Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant a
Trang 1Inorganic pyrophosphatase in the roundworm Ascaris
and its role in the development and molting process
of the larval stage parasites
M Khyrul Islam1, Takeharu Miyoshi1, Harue Kasuga-Aoki1, Takashi Isobe1, Takeshi Arakawa2,
Yasunobu Matsumoto3and Naotoshi Tsuji1
1
Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, 3-1-5, Kannondai, Tsukuba, Ibaraki, Japan;2Division of Molecular Microbiology, Center of Molecular Biosciences, University of the Ryukyus, Senbaru, Okinawa, Japan;3Laboratory of Global Animal Resource Science, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo, Japan
Inorganic pyrophosphatase (PPase) is an important
enzyme that catalyzes the hydrolysis of inorganic
pyro-phosphate (PPi) into ortho-phosphate (Pi) We report
here the molecular cloning and characterization of a gene
encoding the soluble PPase of the roundworm Ascaris
suum The predicted A suum PPase consists of 360 amino
acids with a molecular mass of 40.6 kDa and a pI of 7.1
Amino acid sequence alignment and phylogenetic analysis
indicates that the gene encodes a functional Family I
soluble PPase containing features identical to those of
prokaryotic, plant and animal/fungal soluble PPases The
Escherichia coli-expressed recombinant enzyme has a
spe-cific activity of 937 lmol PiÆmin)1Æmg)1 protein
corres-ponding to a kcatvalue of 638 s)1at 55C Its activity was
strongly dependent on Mg2+and was inhibited by Ca2+
Native PPases were expressed in all developmental stages of
A suum A homolog was also detected in the most closely related human and dog roundworms A lumbricoides and Toxocara canis, respectively The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus
of adult female worms We observed that native PPase activity together with development and molting in vitro of
A suum L3 to L4 were efficiently inhibited in a dose-dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble- and membrane-bound H+-PPases The studies provide evi-dence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process
Keywords: roundworm; inorganic pyrophosphatase; sodium fluoride; imidodiphosphate; molting
Geohelminth parasites are among the commonest and
widespread of human infections, particularly in the regions
where public health hygiene and nutritional status are
poorly maintained The most prevalent geohelminth is
Ascaris lumbricoides (originally described by Linnaeus in
1758), which colonizes the small intestine of children, and is
estimated to infect a quarter of the world’s population [1]
Ascaris suum(originally described by Goeze in 1782) of pigs
is a very closely related species to A lumbricoides, which can
develop in human hosts, indicating its zoonotic significance [2,3] Childhood infections with Ascaris worms are reported
to be associated with stunting growth, malabsorption, deficiencies of macro- and micro-nutrients and damage of the small intestinal mucosa [4,5] In addition, concurrent Ascarisinfection may have potential immunomodulatory effects on the immune response to other infections [6,7] It
is therefore of considerable interest to investigate the biochemical aspects of Ascaris worms to identify potential drug targets and vaccine candidates
Inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of inorganic pyrophosphate (PPi) into inorganic ortho-phosphate (Pi), are widely distributed among living cells The enzymes play an important role in energy metabolism, providing a thermodynamic pull for many biosynthetic reactions [8], and have been shown to be essential to life [9–11] There are two major categories of PPases, the soluble PPases and the membrane-bound H+ -translocating PPases (H+-PPase) Two families of soluble PPases have been recognized to date, Family I includes most
of the currently known soluble PPases [12], and Family II comprises recently discovered Bacillus subtilis PPase as well
as PPases of four other putative members, two streptococcal and two archeal [13,14] These two families do not show any sequence similarity to each other Family I soluble PPases have been further divided into three subfamilies,
Correspondence to N Tsuji, Laboratory of Parasitic Diseases,
National Institute of Animal Health, National Agricultural Research
Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
Fax: + 81 29 8387749, Tel.: + 81 29 8387749,
E-mail: tsujin@affrc.go.jp
Abbreviations: PPase, inorganic pyrophosphatase; H+-PPase,
proton-translocating pyrophosphatase; AsPPase, Ascaris suum inorganic
pyrophosphatase; rAsPPase, recombinant A suum inorganic
pyro-phosphatase; L3, third-stage infective larvae; L4, fourth-stage larvae;
ES, excretory and secretory; IDP, imidodiphosphate.
Enzyme: Soluble inorganic pyrophosphatase (EC 3.6.1.1).
Note: The nucleotide sequences reported in this paper has been
submitted to the DDBJ/EMBL/GenBank with accession number
AB091401.
