstrated that the catalytic and peripheral active site
residues in S. japonicum, S. mansoni, S. haematobium
and S. bovis are all conserved, especially when taking
into consideration the accepted standard primary AChE
(1EA5_A) sequence from the ray Torpedo californica. It
has been shown that the active site of T. californica
AChE consists of a catalytic triad (S200-H440-E327, in
red stars, Fig. 2) which lies close to the bottom of a deep
and narrow tertiary structure gorge, which is lined with
the rings of 14 aromatic amino acid residues [
conserved catalytic triad is present in S. japonicum
(S280-H54-E327), while the nine residues (W148, W186,
W193, Y202, W304, F371, F404, Y407, Y537, in dark red
triangles, Fig. 2) in the rings of the 14 aromatic amino
acid residues of T. californica AChE, are conserved in
the appropriate locations in SjAChE.
The tertiary protein structure for SjAChE was predicted
using Phyre2 (Fig. 3a). Model dimensions for SjAChE (Å)
(X:61.705 Y:62.361 Z:71.856) are the same as those of S.
haematobium AChE. Of note, we found four predicted
NAcetylglucosamine (NAG) binding sites located at (i)
M123, D125; (ii) P423, K245, M428; (iii) R507-T510, P512;
and (iv) Q550-F551, A553-Y556 in SjAChE (Fig. 3b).
NAcetylglucosamine, a monosaccharide derivative of
glucose, is directly incorporated into glycosaminoglycans and
glycoproteins, acting as a substrate for tissue repair
]. The predicted four NAG binding sites in
SjAChE are in line with previous findings which revealed
the presence of NAG in all forms of cholinesterases
], providing evidence for N-linked glycosylation
(See figure on previous page.)
Fig. 2 Alignment of acetylcholinesterases from S. mansoni, S. haematobium, S. bovis, Homo sapiens and T. californica. Red boxes indicate the two
conserved subdomains including carboxylesterase type-B signature 2 (E156-P166) and carboxylesterase type-B serine active site (F258-G273).
Several motifs are found in SjAChE: N-glycosylation sites underlined (N42-I45, N171-H174, N314-Q317, N418-D421, N630-K633); N-myristoylation sites
boxed in purple (G71-Q76, G95-Q100, G298-N303, G305-E310, G395-E400, G532-Y537); Casein kinase II phosphorylation site boxed in brown
(S88D91, S200-D203, S329-D332, S341-E344, T351-D354, T456-E459, S471-E474, T559-E562, T592-E595); Protein kinase C phosphorylation sites boxed in
blue (S105-R107, T316-R318, S379-R381, T473-R475, S481-K486, S631-K633) which are specific for schistosome; Tyrosine kinase phosphorylation site
boxed in green (R454-Y460); amidation site (P503-R506). The conserved catalytic active catalytic triad site is observed S. japonicum
(S280-H54E327, in red stars), while the 9 residues (W148, W186, W193, Y202, W304, F371, F404, Y407, Y537, in dark red triangles) in the rings of 14 aromatic
amino acid residues of T. californica AChE are conserved in the appropriate locations in S. japonicum AChE. The coloured boxes which covered
only sequences from four species of schistosomes indicated the specific motifs for schistosome. Note: AChE from S. mansoni (SmAChE), S.
haematobium (ShAChE), S. bovis (SbAChE), Homo sapiens (HsAChE) and T. californica (TcAChE)
in SjAChE. The predicted protein structure for SjAChE
also suggests that it may be fucosylated on the innermost
N-acetylglucosamine residue of the core [
SDS-PAGE showed the purified rSjAChEC migrated as a
single band with the predicted size of 30 kDa (Fig. 3c).
The specificity of the rabbit anti-SjAChEC antibody was
confirmed as it bound a band of approximately 76 kDa
in adult S. japonicum SWAP, thereby matching well with
the calculated molecular size for SjAChE (Fig. 3c).
Control serum from the pre-immunized control rabbit did
not bind any protein component in S. japonicum SWAP.
