Eur J Biochem 270, 2814–2826 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03658.x Inorganic 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 Matsumoto3 and Naotoshi Tsuji1 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 pyrophosphate (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 specific activity of 937 lmol PiỈmin)1Ỉmg)1 protein corresponding to a kcat value of 638 s)1 at 55 °C 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 dosedependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble- and membrane-bound H+-PPases The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process 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 Ascaris infection 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 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, protontranslocating pyrophosphatase; AsPPase, Ascaris suum inorganic pyrophosphatase; rAsPPase, recombinant A suum inorganic pyrophosphatase; 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 March 2003, revised May 2003, accepted May 2003) Keywords: roundworm; inorganic pyrophosphatase; sodium fluoride; imidodiphosphate; molting Ó FEBS 2003 Roundworm pyrophosphatase (Eur J Biochem 270) 2815 prokaryotic, plant and animal/fungal PPases Among the subfamilies, plant PPases bear a closer similarity to prokaryotic than to animal/fungal PPases [12] The H+-PPases, which appear to work as a reversible H+-pump, are much larger and 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 mutagenesis 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 described 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 suum PPase (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 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) Materials and methods Parasites Adult A suum were obtained from infected pigs at a slaughterhouse in Shimotsuma, Japan Adult A lumbricoides and T canis were obtained from patients after treatment with piperazine in Bac Gian, Vietnam and, from an infected dog in Miyazaki, Japan, respectively Unembryonated and embryonated 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 Committee (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 suum adult female worm cDNA expression library was Immunoscreening of a cDNA expression library An adult female worm cDNA expression library was immunoscreened with rabbit antibodies raised against A suum embryonated 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 37 °C for h When plaques were visible, isopropyl thio-b-D-galactoside-impregnated filters were placed on the plates for 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 °C Antibody reactivity with recombinant proteins was revealed by incubation of the filters with alkaline phosphate-conjugated goat anti-rabbit IgG (ICN) for h and developed with 5-bromo-4-chloro-3-indolylphosphate/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 ExAssistTM helper 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 PRISMTM Ready Dye Terminator Cycle Sequencing Kit (PerkinElmer) DNA samples were analyzed 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 The GENETYX-WINTM DNA 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 the PROSITE 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 CLUSTALW 1.8 (http://www.ebi.ac.uk/clustalw/) with the BLOSUM amino 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 branching order was verified by making 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 Ó FEBS 2003 2816 M K Islam et al (Eur J Biochem 270) 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 (5¢-CCGAGCTCGAGACGTGAAGCGACAATCTCGC AATCT-3¢) containing an XhoI (Promega) site upstream of the start codon and an antisense primer (5¢-CAGCCAA GCTTCTCACTCTTTGATGAAATGCATCT-3¢) containing 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 manufacturer’s instructions The resultant plasmid was transformed into E coli strain TOP10F¢ (Invitrogen) The transformed cells were grown to a D600 at 37 °C in SOB medium supplemented with 50 lgỈmL)1 ampicillin To induce protein expression, isopropyl thio-b-D-galactoside was then added to a final concentration of mM and the culture was grown for an additional h at 37 °C The E coli cells were pelleted and resuspended in lysis buffer [50 mM NaH2PO4 (pH 8.0), 10 mM Tris/HCl (pH 8.0), 100 mM NaCl] Lysozyme was added at 100 lgỈmL)1, and the cell suspension was incubated on ice for 15 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 at °C The supernatants containing recombinant proteins of AsPPase were purified using ProBondTM resin (Invitrogen) 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 °C with several successive changes of 20 mM