Parasitol Res DOI 10.1007/s00436-014-3799-7
ORIGINAL PAPER
Molecular characterization and serological reactivity of a vacuolar ATP synthase subunit ε-like protein from Clonorchis sinensis Xiaoli Lv & Lisi Huang & Wenjun Chen & Xiaoyun Wang & Yan Huang & Chuanhuan Deng & Jiufeng Sun & Yanli Tian & Qiang Mao & Huali Lei & Xinbing Yu
Received: 28 November 2013 / Accepted: 28 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The vacuolar ATPase enzyme complex (V-ATPase) pumps protons across membranes, energized by hydrolysis of ATP. Extensive investigations on structural and biochemical features of these molecules have implied their importance in the physiological process. In this study, a full-length sequence encoding a vacuolar ATP synthase subunit ε-like protein of Clonorchis sinensis (CsATP-ε) was isolated from our cDNA library. The hypothetical 226 amino acid sequence shared 76 % identity with ATP-ε proteins of Schistosoma japonicum and above 55 % identity with ATP-ε proteins from human and other eukaryotes. Characteristic Asp140 amino acid residues and seven B-cell epitopes were predicted in this sequence. The complete coding sequence of the gene was expressed in Escherichia coli. Recombinant CsATP-ε (rCsATP-ε) protein could be probed by anti-rCsATP-ε rat serum and C.sinensisinfected human serum in Western blotting experiment, indicating that it is an antigen of strong antigenicity. The high Xiaoli Lv and Lisi Huang contributed equally to this study X. Lv Department of Medical Laboratory and Research Center, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shannxi 710038, China L. Huang Department of Clinical Laboratory, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510120, China W. Chen : X. Wang : Y. Huang : C. Deng : J. Sun : Y. Tian : Q. Mao : H. Lei : X. Yu (*) Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, China e-mail: [email protected] W. Chen : X. Wang : Y. Huang : C. Deng : J. Sun : Y. Tian : Q. Mao : H. Lei : X. Yu Key Laboratory for Tropical Diseases Control, Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong 510080, China
level of antibody titers (1:204,800) showed that CsATP-ε has a powerful immunogenicity. Both the increased level and the change trend of IgG1/IgG2a subtypes in serum showed that the rCsATP-ε can induce strong combined Th1/Th2 immune responses in rats and stimulate the immune response changes to the dominant Th2 from Th1 along with long time infection. The results of immunoblot and immunolocalization demonstrated that CsATP-ε was consecutively expressed at various developmental stages of the parasite, which was supported by real-time PCR analysis. In immunohistochemistry, CsATP-ε was localized on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of metacercaria, implying that CsATP-ε may relate to energy intake and metabolism. This fundamental study would contribute to further researches that are related to growth and development and immunomodulation of C. sinensis.
Introduction Clonorchis sinensis (C. sinensis), the oriental liver fluke, causes clonorchiasis and is one of the most important trematode parasites in China, Korea, Japan, and Southeast Asian countries (Lun et al. 2005). Increasing researches demonstrate the far-reaching socioeconomic impact of this tropical parasite, which afflicts more than 35 million people worldwide and approximately 15 million in China alone (Young et al. 2010). Human clonorchiasis has been increasingly prevalent in recent years, resulting from more consumption of raw or undercooked freshwater fish containing the infective C. sinensis metacercariae. The metacercariae are excysted in the host’s duodenum and migrated through the ampulla of Vater into the bile duct where they grow into adult worms and cause clonorchiasis characterized by cholangitis, cholelithiasis, and cholangectasis (Choi et al. 2004; Lim et al. 2006;
Parasitol Res
Wang et al. 2009). More importantly, liver fluke infection is one of the most significant causative agents of cholangiocarcinoma (Shin et al. 2010; Sripa et al. 2007; Fried et al. 2011). Despite the invaluable whole-genome sequence information for this neglected parasite obtained (Wang et al. 2011), much more about the biochemical characteristics of this parasite need to be known. The vacuolar ATPase (V-ATPase) is a multi-subunit enzyme that consists of two domains. The peripheral V1 domain is responsible for the hydrolysis of ATP, and the integral V0 domain is responsible for the transport of protons (Forgac 1999; Nishi and Forgac 2002). The structure and catalytic mechanism of V-ATPase is thought to be similar to that of F0F1-ATPase (Futai et al. 2000). Within many intracellular compartments including endosomes, lysosomes, and secretory vesicles, V-ATPase function in the acidification of these compartments, receptor-mediated endocytosis, protein processing, and transport of small molecules (Balklava et al. 2007; Hinton et al. 2009a). In the plasma membranes of certain cells, including renal intercalated cells, osteoclasts, and macrophages, they are associated with acid secretion, bone resorption, and control of cytoplasmic pH (Brown and Breton 2000; Li et al. 1999; Hinton et al. 2009a). Studies show that V-ATPases are also functionally expressed in plasma membranes of some human tumor cells, especially in highly metastatic cells, and play important roles in tumor migration and invasion. In the aspect of a parasite, numerous researches show that the V-ATPase have a key role in nematode biology, particularly in nematode nutrition, osmoregulation, cuticle synthesis, and reproduction. (Allman et al. 2009; Liegeois et al. 2007, 2006). In C. elegans, several genes encoding VATPase subunits have been characterized. The E subunit is required for embryogenesis and yolk transfer (Ji et al. 2006). In yeast, the E subunit of V1 sector forms a part of the stalk of V-ATPase which is important for cell viability (Zhang et al. 1998). As a consequence of its varied role, changes in the VATPase function have been implicated in many diseases, including cancer and osteoporosis (Hinton et al. 2009b; Frattini et al. 2000; Hinton et al. 2009a). The function of the V-ATPase in a broader sense has been reported extensively (Forgac 2007; Jefferies et al. 2008; Lun et al. 2005), but the role of this complex in C.sinensis, which cause a significant burden on health, has been far less discussed. In C.sinensis, the previous research showed that the immune response changes to the dominant Th2 from Th1 along with the prolonged infection, which makes the liver inflammatory reaction decreased gradually and turn for chronic liver fluke disease (Choi et al. 2003). However, what kind of antigen stimulates the immune status changes still remains to be further studied. In the present study, we first recognized a complete sequence encoding a vacuolar ATP synthase subunit ε-like protein from the C.sinensis metacercaria complementary
DNA (cDNA) library, expressed and purified the recombinant CsATP-ε, and described the sequence analysis, mRNA and protein expression levels of four life stages, and immunolocalization and serological reactivity of CsATP-ε. We aimed to seek out the possible biological functions and the immune responses of CsATP-ε during the C.sinensis infection. This fundamental study might pave the way for further studies on growth and immunomodulation of C. sinensis.