Distribution of SjAChE in adults and schistosomula
Indirect immunohistochemistry, incorporating HRP
labelling, indicated that SjAChE immunoreactivity
occurred in the tegument, the underlying musculature but
also throughout the parenchyma and tissues of both
males (Fig. 4a) and females (Fig. 4b). To better
understand how the anti-SjAChE serum interacted with
schistosomula, we used immunofluorescence to show
SjAChE is also localized on the tegumental surface of
live 4-day-old schistosomula (Fig. 4d) and the
parenchyma; the latter observation may be indicative of
damage to the schistosomula during labelling process. By
using two different immunolocalisation methods
involving HRP labelling and immunofluorescence, we showed
a similar distribution of SjAChE in early (schistosomula)
and late (adult males and females) developmental stages
in the mammalian host.
Inhibition of SjAChE activity
SjAChE sensitivity to chemical inhibition, in extracts of
adult worms, was assessed by the pre-incubation of
tegument or carcass proteins with BW284c51 at a
concentration range of 0–1,000 μM. SjAChE, present both
in the worm tegument or carcass extract, was sensitive
to BW284c51, and its activity exhibited a linear response
to concentration changes up to 1000 μM of BW284c51
(Fig. 5a). SjAChE activity in the tegument extract was
significantly higher (about 10-fold; t-test, t = 1.881, df =
6, P < 0.0001) than in the carcasses of adult worms,
suggesting the majority of the enzyme is located on the
tegument of paired adult S. japonicum. The IC50 (50 %
inhibition) of BW284c51 on SjAChE in the tegumental
protein extract of adult worms occurred at a
concentration of 16 μM which indicates a substantially higher
sensitivity than that reported for the AChEs from S.
mansoni, S. bovis and S. haematobium [
sensitivity of SjAChE in the tegument and carcasses isolated
from cultured adult worms in the presence of 200 μM
BW284c51 (IC80, 80 % inhibitory concentration) are
shown in Fig. 5b. With the same concentration of
tegument protein, paired worms had a higher SjAChE
activity than single-sex worms (t-test, t = 3.903, df = 4, P =
0.0175) with male worms having a higher SjAChE
activity than females (t-test, t = 18.66, df = 4, P < 0.0001).
After being treated with 200 μM BW284c51, the SjAChE
enzyme activity in tegument protein extracts of paired
worms, males and female worms decreased by 59 %,
22 % and 50 %, respectively (t-test, t = 40.52,; 17.28; and
39.56, respectively, df = 4, P < 0.0001). Compared with
the tegumental protein extract, there was much less
SjAChE activity in the carcass protein extract, with a
relatively higher activity in males compared with that in
pairs and females (t-test, t = 29.41 and 39.07,
respectively, df = 4, P < 0.0001), with the latter having the lowest
level of SjAChE activity. SjAChE activity in the carcass
protein extracts of males and paired worms was
inhibited by 77 % (t-test, t = 32.69, df = 4, P < 0.0001) and
45 % (t-test, t = 15.07, df = 4, P < 0.0001), respectively in
the presence of 200 μM BW284c51.
Previous studies on AChE in schistosomes have focused
mainly on S. mansoni, S. haematobium and S. bovis and,
prior to this study, very limited information was
available for the enzyme in S. japonicum. Here, we report the
cloning and expression of the complete cDNA encoding
S. japonicum AChE (SjAChE). To better understand its
functions, we performed sequence and phylogenetic
analysis on SjAChE and predicted its tertiary molecular
structure. As might be expected, the protein has high
sequence identity (88 %) with the AChEs in S. mansoni,
S. haematobium and S. bovis. The key residues that are
important for the formation of the three disulphide
bonds and two salt bridges characteristic of AChE [
in substrate binding and for catalytic activity are
conserved across the four species. These residues comprise
important structural features including the peripheral
anionic site [
], the catalytic triad [
] and residues that
line the catalytic gorge [
]. One particularly noteworthy
feature of the AChE protein sequence in schistosomes is
two “missing” residues that form part of the peripheral
active site. Within the AChE of Torpedo californica,
residue F330 has a neighbouring F residue in the same
secondary structure that is not indicated as playing a role in
the catalysis of acetylcholine. However, whereas in
schistosomes, the equivalent of F330 is not present (Fig. 2),
the neighbouring F residue is. It may be possible that
this neighbouring F residue has taken over the catalytic
role, or that this role has been lost altogether in
schistosomes. Similarly, an equivalent residue could not be
found at the position expected for W279 (Fig. 2),
another peripheral active site residue. Considering these
residues are only part of the peripheral active site, they
may not be essential for the function of AChE in
schistosomes and have been lost over time through mutational
As with the other schistosomes, immunolocalisation
showed that SjAChE is located on the tegumental
surface and parenchyma of adult worms and 4-day-old
]. Previous work showed the existence
of two principal molecular forms (external and internal)
of S. mansoni AChE, with approximately half of the
AChE activity being found on the tegumental membrane
via a covalently attached glycosylphosphatidylinositol
(GPI) anchor and which may function in signal
transduction, with the remainder mainly associated with
muscle tissue and involved in cholinergic processes [
These two forms of AChE were also shown to differ in
their heparin-binding properties (only the internal form
interacted with heparin) and in immunological
specificity (being located on the surface the GPI-anchored
form may be susceptible as an immunological target)
]. Further investigation is required to determine
whether there are also different molecular forms of
SjAChE and if so whether they have discrete functional
roles in S. japonicum.
