Parasitol Res DOI 10.1007/s00436-014-3816-x

ORIGINAL PAPER

Identification, sequence analysis, and characterization of serine/threonine protein kinase 17A from Clonorchis sinensis Lisi Huang & Xiaoli Lv & Yan Huang & Yue Hu & Haiyan Yan & Minghui Zheng & Hua Zeng & Xuerong Li & Chi Liang & Zhongdao Wu & Xinbing Yu

Received: 12 January 2014 / Accepted: 7 February 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract This is the first report of a novel protein from Clonorchis sinensis (C. sinensis), serine/threonine protein kinase 17A (CsSTK17A), which belongs to a member of the death-associated protein kinase (DAPK) family known to regulate diverse biological processes. The full-length sequence encoding CsSTK17A was isolated from C. sinensis adult cDNA plasmid library. Two transcribed isoforms of the gene were identified from the genome of C. sinensis. CsSTK17A contains a kinase domain at the N-terminus that shares a degree of conservation with the DAPK families. Besides, the catalytic domain contains 11 subdomains conserved among STKs and shares the highest identity with STK from Schistosoma mansoni (55.9 %). Three-dimensional structure of CsSTK17A displays the canonical STK fold, including the helix C, P-loop, and the activation loop. We

obtained recombinant CsSTK17A (rCsSTK17A) and antirCsSTK17A IgG. The rCsSTK17A could be probed by antirCsSTK17A rat serum, C. sinensis-infected rat serum and the sera from rats immunized with C. sinensis excretory-secretory products, indicating that it is a circulating antigen possessing a strong immunocompetence. Moreover, quantitative RT-PCR and western blotting analyses revealed that CsSTK17A exhibited the highest mRNA and protein expression level in eggs, followed by metacercariae and adult worms. Intriguingly, in the immunolocalization assay, CsSTK17A was intensively localized to the operculum region of eggs in uterus, as well as the vitelline gland of both adult worm and metacercaria, implying that the protein was associated with the reproduction and development of C. sinensis. Overall, these fundamental studies might contribute to further researches on signaling systems of the parasite.

Lisi Huang, and Xiaoli Lv contributed equally to this work. L. Huang : H. Yan : M. Zheng : H. Zeng Department of Clinical Laboratory, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, People’s Republic of China X. Lv Department of Medical Laboratory and Research Center, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, People’s Republic of China Y. Huang : Y. Hu : X. Li : C. Liang : Z. Wu : X. Yu Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China Y. Huang : X. Li : C. Liang : Z. Wu : X. Yu (*) Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou 510080, People’s Republic of China e-mail: [email protected] Y. Hu Department of Clinical Laboratory, The Second People’s Hospital of Hefei City, Hefei 230011, People’s Republic of China

Keywords Clonorchis sinensis kinase 17A . Sequence analysis Immunolocalization

. Serine/threonine protein . Characterization .

Introduction Clonorchis sinensis (C. sinensis) is the causative agent of clonorchiasis, which is one of the most important foodborne diseases in China, Korea, Vietnam, and other Southeast Asian countries. It is estimated that approximately 35 million people are infected with C. sinensis globally (Lun et al. 2005). Human infection usually occurs by eating raw or inappropriately cooked freshwater fish infected with metacercariae of C. sinensis. The patients suffer chronically from epigastric discomfort and dull pain, fatigue, indigestion, diarrhea, and jaundice (Wang et al. 2011; Sripa et al. 2007; Yoo et al. 2011). Moreover, clonorchiasis has epidemiologically been reported to be associated with cholangiocarcinoma by the International

