International Journal for Parasitology 43 (2013) 891–900

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International Journal for Parasitology

journal homepage: www.elsevier .com/locate / i jpara

Expression pattern and substrate specificity of Clonorchis sinensistyrosinases q

0020-7519/$36.00 � 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.ijpara.2013.05.006

q Nucleotide sequence data reported in this paper are available in the GenBankunder the accession numbers KC195913, KC195914, KC195915 and KC195916.⇑ Corresponding author. Tel.: +82 31 299 6251; fax: +82 31 299 6269.

E-mail address: kongy@skku.edu (Y. Kong).

Young-An Bae a,b, Guo-Bin Cai a, Seon-Hee Kim a, Woon-Mok Sohn c, Yoon Kong a,⇑a Department of Molecular Parasitology, Sungkyunkwan University School of Medicine and Center for Molecular Medicine, Samsung Biomedical Research Institute, 300Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Koreab Department of Microbiology, Graduate School of Medicine, Gachon University, Incheon, Republic of Koreac Department of Parasitology and Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 March 2013Received in revised form 8 May 2013Accepted 9 May 2013Available online 14 June 2013

Keywords:Clonorchis sinensisClonorchiasisTyrosinaseDiphenol oxidaseSexual maturationEggshell formation

Tyrosinase (TYR) is a copper-containing glycoenzyme that mediates hydroxylation of tyrosine into dihy-droxyphenylalanine and oxidation of dihydroxyphenylalanine into dihydroxyphenylalanine quinone.TYRs play pivotal roles in eggshell sclerotisation of trematode parasites, while their comprehensive bio-chemical properties remain elusive. We characterised genes encoding four TYRs (CsTYR1–4) of Clonorchissinensis, a causative agent of human hepatobiliary disease. These genes shared tightly conserved aminoacid residues, two copper binding catalytic motifs and a cysteine-rich epidermal growth factor-likedomain. The native and recombinant CsTYRs showed high reactivity against diphenol compounds, espe-cially those with hydroxyl groups in ortho-positions (catechol and L-dihydroxyphenylalanine), butshowed minimal activity toward monophenol compounds. Diphenolase activity was enhanced byincreased pH of the reaction buffer from 5.0 to 7.0. The temporal induction of CsTYR expression coordi-nated with the sexual maturation of the worm; enzyme activity was mainly in the vitelline glands andintrauterine immature eggs proximal to the ovary. The primary structures and functional domains ofCsTYRs showed significant similarities to those of the vertebrate orthologs, whereas the amino acidsshared with the nematode and insect proteins were largely restricted in the bicopper active center. Unlikehighly diverged TYR homologs in vertebrates, multiple paralogs have not yet evolved into the separatelineages in trematode genomes, suggesting that duplication of TYR genes might relate to increased genicdosage/redundancy in trematodes. In vitro treatment of copper chelator, diethyldithiocarbamic acid,inhibited generation of phenotypically normal egg. TYR proteins are essential for C. sinensis reproduction,thus might be targeted for therapeutic and vaccine strategies against clonorchiasis, which is prevalent inseveral Asian countries and is one of the most important predisposing factors for human cholangiocarci-noma. The close phylogenetic relationships between trematode and vertebrate homologs also provide amolecular clue to understand the multifaceted evolutionary pathway of TYR homologs across animaltaxa.

� 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Multiple enzymes classified into the tyrosinase (TYR) familyparticipate in the hydroxylation of tyrosine into dihydroxyphen-ylalanine (DOPA; cresolase or monophenol oxidase activity; EC1.14.18.1) and oxidation of DOPA into DOPA quinone (catecho-lase or diphenol oxidase activity; EC 1.10.3.1) (Solomon et al.,1996; Garcia-Borron and Solano, 2002). TYR and its evolutionaryneighbours are expressed in a wide range of organisms andmediate biological processes including wound healing, immune

response, oxygen transportation, pigmentation and proteincross-linking (van Gelder et al., 1997; Burmester, 2001; Jaenickeand Decker, 2004). The proteins are intimately involved in trem-atode eggshell formation; quinone tanning or sclerotisation pro-cess, in which oxidation of tyrosine residues to quinones occursby the formation of DOPA, is a critical biochemical feature (Cord-ingley, 1987).

Trematode eggs are composed of 30–40 vitellocytes and a ferti-lised ovum encapsulated within closed proteinaceous eggshell inthe ootype (Smyth and Halton, 1983). The vitellocytes that origi-nate from vitelline follicles provide nutrients to the developingembryo and resources for the enclosing eggshell (Threadgold,1982). Eggshell proteins are targeted to the vitelline droplets ofmature vitellocytes and are exocytosed on the surface of a hydro-phobic membrane, which might be delivered by the Mehlis’ gland,

892 Y.-A. Bae et al. / International Journal for Parasitology 43 (2013) 891–900

leading to the alignment of the shell droplets in the ootype. Duringthis process, the tyrosine residues of the precursor proteins arerapidly oxidised into DOPA quinones and cross-linked with aneighbouring tyrosine or lysine on adjacent proteins. In additionto the eggshell proteins, the vitelline droplets contain a zymogenicform(s) of TYR, which participates in the oxidation of tyrosine inthe eggshell proteins (Smyth and Halton, 1983 and referencestherein). The eggshell forms a barrier to maintain the biophysico-chemical integrity of eggs and to protect the fertilised ovumagainst exogenous chemical attacks and aridity.

TYR activities are important in eggshell formation in trematodeparasites such as Schistosoma mansoni (Seed et al., 1978), Schisto-soma japonicum (Cai et al., 2009), Fasciola hepatica (Mansour,1958; Colhoun et al., 1998) and Clonorchis sinensis (Ma, 1963). Se-quence information of TYRs and their biological impact on thereproductive capability of the parasite were recently investigatedin schistosome species. Inhibition of phenol oxidase activity of S.mansoni and S. japonicum during in vitro culture of adult worms in-duced a significantly reduced production of morphologically intacteggs (Fitzpatrick et al., 2007; He et al., 2012). In vivo treatment ofmice infected with S. japonicum with the copper-chelator allyl thio-urea resulted in the complete failure of egg production (Cai et al.,2009). These observations indicate a direct relationship betweenTYR activity and reduced/disrupted fecundity of the schistosomes.However, similar information is not currently available for othertrematode parasites. More importantly, the detailed biochemicalproperties of these proteins remain unclear at the molecular level.