To quantify the relative activity of SjAChE present
within the tegument and in the musculature of adult S.
japonicum, we separated the tegumental protein from
the parasite carcass, and performed enzyme activity
assays. We found that most of the SjAChE activity was
concentrated in the tegument, having 10-fold the activity
of the carcass (Fig. 5a), suggesting that SjAChE has
potential as a drug or immunological target. We also
showed that SjAChE activity was highly enriched in the
male tegument and this observation is understandable as
male parasites, being larger in size, and having an
increased tegumental volume [
]. One established
function of tegumental AChE in schistosomes is in the
regulation of glucose uptake across the tegument in
response to ACh present in the mammalian host
]. Given that male schistosomes play a more
important role in host glucose uptake [
], it is
reasonable to consider that AChE activity would also be higher
in male S. japonicum, as we have shown. The distribution
of AChE in S. japonicum we established correlates with
that reported in the other schistosome species [
It has been shown that AChE activity and its
sensitivity to the inhibitor BW284c51 is dependent on the
relative amount of AChE expressed on the surface of adult
], since the inhibitor does not readily
penetrate membranes of the adult worms [
showed a protein extract of the tegument of adult S.
japonicum had an IC50 with BW284c51 of 16 μM,
which is much lower than the reported IC50 for other
schistosome species (0.1–5.0 mM) [
]. Those results
may reflect a relatively larger amount of AChE activity
presented on the surface of adult S. japonicum compared
to the other schistosome species, indicating the AChE
inhibitor may be more effective against S. japonicum.
We also found that live adults of S. japonicum incubated
with Bw284c51 (200 μM) displayed reduced AChE
activity in tegumental protein by 50 % in females, but only
22 % in males, suggesting that AChE present on the
surface of females is more sensitive to the inhibitor than
that on males. Previous work has shown that AChE is
associated with the AChR on cell surfaces [
] and in
schistosomes the expression of AChR is increased in
sexually paired worms when female parasites mature
into the egg producing stage [
]. The increased level of
AChR expression may require increased AChE activity
on the surface of female worms to maintain
cholinesterase receptor fidelity. A similar situation occurred in
paired worms, where a 59 % decrease in SjAChE activity
was observed when paired incubated worms were
treated with 200 μM of Bw284c51.
The relatively high level of SjAChE activity distributed
within the carcass protein of males, when compared
with female and paired worms, may be indicative of its
involvement in muscle function [
], since male worms
have more muscle tissue. The SjAChE activity in male
carcasses was decreased by 77 % after incubation of live
male parasites with BW284c51 for 24 h, suggesting that
the inhibitor can penetrate the tegument of male S.
japonicum, which is a contradiction to previous reports
stating the inhibitor cannot cross membranes [
It has been reported that AChE expression is induced
during apoptosis and is regulated by the mobilization of
intracellular Ca2+ in various mammalian cell types [
Promoting apoptosis appeared to be a feature of the
mode of action of two already established
antischistosomal drugs, the artemisinins [
], and drug targeting schistosome AChE may also
be effective by inducing apoptosis. Further, it has been
demonstrated that purified polyclonal antibodies raised
against S. mansoni AChE were cytotoxic and caused
almost total complement-dependent killing of parasites in
], while not cross-reacting with human AChE.