Materials and methods Parasites and animals Living metacercariae and cercariae of C.sinensis were isolated from our ecologic pool as previously described (Liang et al. 2009; Chen et al. 2011). Eight-week-old Sprague-Dawley (SD) rats, purchased from the Laboratory Animal Center of Sun Yet-sen University, were infected orally with 50 metacercariae each. The adult worms were harvested from the biliary tracts of rats 9 weeks after experimental infection. Excysted juveniles were obtained from metacercariae treated with trypsin (0.1 %, pH 7.4). All of the procedures involving animals and their care in this study were approved by the Institutional Animal Care and Use Committee of Sun Yet-sen University in accordance with institutional guidelines for animal experiments (Permit Numbers: SCXK (Guangdong)2009-0011). Identification and molecular characterization of cDNA sequence encoding CsATP-ε From 3,475 unigenes in our C. sinensis cDNA library, a clone (No. Cs86B12) encoding a CsATP-ε (accession No. DF144297) was identified at the National Center for Biotechnology Information website (http://www.ncbi.nlm. nih.gov/BLAST). Characteristics of deduced protein sequence were predicted by online tools on Expasy website (http://web.expasy.org/). B-cell and T-cell linear epitopes were analyzed by the tools at http://www.cbs.dtu.dk/services/. Based on similarities, the sequences from different species including Schistosoma japonicum (S. j, AAP06177), Drosophila yakuba (D. y, XP002098037), Caenorhabditis briggsae (C. b, XP002633510), Mus musculus (M. m, NP031536.2), Homo sapiens (H. s, CAA50592), Trichinella spiralis (T. s, XP003376773), Schistosoma mansoni (S. m, XP002579650), Xenopus (Silurana) tropicalis (X. t, NP989123), Culex quinquefasciatus (C. q, XP001849126), Meleagris gallopavo (M. g, XP003202515), and Brugia malayi (B. m, XP001902068) were aligned by the bioinformatics analysis software Vector NTI suite 8.0. The phylogenetic tree was constructed using the neighbor-joining method with MEGA4. Bootstrap proportions were used to
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assess the robustness of the tree with 500 bootstrap replications. Cloning, expression, and purification of CsATP-ε
for 2 h. After washing, the membrane was incubated with rabbit anti-rat IgG HRP-conjugated horse radish peroxidase (1:2,000 dilution; Boster; PR China) at 37 °C for 1 h. Diaminobenzidine (DAB) substrate solution (Invitrogen, USA) was used to visualize the reaction according to the manufacturer's instructions.
Gene sequence encoding CsATP-ε was amplified from cDNA of C. sinensis by polymerase chain reaction (PCR) using the forward primer 5′-CTAGGATCCATGGCGCTCAATGAAA C-3′ and the reverse primer 5′-CGACTCGAGCTAATCAC GGAACTTCC-3′ with BamHI/XhoI restriction enzyme sites (underlined). PCR was carried out for 30 cycles at 94 °C for 45 s, 57 °C for 45 s, and 72 °C for 60 s, and the reaction continued for 10 min at 72 °C after the last cycle. Purified PCR products were cloned into the His6 tag expression vector pET-28a (+) (Novagen; USA) with corresponding incision enzymes. The recombinant plasmid was transformed into E. coli for expression and insertion confirmed by digestion with restriction enzyme and DNA sequencing. Expression of recombinant CsATP-ε protein was induced by isopropyl-β-Dthiogalactoside (IPTG) at a final concentration of 1 mM for 5 h at 37 °C. The bacterial cells were collected by centrifugation at 4 °C, and the inclusion bodies containing the recombinant fusion protein were solubilized completely with 6 M urea in 20 mM Tris-HCl buffer (pH 8.0) followed by purification with the His Bind Purification kit (Novagen; USA) and elution with 100 mM imidazole. Renaturation was carried out by stepwise diluting urea in dialysate buffer (20 mM Tris-HCl, 5 mM EDTA buffer, pH 8.0). Purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue, the final recombinant protein concentration was estimated by Bradford assay using BSA method used as a standard.
In order to analyze the immune responses to CsATP-ε, the antibody titers of total IgG in immunized sera were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, the microtiter plates were coated with 1 μg/ml purified rCsATP-ε in coating buffer (0.1 M carbonate-bicarbonate, pH 9.6) and incubated at 4 °C overnight. Subsequently, the plates were blocked with 5 % skimmed milk in PBS containing 0.05 % Tween 20 (pH 7.4) for 2 h at 37 °C. After washing, the plate was incubated with different dilutions of the immune sera (week 6) raised by rCsATP-ε. Rat sera immunized with PBS was measured as negative controls. HRP-conjugated IgG (1:20,000 dilutions in 0.1 % BSA-PBST, Proteintech Group) was used as the secondary antibodies. After a 1-h incubation, the plate was washed three times with PBST, and the reactions were developed by adding a 100-ml substrate solution (TMB, BD biosciences, San Diego, USA). After a 10-min incubation in the dark, the absorbance was measured at 450 nm after adding 2 M H2SO4 to stop the reaction. After measuring the antibody titer, the level of IgG isotype was tested to investigate the tendency of circulation antibodies and the profiles of immune responses. The rat sera of week 2, 4, 6, and 8 were diluted at 1:400. IgG1 and IgG2a (1:1,000 dilutions, Bethyl, TX, USA) were employed as secondary antibodies.
