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Experimental Parasitology 119 (2008) 117–124

Expression, immunolocalization and serodiagnostic valueof a myophilin-like protein from Schistosoma japonicum

Hailin Peng a,b,e, Kai Song b, Chengyu Huang d, Sai Ye b, Huaiguang Song b,Wei Hu d, Zeguang Han c, Donald P. McManus f, Guoping Zhao b, Qinghua Zhang a,b,*

a Shanghai Institute of Hematology, Ruijin Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiao

Tong University School of Medicine, Shanghai, Chinab National Engineering Research Center of Biochip at Shanghai, Shanghai, China

c Chinese National Human Genome Center at Shanghai, Shanghai, Chinad Institute of Parasitic Disease, Chinese Center for Disease Control and Prevention, Shanghai, China

e Department of Clinical Laboratory, Taizhou People’s Hospital, Jiangsu, Chinaf Molecular Parasitology Laboratory, Queensland Institute of Medical Research, Brisbane, Australia

Received 26 June 2007; received in revised form 24 December 2007; accepted 3 January 2008Available online 8 February 2008

Abstract

The cDNA of a Schistosoma japonicum myophilin-like protein was cloned, sequenced, and expressed in Escherichia coli as a recom-bined protein (rSj myophilin-like protein), and the protein was purified by affinity chromatography. The deduced amino acid sequencesof the Sj myophilin-like protein showed significant homology to myophilin, calponin, Np22 and Mp20. Northern blot and RT-PCR ana-lyzes revealed expression of the Sj myophilin-like protein mRNA in eggs, sporocysts, cercariae, hepatic schistosomula and adult worms.Confocal fluorescence microscopy localized the native protein to the muscle of the adult worm. In schistosome-infected rabbits, the rSjmyophilin-like protein antibody level, assessed by ELISA, was elevated after infection but was reduced after praziquantel treatment. Inhumans, the myophilin-like protein antibody level was evaluated by ELISA in sera from 33 non-infected humans and 61 schistosomiasispatients; the results showed a highly significant difference between the two groups with a sensitivity of 57.4%. Taken together, the myo-philin-like protein may prove useful for monitoring the therapeutic effect of praziquantel rather than in serodiagnosis of schistosomiasis.� 2008 Elsevier Inc.

Index Descriptors and Abbreviations: Schistosoma japonicum; Protein Sj myophilin-like; Myophilin-like protein; Schistosomiasis; Serodiagnosis; Immu-nofluorescence; ELISA, enzyme-linked immunosorbent assay; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; RT-PCR, reversetranscription-polymerase chain reaction; OCT, optimal cutting temperature; PVDF, polyvinylidene difluoride; PBST, phosphate buffer solution Tween;AP, alkaline phosphatase; HRP, horseradish peroxidase; IPTG, isopropyl-b-D-galactopyranoside; FITC, fluorescein sothiocynate

Open access under CC BY license.

1. Introduction

Schistosomiasis remains a serious public health prob-lem worldwide, infecting more than 200 million people,

0014-4894 � 2008 Elsevier Inc.

doi:10.1016/j.exppara.2008.01.017

* Corresponding author. Address: Shanghai Institute of Hematology,Ruijin Hospital, State Key Laboratory of Medical Genomics, ShanghaiJiao Tong University School of Medicine, Shanghai, China. Fax: +86 2151320297.

E-mail address: qinghua_zhang@shbiochip.com (Q. Zhang).

Open access under CC BY license.

mostly in tropical regions, and is endemic in 74 develop-ing nations. Schistosoma japonicum is endemic in Chinaand the Philippines, and is also found in Sulawesi, Indo-nesia (Savioli et al., 2002; Ross et al., 2002; Engels et al.,2002; Chitsulo et al., 2004). Correct diagnosis is funda-mental for controlling schistosomiasis, including casedetection and resulting community treatment, assessmentof morbidity and the evaluation of control strategies. Atpresent, parasitological and immunological tests are con-sidered as the main methods in schistosomiasis diagnosis.