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Agency for Research on Cancer (IARC) in 2009 (Shin et al. 2010). Despite of its public health threat, there are still few effective measures to prevent this neglected tropical disease. Nowadays, a great many of fundamental researches on C. sinensis have been conducted to learn more about biological and biochemical characteristics of the parasite. Considering that the proteins implicated in cellular signal transduction might provide specific and vital targets for novel control strategies, we sought a signal transduction molecule of the parasite in this study, C. sinensis serine/threonine protein kinase 17A (CsSTK17A), which displayed homology to the death-associated protein kinase (DAPK) family. During the life cycle of C. sinensis, the parasite should experience temperature change, osmotic stress, nutrient starvation, damaged mitochondria, protein aggregation, and pathogens invasion. These environmental stresses may represent signals to regulate cell survival and differentiation between different life stages. However, the molecular mechanisms involved remain obscure. In higher eukaryotes, the reversible phosphorylation of proteins on serine, threonine, and tyrosine play an important role in maintaining cellular homeostasis (Hunter 1995; Bononi et al. 2011). The DAPK family is a case in point, which has been reported to involve in regulation of cell death and growth control. The family contains three closely related STKs, named DAPK1, DAPK-related protein 1 (DRP-1, also known as DAPK-2), and Zipper-interacting kinase (ZIPK, also named DAPK-3), which share around 80 % identity in their catalytic domains (Kogel et al. 2001). Two more distant DAPK-related proteins are STK17A and STK17B, also named DAPK-related apoptosis-inducing protein kinase-1 and kinase-2 (DRAK-1 and DRAK-2), which share about 50 % identity with the kinase domains of the other three members. Interestingly, the extracatalytic domains of these five proteins differ markedly, indicating that there should be some biological functional divergences among the family members (Bialik and Kimchi 2006). DAPK1 is the prototypic member of the family. It was initially discovered because of its role in cell death induced by interferon-γ (Deiss et al. 1995). As well as regulating apoptosis, DAPK1 also plays a key role in a diverse range of biological processes, such as cytokinesis, autophagy, and immune responses (Lin et al. 2010). Likewise, CsSTK17A, as a member of the death-associated protein family of serine/ threonine kinases, was supposed to possess similar functions and play essential roles in the growth and development of C. sinensis. However, few proteins of the DAPK family have been well studied in helminthes until now, let alone STK17A from C. sinensis. In the present study, CsSTK17A was firstly expressed and purified. We demonstrated sequence analysis, structural modeling, and immunohistochemical localizations, as well as expression levels of CsSTK17A mRNA and protein in

different life stages. Our study might lay the groundwork to discover the expression pattern and probable role of CsSTK17A in the development of C. sinensis.

Materials and methods Preparation of C. sinensis adult worms, metacercariae, and eggs C. sinensis metacercariae were obtained from naturally infected Pseudorasbora parva, which were digested with pepsinHCl (pH 2.0). Each Sprague-Dawley (SD) rat was then orally infected with one hundred metacercariae. C. sinensis adult worms were recovered from bile ducts of the SD rats infected 8 weeks ago. After being washed by phosphate-buffered saline (PBS, pH 7.4) containing antibiotics for several times, live adult worms were incubated in RPMI-1640 (Gibco, USA) culture medium and maintained at 37 °C under 5 % CO2. The culture medium was then centrifuged at 2,000g for 10 min and the sediment containing C. sinensis eggs was harvested. Finally, the worms, metacercariae, and eggs were frozen immediately in liquid nitrogen and stored at −80 °C until RNA and protein extraction. RNA extraction and complementary DNA (cDNA) synthesis Total RNA from adult worms, metacercariae, and eggs was isolated and further purified by the TRIzol reagent (Invitrogen) according to manufacturer’s protocol. RNA concentration and quality were determined by nucleic acid/protein analyzer (Beckman Coulter, USA) and agarose gel electrophoresis, respectively. Total RNA was then reverse transcribed, the reaction was performed in a final volume of 20 μl, which contained 2 μg of total RNA, 2.5 μM oligo dT primer, and 5 units of avian myeloblastosis virus reverse transcriptase (TaKaRa, Japan). The reaction mixture was then incubated for 1 h at 42 °C and stopped by heating for 5 min at 95 °C. Sequence analysis and structural modeling CsSTK17A was isolated from our cDNA plasmid library of adult C. sinensis. The coding region sequence and open reading frame (ORF) were determined by ORF Finder program at NCBI (http://www.ncbi.nlm.nih.gov/). The locus and transcribed isoforms of CsSTK17A were identified via mapping the cDNA sequence back to our database of de novo genome and transcriptome sequencing (http://fluke. sysu.edu.cn/) (Wang et al. 2011) by BLAST. Besides, the translated amino acid sequence was analyzed for the physicochemical parameters, signal peptide, transmembrane regions, and characteristic motifs using Proteomics tools in ExPaSy

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website (http://www.expasy.org/). The linear epitopes of B cell were predicted online (http://tools.immuneepitope.org/ main/). Multiple sequence alignment of the serine/threonine kinase domains within the related proteins and phylogenetic analysis were performed using the bioinformatics analysis software Vector NTI suite 8.0, and the tree was visualized by TreeView software. In addition, three-dimensional structure of CsSTK17A was predicted based on comparative modeling with the use of SWISS-MODEL.

Production of polyclonal antiserum Anti-rCsSTK17A antibodies were produced in the SD rats. The purified rCsSTK17A was diluted 1:1 with Freund’s adjuvant and emulsified. Two hundred micrograms of the protein was injected subcutaneously into each rat at the first immunization and boosted with 100 μg of rCsSTK17A in Freund’s incomplete adjuvant at 2 weeks interval. Antisera were collected before each immunization. And then serum specificity and antibody titers were determined using enzyme-linked immunosorbent assay (ELISA) developed against the protein.