This observation and the results presented here
strengthen the view that immunological targeting of
schistosome AChEs may be a highly suitable avenue for
future vaccine development and the prevention of
In this paper, we have described the phylogenetic and
molecular/structural characterisation of the AChE
protein from S. japonicum. These findings improved the
understanding of the biological function of AChE in
schistosomes. The relative abundance of AChE activity
(90 %) present on the surface of adult S. japonicum
when compared with that reported in other
schistosomes, suggests SjAChE may be a more effective drug or
immunological target against thus species. Furthermore,
we show that the AChE activity in tegumental extracts
of adult S. japonicum can be significantly inhibited by
the classical inhibitor of AChE (BW285c51) after
incubation with adult worms. The results we present support
the potential of AChE as a future drug target against S.
japonicum and also strengthens the view that
immunological targeting of schistosome AChEs may be a highly
suitable avenue for future vaccine development and the
prevention of schistosomiasis.
ACh, acetylcholine; AChE, acetylcholinesterase; ELISA, enzyme-linked
immunosorbent assay; GPI, glycosylphosphatidylinositol; HRP, horseradish
peroxidise; nAChR, nicotinic acetylcholine receptor; NAG, N-Acetylglucosamine;
PSMD4, proteasome non-ATPase regulatory subunit 4; SjAChE, Schistosoma
japonicum acetylcholinesterase; SWAP, soluble adult worm antigen preparation
We are grateful for funding provided by an Australian Infectious Disease
Research Centre Seed Grant and a Program Grant from the National Health
and Medical Research Council (NHMRC) of Australia (APP 1037304).
We are grateful for funding provided by an Australian Infectious Disease
Research Centre Seed Grant and a Program Grant from the National Health
and Medical Research Council (NHMRC) of Australia (APP 1037304).
Availability of data and material
The complete SjAChE cDNA sequence is submitted to the GenBank database
Conceived and designed the experiments: HY GNG DPM. Performed the
experiments: HY XD, GP. Analysed the data: HY GNG PC MKJ DPM.
Contributed reagents/materials/analysis tools: HY XD GP PC. Wrote the
paper: HY GNG MKJ DPM. All authors read and approved the final version of
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The conduct and procedures involving animal experiments were approved
by the Animal Ethics Committee of the QIMR Berghofer Medical Research
Institute (project number P288). This study was performed in accordance
with the recommendations in the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health.
1. Beaumier CM , Gillespie PM , Hotez PJ , Bottazzi ME . New vaccines for neglected parasitic diseases and dengue . Transl Res . 2013 ; 162 : 144 - 55 .
2. Camacho M , Tarrab-Hazdai R , Espinoza B , Arnon R , Agnew A . The amount of acetylcholinesterase on the parasite surface reflects the differential sensitivity of schistosome species to metrifonate . Parasitology . 1994 ; 108 (Pt 2): 153 - 60 .
3. Salafsky B , Fusco AC , Whitley K , Nowicki D , Ellenberger B . Schistosoma mansoni: analysis of cercarial transformation methods . Exp Parasitol . 1988 ; 67 : 116 - 27 .
4. Wilson RA . Proteomics at the schistosome-mammalian host interface: any prospects for diagnostics or vaccines? Parasitology . 2012 ; 139 : 1178 - 94 .
5. Levi-Schaffer F , Tarrab-Hazdai R , Schryer MD , Arnon R , Smolarsky M. Isolation and partial characterization of the tegumental outer membrane of schistosomula of Schistosoma mansoni . Mol Biochem Parasitol . 1984 ; 13 : 283 - 300 .
6. Ribeiro-dos-Santos G , Verjovski-Almeida S , Leite LC . Schistosomiasis - a century searching for chemotherapeutic drugs . Parasitol Res . 2006 ; 99 : 505 - 21 .
7. Camacho M , Agnew A . Schistosoma: rate of glucose import is altered by acetylcholine interaction with tegumental acetylcholine receptors and acetylcholinesterase . Exp Parasitol . 1995 ; 81 : 584 - 91 .