(Received 4 March 2003, revised 7 May 2003, accepted 9 May 2003)
Trang 2prokaryotic, plant and animal/fungal PPases Among the
subfamilies, plant PPases bear a closer similarity to
prok-aryotic than to animal/fungal PPases [12] The H+-PPases,
which appear to work as a reversible H+-pump, are much
larger and do not have any sequence similarity to either of
the two families of soluble PPases [15–17] All known soluble
PPases are homologous proteins, whose active site residues
are highly conserved evolutionarily [18] Site-directed
mut-agenesis and high-resolution X-ray crystallography studies
on PPases from E coli and Saccharomyces cerevisiae have
implicated 17 amino acid residues as being important for
enzyme activity [19,20] However, depending on the choice
of alignment parameters, 14–17 of the putative active site
residues described by Terzyan et al [21] are conserved in
sequence alignments of E coli and yeast PPases [18] Recent
studies have demonstrated that only 17 residues are
conserved in all currently known Family I soluble PPases,
of which 13 are functionally important active site residues
[12,13] PPases are strongly divalent metal ion-dependent,
with Mg2+ conferring the highest PPi hydrolysis activity
[22] Mg2+has several roles: it activates the enzyme and, as a
Mg2+-PPi complex, forms a true substrate for soluble
PPases; Mg2+also stabilizes the enzyme Ca2+is reported
to be a potent inhibitor of soluble PPases [23]
While PPases from diversified sources have been
des-cribed in some detail, no PPase has ever been studied in any
metazoan helminth parasite including the roundworm
Ascaris To address this, we describe here the cloning,
sequencing and heterologous expression in E coli of a gene
encoding PPase of A suum The amino acid sequence of
A suumPPase (AsPPase) indicates that it is an authentic
member of the Family I soluble PPases We also provide
information concerning the kinetics and properties of the
enzyme More strikingly, we show a novel role of the PPase
enzyme in the development and molting process of A suum
larvae in vitro
Materials and methods
Parasites
Adult A suum were obtained from infected pigs at a
slaughterhouse in Shimotsuma, Japan Adult A
lumbric-oides and T canis were obtained from patients after
treatment with piperazine in Bac Gian, Vietnam and, from
an infected dog in Miyazaki, Japan, respectively
Unem-bryonated and emUnem-bryonated eggs were obtained essentially
as described elsewhere [24] Third-stage infective larvae (L3)
from embryonated eggs and lung-stage L3 were obtained as
described previously [25,26] Excretory and secretory (ES)
products from L3, lung-stage L3 and adult worms were
collected as described previously [27] Animal studies were
performed in accordance with the approval of the National
Institute of Animal Health Animal Care and Use
Commit-tee (Approval no 23)
RNA was isolated from embryonated eggs using an
RNA isolation kit (Clontech) Poly(A)+ mRNA was
prepared from total RNA using a polytract mRNA
isolation kit (Clontech) and first-strand cDNA synthesis
was performed using a cDNA synthesis kit and an oligo
(dT)15 primer from Amersham Pharmacia Biotech An
A suumadult female worm cDNA expression library was
constructed in UniZap XR vector (Stratagene) according to the manufacturer’s instructions as previously described [28] Protein concentrations of NaCl/Pi-soluble parasite antigens and ES products were measured using Micro BCA protein assay reagent (Pierce)
Immunoscreening of a cDNA expression library
An adult female worm cDNA expression library was immunoscreened with rabbit antibodies raised against
A suumembryonated egg trickle inoculations Phages were plated onto a lawn of E coli XL-1 Blue at a density of
50 000 phage per dish and grown at 37C for 4 h When plaques were visible, isopropyl thio-b-D -galactoside-impreg-nated filters were placed on the plates for 3 h to obtain a plaque lift After blocking in Tris/HCl, pH 8.0, with 0.05% Tween 20, the filters were incubated in rabbit immune sera overnight at 4C Antibody reactivity with recombinant proteins was revealed by incubation of the filters with alkaline phosphate-conjugated goat anti-rabbit IgG (ICN) for 1 h and developed with 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (Gibco/BRL) Clones that were reactive with the antibody were plaque-purified by repeated cycles of immune selection Plaque-purified clones were converted using ExAssistTMhelper phages and SOLR
E coli (Stratagene) according to the in vivo excision protocol described in the Stratagene ZAP-cDNA Synthesis Kit (Stratagene) The nucleotide sequences of the cDNAs were determined by the Sanger dideoxy chain termination method using a PRISMTMReady Dye Terminator Cycle Sequencing Kit (PerkinElmer) DNA samples were ana-lyzed using an automated sequencer (373A DNA sequencer, Applied Biosystems).BLASTX(NCBI, National Institute of Health) searches were performed to obtain cDNA clones coding low similarity against mammalian proteins stored at the current database TheGENETYX-WINTMDNA Sequence Analysis Software System (Software Inc) and the BLAST network server of the National Center for Biotechnology Information (NCBI) were used to analyze the nucleotides and deduce the amino acid sequences in determining similarities with previously reported sequences in GenBank
A primary sequence motif was identified using thePROSITE network server at EMBL Analysis of the signal sequence was performed using SIGNALP v1.1 at the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/ services/SignalP/index.html) Sequences were aligned by the program CLUSTALW1.8 (http://www.ebi.ac.uk/clustalw/) with theBLOSUMamino acid substitution matrix using gap penalties of 10.0 and 0.05 for gap opening and extension, respectively Phylogenetic trees were generated from homologies of the PPase amino acid sequences by the neighbor-joining method and the confidence of the branch-ing order was verified by makbranch-ing 1000 bootstrap replicates using the program CLUSTALW 1.8 The tree was viewed and converted to graphic format with TREEVIEW (http:// taxonomy.zoology.gla.ac.uk/rod/treeview.html)