Acquirement of rat anti-rCsATP-ε serum and identification of CsATP
Real-time PCR and Western blotting analysis of CsATP-ε at different life stages of C. sinensis
Polyclonal anti-serum to rCsATP-ε was produced by immunizing SD rats. Briefly, 200 μg of purified rCsATP-ε mixed with an equal volume of complete Freund′s adjuvant (Sigma, USA) was injected subcutaneously into SD rats and two boosters of 100 μg rCsATP-ε with an equal volume of incomplete Freund′s adjuvant were given at a 2-week interval. Immune sera were collected 2 weeks post the injection. The recombinant protein (1 μg per lane) was subjected to SDS-PAGE and electrotransferred onto polyvinylidene difluoride membrane (PVDF, Whatman; USA) at 100 v for 1 h. The membrane was blocked with 5 % skimmed milk in phosphate-buffered saline Tween-20 (PBST, pH 7.4) at 37 °C for 2 h, washed five times with PBST, then incubated with anti-His-tag monoclonal antibody (1:3,000 dilutions), antiCsATP-ε rat serum (1:200 dilutions), C. sinensis-infected human serum (1:200 dilutions), preimmune rat serum (1:200 dilutions), and healthy people serum (1:200 dilutions) at 37 °C
Total RNA was extracted, respectively, from eggs, metacercariae, excysted juveniles, and adult worms with TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. First-strand cDNA was performed with the reverse transcriptase Superscript (Takara, Japan) with oligo(dT) primers using total RNA as template. Real-time PCR amplification was carried out on LightCycler 480 instrument (Roche, Switzerland) using SYBR Premix Ex Taq Kit (Takara, Japan). The LightCycler 480 software (version 1.5) was used to analyze the data according to 2−ΔΔCt method (Pfaffl 2001). The β-actin (accession No.: EU109284) amplification signal was employed as transcriptional control (Yoo et al. 2009; Wang et al. 2012), and signal of the egg was used as the calibrator. Western blotting was carried out to investigate the differential expression of CsATP-ε protein during the parasite life cycle. Parasites from four stages were suspended in the RIPA
Antibody titers and IgG isotype measurement
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lysis buffer (containing 1 mM proteinase inhibitor PMSF, Bioteke, China). The protein content of the supernatant was obtained after centrifugation at 100,000g for 5 min. Protein concentrations were determined by means of the BCA protein assay kit (Novagen, USA). Forty micrograms of total proteins from each life-cycle stage was separated on SDS-PAGE (12 % gel) and blotted onto the PVDF membrane. The blots were developed with anti-CsATP-ε rat serum (1:200 dilutions) and HRP-conjugated goat anti-rat IgG (1:2,000 dilutions). Detection was then carried out by chemiluminescence. Immunolocalization of CsATP-ε in C. sinensis adult worm, metacercaria, and cercaria Sectioned worms and metacercariae in paraffin wax were deparaffinized and incubated in the rat anti-CsATP-ε sera (1:200 dilutions). The collected cercariae were immersed in PBS 0.3 % TritonX-100 for 1 h at room temperature (RT) before being fixed with cold acetone on a glass slide for immunohistochemical analysis. Preimmune rat serum was applied as a negative control. The sections were subsequently incubated with goat anti-rat IgG (1:400 dilutions with 0.1 % BSA in PBS, Alexa Fluor 594, Molecular Probe) for 1 h at RT in the dark, then imaged using a ZEISS Axio Imager Z1 fluorescent microscope.
Results Bioinformatics analysis of CsATP-ε The complete coding sequence of CsATP-ε was comprised of 678 bp encoding a putative protein of 226 amino acids with a predicted molecular mass of 26.5 kDa. The estimated half-life of rCsATP-ε was more than 10 h in E. coli. The indexes of instability in solution, aliphatic, and grand average of hydropathicity were 61.52, 91.02, and −0.728, respectively, which indicated that rCsATP-ε was a stable protein with a hydrophobic core in solution. Blastx analysis revealed that the deduced amino acid sequence of this subunit is one of the most well conserved subunits sharing approximately 76 % identity with ATP-ε proteins of S.japonicum and above 55 % identity with ATP-ε proteins from human and other eukaryotes. The conserved Asp140 amino acid and V-ATPase subunit-ε functional domain were found. Neither signal peptide nor transmembrane domains was found in the sequence. Linear B-cell epitopes were found by IEDB analysis resource, namely: aa22–39, aa70–76, aa106–111, aa155–160, aa170– 179, aa181–187, and aa220–224, suggesting that the rCsATP-ε maintains good immunogenicity (Fig. 1). The relationships displayed in the phylogenetic tree were in good agreement with traditional taxonomy (Fig. 2).
Cloning, expression, and purification of rCsATP-ε The recombinant pET-28a (+) plasmid containing the ATP-ε coding region was confirmed by digestion with restriction enzyme. DNA sequencing revealed that the construct was correct with His6-tag at the N terminus of the recombinant protein. The rCsATP-ε was expressed in E. coli (BL-21) after induction by IPTG, and a purified fusion protein with an approximate molecular weight around 30 kDa was detected, corresponding to the predicted size of 30.073 kDa (including 34 amino acids of vectors) originated from the primary gene sequence. After purification and renaturation, the concentration of the purified CsATP-ε protein was about 0.4 mg/ml (Fig. 3, lane 7). Western blotting analysis of recombinant CsATP-ε protein Purified CsATP-ε could be probed by mouse anti-His-tag monoclonal antibody, anti-CsATP-ε rat serum, and C. sinensis-infected human serum in the Western blot analysis, while not recognized by preimmune rat serum and healthy people serum (Fig. 4). Antibody titers and immune responses to CsATP-ε As shown in Fig. 5, we measured the antibody titers of total IgG in immunized sera of rCsATP-ε. Antibody titers peaked to 1:204,800 (Fig. 5), showing the high immunogenicity of CsATP-ε. Additionally, we explored the Th1-/Th2-type immune responses to CsATP-ε by measuring IgG1 and IgG2a from weeks 2 to 8 post immunization. The ELISA results showed that combined Th1/Th2 immune responses were provoked by rCsATP-ε for both IgG1 and IgG2a level increase (Fig. 6). The increased IgG isotype demonstrated that combined cellular immunity and humoral immunity had been successfully induced by CsATP-ε. Gene/protein expression level of CsATP-ε at different developmental stages of C. sinensis Real-time PCR was performed to detect the CsATP-ε transcripts at life stages of egg, metacercaria, excysted juvenile, and adult worm. The-fold changes of CsATP-ε gene transcriptional level in adult worm, excysted juvenile, and metacercaria were calculated compared with egg. The results showed that the expression levels of CsATP-ε gene were 9.10, 2.58, and 6.78-fold in metacercaria, excysted juvenile, and adult worm, respectively (Fig. 7a). Specific binding bands were detected with anti-CsATP-ε mouse serum by western blotting assay. The expression level of CsATP-ε protein was varied within different developmental stages. Adult worm showed the highest expression level of CsATP-ε protein, followed by metacercariae, excysted juvenile, and egg (Fig. 7b).