Tris/HCl, pH 7.5 and a decreasing concentration of NaCl in a SlideA-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) Adult females of A suum and A lumbricoides were fixed in 4% paraformaldehyde in 0.1 M phosphate 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 in 1% H2O2 in NaCl/Pi containing 10% ethanol to inactivate endogenous peroxidases For immunolocalization, the slides were blocked in NaCl/Pi containing 10% (v/v) goat serum (Wako) for 30 at room temperature They were then flooded with anti-(mouse rAsPPase) Ig diluted : 100 in NaCl/Pi/E coli lysate, overnight at °C 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) Production of mouse polyclonal antibodies Enzyme assay 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 weeks later in the same adjuvant The mice were bled 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 UltraLinkTM immobilized protein G according to manufacturer’s instructions (Pierce) and used for evaluating the native AsPPase-neutralizing activity The rAsPPase activity was determined spectrophotometrically 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 mM Mg2+, 100 mM Tris/HCl (pH 7.5) and mM PPi (Na4P2O7), in a total volume of 200 lL together with the protein solution, at 55 °C The assay was started by adding 10 lL of diluted rAsPPase solution into the standard reaction mixture The reaction was stopped by adding mL of 200 mM glycine/HCl, pH 3.0 Then, 125 lL of 1% ammonium molybdate (in 25 mM H2SO4) and 125 lL of 1% ascorbic acid (in 0.05% KHSO4) were added to the mixture, and incubated for 30 at 37 °C 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 Pi sample using a Two-dimensional electrophoresis Parasite extracts were treated with an equal volume of urea mixture consisting of M urea, 4% Nonidet P-40, 0.8% ampholine (pH 3.5–10; Pharmacia) and 2% 2-mercaptoethanol, 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 h, the gels were incubated in the equilibration buffer for 10 on a shaker Electrophoresis in the second Immunoblot analysis Immunoblot analysis was carried out as previously described [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-indolylphosphate/nitroblue tetrazolium Immunohistochemistry Ó FEBS 2003 Roundworm pyrophosphatase (Eur J Biochem 270) 2817 spectrophotometer (Model 600, Shimadzu) at an optical density of 700 nm The specific activity of rAsPPase was defined as lmol Pi released min)1 Æmg)1 of protein 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 Enzyme kinetic study The Km (Michaelis constant) and Vmax (maximum velocity) values were determined by incubating the diluted recombinant proteins in the standard reaction mixture in the presence of increasing concentrations of PPi (0.05–0.5 mM) at 55 °C Data were fit to the appropriate equation using GRAFIT version 3.09b (Erithacus Software) Km and Vmax values 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 proteins or A suum L3 extracts were preincubated in the standard reaction mixture containing lgỈmL)1 preimmune or anti-(mouse rAsPPase) IgG (15 min, 37 °C) 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 PPi hydrolysis 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 lungstage L3 to fourth-stage larvae (L4) in vitro The lung-stage L3 were obtained from the lungs of New Zealand white rabbits days after inoculation with 2.5 · 105 embryonated infective eggs of A suum [26] Briefly, the rabbits were killed by an overdose of ketamine hydrochloride (50 mgỈkg)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 lgỈmL)1 penicillin/ streptomycin at 37 °C for 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/Pi containing 50 lgỈmL)1 penicillin/ streptomycin, and subjected to molting inhibition assay Briefly, 50–100 L3 in mL of RPMI 1640 medium (Gibco/ BRL), pH 6.8 supplemented with 10% (v/v) fetal bovine serum (Sigma), 50 lgỈmL)1 penicillin/streptomycin were cultured in 24-well flat-bottomed tissue culture plates (CostarÒ) The cultures were incubated at 37 °C in a humidified 5% CO2 incubator 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 Results Identification of cDNA encoding A suum inorganic pyrophosphatases A clone designated AdR44 was isolated initially by immunoscreening an A suum female worm cDNA library with serum obtained from a rabbit immunized with A suum infective eggs AdR44 was selected for further characterization 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 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 2818 M K Islam et al (Eur J Biochem 270) Ó FEBS 2003 using pTrcHisBTM vector, to test whether the clone indeed has an inorganic pyrophosphatase activity Recombinant AsPPase was expressed in E coli with