Clonorchis sinensis thrives in the mammals’ bile ducts includinghumans. Human infection occurs through the consumption ofmetacercaria-laying raw freshwater fish. Approximately 35 millionpeople are infected worldwide (Keiser and Utzinger, 2009). Chronicinfections cause easy fatigue, abdominal pain and mechanicalobstruction of the hepatibiliary duct, cholangiectasis and biliarystones (Lun et al., 2005). Increasing evidence has established thatclonorchiasis is closely related to the cholangiocarcinoma (Shinet al., 2010). The worm was categorised in the biocarcinogen group1 (Bouvard et al., 2009). The eggs of C. sinensis are the major re-sources responsible for maintenance of the parasitic life cycleand spread of the disease. Elucidation of the molecular events rel-evant to egg production is crucial for the understanding and con-trol of clonorchiasis.

In the present study, we isolated novel genes encoding paralo-gous TYR proteins of C. sinensis. Enzymatic activity of native and re-combinant Clonorchis TYR(s) was determined against a series ofphenol compounds. Temporal and spatial expression patterns ofthese genes specific to the development of the vitelline follicleswere examined in accordance with the growth and maturation ofthe sexual reproductive system of the worm.

2. Materials and methods

2.1. Parasite samples

Clonorchis sinensis metacercariae collected from naturally in-fected freshwater fish (Pseudorasbora parva) in endemic areas ofKorea were used to orally infect Sprague–Dawley rats through agavage needle (100 metacercariae/rat). Worms were harvestedfrom the bile ducts at 1, 2, 2.5, 3, 3.5 and 4 weeks p.i. The wormswere washed more than 10 times with physiological saline at4 �C. The worms were immediately homogenised with PBS(100 mM, pH 7.4) in the presence of a protease inhibitor cocktail(one tablet/25 ml; Complete, Roche, Basel, Switzerland) and 5% Tri-ton X-100 to prepare crude extracts, or in TriZol reagent (Invitro-gen, Carlsbad, CA, USA) to extract total RNA. Some freshlyperfused worms were fixed in 4% neutral formaldehyde, stained

with 2.5% hydrochloric carmine (Merck, Darmstadt, Germany)and destained in acidic 70% ethanol. Specimens were observedusing an Axiophor light microscope (Carl Zeiss, Jena, Germany).

Animals were housed in accordance with guidelines from theAssociation for the Assessment and Accreditation of LaboratoryAnimal Care (Thailand). Protocols for parasite infection and anti-body generation were approved by the Institutional Review Boardand conducted in the Laboratory Animal Research Center of Sung-kyunkwan University, Korea (protocol 2008-8-18).

2.2. Isolation of Clonorchis genes encoding TYR

The expressed sequence tags (ESTs) of adult C. sinensis wereanalysed by a series of BLAST searches (Cai et al., 2008) and thoseclones showing high-level similarities to various TYRs were se-lected for further characterisation. Gene-specific primers were pre-pared to amplify 50- and 30-regions of cDNAs encompassing each ofthe EST clones from each potential gene (Supplementary Table S1).After cloning into a pGEM-T Easy vector (Promega, Madison, WI,USA), the nucleotide sequences of amplicons were automaticallydetermined using a BigDye Terminator Cycle Sequencing Core Kit(ver. 3.0; Perkin Elmer, Foster City, CA, USA) and ABI PRISM 377Aautomated DNA sequencer (Applied Biosystems, Foster City, CA,USA). These nucleotide sequences were overlapped to constructthe full-length cDNA genes and their integrity was verified byPCR using primers designed from both ends of the assembled con-tig sequences.

In addition, the nucleotide sequences of schistosome TYRs re-trieved from GenBank were aligned with the C. sinensis genes toprepare degenerate primers from the highly conserved catalyticsites (CuA and CuB). The primers (Tyr-deg-F, 50-YNDSWTTYSY-WACNTGGCAYMG-30 and Tyr-deg-R, 50-YCMRYCCARTCCCAR-TADGG-30) were used in the PCR screening of the cDNA libraryfor further isolation of TYR-related genes encoded in the C. sinensisgenome. The thermal cycle profile included 4 min at 94 �C, 12 cy-cles of 40 s at 94 �C, 50 s at 48 �C and 1.5 min at 72 �C, 25 cyclesof 40 s at 94 �C, 50 s at 60 �C and 1.5 min at 72 �C, with a finalextension for 10 min at 72 �C. The PCR products were cloned intothe pGEM-T Easy vector and their similarity patterns were ana-lysed by BLAST searches after sequencing. Full-length cDNAs cov-ering the degenerate clones were similarly determined by PCR.

2.3. Sequence analysis

The putative amino acid (aa) sequences encoded by the Clonor-chis genes, designated C. sinensis TYRs (CsTYRs), were deduced bythe ORF Finder program at the National Center for BiotechnologyInformation (NCBI; http://www.ncbi.nlm.nih.gov/). Similarity pat-terns of the CsTYRs were analysed against the non-redundant geno-mic/proteomic databases of GenBank using BLAST programs at thenucleotide and aa sequence levels. The aa sequences were also em-ployed as queries during sequence analyses based on the HiddenMarkov Models (InterProScan, http://www.ebi.ac.uk/Tools/Inter-ProScan/). Signal peptide and transmembrane domain(s) were pre-dicted by the SignalP (http://www.cbs.dtu.dk/services/SignalP/)and TMpred (http://www.ch.embnet.org/software/TMPRED_-form.html) programs, respectively.