Expression and purification of recombinant AsPPase proteins
A full length cDNA (lacking signal peptides) was amplified
by PCR as previously described [29] A sense primer
Trang 3AATCT-3¢) containing an XhoI (Promega) site upstream of
the start codon and an antisense primer (5¢-CAGCCAA
GCTTCTCACTCTTTGATGAAATGCATCT-3¢)
con-taining a HindIII (Promega) site just downstream of amino
acid residue were used The PCR fragments digested with
XhoI and HindIII were ligated into plasmid expression
vector pTrcHisBTM (Invitrogen), which had also been
digested with the same enzymes according to the
manufac-turer’s instructions The resultant plasmid was transformed
into E coli strain TOP10F¢ (Invitrogen) The transformed
cells were grown to a D600 at 37C in SOB medium
supplemented with 50 lgÆmL)1ampicillin To induce
pro-tein expression, isopropyl thio-b-D-galactoside was then
added to a final concentration of 1 mMand the culture was
grown for an additional 4 h at 37C The E coli cells were
pelleted and resuspended in lysis buffer [50 mMNaH2PO4
(pH 8.0), 10 mMTris/HCl (pH 8.0), 100 mMNaCl]
Lyso-zyme was added at 100 lgÆmL)1, and the cell suspension
was incubated on ice for 15 min The cell suspension was
disrupted using an ultrasonic processor (VP-5, TAITEC) on
ice The E coli lysate was centrifuged at 26 000 g for 30 min
at 4C The supernatants containing recombinant proteins
of AsPPase were purified using ProBondTMresin
(Invitro-gen) under nondenaturing conditions and subsequently
eluted with a stepwise gradient of imidazole (50–500 mM)
The eluted fractions were concentrated by Centrisart I (cut
off MW 10 000; Sartorius) and then dialyzed extensively at
4C with several successive changes of 20 mM Tris/HCl,
pH 7.5 and a decreasing concentration of NaCl in a
Slide-A-Lyzer Dialysis Cassette (Pierce) Fractions were collected
and the presence and purity of recombinant protein was
detected by 10% SDS/PAGE [30] and immunoblot [31]
using anti-T7 tag Ig (Invitrogen) Protein concentrations
were measured with the Micro BCA protein assay reagent
(Pierce)
Production of mouse polyclonal antibodies
BALB/c mice were immunized first with a subcutaneous
injection of 50 lg of recombinant AsPPase (rAsPPase)
emulsified with TiterMax GoldTM (CytRx), followed by
another injection 2 weeks later in the same adjuvant The
mice were bled 2 weeks after the second injection The
antisera from the mice were mixed and stored at)20 C
until used Anti-(mouse rAsPPase) IgG from immune sera
and mouse preimmune IgG were affinity purified using
UltraLinkTMimmobilized protein G according to
manu-facturer’s instructions (Pierce) and used for evaluating the
native AsPPase-neutralizing activity
Two-dimensional electrophoresis
Parasite extracts were treated with an equal volume of urea
mixture consisting of 9M urea, 4% Nonidet P-40, 0.8%
ampholine (pH 3.5–10; Pharmacia) and 2%
2-mercapto-ethanol, and then subjected to 2D PAGE Nonequilibrium
pH gradient electrophoresis was performed [32] in the first
dimension using a rectangular gel electrophoresis apparatus
(AE-6050 A; ATTO) After electrophoresis at 400 V for
2 h, the gels were incubated in the equilibration buffer
for 10 min on a shaker Electrophoresis in the second
dimension was performed on 8% SDS/PAGE gels under reducing conditions The proteins were either stained using
a silver staining kit (Dai-ichi Pure Chemicals) or transferred
to nitrocellulose membranes
Immunoblot analysis Immunoblot analysis was carried out as previously des-cribed [29] Anti-(mouse rAsPPase) serum was used at a dilution of 1: 500 The proteins bound to the secondary antibody were visualized with 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium
Immunohistochemistry Adult females of A suum and A lumbricoides were fixed in 4% paraformaldehyde in 0.1Mphosphate buffer, pH 7.2, overnight and embedded in paraffin Thin transverse sections were made from paraffin-embedded fixed worms The sections on glass slides were deparaffinized and dehydrated using a graded series of alcohol and then rehydrated in NaCl/Pi The slides were blocked for 30 min
in 1% H2O2in NaCl/Picontaining 10% ethanol to inacti-vate endogenous peroxidases For immunolocalization, the slides were blocked in NaCl/Picontaining 10% (v/v) goat serum (Wako) for 30 min at room temperature They were then flooded with anti-(mouse rAsPPase) Ig diluted 1 : 100
in NaCl/Pi/E coli lysate, overnight at 4C Afterwards, the slides were rinsed thoroughly with NaCl/Pi, and the antibody binding was resolved with a peroxidase-labeled anti-mouse IgG and the substrate 3¢,3¢-diaminobenzidine tetrahydrochloride (Sigma FastTM tablets, Sigma) After color development, the slides were dehydrated in a graded series of alcohol and cleared in xylene The slides were then covered with cover slips and observed with a microscope (Axiophot; Carl Zeiss)
Enzyme assay The rAsPPase activity was determined spectrophotometri-cally by measuring the rate of liberation of Pi from PPi using a molybdate-blue based colorimetric assay [33] The recombinant protein was assayed in the standard reaction mixture containing 5 mM Mg2+, 100 mM Tris/HCl (pH 7.5) and 1 mM PPi (Na4P2O7), in a total volume of
200 lL together with the protein solution, at 55C The assay was started by adding 10 lL of diluted rAsPPase solution into the standard reaction mixture The reaction was stopped by adding 1 mL of 200 mM glycine/HCl,