118 H. Peng et al. / Experimental Parasitology 119 (2008) 117–124

The appearance of parasite eggs in urine or feces directlyindicates the presence of the worms. However, disadvan-tages of this relatively time-consuming approach are wellrecognized, including high fluctuation in egg counts andeasily missed low infections, (Kongs et al., 2001; Rosset al., 2001). Immunodiagnostic techniques, on the otherhand, have the advantages of much higher sensitivityand ease of performance (Ross et al., 2001; Bergquist,1992; Wu, 2002) and many antibody assays have beendeveloped (Xiao et al., 2003; van Dam et al., 2004; Stot-hard et al., 2006). However, few serological tests are com-mercially available, and the preparation of antigens fromparasites requires the maintenance of the complete para-sitic cycle and often tedious laboratory extraction proce-dures. In the past decade, recombinant schistosomeantigen-based serodiagnostic assays have been developed(Moser et al., 1992; Liu et al., 1993; Hu et al., 2003a,b),but more specific and sensitive antigen preparations arerequired to develop assays with improved performance.In addition, although praziquantel-based chemotherapy(Gonnert and Andrews, 1977; Kihara et al., 2007) is thebest regimen available today for the clinical managementof schistosomiasis and is highly effective against all of thehuman schistosomes (Geerts and Gryseels, 2000; Kiharaet al., 2007), few simple and effective methods formonitoring the therapeutic effect of praziquantel areavailable.

Using data from the published genomic and cDNAsequences for S. japonicum (Hu et al., 2003a,b), we identi-fied a gene encoding a myophilin-like protein (GenBankAccession No.: AY223463). Here we report the prokary-otic expression and purification of this S. japonicum myo-philin (rSj myophilin), its functional evaluation of itspotential rabbit models. In addition, immunolocalizationof the Sj myophilin-like was reported.

2. Materials and methods

2.1. Preparation of schistosome samples for analysis

A Chinese isolate of S. japonicum was collected fromAnhui province, China, and the life cycle was maintainedin Oncomelania hupensis snails and Kunming strain miceat the Institute of Parasitic Disease, Shanghai. mRNAsamples from eggs, sporocysts, cercaria, hepatic schisto-somula and adult worms, and the native solubleproteins from adult worms of S. japonicum were preparedas previously described (Hu et al., 2003a,b; Liu et al.,2006, 2007).

2.2. Sequence similarity comparison

Online BLAST was used to find homologous sequencesof the S. japonicum myophilin-like cDNA and protein.The sequences were aligned using CLUSTAL(1.0)multiple sequence alignment programs (http://us.exp-asy.org/).

2.3. Construction of recombined plasmid pET22b-Sj

myophilin-like and pBluescript-Sj myophilin-like

The S. japonicum myophilin-like cDNA was PCR ampli-fied with primers flanked with BamHI and XhoI (F 50-GCGTCAGGGATCCGATGGCTAATCAACCT TTAG-30,R 50-GCGTCTGCTCGAGATCAAGAATCATACGTTG-30) from the S. japonicum cDNA clone pBluescript IISK(+) sjs2-011 C10 and subcloned into the pET22b expres-sion vector and pBluescript II SK(+), respectively. The sub-cloned protein coding sequences (CDS) were verified to bemutation free by sequencing.

2.4. Heterogeneous expression and purification of rSj

myophilin-like protein

Recombinant CDS expression was performed followingpublished procedures (Sambrook and Russell, 2001) withminor modifications. Briefly, the recombinant plasmidpET22b-Sj myophilin-like was transformed into Esche-

richia coli BL21 (DE3), and then the cells were grown,reaching an optical density of 0.6 at 600 nm, and inducedwith 0.8 mM IPTG at 30 �C for an additional 4 h. Afterincubation, the culture was harvested and processedaccording to published protocols (Marshak et al., 1996)to obtain a pellet of inclusion bodies. Then the inclusionbodies were dissolved in loading buffer containing 6 M gua-nidine hydrochloride, after centrifugation at 4 �C(8000 rpm, 30 min), the supernatant was filtered througha 0.45-lm-pore-size filter before being loaded onto a Che-lating Sepharose Fast Flow column (GE Healthcare,USA). After washing the column, the adsorbed recombi-nant protein was eluted using four steps of increased imid-azole concentration in elution buffer. Fractions wereanalyzed by SDS–PAGE and stained with Coomassie blueto assess their purity.