a yield of mgỈL)1 of 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 PPi hydrolysis assay in a standard reaction mixture containing mM Mg2+, 100 mM Tris/HCl, pH 7.5 and mM PPi The recombinant protein showed a specific activity of 937 lmol PiỈmin)1Ỉmg)1 protein corresponding to a kcat value of 638 s)1 that could be abolished by Ca2+ or removal of Mg2+ (Table 1) This activity could not be due to copurification of endogenous E coli PPase 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 Fig Sequence alignment of representative members of Family I soluble 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 numbering 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 and 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 Furthermore, 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¢ 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 polyvinylidene difluoride membranes and subjected them to analysis by the automatic Edman degradation method The sequence 1MALAASATIS-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 PiỈmin)1Ỉmg)1 protein with a Km (Michaelis Constant) value of 0.117 ± 0.006 mM for PPi from three independent experiments (Fig 5A) The Km value is significantly higher than the values of Ó FEBS 2003 Roundworm pyrophosphatase (Eur J Biochem 270) 2819 Fig 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 0.0009–0.00147 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 Km values for PPi are not entirely surprising in view of the many differences in enzyme purity and assay methodology The rAsPPase was shown to absolutely require Mg2+ for PPi hydrolysis The maximum enzyme activity was found with mM Mg2+ using mM PPi, which then gradually declined with increased concentrations of Mg2+ (Fig 5B) A drop in enzyme activity due to increased concentrations of Mg2+ (>5 mM Mg2+) 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 Ó FEBS 2003 2820 M K Islam et al (Eur J Biochem 270) Table 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 A suum PPase +5.0 mM Mg2+ A suum PPase +0.0 mM Mg2+ A suum PPase +5.0 mM Ca2+ Yeast-PPase +5.0 mM Mg2+ (positive control) As14 + 5.0 mM Mg2+ (negative control) Activity (lmol PiỈmin)1Ỉmg protein)1) 937.76 ± 39.76 – – 13232.36 ± 183.42 – Fig 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 equivalents 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 catalysis to proceed, whereas binding at M4 causes inhibition [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 hydrolysis The L3 extracts showed an activity of 1.58 ± 0.02 lmol PiỈmin)1Ỉmg)1, whereas L3 ES exhibited that of 0.51 ± 0.03 lmol Pi Æmin)1Æmg)1 protein for PPi hydrolysis Anti-(mouse rAsPPase) IgG partially inhibited recombinant AsPPase (6 ng) activity up to 22% in the presence of lgỈmL)1 anti-(mouse rAsPPase) IgG relative to AsPPase activity determined in the presence of lgỈmL)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 PPi in 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 dosedependent 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 nonhydrolyzable PPi analogue, and NaF for their possible in vivo ability to inhibit/arrest development and the molting process by blocking the PPase activated PPi hydrolysis, 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 suum L3 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 mM IDP without affecting the growth and viability of the L3 In contrast, NaF inhibited 65% molting at mM concentration However, at higher concentrations (>1 mM NaF) molting inhibition was increased drastically up to 100% with an apparent growth inhibition of the L3 observed on day postculture and onwards A mild larvicidal effect of 10 mM NaF with progressive damage of the body wall and intestine was seen on day 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 Ó FEBS 2003 Roundworm pyrophosphatase (Eur J Biochem 270) 2821 Fig 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 from mammalian tissues In contrast, we 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 evolutionarily 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 functioning 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 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 mammalian PPases (Fig 2) Moreover, E coli-expressed purified rAsPPase protein has shown enzymatic activity (937 lmol PiỈmin)1Ỉmg protein)1) by PPi hydrolysis assay that was found to be closer to those of the highly purified and crystallized E coli (2000 lmol PiỈmin)1Ỉmg)1 [50]), yeast (655 lmol PiỈmin)1Ỉmg)1 [51]), rat liver 600– 700 lmol PiỈmin)1Ỉmg)1 [36]) and bovine retinal PPases (>885 lmol PiỈmin)1Ỉmg)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 