The non-redundant genomic/proteomic databases of GenBankat NCBI were surveyed for the retrieval of CsTYR homologs usingBLASTp and tBLASTn. The aa sequences were aligned with the Clu-stalX program and optimised with the GeneDoc program. Only theaa strings encompassing the catalytic CuA and CuB domains wereselected and used in a phylogenetic analysis after concatenation(approximately 161 aa positions). Divergence rates were calculatedusing the Jukes–Cantor–Thornton model and a phylogenetic treewas constructed by a neighbour-joining algorithm in the PHYLIP

Y.-A. Bae et al. / International Journal for Parasitology 43 (2013) 891–900 893

package (ver. 3.69). Statistical significance of branching nodes waspredicted by observing their frequencies in 1000 bootstrappingtrees using the Seqboot program.

2.4. Generation of recombinant proteins and specific antibodies

The putative mature domains of CsTYRs were predicted by sim-ulating their tertiary structure using the ESyPred3D program in theEXPASy Proteomic Server (http://www.espasy.ch/), based on thecrystal structure of a Streptomyces castaneoglobisporus TYR(1WX2_A; Matoba et al., 2006). Nucleotide fragments correspond-ing to mature domains were amplified from the adult cDNA libraryby PCR using primers containing restriction sites for BamHI or Hin-dIII (Supplementary Table S1). The PCR products were digestedwith the restriction enzymes and ligated into the pET-28a vector(Novagen, Madison, WI, USA), which was pre-restricted with iden-tical enzymes. The nucleotide sequence of the plasmids in Esche-richia coli DH5a were confirmed by sequencing, after which theconstructs were introduced into competent E. coli BL21 (DE3) cells.The bacteria were cultured in Luria–Bertani media and proteinexpression was induced by 0.5 mM isopropyl-b-D-thiogalactopyra-noside (IPTG) for 4 h at 37 �C. E. coli cells were harvested, sonicatedand the recombinant CsTYR (recCsTYR) proteins were purified bynickel–nitrilotriacetic acid (Ni–NTA) agarose chromatography(Qiagen, Valencia, CA, USA). The molecular weights and purity ofthe proteins were examined by 12% SDS–PAGE under reducingconditions.

The proteins (30 lg) were mixed with Freund adjuvants (Sig-ma–Aldrich, St. Louis, MO, USA) and subcutaneously injected intospecific pathogen-free BALB/c mice three times at 2 weekly inter-vals. The mice were finally boosted with 10 lg of proteins in PBSthrough their tail vein. Seven days later, the blood was collectedby heart puncture and centrifuged for 10 min at 3000g. The recCs-TYR-specific mouse antisera were stored at �80 �C until use.

2.5. Detection of native TYR activity in C. sinensis

Native CsTYR proteins in the crude extracts of adult C. sinensiswere examined by 12% SDS–PAGE under reducing conditions andfollowing Western blotting with the specific mouse antiseraagainst recCsTYR2 and recTYR3. For in-gel zymography of TYRactivity, the extracts (50 lg) were resolved by 7.5% native PAGEat 4 �C, after which the gel was incubated with 25 mM catecholor tyrosine methyl ester (TME) for 10 min at 37 �C. The color reac-tion was developed with 0.3% 3-methyl-2-benzothiazoline hydro-chloride hydrate (MBTH) dissolved in 90% ethanol (Nellaiappanand Vinayagam, 1986).

To quantitatively estimate the enzymatic activity, the crude ex-tracts (100 lg) were incubated in PBS (pH 7.0) with 0.5 mM cate-chol or TME supplemented with 0.3% MBTH in a final volume of200 ll for 4 min at 37 �C. Absorbance was monitored at 1 minintervals for 7 min at 488 nm (Nellaiappan et al., 1989) using anInfinite M200 spectrophotometer (Tecan, Grödig, Austria). Thepreparations containing diethyldithiocarbamic acid (DDC, 1 and10 mM) were also included during the measurement. Each reactionwas independently assayed in triplicate and the enzymatic activitywas expressed as the mean ± S.D. of absorbance per mg of proteinper min.

Fresh live worms were incubated at 37 �C for 1 h in RPMI-1640supplemented with catechol or TME (each 250 mM) for in situzymography. Worms were placed on a glass slide with two dropsof PBS containing 0.05% sodium phenobarbital and observed usingan Axiophor fluorescence microscope (Carl Zeiss, Jena, Germany).For comparison, the worms were simultaneously incubated inRPMI-1640 only (negative control) or RPMI-1640 containing cate-chol/DDC (each 250 mM).

2.6. Enzymatic characterisation of recCsTYR proteins

Purified recCsTYR proteins dissolved in 8 M urea (pH 4.5) wereincubated at room temperature for 1 h in the presence of 10 mMDTT and diluted fivefold in an oxidation buffer (50 mM Tris–HCl(pH 8.5), 5 mM cysteine, 1 mM L-arginine, 5 mM CaCl2, 5 lM CuSO4

and 8 M urea). The solution was kept overnight at 4 �C and thedenaturing urea was removed by serial dialysis against PBS witha step-wise decrease in urea concentration from 6 M to 0 M. Theproteins were finally dialysed against a phosphate buffer(100 mM, pH 7.0) and centrifuged at 20000g for 30 min to removeun-refolded protein aggregates.

Only the recCsTYR3 retained its enzymatic activity during therefolding procedure. Therefore, recCsTYR3 protein (1 lg) was usedin the spectrophotometric assay of specific TYR activity againstvarious mono- and dihydroxyphenol compounds, as described inSection 2.5 (tyramine (4-hydroxyphenethylamine), tyrosine andTME for monohydroxy phenol substrates; 4-methylcatechol (4-MC), dihydroxybenzene (catechol), L-DOPA and dopamine(dihydroxyphenethylamine) for o-phenols; 2-methylhydroqui-none (2-MHQ) and hydroquinone (HQ) for p-phenols). The effectof pH on the diphenolase activity of recCsTYR3 toward catecholand L-DOPA was further examined by changing the pH of the reac-tion buffer from 5 to 7. For steady-state kinetic studies, dihydroxyphenol compounds were used, ranging in concentrations from1–60 mM (catechol) or 1–15 mM (L-DOPA). All of the chemicalsused in the biochemical assay of CsTYR were purchased fromSigma–Aldrich.