pH 3.0 Then, 125 lL of 1% ammonium molybdate (in
25 mMH2SO4) and 125 lL of 1% ascorbic acid (in 0.05% KHSO4) were added to the mixture, and incubated for
30 min at 37C Protein concentrations and reaction times were chosen in order to obtain the linearity of the reactions
As positive and negative controls, pure yeast-soluble PPase from Sigma (1-1643) and an unrelated A suum 14-kDa recombinant protein (As14; [28]) were used, respectively The concentrations of individual components were varied as indicated for the determination of Mg2+and pH dependent rAsPPase activity The amount of Pi liberated from the hydrolysis of PPi during the course of the reaction was measured in comparison to a standard P sample using a
Trang 4spectrophotometer (Model 600, Shimadzu) at an optical
density of 700 nm The specific activity of rAsPPase was
defined as lmol Pireleased min)1Æmg)1of protein
Enzyme kinetic study
The Km(Michaelis constant) and Vmax(maximum velocity)
values were determined by incubating the diluted
recombin-ant proteins in the standard reaction mixture in the presence
of increasing concentrations of PPi(0.05–0.5 mM) at 55C
Data were fit to the appropriate equation using GRAFIT
version 3.09b (Erithacus Software) Kmand Vmaxvalues were
reported with their standard errors derived from the fit
Native AsPPase activity and NaF sensitivity
To investigate the native AsPPase activity during A suum
larval development and molting, the L3 soluble extracts (in
20 mM Tris/HCl, pH 7.5) and the L3 ES products in the
culture fluids (dialyzed against 20 mM Tris/HCl, pH 7.5),
were assayed in the standard reaction mixture as described
above Anti-(mouse rAsPPase) IgG were evaluated for
AsPPase-neutralizing activity Recombinant AsPPase
pro-teins or A suum L3 extracts were preincubated in the
standard reaction mixture containing 5 lgÆmL)1
pre-immune or anti-(mouse rAsPPase) IgG (15 min, 37C)
before adding PPi The sensitivity of the native AsPPase to
inhibition by sodium fluoride (NaF, S-7920; Sigma) was
tested in the present study The L3 extracts were assayed in
the standard reaction mixture for PPi hydrolysis, in the
presence of increasing concentrations of NaF The PPase
activated PPihydrolysis rate was then calculated
Larval molting inhibition assay
To confirm whether the native PPase enzyme is involved in
the molting process, we examined the effects of two PPase
specific inhibitors, imidodiphosphate (IDP, 1-0631; Sigma)
and NaF on development and molting of A suum
lung-stage L3 to fourth-lung-stage larvae (L4) in vitro The lung-lung-stage
L3 were obtained from the lungs of New Zealand white
rabbits 7 days after inoculation with 2.5· 105embryonated
infective eggs of A suum [26] Briefly, the rabbits were killed
by an overdose of ketamine hydrochloride (50 mgÆkg)1
body weight, i.v.) followed immediately by decapitation and
the lungs were removed and minced with a surgical knife
The minced tissue was wrapped in cotton gauze and
suspended in NaCl/Pi containing 100 lgÆmL)1 penicillin/
streptomycin at 37C for 3 h After incubation the tissues
were removed, and the larvae were collected from the
bottom of the tube The recovered L3 were washed several
times with warm NaCl/Picontaining 50 lgÆmL)1penicillin/
streptomycin, and subjected to molting inhibition assay
Briefly, 50–100 L3 in 1 mL of RPMI 1640 medium (Gibco/
BRL), pH 6.8 supplemented with 10% (v/v) fetal bovine
serum (Sigma), 50 lgÆmL)1 penicillin/streptomycin were
cultured in 24-well flat-bottomed tissue culture plates
(Costar) The cultures were incubated at 37 C in a
humidified 5% CO2incubator in the absence (control) and
presence of increasing concentrations of inhibitors for
10 days, and the number of molting larvae was determined
Molting was manifested by shedding of the L3 cuticle
Numbers of molted larvae in a culture well were therefore determined by counting the L3 cuticles shed from the larvae Furthermore, molted larvae (that had already shed their cuticles) exhibited an intense motility compared with unmolted larvae (that had not shed their cuticle) Aliquots
of larvae were removed at different days of postcultures and photographs were taken
Results
Identification of cDNA encodingA suum inorganic pyrophosphatases
A clone designated AdR44 was isolated initially by immu-noscreening an A suum female worm cDNA library with serum obtained from a rabbit immunized with A suum infective eggs AdR44 was selected for further characteriza-tion because of its sequence homology to the inorganic pyrophosphatase family of proteins Sequence analysis showed that AdR44 was 1,375-bp long with an open reading frame (ORF) coding for 360 amino acids The ATG initiation codon is predicted to be at nucleotides 79–81 and
is followed by a region encoding a hydrophobic sequence of
17 amino acids, which may function as a signal peptide The 3¢ untranslated region contained 224 bp and ended with 17-bp poly(A)+tail that began 14-bp down-stream from the sequence AATAAA, which is the eukaryotic consensus polyadenylation signal An entire ORF of the AdR44 cDNA encodes a sequence of 360 amino acids, predicting a
40 600-Da polypeptide with an isoelectric point of 7.1 Removal of the signal peptide resulted in a putative mature protein with molecular weight 38 771 Da Two potential sites for N-glycosylation (residues 50–53, 246–249) were predicted in AsPPase The three conserved aspartates that are involved in the binding of cations in PPases (D-[SGDN]-D-[PE]-[LIVMF]-D-[LIVMGAC]) were found at position 192–197 A search of the protein database conducted using the information obtained from the NCBI revealed that AsPPase shared a high degree of sequence similarity to those