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Fig. 1 Amino acid sequence alignment of CsATP-ε and other V-ATPase ε subunit homologues. The protein sequence of CsATP-ε is depicted and aligned with Schistosoma japonicum (S. j, AAP06177.1), Caenorhabditis briggsae (C. b, XP_002633510.1), Drosophila yakuba (D. y, XP002098037.1), Mus musculus (M. m, NP031536.2), and Homo sapiens (H. s, CAA50592.1) homologues. Numbers in parentheses stand
for GenBank accession number for each organism. Dark-shaded boxes represent amino acid residues identical in all six proteins and light-shaded boxes represent residues conserved in four or five proteins. An asterisk indicates the conserved amino acid, Asp140. B-cell and T-cell linear epitopes were indicated with full lines and dotted lines, respectively
Fig. 2 Phylogenetic analysis of CsATP-ε and other related enzymes. Trichinella spiralis (T. s, XP003376773), Schistosoma mansoni (S. m, XP002579650), Xenopus (Silurana) tropicalis (X. t, NP989123), Culex quinquefasciatus (C. q, XP001849126), Meleagris gallopavo (M. g, XP003202515), and Brugia malayi (B. m, XP001902068). The tree was
constructed with the neighbor-joining method using MEGA4 program. Numbers on the branches indicated bootstrap proportions (500 replicates). The relationships displayed in the phylogenetic tree were in good agreement with traditional taxonomy
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Fig. 3 Expression and purification of CsATP-ε by 12 % SDS-PAGE. Protein molecular weight markers (M), lysate of E. coli with pET-28a(+) before induction with IPTG (lane 1) and after induction (lane 2), lysate of E. coli with pET28a(+)-CsATP-ε before induction (lane 3) and after induction (lane 4), supernatant of induced pET28a(+)-CsATP-ε (lane 5), and sediment (lane 6) and purified rCsATP-ε (lane 7)
Immunolocalization of CsATP-ε at adult worm and metacercaria of C. sinensis Using the rat anti-rCsATP-ε serum as the primary antibody and red-fluorescent Cy3 labeling IgG as the secondary antibody, results of immunochemistry showed that CsATP-ε was distributed both in adult worms and metacercariae. Specific fluorescence was detected on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of the metacercaria. No fluorescence was visualized in other organs or in controls treated with naïve serum (Fig. 8).
Discussion In the present study, a full-length gene encoding an ATP synthase subunit ε-like protein from C. sinensis (CsATP-ε) was identified, cloned, and overexpressed in E. coli. It contains 678 bp encoding 226 amino acids with a predicted
Fig. 4 Western blot analysis of rCsATP-ε. Protein molecular weight markers(M), rCsATP-ε probed with mouse anti-His-tag monoclonal antibody (lane 1), rCsATP-ε probed with anti-CsCP rat serum (lane 2), rCsATP-ε probed with C. sinensis-infected human serum (lane 3), rCsATP-ε probed with preimmune rat serum (lane 4), and human normal serum (lane 5)
Fig. 5 Antibody titers of IgG induced by CsATP-ε measured by ELISA. Briefly, 1 mg/well recombinant pET-28a-CsATP-ε protein was coated on the plates and blocked with 5 % skimmed milk. The plate was incubated with different dilutions of the immune sera (week 6) raised by pET-28aCsATP-ε. Rat sera immunized with PBS were measured under the same conditions as negative controls. HRP-conjugated IgG (1:20,000 dilutions) was used as the secondary antibodies. The reactions were developed with substrate solution TMB, stopped by 2 M H2SO4, and measured at 450 nm
molecular mass of 26.5 kDa. Blastx in NCBI reveals that it shares 76 % identity with ATP-ε proteins of S. japonicum and above 55 % identity with ATP-ε proteins from human and other eukaryotes. The high identity of this ε subunit with other eukaryotic organisms indicates its crucial role in the function of the enzyme. The conserved Asp140, known to be essential for E subunit function (Zhang et al. 1998), was found in the sequence. Bioinformatics analysis indicated that it is a novel complete gene of C.sinensis. ATP synthases are membrane-bound enzyme complexes/ ion transporters that combine ATP synthesis and/or hydrolysis with the transport of protons across a membrane. There are different types of ATPases including F-ATPases, V-ATPases,
Fig. 6 IgG isotype induced by CsATP-ε measured by ELISA. 1 mg/well recombinant pET-28a-CsATP-ε protein was coated on the plates and blocked with 5 % skimmed milk. Immune sera from weeks 2 to 8 were diluted at 1:400. Rat sera immunized with PBS were measured under the same conditions as negative controls. IgG1 and IgG2a (1:1,000 dilutions) were used as secondary antibodies
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Fig. 7 a Stage-specific transcription levels of CsATP-ε gene revealed by real-time PCR. CsATP-ε PCR products are using metacercaria cDNA (lane 1), adult worm cDNA (lane 2), excysted juvenile cDNA (lane 3), and egg cDNA (lane 4) as template. β-actin was employed as the transcriptional control, and the sample of the egg was used as the calibrator. Each bar represented the mean value from three experiments with standard deviation (*p
ORIGINAL PAPER
Molecular characterization and serological reactivity of a vacuolar ATP synthase subunit ε-like protein from Clonorchis sinensis Xiaoli Lv & Lisi Huang & Wenjun Chen & Xiaoyun Wang & Yan Huang & Chuanhuan Deng & Jiufeng Sun & Yanli Tian & Qiang Mao & Huali Lei & Xinbing Yu