2.7. Semiquantitative reverse transcription-PCR (RT-PCR)

Total RNAs were isolated from C. sinensis worms in differentdevelopmental stages using TriZol reagent. The RNAs were treatedwith RNase-free DNase (GIBCO BRL, Rockville, MD, USA) to removeany contaminating DNA. The CsTYR transcripts in the RNA solutions(1 lg) were reverse transcribed into the first-strand cDNAs usingthe CsTYR-specific reverse primers (Supplementary Table S1)employing a RNA PCR Kit (AMV, ver2.1) according to the manufac-turer’s instructions (Takara, Shiga, Japan). The cDNAs were ampli-fied by adding each of the gene-specific forward primers. Thethermal cycling profile of the subsequent PCR included a preheat-ing for 2 min at 94 �C, 25 cycles of 50 s at 94 �C, 1 min at 60 �C,1.5 min at 72 �C and a final extension for 10 min at 72 �C. The cy-cling number was empirically determined to ensure the amplifica-tion reactions in their exponential phases. Genes encoding eggshellprecursor proteins (CsEPP) (Ebersberger et al., 2005) were also in-cluded in the PCR examination of mRNA transcripts (CsEPP1,AY520541 and CsEPP2, AY150340). A primer pair for the tropomy-osin gene (CsTROP, L43918) was selected as a housekeeping con-trol, as previously described (Bae et al., 2011). Products wereanalysed by 1% agarose gel electrophoresis and staining with ethi-dium bromide.

2.8. Immunohistochemical staining

Adult worms fixed in 4% neutral paraformaldehyde wereembedded in paraffin. Tissue sections (4 lm thick) were mountedon electrostatically-charged glass slides. After deparaffinisation,the sections were treated with 3% hydrogen peroxide for 5 min,after which they were blocked with 1% BSA for 1 h. The sectionswere incubated overnight with the anti-recCsTYR antibodies di-luted to 1:2000 in PBS containing 1% BSA at 4 �C. Preimmunemouse serum (1:2000 dilutions) was included as a negative con-trol. The slides were incubated with goat anti-mouse IgG antibodyconjugated with rhodamine (Abcam, Cambridge, UK). The staining

894 Y.-A. Bae et al. / International Journal for Parasitology 43 (2013) 891–900

patterns were observed using a model LSM510 Meta DuoScan con-focal fluorescent microscope (Carl Zeiss).

2.9. In vitro effect of TYR inhibitor on C. sinensis eggshell formation

Ten fresh, viable adults were incubated in 5 ml of RPMI-1640supplemented with 10% heat inactivated FCS, 2 mM L-glutamineand 100 lg/ml of penicillin/streptomycin for 24 h at 37 �C in a5% CO2 incubator in the presence/absence of TYR inhibitor,DDC (100 lg/ml). Incubated worms were washed more than 10times with cacodylate buffer (200 mM, pH 7.2) and fixed in2.5% glutaraldehyde at 4 �C. After washing with the same buffer,the worms were dehydrated through a graded alcohol series(50%, 70%, 80%, 90%, 95% and absolute alcohol) and dried in acritical point dryer. The worms were teased and the eggs liber-ated from the uterus were coated with gold in the JFC-1100Eion sputtering device (JEOL, Tokyo, Japan). The eggs were ob-served using a scanning electron microscope (SEM; Philips XL-30S, Amsterdam, Netherlands) with an accelerating voltage of20 kV.

3. Results

3.1. Isolation and molecular characterisation of Clonorchis TYR genes

We identified three cDNA fragments, which exhibited strongcladistic affinities to vertebrate TYRs, during analysis of adult C. sin-ensis ESTs based on the BLAST algorithms. Nucleotide sequences offull-length genes encompassing each of the partial clones weredetermined from an adult C. sinensis cDNA library by adapting aseries of PCRs. The cDNAs were designated CsTYR1 (KC195913),CsTYR3 (KC195915) and CsTYR4 (KC195916). The sequences were

MAT R . . . . . . I I GP AT L L VL CWL S . . HVR AL I P S E CAK NVT T P . . E K VCCP R DP HNMS T S R Y VVAGAI VP AT L L VL CWL S . . HVR AL I P S E CVK NVT R H. . GQI CCP QNP HNMS K I DE L T . . T VCCL VL L GI VT L K T NHVT AL I P E QCAHNVS R P . . F S T CCP T DP F NMQY I N. . . . . . . HI I S VWL L I T I S QWE ADAL I P K VCVHNI T AT GGS GVCCP I P K GA. . . . . . . . . . . . ML L AVL Y CL L WS F QT S AGHF P R ACVS S K NL M. . E K E CCP P WS GDMS AP . . . . . . K L L S L GCI F F P L L L F QQAR AQF P R QCAT VE AL R . . S GMCCP DL S P A. MS P . . . . . . L WWGF L L S CL GCK I L P GAQGQF P R VCMT VDS L V. . NK E CCP R L GAE

F GVT CE QCWF GWT GP NCDQP . E K R I R R DI R T Y S P DE L NI F K DVL AR S WS WP S K Y MIF GVACE QCWF GWT GP NCDQP . E K R I R R DI R T Y S P DE L NI S K DVL AR S WS WP S K Y MIY GVACE QCWF GWT GP HCDQK . E I R I R R NI R S L S E DE L E L F K DVMY R S QT WP S GF WVF GVGCE E CY Y GWK GP L CNK R . E K VL R R NVMS F T K K E K R MF VDI VAHMP L T Y T DY VIMGF NCGNCK F GF WGP NCT E R . R L L VR R NI F DL S AP E K DK F F AY L T L AK HT I S S DY VS GHNCGT CR P GWR GAACDQR . VL I VR R NL L DL S K E E K NHF VR AL DMAK R T T HP L F VAGY NCGDCK F GWT GP NCE R K K P P VI R QNI HS L S P QE R E QF L GAL DL AK K R VHP DY V