of animal/fungal PPases in Family I soluble PPases Figure 1 shows a comparison of the AsPPase sequence to five other sequences of animal/fungal soluble PPases The deduced amino acid sequence of AsPPase shows 74% similarity (56% identical) to the Drosophila melanogaster PPase sequence, 69% similarity (55% identical) to the sequence of Caenorhabditis elegans, 72% similarity (55% identical) to the sequence of Schizosaccharomyces pombe, 70% similarity (51% identical) to the sequence of Bos taurus and 67% similarity (51% identical) to the sequence of
S cerevisiae Sequence similarities occur throughout the protein but few are at both ends Sequence analysis revealed that all 13 functionally important active site residues (AsPPase numbering: E-125, K-133, E-135, R-155, Y-170, D-192, D-194, D-197, D-224, D-229, K-231, Y-269 and K-270) (Fig 1), which have been reported previously to be evolutionarily well conserved in Family I soluble PPases [12,13,18,34,35], are identical in AsPPase
Phylogenetic analysis of available PPases
We have constructed a phylogenetic tree using Family I soluble PPase sequences by the neighbor-joining method
Trang 5and the confidence of the branching order was verified by
making 1000 bootstrap replicates with the CLUSTALW
program (Fig 2) The neighbor-joined trees reveal that
animal and fungal PPases including AsPPase represent a
separate group from plant and prokaryotic PPases
Fur-thermore, within the animal/fungal subgroup, AsPPase is
more closely clustered with PPases from the free-living
model nematode C elegans and the insect D melanogaster
Characterization of recombinant AsPPase
The gene encoding the soluble PPase of A suum was
amplified by PCR with A suum female worm cDNA
AsPPase was then overexpressed in E coli strain TOP10F¢
using pTrcHisBTMvector, to test whether the clone indeed has an inorganic pyrophosphatase activity Recombinant AsPPase was expressed in E coli with a yield of 1 mgÆL)1of bacterial culture The rAsPPase was 99% pure as determined
by SDS/PAGE analysis The observed molecular mass of rAsPPase corresponded well to the calculated mass of the AdR44 cDNA (data not shown) The functional activity of the purified rAsPPase was determined using a PPihydrolysis assay in a standard reaction mixture containing 5 mMMg2+,
100 mMTris/HCl, pH 7.5 and 1 mMPPi The recombinant protein showed a specific activity of 937 lmol PiÆmin)1Æmg)1 protein corresponding to a kcatvalue of 638 s)1that could be abolished by Ca2+or removal of Mg2+ (Table 1) This activity could not be due to copurification of endogenous
E coliPPase as, the rAsPPase contained a His-tag that was used for purification and the recombinant protein was determined to be pure by SDS/PAGE analysis
Expression and immunohistochemical detection
of native AsPPase
We performed 2D immunoblot analysis to identify native AsPPase in adult female A suum Anti-(mouse rAsPPase) serum reacted strongly with a protein having a molecular mass of 39 kDa with a pI of 7.1 (Fig 3A) confirming that it corresponded to the predicted size of the putative mature protein (38.771 kDa) calculated from the AsPPase amino acid sequence except for a signal peptide In addition, a native AsPPase was identified on silver-stained 2D gels on which more than 200 visible protein spots appeared (data not shown) To determine the N-terminal residues, we excised the native AsPPase spots from 2D immunoblotted polyvinylid-ene difluoride membranes and subjected them to analysis by the automatic Edman degradation method The sequence 1-MALAASATIS-10 of native AsPPase was identical to that
of the putative mature protein This confirmed that our clone encoded a soluble PPase of A suum A spot reacting with the anti-mouse rAsPPase was also seen in parasite extracts and
ES products from various developmental stages, including embryonated eggs, L3, lung-stage L3 and adult male and female worms, indicating that native AsPPases were expressed in all lifecycle stages of A suum (data not shown) Interestingly, enzyme homologs were also expressed in the human roundworm A lumbricoides and the dog roundworm
T canis(Fig 3B) Native AsPPases that reacted with mouse polyclonal antibodies against rAsPPase were localized in various structures such as the hypodermis, dorsal and lateral hypodermalchord, in muscle cells, gut epithelium and, in the uterus and ovary of adult female A suum (Fig 4B–D) No labeling was, however, seen in sections probed with mouse preimmune sera (Fig 4A) This study also detected the ubiquitous presence of AsPPase homologs in various organs
of A lumbricoides (data not shown)
Enzymatic properties of recombinant proteins The PPi dependence of the maximum hydrolytic velocity (Vmax) of the recombinant AsPPase protein was shown to
be 849.005 ± 14.635 lmol PiÆmin)1Æmg)1 protein with a
Km (Michaelis Constant) value of 0.117 ± 0.006 mM for PPi from three independent experiments (Fig 5A) The K value is significantly higher than the values of
Fig 1 Sequence alignment of representative members of Family I
sol-uble PPases CLUSTALW alignment of soluble PPases (GenBank
accession numbers are indicated in parentheses): A suum (AB091401),
C elegans (CAA93107), D melanogaster (O77460), Bos taurus
(P37980), S cerevisiae (2781300) and Schizosaccharomyces pombe
(P19117) Identical residues among PPases are marked with asterisks.
The 13 essential, active site residues that are conserved in all Family I
soluble PPase sequences currently available in the GenBank are further
emphasized by bold typeface The signal peptides are underlined.
Dashes indicate gaps inserted to optimize the alignment The
num-bering is for the sequence of A suum (As) PPase As, A suum, Ce,
C elegans, Dm, D melanogaster, Bt, B taurus, Sc, S cerevisiae, Sp,
S pombe.