2.5. RT-PCR

Total RNA (1–5 lg) from each S. japonicum stage wasreverse-transcribed using a SuperScriptTM II Reverse Tran-criptase system (Invitrogen, USA) and oligo (dT). Theresulting cDNAs and specific oligonucleotide primers (F50-GCGTCAGGGATCCGATGGCTAATCAACCTTTAG-30 and R 50-GCGTCTGCTCGAGATCAAGAATCATACGTTG-30) were used in a PCR reaction to amplifya 570 bp fragment using an EX-Taq DNA polymerase sys-tem (TAKARA, Dalian, China). The PCR products wereanalyzed on a 1% agarose gel.

2.6. Northern blot analysis

Northern blot analysis was performed using a non-Iso-topic DIG Northern Starter Kit (Roche Diagnostics,USA) as previously described (Li et al., 2006). Specifically,3 lg of total RNA (from adult male worms) per lane wereloaded for electrophoresis and the pBluescript-Sj myophi-

H. Peng et al. / Experimental Parasitology 119 (2008) 117–124 119

lin-like plasmid was linearized with XbaI for probegeneration.

2.7. Preparation of polyclonal mouse anti-Sj myophilin-like

serum

Polyclonal mouse antisera were prepared by genetic-immunization using a DNA prime-protein boost strategy(Peng et al., 2007). Briefly, mice (Balb/c, female, 6 weeks)were injected intramuscularly 3 times with 100 lg recom-bined-plasmid pVAX1-Sj myophilin-like followed byboosting with 50 lg of rSj myophilin-like protein at 2weeks intervals. Serum was collected 3 days after the finalboost.

2.8. Immunofluorescence assay

Immunofluorescence assays were performed as previ-ously described (Liu et al., 2006) with some modifications.Four-micron-thick frozen sections of adult worms wereembedded in OCT compound and fixed with methanolfor 20 min at room temperature. Slides were blocked withgoat serum overnight at 4 �C, and incubated in a humidatmosphere at room temperature for 2 h with the anti-Sjmyophilin-like serum (1:400 in 0.1% PBST) or pre-immu-nized mouse serum. The slides were washed twice and incu-bated for 2 h with 1:80 FITC-conjugated goat anti-mouseIgG (Sigma, USA), containing 20 lg/ml Evans blue in ahumid atmosphere2 at room temperature. Confocal fluo-rescence microscopy (Zeiss 510 Meta, German) was usedin visualization of antibody staining.

2.9. Human and rabbit sera

Human sera were obtained from 61 patients with schis-tosomiasis japonica, confirmed by Kato–Katz stool exam-ination (Katz et al., 1970), from endemic area in JiangxiProvince. Control sera were obtained from 33 volunteerswithout schistosomiasis from a non-endemic area inShanghai.

Seventeen rabbits were exposed to an average of 1000cercariae per rabbit; nine of these infected rabbits weretreated with praziquantel (administered orally at a doseof 200 mg/kg body weight) 10 weeks after infection. Serumsamples from the rabbits were collected by lateral ear veinpuncture at 3 weekly intervals.

2.10. Western blotting

SDS–PAGE (10%) was used to analyze the recombi-nant or native Sj myophilin-like proteins. Aliquots ofpurified rSj myophilin-like (50 ng/well) or crude extracts(10 lg/well) were loaded onto gels. After electrophoresis,the gels were either Coomassie blue-stained to visualizethe protein bands or electro-transferred onto PVDF mem-branes (Millipore, USA) and reacted with antibodyprobes. PVDF membranes were blocked for 1 h in 0.1%-

PBST containing 5% milk powder and immunostainedwith human sera (1:100 in 0.1%-PBST) or mouse poly-clonal antiserum (1:4000 in 0.1%-PBST). Then, the mem-branes were washed 3 times in 0.1%-PBST for 15 min andincubated with affinity purified HRP-conjugated goatanti-human or anti-mouse IgG (1:5000, Sigma, USA)and developed in an ECL Western blotting system (GEHealthcare, USA).