DGP E F P T WHR Y L ML I WE R L L AE I AWK T HGI K DF T L P Y WDWVGL L K . CDI CDNK Y VGDGP T F P T WHR Y L ML I WE R ML AE I AWK T HGI K NF AL P Y WDWVGL L K . CDI CDNK Y VGDGVT F P T WHR Y F QL I WE R ML S NI AL E VHGI HDF AVP Y WDMI GL E K . CDI CT DDY VGHVL GF AT WHR Y F ML VWE R QL R K I AT R L Y GWK DF AVP Y WDWI DADK . CDVCVNS L VGE AP AF L P WHR L F L L R WE QE I QK L T GDE N. . . . F T I P Y WDWR DAE K . CDI CT DE Y MGE GP AF L T WHR Y HL L R L E K DMQE ML QE P S . . . . F S L P Y WNF AT R K NVCDI CT DDL MGQGP AF VT WHR Y HL L CL E R DL QR L I GNE S . . . . F AL P Y WNF AT GR NE CDVCT DQL F G

S . . E VF P T QADVAF VL DL K NY F VR G. E R DNP R CE S F HMAL E GF CGR . . P GADS T GLS . . E VF P NQADI AF I L DL K NY F T S G. E R DT P R CE S F HMAL E GF CGR . . P GADS NGLT . . NAF P NQE DL Y F T L NL QDY F VP G. E R DS E E CR S F HMAL E GY CGR . . P DT DP T Y RQ. . T AF P T T K DL QF T L S R GS F Y L P QK E E DDK K CR GF HQAL E GF CAA. . P GT NE E NLS R T P R L P S S ADVE F CL S L T QY E S GS . . MDK AANF S F R NT L E GF AS P L T GI ADAS QSP MVQR L P E P QDVAQCL E VGL F DT P P . . F Y S NS T NS F R NT VE GY S DP . T GK Y DP AVR. . . MK L P T L K DI R DCL S L QK F DNP P . . F F QNS T F S F R NAL E GF DK A. DGT L DS QVM

P GHK R DAF L T AI F P L VR NGDMF T DV. NNL GY DY DQP DMVGL F AHNGE K P F HL Y Q. .P GHK R DAF L AAI F P L AR NS DMF T DV. R NL GY DY DK P NI VGL F AQNGGK P L Y L . . . .P GHS R DAF MVAL MP L L R NADMF VDS . L QL GY DY DN. MMF GQF AQNGVP P I VVE I . .L GS CR E CNI I GF I P T I R HI QMF VDL . R QL GI Y Y DN. Y HF GK HGY R GE E F I K HGP S YI GHNR E S Y MVP F I P L Y R NGDF F I S S . K DL GY DY S Y L QDS DP DS F QDY I K S Y L E QASI GHNR QY NMVP F WP P VT NT E MF VT AP DNL GY T Y E I QWP S R . . . . . . . . . . . . E F S VI GHNR MY NMVP F F P P VT NE E L F L T S . DQL GY S Y AI DL P VS . . . . . . . . . . . . VE E T

Signal peptide

ELD

CsTYR1 CsTYR2 CsTYR3 CsTYR4 HsTYR HsTYRP1 HsTYRP2

CsTYR1 CsTYR2 CsTYR3 CsTYR4 HsTYR HsTYRP1 HsTYRP2

CsTYR1 CsTYR2 CsTYR3 CsTYR4 HsTYR HsTYRP1 HsTYRP2

CsTYR1 CsTYR2 CsTYR3 CsTYR4 HsTYR HsTYRP1 HsTYRP2

CsTYR1 CsTYR2 CsTYR3 CsTYR4 HsTYR HsTYRP1 HsTYRP2

: : : : : : :

: : : : : : :

: : : : : : :

: : : : : : :

: : : : : : :

Fig. 1. Primary structures of Clonorchis sinensis tyrosinases (CsTYRs). The amino acid seqand tyrosinase-related proteins (HsTYRP1, CAG28611 and HsTYRP2, CAC19460). Diffeindividual positions. Gaps were introduced in the alignment to increase the identity valuea dinuclear copper center (CuA and CuB, blue (dotted) boxes), epidermal growth factor (E(solid) box) are indicated. Blue (closed) circles demonstrate cysteine residues which mistructures. Histidine residues, which possibly involved in binding of copper, are marked whighlighted by a green (dashed and dotted) box. The red (black) vertical line combined wand CsTYR4 proteins generated in this study. (For interpretation of the references to co

aligned to those of the schistosome orthologs and a degenerate pri-mer pair was designed from the highly conserved nucleotide se-quences (data not shown). The primers were used to additionallyisolate a paralogous gene, designated CsTYR2 (KC195914), fromthe cDNA library. The four Clonorchis genes were 1493, 1508,1570 and 1661 bp in size, and coded for a single open readingframe (ORF) for a 472 (CsTYR1), 476 (CsTYR2), 477 (CsTYR3) and482 aa (CsTYR4) polypeptide, respectively.

The deduced aa sequences of the CsTYRs contained a hydropho-bic N-terminal region, which is likely to act as target signal for thetranslocation of the protein into vitelline droplets (Smyth and Hal-ton, 1983). Typical aa signatures for laminin-type epidermalgrowth factor (EGF)-like (InterPro entry accession numberIPR002049) and di-copper center (IPR008922)/tyrosinase(IPR002227) domains (CuA and CuB) were recognised in these pro-teins, based on the Hidden Markov Model (HMM). The tertiarystructures of CsTYRs simulated based on a homology model witha bacterial tyrosinase were also similar to those of human tyrosi-nase and tyrosinase-related proteins (data not shown).