Received: 28 November 2013 / Accepted: 28 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The vacuolar ATPase enzyme complex (V-ATPase) pumps protons across membranes, energized by hydrolysis of ATP. Extensive investigations on structural and biochemical features of these molecules have implied their importance in the physiological process. In this study, a full-length sequence encoding a vacuolar ATP synthase subunit ε-like protein of Clonorchis sinensis (CsATP-ε) was isolated from our cDNA library. The hypothetical 226 amino acid sequence shared 76 % identity with ATP-ε proteins of Schistosoma japonicum and above 55 % identity with ATP-ε proteins from human and other eukaryotes. Characteristic Asp140 amino acid residues and seven B-cell epitopes were predicted in this sequence. The complete coding sequence of the gene was expressed in Escherichia coli. Recombinant CsATP-ε (rCsATP-ε) protein could be probed by anti-rCsATP-ε rat serum and C.sinensisinfected human serum in Western blotting experiment, indicating that it is an antigen of strong antigenicity. The high Xiaoli Lv and Lisi Huang contributed equally to this study X. Lv Department of Medical Laboratory and Research Center, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shannxi 710038, China L. Huang Department of Clinical Laboratory, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510120, China W. Chen : X. Wang : Y. Huang : C. Deng : J. Sun : Y. Tian : Q. Mao : H. Lei : X. Yu (*) Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, China e-mail: [email protected] W. Chen : X. Wang : Y. Huang : C. Deng : J. Sun : Y. Tian : Q. Mao : H. Lei : X. Yu Key Laboratory for Tropical Diseases Control, Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong 510080, China
level of antibody titers (1:204,800) showed that CsATP-ε has a powerful immunogenicity. Both the increased level and the change trend of IgG1/IgG2a subtypes in serum showed that the rCsATP-ε can induce strong combined Th1/Th2 immune responses in rats and stimulate the immune response changes to the dominant Th2 from Th1 along with long time infection. The results of immunoblot and immunolocalization demonstrated that CsATP-ε was consecutively expressed at various developmental stages of the parasite, which was supported by real-time PCR analysis. In immunohistochemistry, CsATP-ε was localized on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of metacercaria, implying that CsATP-ε may relate to energy intake and metabolism. This fundamental study would contribute to further researches that are related to growth and development and immunomodulation of C. sinensis.
Introduction Clonorchis sinensis (C. sinensis), the oriental liver fluke, causes clonorchiasis and is one of the most important trematode parasites in China, Korea, Japan, and Southeast Asian countries (Lun et al. 2005). Increasing researches demonstrate the far-reaching socioeconomic impact of this tropical parasite, which afflicts more than 35 million people worldwide and approximately 15 million in China alone (Young et al. 2010). Human clonorchiasis has been increasingly prevalent in recent years, resulting from more consumption of raw or undercooked freshwater fish containing the infective C. sinensis metacercariae. The metacercariae are excysted in the host’s duodenum and migrated through the ampulla of Vater into the bile duct where they grow into adult worms and cause clonorchiasis characterized by cholangitis, cholelithiasis, and cholangectasis (Choi et al. 2004; Lim et al. 2006;
Parasitol Res
Wang et al. 2009). More importantly, liver fluke infection is one of the most significant causative agents of cholangiocarcinoma (Shin et al. 2010; Sripa et al. 2007; Fried et al. 2011). Despite the invaluable whole-genome sequence information for this neglected parasite obtained (Wang et al. 2011), much more about the biochemical characteristics of this parasite need to be known. The vacuolar ATPase (V-ATPase) is a multi-subunit enzyme that consists of two domains. The peripheral V1 domain is responsible for the hydrolysis of ATP, and the integral V0 domain is responsible for the transport of protons (Forgac 1999; Nishi and Forgac 2002). The structure and catalytic mechanism of V-ATPase is thought to be similar to that of F0F1-ATPase (Futai et al. 2000). Within many intracellular compartments including endosomes, lysosomes, and secretory vesicles, V-ATPase function in the acidification of these compartments, receptor-mediated endocytosis, protein processing, and transport of small molecules (Balklava et al. 2007; Hinton et al. 2009a). In the plasma membranes of certain cells, including renal intercalated cells, osteoclasts, and macrophages, they are associated with acid secretion, bone resorption, and control of cytoplasmic pH (Brown and Breton 2000; Li et al. 1999; Hinton et al. 2009a). Studies show that V-ATPases are also functionally expressed in plasma membranes of some human tumor cells, especially in highly metastatic cells, and play important roles in tumor migration and invasion. In the aspect of a parasite, numerous researches show that the V-ATPase have a key role in nematode biology, particularly in nematode nutrition, osmoregulation, cuticle synthesis, and reproduction. (Allman et al. 2009; Liegeois et al. 2007, 2006). In C. elegans, several genes encoding VATPase subunits have been characterized. The E subunit is required for embryogenesis and yolk transfer (Ji et al. 2006). In yeast, the E subunit of V1 sector forms a part of the stalk of V-ATPase which is important for cell viability (Zhang et al. 1998). As a consequence of its varied role, changes in the VATPase function have been implicated in many diseases, including cancer and osteoporosis (Hinton et al. 2009b; Frattini et al. 2000; Hinton et al. 2009a). The function of the V-ATPase in a broader sense has been reported extensively (Forgac 2007; Jefferies et al. 2008; Lun et al. 2005), but the role of this complex in C.sinensis, which cause a significant burden on health, has been far less discussed. In C.sinensis, the previous research showed that the immune response changes to the dominant Th2 from Th1 along with the prolonged infection, which makes the liver inflammatory reaction decreased gradually and turn for chronic liver fluke disease (Choi et al. 2003). However, what kind of antigen stimulates the immune status changes still remains to be further studied. In the present study, we first recognized a complete sequence encoding a vacuolar ATP synthase subunit ε-like protein from the C.sinensis metacercaria complementary
DNA (cDNA) library, expressed and purified the recombinant CsATP-ε, and described the sequence analysis, mRNA and protein expression levels of four life stages, and immunolocalization and serological reactivity of CsATP-ε. We aimed to seek out the possible biological functions and the immune responses of CsATP-ε during the C.sinensis infection. This fundamental study might pave the way for further studies on growth and immunomodulation of C. sinensis.