Trang 60.0009–0.00147 mM from bovine retinal PPase [23],
0.005 mM from rat liver PPase [36] and 0.026 mM from
bovine rod outer segment PPase [37] These discrepancies in
Kmvalues for PPiare not entirely surprising in view of the
many differences in enzyme purity and assay methodology
The rAsPPase was shown to absolutely require Mg2+for
PPihydrolysis The maximum enzyme activity was found
with 5 mM Mg2+using 1 mM PP, which then gradually
declined with increased concentrations of Mg2+(Fig 5B)
A drop in enzyme activity due to increased concentrations
of Mg2+(>5 mMMg2+) has not been investigated in the present study Although excess of Mg2+is known to inhibit the PPases, however, the mechanism is yet unclear Both
E coli and yeast PPases have four (M1–M4) subsites for binding metal ions for catalysis [38,39] It has been urgued that binding of Mg2+ at three subsites is required for
Fig 2 Phylogenetic tree based on alignment of available Family I soluble PPase sequences The sequences shown are those from (GenBank accession numbers are indicated in parentheses): A suum (AB091401), S cerevisiae (2781300), Kluyveromyces lactis (P13998), Pichia pastoris (O13505),
S pombe (P19117), D melanogaster (O77460), C elegans (CAA93107), B taurus (P37980), Homo sapiens, from ([12]), S cerevisiae mitochondria (P28239), Hordeum vulgare (O23979), Zea mays (O48556), Solanum tuberosum (O43187), Arabidopsis thaliana (AAC33503), Oryza sativa (AAC78101), Chlamydia pneumoniae (AAD19056), Chlamydia trachomatis (O84777), Mycoplasma pneumoniae (P75250), Mycoplasma genitalium (P47593), Bacillus stearothermophilus (BAA19837), Synechocystis (PCC6803, P80507), Thermoplasma acidophilum (P37981), Methanobacterium thermoautotrophicum (O26363), Thermococcus litoralis (P77992), Pyrococcus horikoshii (O59570), T thermophilus (P38576), Mycobacterium leprae (O69540), Mycobacterium tuberculosis (CAB08851), Haemophilus influenzae (1170585), Sulfolobus acidocaldarius (P50308), Aquifex aeolicus (O67501), Helicobacter pylori (P56153), Gluconobacter suboxydans (O05545), Bartonella bacilliformis (P51064), Rickettsia prowazekii (CAA15034), Legionella pneumophila (O34955) and E coli (P17288) The bar indicates the numbers of substitutions per site Unrooted neighbor-joining trees were generated from homologies of soluble PPase sequences and the confidence of the branching order was verified by making 1000 bootstrap replicates using the CLUSTALW program The tree was viewed and converted to graphic format with TREEVIEW
Trang 7catalysis to proceed, whereas binding at M4 causes
inhibi-tion [38,40]) Omission of Mg2+from the reaction medium
abolished PPase-mediated PPi hydrolysis The enzyme
activity was found to be optimum in the pH range 7.0–8.0
(Fig 5C) The pH profile showed a dramatic drop in
activity at high pH These results are consistent with other
soluble PPases from various sources [35,36,41]
Detection of native AsPPase activity and inhibition
by IDP and NaF of larval development and molting Native enzyme activity in L3 soluble extracts and in
ES products was detected by PPase-activated PPi hydroly-sis The L3 extracts showed an activity of 1.58 ± 0.02 lmol
PiÆmin)1Æmg)1, whereas L3 ES exhibited that of 0.51 ± 0.03 lmol PiÆmin)1Æmg)1protein for PPihydrolysis Anti-(mouse rAsPPase) IgG partially inhibited recombinant AsPPase (6 ng) activity up to 22% in the presence of
5 lgÆmL)1anti-(mouse rAsPPase) IgG relative to AsPPase activity determined in the presence of 5 lgÆmL)1 mouse preimmune IgG (data not shown) Also, native AsPPase activity in L3 extracts was shown to be partially inhibited (25%) by anti-(mouse rAsPPase) IgG (data not shown) indicating that AsPPase is responsible for the hydrolyzing activity of PPiin A suum L3 extracts NaF, an anion, is a potent inhibitor of PPases and was able to inhibit native AsPPase activity at micromolar concentrations in a dose-dependent manner (Fig 6A) This agent is also known to inhibit the H+-PPases from plants [42], trypanosomatids [43,44] and apicomplexan protozoa [45]
The L3 of A suum develop and molt to L4 in the lungs of their vertebrate hosts that can also occur during in vitro cultivation To determine whether this complex process is regulated by PPase enzyme, we examined IDP, a non-hydrolyzable PPi analogue, and NaF for their possible
in vivoability to inhibit/arrest development and the molting process by blocking the PPase activated PPihydrolysis, as the native PPases in L3 extracts were found to be very sensitive to inhibition by NaF (Fig 6A) As IDP interferes with the colorimetric assay, it was, however, not possible
to examine enzyme sensitivity with this compound in the present study In vitro molting inhibition experiments demonstrated that IDP and NaF inhibited molting of
A suumL3 to L4 with varying success, in a dose-dependent manner (Fig 6B,C) Up to 55% molting was inhibited at a maximum concentration of 10 mMIDP without affecting the growth and viability of the L3 In contrast, NaF inhibited 65% molting at 1 mMconcentration However, at higher concentrations (>1 mMNaF) molting inhibition was increased drastically up to 100% with an apparent growth inhibition of the L3 observed on day 5 postculture and onwards A mild larvicidal effect of 10 mM NaF with progressive damage of the body wall and intestine was seen
on day 5 postculture and onwards (data not shown) The molted L3 developed well to L4 in control culture with increased body length and width (data not shown), and changes in the structure of the head and tail (Fig 7A–C) compared with unmolted L3 which achieved little or no development, being inhibited by IDP/NaF (Fig 7D,E) Under light microscopy, it was however, not possible to detect the formation and/or separation of new cuticles of unmolted L3 exposed to inhibitors that might be carried out
by electron microscopy The mean molting percentage in control culture was recorded as, 52.59 ± 4.12
Discussion
Although PPases are distributed widely among living cells, most of the previous studies have focused on microbial and plant enzymes, and very little is known about the enzyme
Fig 3 Identification of A suum native PPase in adult female worm.