The CsTYR sequences showed identity values ranged from 53%(CsTYR2 versus CsTYR4) to 88% (CsTYR1 versus CsTYR2). We wereable to retrieve more than 250 orthologous entries from the non-redundant genomic/proteomic databases of GenBank in BLASTsearches (identity values > 30%, E-values < 2e�18). Almost all ofthe matched proteins originated from deuterostomian genomes,including human. The proteins also exhibited significant similari-ties to TYR-related protein (TYRP) 1 and TYRP2 found in verte-brates. Fig. 1 shows a multiple alignment of the entire aasequences of CsTYRs and their human orthologs, HsTYR, HsTYRP1and HsTYRP2. The functional domains of TYR predicted by HMM,such as EGF (orange box) and CuA/CuB (blue boxes) were highlyconserved in each of the respective regions. The positions of cys-teine residues involved in the formation of disulfide bonds which

G. . . . MI CGG. P GR GF CQHL E AGK E DI P K VL WV. . . DDR I E WP T R F I K HACQCGE R F G. . . . MT CGG. S GR GF CQHI AAGK E S I P K VL WV. . . DDR VE WP T R F I QY ACQCT GHF H. . . . L VCGG. P E R GS CQQL T I HR E Y VP R VF L M. . . DDR L F WP AR F F DHVCHCK DK F I . . . . HP CGG. VGI GT CQR QY I QF E K I P K HNL R . . . DDR L HWP S R F F K Y MCQCE GNY R . . . . S P CGQL S GR GS CQNI L L S NAP L GP QF P F T GVDDR E S WP S VF Y NR T CQCS GNF S GP GT DR CGS S S GR GR CE AVT ADS R P HS P QY P HDGR DDR E VWP L R F F NR T CHCNGNF S . . . ANVCGS QQGR GQCT E VR ADT R P WS GP Y I L R NQDDR E L WP R K F F HR T CK CT GNF

L DE S T NMR S D. . P L NNP K F MF AS VQY Y I T F L HNY S S R T T L Y K S K F MCE E Y GI L DF S H L DE S T NMR S D. . P L K NP K F L S AS VQY Y I T F L NS Y GS R T T L Y S NR HK CE E Y S I L DF NH L DE S T NNR S D. . P L F K P R L K P AS VQY WAAF I HR Y GAR P T L Y E T E E E CS R F GI L NF S H I HE GDR Y HS D. . P L WK P R F MDVHL QY L I AY L HE Y AS R GT L Y K DDF NCL F R K K L DNNH I P I GT Y GQMK . . NGS T P MF NDI NI Y DL F VWMHY Y VS MDAL L G. . . GS E I WR DI DF AH I AT R R S E E I L GP DGNT P QF E NI S I Y NY F VWT HY Y S VK K T F L GV. . GQE S F GE VDF S H I T T QHWL GL L GP NGT QP QF ANCS VY DF F VWL HY Y S VR DT L L GP . . GR . P Y R AI DF S H

AP GR R DK DGT R L DS E . S I F Y NVT NY CW. . . . E P E P GMVCS GCQAGGR AGK . L VR R F V AP GK K DR DGI R L DP R . S I F Y NAT NY CW. . . . Y P K S DAL CVGCQAGGR VGK . L VR K F V GP GHI DR Y GVHL S P N. S AF T GF QE Y CL . . . . E P E GDE VCF GCQGT K P NT T . I T R QF L AP G. P F VDGI R L I HR DS P F S NWT E QCS . . . . P P R F GGGCI S CHS AWP NF K P L NR HY K GQH. . . P T NP NL L S P AS F F S S WQI VCS R L E E Y NS HQS L CNGT P E GP L R R NP GNHDK . S R S . . . NF DS T L I S P NS VF S QWR VVCDS L E DY DT L GT L CNS T E DGP I R R NP AGNVAR AAR . . . P DDP T L I S R NS R F S S WE T VCDS L DDY NHL VT L CNGT Y E GL L R R NQMGR NS .

WMHNK VHNMI DGAMR S T AT AT NDP F F I L HHNF VDK L L S MWY R R HK P P F DAY P NHNVR WT NNK VHNMI E GS MQR T AT AT NDP I Y I L HQVF I DK L L S MWY R R HK P P F DAY P NHNVR WMHNR L HR MI NGS MCCT S T AT NDP I F I VL HNF VDK I F NAWL R L Y HP P I E AY P R DNVR F MHNK VHNMVHGS F CCAS T AANDP L F L L HHS QI DR I MQVWF E HY R P R P T E Y P NHGVD S MHNAL HI Y MNGT MS QVQGS ANDP I F L L HHAF VDS I F E QWL QR HR P L QE VY P E ANAP S L HNL AHL F L NGT GGQT HL S P NDP I F VL L HT F T DAVF DE WL R R Y NADI S T F P L E NAP S L HNL VHS F L NGT NAL P HS AANDP I F VVL HS F T DAI F DE WMK R F NP P ADAWP QE L AP

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y R DL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R I WS WL L GAAMVGAVL T AL L AGL VS L L CR HK R K QL P E E K QP L L ME K E DY HS L Y QS HL P . . E I I AI AVVGAL L L VAL I F GT AS Y L I R AR R . S MDE ANQP L L T DQY . . QCY AE E R I P GWP T T L L VVMGT L VAL VGL F VL L AF L QY R R . . . L R K GY T P L ME T HL S S K R Y T E E A.

CuA

CuB

TMD

95 101 101 98 95

105 101

205 211 211 208 202 215 211

311 317 317 315 306 321 316

419 425 425 424 417 431 423

472 476 477 482 529 527 519

uences of CsTYRs were aligned with those of human tyrosinase (HsTYR, AAB60319)rent shades of gray indicate the degree of similarity among the proteins for thes. Amino acids comprising the tightly conserved functional protein domains, such asGF)-like domain (ELD, orange (dashed) box) and transmembrane domain (TMD, redght be involved in the formation of disulfide bonds to conform the correct tertiaryith red (open) circles. Putative signal peptides predicted by the SignalP program areith a red (black) arrow marks the N-terminal ends of recombinant CsTYR2, CsTYR3

lour in this figure legend, the reader is referred to the web version of this article.)