Materials and methods Parasites and animals Living metacercariae and cercariae of C.sinensis were isolated from our ecologic pool as previously described (Liang et al. 2009; Chen et al. 2011). Eight-week-old Sprague-Dawley (SD) rats, purchased from the Laboratory Animal Center of Sun Yet-sen University, were infected orally with 50 metacercariae each. The adult worms were harvested from the biliary tracts of rats 9 weeks after experimental infection. Excysted juveniles were obtained from metacercariae treated with trypsin (0.1 %, pH 7.4). All of the procedures involving animals and their care in this study were approved by the Institutional Animal Care and Use Committee of Sun Yet-sen University in accordance with institutional guidelines for animal experiments (Permit Numbers: SCXK (Guangdong)2009-0011). Identification and molecular characterization of cDNA sequence encoding CsATP-ε From 3,475 unigenes in our C. sinensis cDNA library, a clone (No. Cs86B12) encoding a CsATP-ε (accession No. DF144297) was identified at the National Center for Biotechnology Information website (http://www.ncbi.nlm. nih.gov/BLAST). Characteristics of deduced protein sequence were predicted by online tools on Expasy website (http://web.expasy.org/). B-cell and T-cell linear epitopes were analyzed by the tools at http://www.cbs.dtu.dk/services/. Based on similarities, the sequences from different species including Schistosoma japonicum (S. j, AAP06177), Drosophila yakuba (D. y, XP002098037), Caenorhabditis briggsae (C. b, XP002633510), Mus musculus (M. m, NP031536.2), Homo sapiens (H. s, CAA50592), Trichinella spiralis (T. s, XP003376773), Schistosoma mansoni (S. m, XP002579650), Xenopus (Silurana) tropicalis (X. t, NP989123), Culex quinquefasciatus (C. q, XP001849126), Meleagris gallopavo (M. g, XP003202515), and Brugia malayi (B. m, XP001902068) were aligned by the bioinformatics analysis software Vector NTI suite 8.0. The phylogenetic tree was constructed using the neighbor-joining method with MEGA4. Bootstrap proportions were used to
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assess the robustness of the tree with 500 bootstrap replications. Cloning, expression, and purification of CsATP-ε
for 2 h. After washing, the membrane was incubated with rabbit anti-rat IgG HRP-conjugated horse radish peroxidase (1:2,000 dilution; Boster; PR China) at 37 °C for 1 h. Diaminobenzidine (DAB) substrate solution (Invitrogen, USA) was used to visualize the reaction according to the manufacturer's instructions.
Gene sequence encoding CsATP-ε was amplified from cDNA of C. sinensis by polymerase chain reaction (PCR) using the forward primer 5′-CTAGGATCCATGGCGCTCAATGAAA C-3′ and the reverse primer 5′-CGACTCGAGCTAATCAC GGAACTTCC-3′ with BamHI/XhoI restriction enzyme sites (underlined). PCR was carried out for 30 cycles at 94 °C for 45 s, 57 °C for 45 s, and 72 °C for 60 s, and the reaction continued for 10 min at 72 °C after the last cycle. Purified PCR products were cloned into the His6 tag expression vector pET-28a (+) (Novagen; USA) with corresponding incision enzymes. The recombinant plasmid was transformed into E. coli for expression and insertion confirmed by digestion with restriction enzyme and DNA sequencing. Expression of recombinant CsATP-ε protein was induced by isopropyl-β-Dthiogalactoside (IPTG) at a final concentration of 1 mM for 5 h at 37 °C. The bacterial cells were collected by centrifugation at 4 °C, and the inclusion bodies containing the recombinant fusion protein were solubilized completely with 6 M urea in 20 mM Tris-HCl buffer (pH 8.0) followed by purification with the His Bind Purification kit (Novagen; USA) and elution with 100 mM imidazole. Renaturation was carried out by stepwise diluting urea in dialysate buffer (20 mM Tris-HCl, 5 mM EDTA buffer, pH 8.0). Purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue, the final recombinant protein concentration was estimated by Bradford assay using BSA method used as a standard.
In order to analyze the immune responses to CsATP-ε, the antibody titers of total IgG in immunized sera were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, the microtiter plates were coated with 1 μg/ml purified rCsATP-ε in coating buffer (0.1 M carbonate-bicarbonate, pH 9.6) and incubated at 4 °C overnight. Subsequently, the plates were blocked with 5 % skimmed milk in PBS containing 0.05 % Tween 20 (pH 7.4) for 2 h at 37 °C. After washing, the plate was incubated with different dilutions of the immune sera (week 6) raised by rCsATP-ε. Rat sera immunized with PBS was measured as negative controls. HRP-conjugated IgG (1:20,000 dilutions in 0.1 % BSA-PBST, Proteintech Group) was used as the secondary antibodies. After a 1-h incubation, the plate was washed three times with PBST, and the reactions were developed by adding a 100-ml substrate solution (TMB, BD biosciences, San Diego, USA). After a 10-min incubation in the dark, the absorbance was measured at 450 nm after adding 2 M H2SO4 to stop the reaction. After measuring the antibody titer, the level of IgG isotype was tested to investigate the tendency of circulation antibodies and the profiles of immune responses. The rat sera of week 2, 4, 6, and 8 were diluted at 1:400. IgG1 and IgG2a (1:1,000 dilutions, Bethyl, TX, USA) were employed as secondary antibodies.