Cloning of CsSTK17A Identification of rCsSTK17A by western blotting The gene-encoding CsSTK17A sequence was amplified by polymerase chain reaction (PCR) using No. 2g07 plasmid clone as the template. Two degenerate oligonucleotide primers corresponding to the ORF of the protein were synthesized: primer 1 (forward), 5′-GCCGGGATCCATGATTACGTTT CAGCAGC-3′ with a BamHI restriction enzyme site (underlined), and primer 2 (reverse), 5′-GCCGAAGC TTAGATCAACACTCGCCATA-3’′with a HindIII restriction enzyme site (underlined). The PCR was performed with an initial denaturation for 5 min at 95 °C. Thirty-five cycles were run, with denaturation for 1 min at 94 °C, annealing for 1 min at 60 °C, and extension for 1 min at 72 °C. The final extension was 10 min at 72 °C. The purified PCR product was subcloned into the corresponding restriction sites of a prokaryotic expression vector pET-28a(+). And the recombinant plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) and sequenced to confirm that the ORF was in frame and in the correct orientation.

Expression and purification of CsSTK17A in E. coli The recombinant CsSTK17A (rCsSTK17A) was expressed by inducing with isopropyl-β- D -thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM at 28 °C for 4 h. The bacteria were collected by centrifugation at 4 °C. And the cells were sonicated and then centrifuged. The pellets were resuspended in lysis buffer (10 mM Tris-HCl, 100 mM NaH2PO4 ·H2O, 6 M guanidine hydrochloride, pH 8.0). The lysates were centrifuged at 10,000g for 15 min at 4 °C. And then the fusion protein was purified with His•Bind Purification kit (Novagen) according to the protocol recommended by the manufacturer. The purified recombinant protein was renatured in PBS (pH 7.4) with decreasing grade guanidine hydrochloride. Protein purity was then monitored by Coomassie blue staining after the sodium dodecyl sulfidepolyacrylamide gel electrophoresis (SDS-PAGE, 12 %). The final concentration of purified protein was detected by the method of Bradford on nucleic acid/protein analyzer (Beckman, USA).

The rCsSTK17A protein was resolved by SDS-PAGE (12 % gel) and electrotransferred to polyvinylidene difluoride (PVDF) membrane (Millipore, USA) at 100 V for 1 h. The membrane was blocked with PBS (pH 7.4) containing 5 % nonfat milk overnight at 4 °C and then probed with preimmune rat serum (1:500 dilutions), anti-rCsSTK17A rat serum (1:500 dilutions), anti His-tag mouse monoclonal antibody (1:2,000 dilutions), the sera from rats infected with C. sinensis (1:200 dilutions), and the sera from rats immunized with C. sinensis excretory-secretory products (CsESPs, 1:200 dilutions) for 2 h at room temperature (RT), respectively. After being washed three times with PBS-0.05 % Tween 20, the membrane was incubated with horse radish peroxidase (HRP)-conjugated goat anti-rat IgG and HRP-conjugated goat anti-mouse IgG (1:2,000 dilutions, ProteinTech Group, USA) for 1 h at RT. Colorimetric signals were developed in the presence of hydrogen peroxide and diaminobenzidine (DAB). Quantitative RT-PCR (qRT-PCR) and western blotting analysis of CsSTK17A expression levels during the C. sinensis life cycle To investigate the mRNA transcriptional levels of CsSTK17A at different developmental stages of C. sinensis (adult worms, metacercariae, and eggs), cDNAs from these three stages were used as templates. The final volume of each qRT-PCR reaction was 20 μl, contained 10 μl 2×SYBR Premix Ex Taq (TaKaRa, Japan), 2 μl diluted cDNA template, 7.2 μl PCRGrade water, and 0.4 μl of each 10 μM primer. PCR conditions were 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 20 s, with a 0.1 °C/s incremental increase from 60 °C to 95 °C. The primers used were as follows: CsSTK17A, 5′CGCCGTTCGTTTCATCCA-3 and 5′-CATCATCTGCCG CTTGTGTAA-3′; C. sinensis β-actin (GenBank accession number EU109284) as an internal standard, 5′-ACCGTGAG AAGATGACGCAGA-3′ and 5′-GCCAAGTCCAAACGAA GAATT-3′. Samples were run in triplicate, and CsSTK17A transcriptional levels were calculated by the 2−ΔΔCt comparative CT method (Pfaffl 2001).