8. Harder A . Chemotherapeutic approaches to schistosomes: current knowledge and outlook . Parasitol Res . 2002 ; 88 : 395 - 7 .
9. Espinoza B , Tarrab-Hazdai R , Himmeloch S , Arnon R . Acetylcholinesterase from Schistosoma mansoni: immunological characterization . Immunol Lett . 1991 ; 28 : 167 - 74 .
10. Bentley GN , Jones AK , Agnew A . Expression and comparative functional characterisation of recombinant acetylcholinesterase from three species of Schistosoma . Mol Biochem Parasitol . 2005 ; 141 : 119 - 23 .
11. Jones AK , Bentley GN , Oliveros Parra WG , Agnew A . Molecular characterization of an acetylcholinesterase implicated in the regulation of glucose scavenging by the parasite Schistosoma . FASEB J . 2002 ; 16 : 441 - 3 .
12. Pearson MS , Becker L , Driguez P , Young ND , Gaze S , Mendes T , Li XH , Doolan DL , Midzi N , Mduluza T , et al. Of monkeys and men: immunomic profiling of sera from humans and non-human primates resistant to schistosomiasis reveals novel potential vaccine candidates . Front Immunol . 2015 ; 6 : 213 .
13. Camacho M , Alsford S , Jones A , Agnew A . Nicotinic acetylcholine receptors on the surface of the blood fluke Schistosoma . Mol Biochem Parasitol . 1995 ; 71 : 127 - 34 .
14. Bentley GN , Jones AK , Oliveros Parra WG , Agnew A . ShAR1alpha and ShAR1beta: novel putative nicotinic acetylcholine receptor subunits from the platyhelminth blood fluke Schistosoma . Gene . 2004 ; 329 : 27 - 38 .
15. Bentley GN , Jones AK , Agnew A . ShAR2beta, a divergent nicotinic acetylcholine receptor subunit from the blood fluke Schistosoma . Parasitology . 2007 ; 134 : 833 - 40 .
16. Jones MK , McManus DP , Sivadorai P , Glanfield A , Moertel L , Belli SI , Gobert GN . Tracking the fate of iron in early development of human blood flukes . Int J Biochem Cell Biol . 2007 ; 39 : 1646 - 58 .
17. Gobert GN , Tran MH , Moertel L , Mulvenna J , Jones MK , McManus DP , Loukas A . Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes . PLoS Negl Trop Dis . 2010 ; 4 : e600 .
18. Dereeper A , Guignon V , Blanc G , Audic S , Buffet S , Chevenet F , Dufayard JF , Guindon S , Lefort V , Lescot M , et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist . Nucleic Acids Res . 2008 ; 36 : W465 - 9 .
19. Kelley LA , Sternberg MJ . Protein structure prediction on the web: a case study using the Phyre server . Nat Protoc . 2009 ; 4 : 363 - 71 .
20. Wass MN , Kelley LA , Sternberg MJ . 3DLigandSite : predicting ligand-binding sites using similar structures . Nucleic Acids Res . 2010 ; 38 : W469 - 73 .
21. You H , Zhang W , Jones MK , Gobert GN , Mulvenna J , Rees G , Spanevello M , Blair D , Duke M , Brehm K , et al. Cloning and characterisation of Schistosoma japonicum insulin receptors . PLoS One . 2010 ; 5 : e9868 .
22. Ranasinghe SL , Fischer K , Gobert GN , McManus DP . Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni . Parasit Vectors . 2015 ; 8 : 408 .
23. McWilliam HE , Driguez P , Piedrafita D , Maupin KA , Haab BB , McManus DP , Meeusen EN . The developing schistosome worms elicit distinct immune responses in different tissue regions . Immunol Cell Biol . 2013 ; 91 : 477 - 85 .
24. Jia X , Schulte L , Loukas A , Pickering D , Pearson M , Mobli M , Jones A , Rosengren KJ , Daly NL , Gobert GN , et al. Solution structure, membrane interactions , and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke Schistosoma mansoni . J Biol Chem . 2014 ; 289 : 7151 - 63 .
25. Zheng H , Zhang W , Zhang L , Zhang Z , Li J , Lu G , Zhu Y , Wang Y , Huang Y , Liu J , et al. The genome of the hydatid tapeworm Echinococcus granulosus . Nat Genet . 2013 ; 45 : 1168 - 75 .