Acquirement of rat anti-rCsATP-ε serum and identification of CsATP
Real-time PCR and Western blotting analysis of CsATP-ε at different life stages of C. sinensis
Polyclonal anti-serum to rCsATP-ε was produced by immunizing SD rats. Briefly, 200 μg of purified rCsATP-ε mixed with an equal volume of complete Freund′s adjuvant (Sigma, USA) was injected subcutaneously into SD rats and two boosters of 100 μg rCsATP-ε with an equal volume of incomplete Freund′s adjuvant were given at a 2-week interval. Immune sera were collected 2 weeks post the injection. The recombinant protein (1 μg per lane) was subjected to SDS-PAGE and electrotransferred onto polyvinylidene difluoride membrane (PVDF, Whatman; USA) at 100 v for 1 h. The membrane was blocked with 5 % skimmed milk in phosphate-buffered saline Tween-20 (PBST, pH 7.4) at 37 °C for 2 h, washed five times with PBST, then incubated with anti-His-tag monoclonal antibody (1:3,000 dilutions), antiCsATP-ε rat serum (1:200 dilutions), C. sinensis-infected human serum (1:200 dilutions), preimmune rat serum (1:200 dilutions), and healthy people serum (1:200 dilutions) at 37 °C
Total RNA was extracted, respectively, from eggs, metacercariae, excysted juveniles, and adult worms with TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. First-strand cDNA was performed with the reverse transcriptase Superscript (Takara, Japan) with oligo(dT) primers using total RNA as template. Real-time PCR amplification was carried out on LightCycler 480 instrument (Roche, Switzerland) using SYBR Premix Ex Taq Kit (Takara, Japan). The LightCycler 480 software (version 1.5) was used to analyze the data according to 2−ΔΔCt method (Pfaffl 2001). The β-actin (accession No.: EU109284) amplification signal was employed as transcriptional control (Yoo et al. 2009; Wang et al. 2012), and signal of the egg was used as the calibrator. Western blotting was carried out to investigate the differential expression of CsATP-ε protein during the parasite life cycle. Parasites from four stages were suspended in the RIPA
Antibody titers and IgG isotype measurement
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lysis buffer (containing 1 mM proteinase inhibitor PMSF, Bioteke, China). The protein content of the supernatant was obtained after centrifugation at 100,000g for 5 min. Protein concentrations were determined by means of the BCA protein assay kit (Novagen, USA). Forty micrograms of total proteins from each life-cycle stage was separated on SDS-PAGE (12 % gel) and blotted onto the PVDF membrane. The blots were developed with anti-CsATP-ε rat serum (1:200 dilutions) and HRP-conjugated goat anti-rat IgG (1:2,000 dilutions). Detection was then carried out by chemiluminescence. Immunolocalization of CsATP-ε in C. sinensis adult worm, metacercaria, and cercaria Sectioned worms and metacercariae in paraffin wax were deparaffinized and incubated in the rat anti-CsATP-ε sera (1:200 dilutions). The collected cercariae were immersed in PBS 0.3 % TritonX-100 for 1 h at room temperature (RT) before being fixed with cold acetone on a glass slide for immunohistochemical analysis. Preimmune rat serum was applied as a negative control. The sections were subsequently incubated with goat anti-rat IgG (1:400 dilutions with 0.1 % BSA in PBS, Alexa Fluor 594, Molecular Probe) for 1 h at RT in the dark, then imaged using a ZEISS Axio Imager Z1 fluorescent microscope.
Results Bioinformatics analysis of CsATP-ε The complete coding sequence of CsATP-ε was comprised of 678 bp encoding a putative protein of 226 amino acids with a predicted molecular mass of 26.5 kDa. The estimated half-life of rCsATP-ε was more than 10 h in E. coli. The indexes of instability in solution, aliphatic, and grand average of hydropathicity were 61.52, 91.02, and −0.728, respectively, which indicated that rCsATP-ε was a stable protein with a hydrophobic core in solution. Blastx analysis revealed that the deduced amino acid sequence of this subunit is one of the most well conserved subunits sharing approximately 76 % identity with ATP-ε proteins of S.japonicum and above 55 % identity with ATP-ε proteins from human and other eukaryotes. The conserved Asp140 amino acid and V-ATPase subunit-ε functional domain were found. Neither signal peptide nor transmembrane domains was found in the sequence. Linear B-cell epitopes were found by IEDB analysis resource, namely: aa22–39, aa70–76, aa106–111, aa155–160, aa170– 179, aa181–187, and aa220–224, suggesting that the rCsATP-ε maintains good immunogenicity (Fig. 1). The relationships displayed in the phylogenetic tree were in good agreement with traditional taxonomy (Fig. 2).
Cloning, expression, and purification of rCsATP-ε The recombinant pET-28a (+) plasmid containing the ATP-ε coding region was confirmed by digestion with restriction enzyme. DNA sequencing revealed that the construct was correct with His6-tag at the N terminus of the recombinant protein. The rCsATP-ε was expressed in E. coli (BL-21) after induction by IPTG, and a purified fusion protein with an approximate molecular weight around 30 kDa was detected, corresponding to the predicted size of 30.073 kDa (including 34 amino acids of vectors) originated from the primary gene sequence. After purification and renaturation, the concentration of the purified CsATP-ε protein was about 0.4 mg/ml (Fig. 3, lane 7). Western blotting analysis of recombinant CsATP-ε protein Purified CsATP-ε could be probed by mouse anti-His-tag monoclonal antibody, anti-CsATP-ε rat serum, and C. sinensis-infected human serum in the Western blot analysis, while not recognized by preimmune rat serum and healthy people serum (Fig. 4). Antibody titers and immune responses to CsATP-ε As shown in Fig. 5, we measured the antibody titers of total IgG in immunized sera of rCsATP-ε. Antibody titers peaked to 1:204,800 (Fig. 5), showing the high immunogenicity of CsATP-ε. Additionally, we explored the Th1-/Th2-type immune responses to CsATP-ε by measuring IgG1 and IgG2a from weeks 2 to 8 post immunization. The ELISA results showed that combined Th1/Th2 immune responses were provoked by rCsATP-ε for both IgG1 and IgG2a level increase (Fig. 6). The increased IgG isotype demonstrated that combined cellular immunity and humoral immunity had been successfully induced by CsATP-ε. Gene/protein expression level of CsATP-ε at different developmental stages of C. sinensis Real-time PCR was performed to detect the CsATP-ε transcripts at life stages of egg, metacercaria, excysted juvenile, and adult worm. The-fold changes of CsATP-ε gene transcriptional level in adult worm, excysted juvenile, and metacercaria were calculated compared with egg. The results showed that the expression levels of CsATP-ε gene were 9.10, 2.58, and 6.78-fold in metacercaria, excysted juvenile, and adult worm, respectively (Fig. 7a). Specific binding bands were detected with anti-CsATP-ε mouse serum by western blotting assay. The expression level of CsATP-ε protein was varied within different developmental stages. Adult worm showed the highest expression level of CsATP-ε protein, followed by metacercariae, excysted juvenile, and egg (Fig. 7b).