2822 M K Islam et al (Eur J Biochem 270) Fig PPi (A), Mg2+ (B) and pH (C) dependence of A suum recombinant PPase activity (A) Diluted recombinant 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 PPi (0.05–0.5 mM) Data were analyzed using a computer assisted program (GRAFIT version 3.09b) The theoretical curve drawn is for the best fit values of Km ¼ 0.117 ± 0.006 mM, and Vmax ẳ 849.005 14.635 lmolặmin)1ặmg protein)1 The inset in (A) represents the linear transformation of the curve (B) Mg2+ dependency was determined as described in (A) in the presence of increasing concentrations of Mg2+ (C) pH dependency of the enzyme was examined as described above using several buffers with increasing pH values The buffers used were (100 mM), 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 Ó FEBS 2003 Fig 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 proteinỈmL)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 PiỈmin)1Ỉmg protein)1 for PPi 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 consistent 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 PPi is a potent inhibitor, and free Mg2+ activates the enzyme and binds with PPi to form a true substrate, Mg2+PPi for 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+ Ó FEBS 2003 Roundworm pyrophosphatase (Eur J Biochem 270) 2823 Fig In vitro development and molting of A suum lung-stage L3 to L4 in the absence of inhibitor (control; A–C) and its presence (D,E) (A) L3 on day culture from control (B) L3 had initiated molting on day 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 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 activates the enzymes preincubated with Mn2+ [13,53] further indicating that AsPPase reported here is an authentic 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 PiỈmin)1Ỉmg)1 and 0.51 ± 0.03 lmol PiỈmin)1Ỉmg)1 protein for PPi hydrolysis, 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 PPi analogue, 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 suum L3 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 probably involved in the development and molting process of A suum L3 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, 2824 M K Islam et al (Eur J Biochem 270) still poorly understood Based on our results presented above, it is assumed that the target molecules of IDP and NaF might be PPase enzymes in the hypodermis of L3 (Fig indicates the abundance of native PPases in the hypodermal cells of sectioned adult worms in immunohistochemical staining), and that the inhibition of PPasecatalyzed PPi hydrolysis by these inhibitors, is likely to prevent the synthesis of the new cuticle from the hypodermis and/or ecdysis of the old cuticle This assumption is supported by the fact that PPase-activated PPi hydrolysis is essential to maintain the forward direction of many biosynthetic reactions like synthesis of DNA, RNA, proteins and polypeptides [8] In addition, several investigators have demonstrated that both IDP and NaF selectively inhibited the plant and protist H+-PPases [42–45] and other potential PPi analogues (bisphosphonates) selectively inhibited the proliferation of acidocalcisome-containing parasites [58] Recent studies have shown that fluoride can inhibit several metabolic and defending enzymes of microbial origin and can alter expression of protein essential for survival and virulence in unicellular bacteria [59–61] It is thought important that the hypodermis of the body wall, which synthesizes components of the cuticle, may offer a useful target for studies into the mechanism of the development and molting process in the roundworms Investigation into the effects of fluoride exposure to A suum L3 on protein expression to explore the PPase-regulated development and molting process of the Ascaris parasites is currently being undertaken in our laboratory We conclude that PPase is a novel enzyme in A suum that may play an important role in the development and molting of the larval stage parasites Furthermore, the biochemical as well as the in vivo/in vitro 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Luengpailin, J., Cao, J., Pierce, W.M., Cai, J., Klein, J.B & Doyle, R.J (2002) Fluoride exposure attenuates expression of Streptococcus pyogenes virulence factors J Biol Chem 277, 16599–16605  ... that the hypodermis of the body wall, which synthesizes components of the cuticle, may offer a useful target for studies into the mechanism of the development and molting process in the roundworms... 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 Results Identification of cDNA... 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 lungstage L3 to fourth-stage