2.11. ELISA

The human and rabbit sera were also tested for specificIgG antibody levels by ELISA using plates coated (1 lg/ml) with the rSj myophilin-like purified protein. The sec-ondary antibody used in the ELISA was AP-conjugatedgoat anti-mouse or goat anti-human IgG (Sigma, USA).Color reaction was developed by the addition of o-phenyl-enediamine (OPD) (Sigma, USA) in phosphate buffer andstopped with sulfuric acid. The absorbance was read at490 nm using a micro plate reader.

The ELISA data were statistically analyzed by a groupt-test, and a P-value <0.05 was regarded as significant.

3. Results

3.1. Molecular characterization of Sj myophilin-like protein

The CDS encoding the Sj myophilin-like protein wassuccessfully subcloned from the cDNA clone pBluescriptII SK(+) sjs2-011 C10 into pET22b. The Sj myophilin-likeclone was 570 bp in length, encoding a protein of 190amino acid residues (Fig. 1), with a molecular mass of21.138 kDa, and its isoelectric point (PI) was 8.66 (http://us.expasy.org/cgi-bin/pi_tool). The amino acid sequenceof Sj myophilin-like is 100% identical to that of Echinococ-

cus granulosus myophilin-like protein (GenBankAAT46028), and 74% identical to the myophilin antigenfrom E. granulosus (GenBank CAA82316), 52% identicalto the calponin of Branchiostoma belcheri (GenBankBAC16745), 52% identical to the neuronal protein 22 fromRattus norvegicus (GenBank AAL66341), and 56% identi-cal to the mp20 from Bombyx mori (GenBankNP_001040476) (Fig. 1).

3.2. Transcript analysis of Sj myophilin-like

To identify the transcript of the myophilin-like proteinin adult worms, a Northern blot was performed with totalRNA from adult male worms. A distinct signal of approx-imately 740 bp was observed (Fig. 2A). The size of the sig-nal corresponds with the molecular size of the full-lengthcDNA clone found in the library.

RT-PCR was performed with various stages in order todetect the expression of myophilin-like mRNA during theS. japonicum life cycle. As shown in Fig. 2B, the expectedsize (�570 bp) myophilin-like fragments were amplifiedfrom all of the cDNA libraries. The RT-PCR results

Fig. 1. Multiple alignment of the deduced amino acid sequences of the Sj myophilin-like protein with the homologs, E. granulosus myophilin-like protein,myophilin antigen from E. granulosus, calponin of B. belcheri, Mp20 from B. mori, and neuronal protein 22 from R. norvegicus. Gaps are represented bydashed lines. ‘‘�” means that the residues in that column are identical in all sequences in the alignment; ‘‘:” means that conserved substitutions have beenobserved; ‘‘.” means that semi-conserved substitutions are observed.

Fig. 2. Gene expression analysis of Sj myophilin-like transcripts. (A)Northern blot analysis of Sj myophilin-like mRNA expression in S.

japonicum adult worms. Northern blot with 3 lg total RNA from adultmale worms. The transcript of the Sj myophilin-like protein band at740 bp. (B) RT-PCR analysis of Sj myophilin-like mRNA in differentdevelopmental stages. M, molecular weight marker (2000, 1000, 750, 500,250, 100 bp); E, eggs; Sp, sporocysts; C, cercariae; Hs, hepatic schisto-somula; A, adults (mixed-gender); P, positive control, plasmid pET22b-Sjmyophilin-like as template; N, no template control. cDNA samples usedwere from individual stage cDNA libraries.

120 H. Peng et al. / Experimental Parasitology 119 (2008) 117–124

showed that the myophilin-like mRNA was expressed ineggs, sporocysts, cercariae, hepatic schistosomula andadult worms.