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Fig. 1 Amino acid sequence alignment of CsATP-ε and other V-ATPase ε subunit homologues. The protein sequence of CsATP-ε is depicted and aligned with Schistosoma japonicum (S. j, AAP06177.1), Caenorhabditis briggsae (C. b, XP_002633510.1), Drosophila yakuba (D. y, XP002098037.1), Mus musculus (M. m, NP031536.2), and Homo sapiens (H. s, CAA50592.1) homologues. Numbers in parentheses stand
for GenBank accession number for each organism. Dark-shaded boxes represent amino acid residues identical in all six proteins and light-shaded boxes represent residues conserved in four or five proteins. An asterisk indicates the conserved amino acid, Asp140. B-cell and T-cell linear epitopes were indicated with full lines and dotted lines, respectively
Fig. 2 Phylogenetic analysis of CsATP-ε and other related enzymes. Trichinella spiralis (T. s, XP003376773), Schistosoma mansoni (S. m, XP002579650), Xenopus (Silurana) tropicalis (X. t, NP989123), Culex quinquefasciatus (C. q, XP001849126), Meleagris gallopavo (M. g, XP003202515), and Brugia malayi (B. m, XP001902068). The tree was
constructed with the neighbor-joining method using MEGA4 program. Numbers on the branches indicated bootstrap proportions (500 replicates). The relationships displayed in the phylogenetic tree were in good agreement with traditional taxonomy
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Fig. 3 Expression and purification of CsATP-ε by 12 % SDS-PAGE. Protein molecular weight markers (M), lysate of E. coli with pET-28a(+) before induction with IPTG (lane 1) and after induction (lane 2), lysate of E. coli with pET28a(+)-CsATP-ε before induction (lane 3) and after induction (lane 4), supernatant of induced pET28a(+)-CsATP-ε (lane 5), and sediment (lane 6) and purified rCsATP-ε (lane 7)
Immunolocalization of CsATP-ε at adult worm and metacercaria of C. sinensis Using the rat anti-rCsATP-ε serum as the primary antibody and red-fluorescent Cy3 labeling IgG as the secondary antibody, results of immunochemistry showed that CsATP-ε was distributed both in adult worms and metacercariae. Specific fluorescence was detected on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of the metacercaria. No fluorescence was visualized in other organs or in controls treated with naïve serum (Fig. 8).
Discussion In the present study, a full-length gene encoding an ATP synthase subunit ε-like protein from C. sinensis (CsATP-ε) was identified, cloned, and overexpressed in E. coli. It contains 678 bp encoding 226 amino acids with a predicted
Fig. 4 Western blot analysis of rCsATP-ε. Protein molecular weight markers(M), rCsATP-ε probed with mouse anti-His-tag monoclonal antibody (lane 1), rCsATP-ε probed with anti-CsCP rat serum (lane 2), rCsATP-ε probed with C. sinensis-infected human serum (lane 3), rCsATP-ε probed with preimmune rat serum (lane 4), and human normal serum (lane 5)
Fig. 5 Antibody titers of IgG induced by CsATP-ε measured by ELISA. Briefly, 1 mg/well recombinant pET-28a-CsATP-ε protein was coated on the plates and blocked with 5 % skimmed milk. The plate was incubated with different dilutions of the immune sera (week 6) raised by pET-28aCsATP-ε. Rat sera immunized with PBS were measured under the same conditions as negative controls. HRP-conjugated IgG (1:20,000 dilutions) was used as the secondary antibodies. The reactions were developed with substrate solution TMB, stopped by 2 M H2SO4, and measured at 450 nm
molecular mass of 26.5 kDa. Blastx in NCBI reveals that it shares 76 % identity with ATP-ε proteins of S. japonicum and above 55 % identity with ATP-ε proteins from human and other eukaryotes. The high identity of this ε subunit with other eukaryotic organisms indicates its crucial role in the function of the enzyme. The conserved Asp140, known to be essential for E subunit function (Zhang et al. 1998), was found in the sequence. Bioinformatics analysis indicated that it is a novel complete gene of C.sinensis. ATP synthases are membrane-bound enzyme complexes/ ion transporters that combine ATP synthesis and/or hydrolysis with the transport of protons across a membrane. There are different types of ATPases including F-ATPases, V-ATPases,
Fig. 6 IgG isotype induced by CsATP-ε measured by ELISA. 1 mg/well recombinant pET-28a-CsATP-ε protein was coated on the plates and blocked with 5 % skimmed milk. Immune sera from weeks 2 to 8 were diluted at 1:400. Rat sera immunized with PBS were measured under the same conditions as negative controls. IgG1 and IgG2a (1:1,000 dilutions) were used as secondary antibodies
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Fig. 7 a Stage-specific transcription levels of CsATP-ε gene revealed by real-time PCR. CsATP-ε PCR products are using metacercaria cDNA (lane 1), adult worm cDNA (lane 2), excysted juvenile cDNA (lane 3), and egg cDNA (lane 4) as template. β-actin was employed as the transcriptional control, and the sample of the egg was used as the calibrator. Each bar represented the mean value from three experiments with standard deviation (*p