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Next, western blotting was performed to analyze the expression levels of CsSTK17A protein at the C. sinensis life cycle stages mentioned above. Parasites from each stage were solubilized in the RIPA lysis buffer (Bioteke, China) containing 1-mM proteinase-inhibitor PMSF. After sonicated three times for 30 s, the samples were centrifuged at 10,000g for 5 min and then the supernatant containing the protein was obtained. The BCA protein assay kit (Novagen, USA) was used to detect the concentrations of proteins. 40 μg of each sample was subjected to 12 % SDS-PAGE and electrophoretically transferred onto PVDF membrane. The sheets were incubated with anti-rCsSTK17A rat serum (1:100 dilutions), preimmune rat serum (1:100 dilutions), and HRP-conjugated goat anti-rat IgG (1:2,000 dilutions). Bound Ig was detected using the SuperSignal West Pico Chemiluminescent Substrate (Applygen, China) and Kodak X-Omat film. Immunolocalization of CsSTK17A in C. sinensis adult worm and metacercaria The adult worms and metacercariae were fixed in 4 % paraformaldehyde and cut by microtome into sections of 3–5 μm in thickness. The sections in paraffin wax were then deparaffinized in xylene and hydrated in a series of ethanol. Nonspecific staining was blocked by incubation in 5 % normal goat serum in PBS (pH 7.4) for 30 min. Then the samples were incubated with anti-rCsSTK17A antibodies diluted 1:800 in 0.1 % bovine serum albumin (BSA) in a moist chamber overnight at 4 °C. The same dilution of preimmune rat serum was served as a negative control. After three washes with PBS (pH 7.4), the sections were probed with goat anti-rat IgG (Alexa Fluor 594, Molecular Probe) diluted 1:400 with 0.1 % BSA for 1 h at RT in dark. The slides were subsequently examined by fluorescent microscope (ZEISS, Germany).

Results Sequence analysis of the CsSTK17A gene The complete cDNA of CsSTK17A contains an ORF of 1,023 bp encoding a protein with 341 amino acids and a theoretical molecular weight of 38.7 kDa (GenBank accession No. GAA54491.1). When mapping cDNA sequence of CsSTK17A back to our database of de novo genome and transcriptome sequencing, two transcribed isoforms of CsSTK17A (Gene ID of locus: csin103413) were identified. Among these, one includes an integrated CsSTK17A gene encoding the N-terminal catalytic domain (including 11 sudomains) and C-terminal domain (csin103413.1); the other owns four subdomains and the whole C-terminal sequence of 49 codons (csin103413.2) (Fig. 1a).

The estimated half-life time of CsSTK17A was more than 10 h in E. coli. And the instability index was computed to be 62.00, indicating that the protein was unstable. No signal peptide was found in the sequence. The translated amino acid sequence from 216 to 234 was predicted to be strong transmembrane helices by TMpred. In addition, structural analysis indicated that CsSTK17A mainly contained a catalytic domain of STK (between 32 and 291 amino acid), including ATP binding site (between 38 and 61 amino acid) and active site of protein kinase (between 152 and 164 amino acid). Besides, the glycine-rich motif and the conserved lysine residue within the ATP-binding site were found. The catalytic domain of CsSTK17A comprised 11 motifs (subdomains I through XI) conserved among the STKs. It shared the highest identity with HsSTK17A (41.8 %) within the DAPK family of Homo sapiens (Fig. 1b). Next, the characterized catalytic subunits of STK17A from different species were aligned (Fig. 1c). Among these, the deduced amino acid sequence of CsSTK17A shared 55.9, 40.2, 43.5, 41.4, and 40.6 % identity with STK from Schistosoma mansoni, and STK17A from Heterocephalus glaber, Strongylocentrotus purpuratus, Xenopus laevis, and Geospiza fortis, respectively. As seen in Fig. 1d, the relationships shown in the phylogenic tree were in correspondence with traditional taxonomy. CsSTK17A was most closely related to STK of S. mansoni and SJCHGC06342 protein of Schistosoma japonicum, followed by DAPK1 of Pediculus humanus corporis, and it was far from STK17A of H. sapiens, Bos taurus, Oryctolagus cuniculus, and so on. Structural modeling of CsSTK17A Three-dimensional structure of CsSTK17A was predicted according to the molecular model of STK17B from H. sapiens by SWISS-MODEL (Fig. 2). The overall fold of the catalytic domain of CsSTK17A conforms closely to those of the eukaryotic protein kinases and is composed of two lobes: an Nterminal subdomain, including a curled β-sheet and a prominent α-helix (helix C), and a C-terminal lobe that predominantly consists of α-helices (Huse and Kuriyan 2002). Notably, the helix C, which ranges from 69 to 85 amino acids in length, is thought to be a critical mediator of conformational changes that take place within the catalytic center (Greenstein et al. 2005). The hexapeptide 39-GRGRFA-44 forms a standard P-loop in which the glycine residues help coordinate the phosphates of ATP. Besides, the active-site loop (residues 152–164) was also labeled in Fig. 2. Cloning, expression, and purification of CsSTK17A in E. coli The rCsSTK17A was expressed as inclusion bodies in E. coli. Its molecular weight containing His-tag was in agreement with