3.3. Expression and purification of rSj myophilin-like protein

IPTG (0.8 mM) efficiently induced expression of the rSjmyophilin-like protein and the protein accumulated ininclusion bodies (Fig. 3A). The apparent molecular massof the rSj myophilin-like protein was about 25 kDa. Thisband is about 3 kDa bigger than the estimated size deducedfrom the sequence data because the recombinant proteincontains an additional 30 amino acids comprising the N-terminal pelB leader and the C-terminal 6� His tagsequence. The recombinant protein was subsequently puri-fied using an affinity chromatography purification kit. Itwas absorbed by the Ni2+ through its 6� His tag whenpassed through the column. The absorbed proteins wereeluted with a linear imidazole gradient as described in Sec-tion 2 (Fig. 3B, lane 4).

Fig. 3. Expression analysis of the recombinant Sj myophilin-like protein.Lane M, molecular mass standards. (A) Lane 1, before induction; lane 2,supernatant after induction; lane 3, precipitate after induction. (B) Lane 4,purified protein after affinity chromatography.

H. Peng et al. / Experimental Parasitology 119 (2008) 117–124 121

3.4. Western blots of Sj myophilin-like protein probed with

human or mouse sera

Purified rSj myophilin-like protein (50 ng/well) andcrude extract protein from adult worm (10 lg/well) wereloaded onto SDS–PAGE gels. After electrophoresis andtransfer, the proteins were probed with human or mousesera. (Fig. 4). Pooled sera from healthy humans did not rec-ognize the rSj myophilin-like protein (Fig. 4B, lane 5). Incontrast, the rSj myophilin-like protein was recognized bya serum pool from schistosomiasis patients (Fig. 4B, lane6). Polyclonal mouse antisera recognized a band of

Fig. 4. Western blot analysis of Sj myophilin-like protein. The samplesloaded were crude protein extract from adult worms (lanes 1 and 2) or rSjmyophilin-like protein (lanes 3–6). (A) Probed with mouse sera. Lanes 1and 4 probed with pre-immune mouse serum; lanes 2 and 3 probed withsera from immunized mice. (B) Probed with human sera. Lane 5 probedwith serum pool from healthy humans; lane 6, probed with serum poolfrom schistosomiasis japonica patients. An arrow indicates the location ofthe rSj myophilin-like protein (25 kDa) or native Sj myophilin-like protein(22 kDa).

�22 kDa in the crude protein extract from adult worms(Fig. 4A lane 2), and this antiserum also recognized therSj myophilin-like protein as a band about 25 kDa(Fig. 4A, lane 3). Neither the crude protein extract northe recombinant protein reacted with the pre-immunemouse serum (Fig. 4A, lanes 1 and 4).

3.5. Immunofluorescence localization of Sj myophilin-like

protein in adult worms

The distribution of rSj myophilin-like protein in adultworms was investigated by indirect immunofluorescence.Cryosectioned S. japonicum adults were incubated withmouse anti-serum and pre-immune mouse serum as con-trol. The myophilin-like protein was found to be distrib-uted in the muscle of adult worms (Fig. 5). Nofluorescence was detected in control sections.

3.6. Sj myophilin-like protein antibodies in human sera

A series of ELISA experiments were conducted to detectantibody levels in human sera using the rSj myophilin-likeprotein as antigen (Table 1). Two of the 33 healthy humanserum samples were positive, giving a false positive rate of6.06% (2/33). Sera (35 of 61) from confirmed schistosomi-asis patients, were positive, giving a sensitivity of 57.4%(35/61). The difference between the two groups was statis-tically significant (P < 0.0001).

3.7. Diminution of antibody levels in treated infected rabbits

Specific antibody levels in infected rabbits were deter-mined over time by ELISA using the rSj myophilin-likeprotein as antigen (Fig. 5). In the praziquantel-treatedgroup, the antibody level increased remarkably 9 weeksafter treatment but then decreased to a level almost equalto that of the pre-infected animals (P > 0.05, data notshown) 11 weeks after treatment (Fig. 6B). In contrast,the antibody level of the un-treated group increasedthroughout the experiment until 18 weeks after infection,and the level was clearly different from that of the pre-infected animals (P < 0.05) (Fig. 6A).