(A) Fifty micrograms of female worm extract was separated by 2D
nonequilibrium pH-gradient gel electrophoresis, and the proteins were
then transferred to a nitrocellulose membrane The native AsPPase
bound to the anti-(mouse rAsPPase) serum was found by alignment of
the stained gel and immunoblot membrane (B) Expression of AsPPase
homologs in ascarid roundworms Sixty or 80 mg of protein
equiva-lents of each parasite extract were electrophoresed on a 10% SDS/
PAGE and blotted onto a nitrocellulose membrane The AsPPase
homologs bound to the anti-(mouse rAsPPase) serum were detected by
5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium Lane 1,
A lumbricoides; lane 2, T canis; lane 3, A suum.
Table 1 Recombinant A suum PPase activity Pyrophosphatase
activity was assayed as described in the Materials and methods section;
–, no detectable activity Data represent the mean ± SE from three
independent experiments An unrelated A suum 14-kDa recombinant
protein was used as the negative control (As 14; [28]).
Assay conditions
Activity (lmol P i Æmin)1Æmg protein)1)
A suum PPase +5.0 m M Mg 2+ 937.76 ± 39.76
A suum PPase +0.0 m M Mg 2+ –
A suum PPase +5.0 m M Ca2+ –
Yeast-PPase +5.0 m M Mg 2+
(positive control)
13232.36 ± 183.42 As14 + 5.0 m M Mg2+
(negative control)
–
Trang 8from mammalian tissues In contrast, we do not have any
evidence of PPases from parasitic helminths We report here
the cloning, sequencing and biochemical and functional
characterization of a novel PPase from the important
pathogenic roundworm A suum The deduced amino acid
sequence of AsPPase shows significant similarity with
animal/fungal PPase sequences in Family I soluble PPases
(Fig 1) All members of Family I soluble PPases currently
available in the database have been shown to contain 13
functionally important active site residues that are
evolu-tionarily well conserved, and were found to be identical in
AsPPase Several highly conserved regions, the most
prominent of which is an eight residue long sequence
(224-DEGETDWK-231), are also seen in the AsPPase
sequence It will be interesting to see the significance of this
highly conserved region in AsPPase structure and
function-ing Over 37 Family I soluble PPases have been identified
The prokaryotic PPases are hexamers of 20 kDa and
reported to contain 162–220 amino acid residues per
subunit, while eukaryotic PPases are dimers of 30–36 kDa
with 211–310 residues per subunit [12,13,18] AsPPase, with
360 amino acid residues having a calculated molecular mass
of 40.6 kDa, resembles eukaryotic PPases and thus is the
largest among the Family I soluble PPases stored in the
current protein database This is largely due to a longer
N-terminal region compared with other PPases (Fig 1)
The membrane-bound H+-PPases that are found in plants
[17], certain bacteria [46], and more recently identified from
trypanosomatids [47] and apicomplexan protozoa [48] differ greatly in structure and function from soluble PPases The
H+-PPases are much larger (660–770 amino acid residues per monomer) and do not have any sequence similarity to soluble forms [15,16,49] The AsPPase described here is clearly a soluble PPase and it does not have any sequence similarity to plant/protist H+-PPases Together, these findings suggest that AsPPase is a distinct Family I soluble PPase Phylogenetically, AsPPase is, within the subfamily
of animal/fungal soluble PPases, closer to C elegans and
D melanogaster PPases than to fungal and mamma-lian PPases (Fig 2) Moreover, E coli-expressed purified rAsPPase protein has shown enzymatic activity (937 lmol PiÆmin)1Æmg protein)1) by PPi hydrolysis assay that was found to be closer to those of the highly purified and crystallized E coli (2000 lmol PiÆmin)1Æmg)1 [50]), yeast (655 lmol PiÆmin)1Æmg)1 [51]), rat liver 600–
700 lmol PiÆmin)1Æmg)1 [36]) and bovine retinal PPases (>885 lmol PiÆmin)1Æmg)1[23]) Thus, AsPPase represents the first member of Family I soluble PPase enzymes to be identified from the parasitic helminths
The rAsPPase activity was shown to be strictly Mg2+ -dependent On the contrary, Ca2+inhibited the activity to some degree in the presence of Mg2+(data not shown) These distinctive features have been well demonstrated for Family I soluble PPases [23,52,53] The rAsPPase enzyme, however, requires a higher concentration of Ca2+ for inhibition (data not shown), and this finding is fairly
Fig 4 Immunohistochemical localization of A suum native PPase in adult female worm A suum female worms were fixed in paraformaldehyde, embedded in paraffin, sectioned (7-lm thickness) and exposed to either mouse preimmune serum as a control (A) or mouse anti-rAsPPase serum diluted 1: 100 (B) (C) and (D) (both 25·) are magnified areas of (B) Arrows indicate antibody-labeled regions; cu, cuticle; hd, hypodermis;
hc, hypodermal chord; mu, muscle; gu, gut; ov, ovary; ut, uterus.