26. Tsai IJ , Zarowiecki M , Holroyd N , Garciarrubio A , Sanchez-Flores A , Brooks KL , Tracey A , Bobes RJ , Fragoso G , Sciutto E , et al. The genomes of four tapeworm species reveal adaptations to parasitism . Nature . 2013 ; 496 : 57 - 63 .
27. Barak D , Ordentlich A , Bromberg A , Kronman C , Marcus D , Lazar A , Ariel N , Velan B , Shafferman A . Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite . Biochemistry . 1995 ; 34 : 15444 - 52 .
28. Shafferman A , Velan B , Ordentlich A , Kronman C , Grosfeld H , Leitner M , Flashner Y , Cohen S , Barak D , Ariel N. Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center . EMBO J . 1992 ; 11 : 3561 - 8 .
29. Sussman JL , Harel M , Frolow F , Oefner C , Goldman A , Toker L , Silman I. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein . Science . 1991 ; 253 : 872 - 9 .
30. Bentley GN , Jones AK , Agnew A . Mapping and sequencing of acetylcholinesterase genes from the platyhelminth blood fluke Schistosoma . Gene . 2003 ; 314 : 103 - 12 .
31. Sussman JL , Harel M , Silman I . Three-dimensional structure of acetylcholinesterase and of its complexes with anticholinesterase drugs . Chem Biol Interact . 1993 ; 87 : 187 - 97 .
32. Salvatore S , Heuschkel R , Tomlin S , Davies SE , Edwards S , Walker-Smith JA , French I , Murch SH . A pilot study of N-acetyl glucosamine, a nutritional substrate for glycosaminoglycan synthesis, in paediatric chronic inflammatory bowel disease . Aliment Pharmacol Ther . 2000 ; 14 : 1567 - 79 .
33. You H , McManus DP , Hu W , Smout MJ , Brindley PJ , Gobert GN . Transcriptional responses of in vivo praziquantel exposure in schistosomes identifies a functional role for calcium signalling pathway member CamKII . PLoS Pathog . 2013 ; 9 : e1003254 .
34. Tarrab-Hazdai R , Levi-Schaffer F , Gonzales G , Arnon R . Acetylcholinesterase of Schistosoma mansoni . Molecular forms of the solubilized enzyme . Biochim Biophys Acta . 1984 ; 790 : 61 - 9 .
35. Arnon R , Silman I , Tarrab-Hazdai R . Acetylcholinesterase of Schistosoma mansoni - functional correlates. Contributed in honor of Professor Hans Neurath's 90th birthday . Protein Sci . 1999 ; 8 : 2553 - 61 .
36. Gobert GN , Stenzel DJ , McManus DP , Jones MK . The ultrastructural architecture of the adult Schistosoma japonicum tegument . Int J Parasitol . 2003 ; 33 : 1561 - 75 .
37. Cornford EM , Fitzpatrick AM . The mechanism and rate of glucose transfer from male to female schistosomes . Mol Biochem Parasitol . 1985 ; 17 : 131 - 41 .
38. Camacho M , Agnew A . Glucose uptake rates by Schistosoma mansoni, S. haematobium, and S. bovis adults using a flow in vitro culture system . J Parasitol . 1995 ; 81 : 637 - 40 .
39. Massoulie J , Pezzementi L , Bon S , Krejci E , Vallette FM . Molecular and cellular biology of cholinesterases . Prog Neurobiol . 1993 ; 41 : 31 - 91 .
40. Zhu H , Gao W , Jiang H , Jin QH , Shi YF , Tsim KW , Zhang XJ . Regulation of acetylcholinesterase expression by calcium signaling during calcium ionophore A23187- and thapsigargin-induced apoptosis . Int J Biochem Cell Bio . 2007 ; 39 : 93 .
41. Ho WE , Peh HY , Chan TK , Wong WS . Artemisinins: pharmacological actions beyond anti-malarial . Pharmacol Ther . 2014 ; 142 : 126 - 39 .
42. Hong Y , Donald PM , Geoffrey NG . Current and prospective chemotherapy options for schistosomiasis . Expert Opin Orphan D . 2015 ; 3 : 195 .