Y.-A. Bae et al. / International Journal for Parasitology 43 (2013) 891–900 895

conform to the correct tertiary structures and histidine residuesthat are directly involved in copper binding were also comparableamong the proteins examined (blue and red circles). The major dif-ference between the trematode and deuterostomian proteins ap-peared to be an absence or presence of the transmembranedomain (TMD) in their C-terminal ends (highlighted by a redbox). Proteins isolated from other protostomians including nema-todes, insects and molluscans displayed considerable similaritiesto the trematode proteins only within the narrow bicopper centerregions (data not shown).

3.2. Phylogenetic positions of CsTYRs

On the basis of the BLAST searches, aa sequences of 48 TYRmembers were selected to examine the phylogenetic position ofCsTYRs. The neighbour-joining method in PHYLIP clearly separatedthese proteins into distinct clades according to the donor’s taxo-nomical positions, such as Mollusca, Nematoda, Trematoda andChordata (a major rule tree is presented in Fig. 2). Consistent withthe similarity patterns, the trematode clade composed of Clonorchisand Schistosoma proteins showed the closest relationship to thatcontaining chordate-specific proteins (bootstrapping value of 99).It seemed that an ancestral TYR gene had independently duplicatedin each of the animal taxa and, thereafter, the progeny copies fol-lowed modular evolutionary pathways leading to the generation

CAAACABAAAAAAACANPCACAQ

100

78

91100

99

99

100

99

99

98

AACANPCABACABACAAAAANPAACA

99

99

100AAABBAAABACABABANPBAAAAAAAAABAXPAA

58

79

100

AAAACs

CsCs

Cs

AAAA

BA

96

Fig. 2. Phylogenetic analysis of Clonorchis sinensis tyrosinases (CsTYRs) with their relateand CuB) of tyrosinase-like proteins were isolated and individually catenated to applyneighbour-joining tree of the amino acid alignment, which was constructed by the PHnumerals at branching nodes indicate their percentages of appearance in 1000 bootstra

of multifaceted proteins with different structural/functional mo-tifs, along with their donor organisms (Supplementary Fig. S1).Genes that are ancestral for each of the TYR and TYRP lineageswould have been separated before the divergence of tunicatesand craniates. The TYRP-lineage gene have evolved further intoTYRP1 and TYRP2 in craniates (or at latest in vertebrates), whilethe multiplied siblings maintained their un-diverged ancestry inthe tunicates.

3.3. Enzymatic characteristics of CsTYRs

TYR activity expressed in adult C. sinensis was examined by in-gel zymography using catechol or TME as a substrate. As shown inFig. 3A, two distinct reactive bands were clearly evident in the gelincubated with catechol. The positive signals completely disap-peared when a copper chelator DDC was supplemented in the incu-bation medium. In contrast, the crude extracts did not exhibit anyobservable reaction against the monohydroxy phenol substrate,TME. The specific activity of CsTYRs biased toward the dihydroxy-benzene was similarly determined during the spectrophotometricassay (Fig. 3B). The diphenolase activity was increased or de-creased in proportion to the amounts of proteins (data not shown)or specific inhibitor added in the reaction (Fig. 3B). The mono- anddiphenolase activities were calculated as 0.38 ± 0.03/min/mgand 2.67 ± 0.15/min/mg. In situ zymography examination

E81941 (Neurospora crassa) - OutgroupZ66340 (Pinctada fucata)C82191 (Sepia officinalis)C87843 (Illex argentinus)K68589 (Caenorhabditis elegans)K21515 (C. elegans)A27909 (C. elegans)B04594 (C. elegans) 491709 (C. elegans)E74225 (C. briggsae)A95805 (C. elegans)

19673 (C. elegans)

TYR - Trematoda

TYR - Mollusca

TYR - Nematoda

F81264 (Danio rerio)G05915 (Tetraodon nigroviridis)_001017161 (Xenopus tropicalis)A44951 (Mus musculus)D99582 (Sus scrofa)C19460 (Homo sapiens)C76423 (Halocynthia roretzi)G28611 (H. sapiens)H76598 (M. musculus)H43815 (Xenopus laevis)_001016476 (X. tropicalis)H76406 (D. rerio)G02287 (T. nigroviridis)

TYRP 1 - Chordata

TYRP 2 - Chordata

N17339 (D. rerio)D73809 (Salmo salar)D15282 (Oncorhynchus mykiss)F20161 (Ictalurus punctatus)C99085 (O. mykiss)G00461 (T. nigroviridis)A02077 (Rana nigromaculata)B79631 (Coturnix japonica) 989491 (Gallus gallus)B79632 (Pelodiscus sinensis)B60319 (H. sapiens)Q17535 (Canis familiaris)F43895 (Oryctolagus cuniculus)L02331 (Bos taurus)D99580 (S. scrofa)_238901 (Rattus norvegicus)H79678 (M. musculus)

TYR - Chordata

W24636 (S. japonicum)W21822 (S. mansoni)TYR4 (C. sinensis)

TYR1 (C. sinensis)TYR2 (C. sinensis)

TYR3 (Clonorchis sinensis)

W26996 (Schistosoma japonicum)P93838 (Schistosoma mansoni)

C76424 (H. roretzi)

TYRP - Tunicata

TYR - Tunicata

d proteins. The amino acid sequences comprising the dinuclear copper center (CuAthe phylogenetic analysis. The majority-role consensus tree was derived from aYLIP program. The tree was rooted with a fungal tyrosinase (CAE81941). Arabic

p replicates.

896 Y.-A. Bae et al. / International Journal for Parasitology 43 (2013) 891–900

demonstrated that the diphenolase activity is expressed mainly invitellaria and immature eggs deposited in the uterus proximal tothe ovary (Fig. 3C). A negligible reaction signal was observed inworms treated with TME (data not shown).