Trang 9consistent with other animal PPases [36,54] but contrasts with the reports on yeast PPase and on porcine brain and bovine retinal PPases [23,41] that demonstrated much lower concentrations of Ca2+were needed for enzyme inhibition Prior studies have shown that free PPiis a potent inhibitor, and free Mg2+activates the enzyme and binds with PPito form a true substrate, Mg2+PPifor soluble PPases [38,55] Family II PPases are easily distinguishable from Family I PPases in having a preference for Mn2+over Mg2+as the activator, and are not inhibited by Ca2+, rather Ca2+
Fig 5 PP i (A), Mg2+(B) and pH (C) dependence of A suum
recom-binant PPase activity (A) Diluted recomrecom-binant proteins were run in the
standard reaction mixture (as described in Materials and methods) for
PPase assays at 55 C, in the presence of increasing concentrations of
PP i (0.05–0.5 m M ) Data were analyzed using a computer assisted
program ( GRAFIT version 3.09b) The theoretical curve drawn is for
the best fit values of K m ¼ 0.117 ± 0.006 m M , and V max ¼
849.005 ± 14.635 lmolÆmin)1Æmg protein)1 The inset in (A)
repre-sents the linear transformation of the curve (B) Mg2+dependency was
determined as described in (A) in the presence of increasing
concen-trations of Mg 2+ (C) pH dependency of the enzyme was examined as
described above using several buffers with increasing pH values The
buffers used were (100 m M ), sodium acetate (pH 5.0–5.5), Mops
(pH 6.0–6.5), Tris/HCl (pH 7.0–8.5) and glycine/NaOH (9.0–10.5).
Data represent mean ± SEM from three independent experiments.
Fig 6 Inhibition of A suum native PPase activity and A suum L3 molting by IDP and NaF (A) Aliquots of A suum L3 soluble extracts,
17 mg proteinÆmL)1 was run in the standard reaction mixture for PPase assays at 55 C, in the presence of increasing concentrations
of NaF Percentage activity compared to the control in the absence
of NaF (100%) Control activities were 1.58 ± 0.01 lmol
P i Æmin)1Æmg protein)1 for PP i hydrolysis Data represent mean ± SEM from three independent experiments (B) Lung-stage A suum L3 were cultured for molting inhibition assays, in the presence of increasing concentrations of IDP and (C) NaF Molting percentage was determined on day 10 Percentage activities are relative to the control in the absence of inhibitor (100%) Molting percentage of control was 52.59 ± 4.12 Data represent mean ± SEM of triplicates.
Trang 10activates the enzymes preincubated with Mn2+ [13,53]
further indicating that AsPPase reported here is an
authen-tic member of Family I soluble PPases
An intense expression of native AsPPase in metabolically
active tissues, such as the body wall, gut epithelium and
reproductive organs, of adult female worms suggests a
critical role of the enzyme in these organs The presence of
AsPPase in embryonated eggs, L3, lung-stage L3, adult
worms and their ES products together with its direct
detection in L3 soluble extracts and in ES products
(1.58 ± 0.02 lmol PiÆmin)1Æmg)1 and 0.51 ± 0.03 lmol
PiÆmin)1Æmg)1protein for PPihydrolysis, for L3 extracts and
ES products, respectively) strongly suggested the important
roles of the PPase enzyme throughout the developmental
cycle of Ascaris parasites The results of neutralization
studies indicate that AsPPase-specific IgG may interfere
with the development and molting process of Ascaris larvae
We showed that the native AsPPases are very sensitive to
inhibition by NaF in the micromolar range (Fig 6A) This
value is much lower than previously reported data for NaF
against H+-PPases from parasitic protozoa [44] These
results prompted us to investigate the role of PPase enzymes
in the development and molting process of A suum larvae
and to test whether this could be targeted by inhibitors We
used IDP, a nonhydrolyzable PPianalogue, and NaF, a well
known inhibitor of Family I and Family II soluble PPases
(NaF competes with the hydroxide ion for binding to Mg2+
in the active site of the enzyme [35,53]), to block enzyme
activity We demonstrated for the first time that NaF is
highly effective in inhibiting the development and molting of
A suumL3 to L4, in a concentration-dependent manner, whereas, IDP has shown only partial inhibitory effect (Fig 6B,C) However, a much higher concentration of NaF (>1 mM) is required to completely block development and molting of L3 as against micromolar concentration is needed for the inhibition of native enzyme in vitro This difference may in part be attributed to the difference between live parasites and their soluble extracts used in the assay system
We observed that during in vitro cultivation, the L3 could not develop and molt to L4 in the presence of inhibitor, even
at the end when the culture had terminated (Fig 7D,E) These observations indicate that PPase enzymes are prob-ably involved in the development and molting process of
A suumL3 to L4 However, the mechanisms of inhibition of this complex process by PPase inhibitors are yet to be elucidated Although aminopeptidase, cysteine protease and hyaluronidase enzymes so far have been reported to be involved in the development and molting process of A suum L3 to L4 in vitro, virtually none had been characterized in relation to the actual mechanism of the molting process in this roundworm [56] The basic structure of the body wall of parasitic roundworms consists of the cuticle, an underlying syncytial or cellular layer called the hypodermis, and the longitudinally oriented somatic musculature The ecdysis of
an old cuticle and deposition of the components of a new cuticle that are synthesized in the hypodermis and are secreted across the hypodermal membrane into the space between it and the old cuticle, occur at each of four molts during the lifecycle of all roundworms [57] The molecular mechanisms regulating this complex process are, however,
Fig 7 In vitro development and molting of A suum lung-stage L3to L4 in the absence of inhibitor (control; A–C) and its presence (D,E) (A) L3 on day 0 culture from control (B) L3 had initiated molting on day 5 postculture from control Arrow indicates an entirely distended L3 cuticle (C) L4 (molted L3) on day 10 postculture from control A cuticle which had shedded from L3 is indicated by an arrow (D) L3 had not initiated molting on day 5 postculture with inhibitors, IDP/NaF (E) L3 had not molted on day 10 postculture with IDP/NaF Photographs were taken using differential interference contrast microscopy.