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Fig. 1 Sequence analysis of CsSTK17A. (a) Two transcript isoforms of CsSTK17A locus csin103413 of C. sinensis genome. csin103413.1, included a full length gene of CsSTK17A; csin103413.2, encoded four subdomains and non-catalytic C-terminal region. The arrow indicated the encoding direction. (b) Alignment of the catalytic domains of CsSTK17A and the DAPK families from Homo sapiens. The 11 subdomains conserved among serine/threonine protein kinases were indicated above the sequences. Identical amino acid residues were shaded in black. The GenBank accession numbers for HsDAPK1, HsDRP-1, HsZIPK, and HsSTK17A/B were NP_004929.2, NP_055141.2, NP_001339.1, NP_004751.2, and NP_004217.1, respectively. (c) Amino acid sequence alignment of the catalytic domains of STK17A from different species. The numbers referred to amino acid residues. Highly conserved residues were shaded in black. The ATP binding site, active site, and helix C of CsSTK17A were respectively illustrated as full lines. The amino acid position of the glycine-rich motif was marked with (triangle), and the residue highly conserved within the ATP binding site was labeled with (star). Besides, the main linear epitopes of B cell were indicated with dotted

lines. SmSTK, Schistosoma mansoni STK (GenBank accession no. XP_002573273.1); HsSTK17A, H. sapiens STK17A (GenBank accession no. BAA34126.1); HgSTK17A, Heterocephalus glaber STK17A (GenBank accession no. XP_004840298.1); BtSTK17A, Bos taurus STK17A (GenBank accession no. NP_001076891.1); AfSTK17A, Apis florea STK17A (GenBank accession no. XP_003695128.1); SpSTK17A, Strongylocentrotus purpuratus STK17A (GenBank accession no. XP_787526.2); AsSTK17A, Alligator sinensis STK17A (GenBank accession no. XP_006017728.1); DrSTK17A, Danio rerio STK17A (GenBank accession no. NP_001082806.1); XlSTK17A, Xenopus laevis STK17A (GenBank accession no. NP_001091414.1); ApSTK17A, Anas platyrhynchos STK17A (GenBank accession no. XP_005014994.1); GfSTK17A, Geospiza fortis STK17A (GenBank accession no. XP_005425760.1). (d) The phylogenetic tree of CsSTK17A and other related DAPK families. The tree was inferred from the amino acid alignment for STKs and illustrated using TreeView software. Distances on the x-axis showed the grade of sequence identity, and distances on the y-axis were arbitrary

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Fig. 1 continued.

the predicted 42.1 kDa (Fig. 3). After purification and renaturation, the concentration of the protein was about 0.25 mg/ml. Identification of rCsSTK17A by western blotting Purified rCsSTK17A could be probed at around 42 kDa by anti-rCsSTK17A rat serum (Fig. 4, lane 2) and anti-His tag

monoclonal antibody (Fig. 4, lane 3), while not recognized by preimmune rat serum (Fig. 4, lane 1), which suggested that CsSTK17A was successfully cloned and expressed in pET28a(+) vector. Moreover, the rCsSTK17A could react with C. sinensis-infected rat serum (Fig. 4, lane 4) and the sera from rats immunized by CsESPs (Fig. 4, lane 5). These data indicated that CsSTK17A might be a component of CsESPs.

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Fig. 1 continued.

Quantitative RT-PCR and western blotting analysis of CsSTK17A at different developmental stages of C. sinensis

Immunolocalization of CsSTK17A in C. sinensis adult worm and metacercaria

CsSTK17A was transcribed in all life cycle stages of C. sinensis which occur in the definitive host, i.e., metacercariae, adult worms, and eggs (Fig. 5a). Transcriptional level of CsSTK17A mRNA was significantly higher in eggs compared with adult worms when normalized with C. sinensis β-actin (14.03-fold, p

threonine protein kinase 17A from Clonorchis sinensis.

This is the first report of a novel protein from Clonorchis sinensis (C. sinensis), serine/threonine protein kinase 17A (CsSTK17A), which belongs to a...
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