4. Discussion

Myophilin was first identified as a muscle-specific pro-tein of E. granulosus, where it appeared to be involved inthe regulation of muscle contraction (Martin et al., 1995,1996). A myophilin-like protein was subsequently identi-fied from E. granulosus with high similarity (74% aminoacid sequence identity) myophilin. In the present study,a myophilin-like protein from S. japonicum was identicalto the ortholog from E. granulosus. By mapping the draftS. japonicum genome sequence, single copy of the myo-philin-like protein gene was annotated in the S. japoni-cum genome; it contains four exons segregated by threeintrons (data not shown). No homolog of the E. granulo-

Fig. 5. Immunolocalization of the Sj myophilin-like in adult male S. japonicum. (A) Detected with pre-immunized mouse sera; (B) Detected with mouseanti-serum against Sj myophilin-like protein. Magnification 400�.

Table 1Anti-Sj22 antibody detection in sera from healths human and schistosomiasis patients

Groups OD490 value ð�x� SDÞ Positive cut-offð�xþ 2SDÞ No. samples Positives no. (%) P-value*

Healths 0.2085 ± 0.0598 0.328 33 2 (6.06) <0.0001Patients 0.4312 ± 0.2291 61 35 (57.4)

* Group t-test.

Fig. 6. Changes in anti-Sj myophilin-like serum antibody levels in infectedrabbits groups. (A) Group without treatment with praziquantel post-infection; (B) Group treated with praziquantel at 10 weeks post-infection.

122 H. Peng et al. / Experimental Parasitology 119 (2008) 117–124

sus myophilin antigen was identified within the S. japon-

icum genome (http://lifecenter.sgst.cn), a situation similar

to Schistosoma mansoni (Haas et al., 2007; Oliveira,2007).

To investigate transcription of the S. japonicum myophi-lin-like protein in different life cycle stages, Northern blot-ting and RT-PCR analysis were undertaken and the resultsrevealed that transcripts of the myophilin-like protein werepresent in eggs, sporocysts, cercariae, hepatic schistosom-ula and adult worms. This corroborated earlier proteomicsanalysis which showed that the myophilin-like protein ispresent in the eggs, cercariae, schistosomula and adultworms of S. japonicum (Liu et al., 2006). We detected themyophilin-like protein in the muscle of adult worms byimmunofluorescence. Taken together, it appears that theS. japonicum myophilin-like protein is a muscle proteinand likely plays an important role throughout the life cyclein the developmental biology of the worm, similar to thatreported for its homolog, calponin, in B. belcheri (Uranoet al., 2003).

The antigenity of rSj myophilin-like protein was exam-ined by Western blotting, and the level of anti-Sj myophi-lin-like antibody in sera was assessed by ELISA inuninfected individuals, schistosomiasis patients, infectedrabbits, including animals treated with praziquantel. TheELISA results showed that the difference in anti-myophi-lin-like protein antibodies between healthy and schistoso-miasis patients was significant (P < 0.0001), and adiagnostic sensitivity of 57.4% (35/61) was obtained.

H. Peng et al. / Experimental Parasitology 119 (2008) 117–124 123

Although, for diagnosis, the sensitivity of the rSj myophi-lin-like protein is insufficiently high, it could be usefulwhen combined with other antigen(s). In rabbits, the antiSj myophilin-like protein antibody levels significantlyincreased after S. japonicum infection. In the group trea-ted with praziquantel, the antibody level had decreased10 weeks after infection, to the pre-infection level,whereas it increased continually in un-treated groups.Together with the antibody level differences betweenhealthy and schistosomiasis patients, these results suggestthat detection of anti-S. japonicum myophilin-like anti-bodies could be used to monitor the treatment efficacyof praziquantel.

In conclusion, we identified a novel muscle myophilin-like protein from S. japonicum. The rSj myophilin-like pro-tein may be useful as an adjunct immunodiagnosis antigenfor schistosomiasis as well as a marker of drug treatmentefficacy.

Acknowledgments

The work received financial support from ShanghaiCommission for Science and Technology (No.05XDB1414) and Chinese National Hi-Tech Program(863) (No. 2004AA2Z1010).

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