We generated recombinant proteins corresponding to the ma-ture domains of CsTYRs (recTYRs; red arrow in Fig. 1) using theE. coli system, to access detailed biochemical features of CsTYRs.All recTYRs were expressed as insoluble forms present in the inclu-sion bodies. Only recCsTYR3 was successfully converted to an ac-tive soluble enzyme after refolding. The protein was used in theenzymological assays employing a series of mono- and dihydroxyphenols. Similar to the unfractionated native enzymes, recTYR3rapidly oxidised dihydroxy phenols, especially those with hydroxylgroups at ortho-positions including 4-MC, catechol, L-DOPA anddopamine under neutral conditions (pH 7.0). Substrates with hy-droxyl groups at para-positions (2-MHQ and HQ) were also, butless effectively, converted into the respective p-quinones by theprotein (laccase activity). However, monohydroxy phenols, suchas tyramine, tyrosine and TME, were barely hydroxylated by therecTYR3 protein (Fig. 4A). The diphenolase activity of recTYR3 ap-peared to be pH-dependent and greatly enhanced under neutral pHat 7.0 (Fig. 4B). The activity could not be properly estimated whenthe pH exceeded 8, mainly due to the auto-oxidation of the phenolcompounds (data not shown). The steady-state kinetic analyses

- DDC+

Catechol TME

A

Uterus distal

Vitel

Uterus proxi

Control Catechol C

B

Tyro

sina

seac

tivity

0

0.5

1.0

1.5

2.0

0

2.5

Fig. 3. Detection of tyrosinase (TYR) activity in adult Clonorchis sinensis. (A) Clonorchissoluble proteins (50 lg) were resolved by 7.5% native-PAGE. The gels were incubated witat 37 �C. The color reaction was developed with 0.3% 3-methyl-2-benzothiazoline (MBspecific tyrosinase inhibitor. (B) The TYR activity in the parenchymal extracts was detersupplemented with 0.3% MBTH in the presence of catechol or TME (each of 0.5 mM) for 4spectrophotometer at 488 nm. The preparations containing DDC (1 and 10 mM) were alsotriplicate preparations. The titration curves for the optical absorption were adapted frommin/mg proteins, were presented as mean ± S.D. (C) The viable C. sinensis adult worms weor catechol/DDC (20 mM). After treating the worms with MBTH, the signals in the wholeonly was included as a control.

demonstrated that the recTYR3-mediated reactions followed theMichaelis–Menten equation. Michaelis constants (Km) for catecholand L-DOPA were calculated as 23.52 mM and 1.62 mM, respec-tively (Fig. 4C).

We assessed effects of tyrosinase inhibitor during eggshell for-mation. When intact viable adults were cocultured in the presenceof DDC, a significant deformity of the eggs was observed. SEMexamination of eggs collected from control culture demonstrateda phenotypically normal sesame seed-like appearance consistingof operculum, shoulder rim with well-developed muskmelon-likewrinkling on the eggshell surface (Fig. 5A). Conversely, eggs fromworms treated with DDC revealed morphological defects includingirregular disruption and under-development of muskmelon-likewrinkling. Moreover, oval-shaped general configuration of theegg was significantly distorted (Fig. 5B).

3.4. Spatiotemporal expression pattern of CsTYR in C. sinensis

We observed developmental expression patterns of these TYRsduring the parasite’s maturation in the definitive host (Fig. 6A). Atthe metacercarial stage, the worms did not reveal any discerniblereproductive systems such as female-specific vitellaria, ovary,seminal receptacle and uterus, and male-specific testis. The densecell masses observed beneath the ventral sucker would be the

from ovary

line

mal to ovary

(250 mM) Catechol + DDC (20 mM)

Time (min)2 4 61 3 5 7

2.67 0.15/min/mg

0.38 0.03/min/mg

Blank

Catechol+DDC (1 mM)CatecholTME

Catechol+DDC (10 mM)

sinensis adult worms were extracted with PBS containing 5% Triton X-100 and theh 25 mM of catechol (dihydroxybenzene) or tyrosine methyl ester (TME) for 10 minTH). The copper-chelator, diethyldithiocarbamic acid (DDC, 5 mM) was used as a

mined against catechol or TME. The protein (100 lg) was incubated in PBS (pH 7.0)min at 37 �C. The absorbance was monitored at 1 min intervals for 7 min by using aincluded during the assay. The measurements were independently conducted with

one of the reactions and the enzymatic activities, which were expressed as DOD488/re incubated overnight in RPMI-1640 media supplemented with catechol (250 mM)body were observed using fluorescence microscopy. The incubation with medium

A

C

[Catechol] (mM)

Vmax = 13.14Km = 23.52 mM

10

0 20 40 600

4

2

6

8

10 30 50 70[L-DOPA] (mM)

Vmax = 1.85Km = 1.62 mM

0 2 4 6 8 10 12 14 160

0.3

0.6

0.9

1.2

1.5

1.8

Spec

ific

activ

ity(Δ

OD

488/m

in/μ

M)

pH5.0 6.0 7.0

0

1

2

3

4

5Catechol

L-DOPA

B

Spec

ific

activ

ity(Δ

OD

488/m

in/μ

M)

0

2

4

6

8

Spec

ific

activ

ity(Δ

OD

488/m

in/μ

M)

OH OH

OH

OH

Fig. 4. Enzymatic property of recombinant Clonorchis sinensis tyrosinase 3 (recCsTYR3). (A) The substrate availability of recCsTYR3 was assessed with various di- andmonophenol compounds. The recCsTYR3 (100 lg) was incubated in PBS (pH 7.0) with catechol (dihydroxybenzene) or tyrosine methyl ester (TME) (each 0.5 mM), and 0.3% 3-methyl-2-benzothiazoline (MBTH) in a final volume of 200 ll. Following incubation for 4 min at 37 �C, the absorbance was spectrophotometrically assayed at 1 min intervalsfor 7 min at 488 nm. Specific activity was defined as DOD488/min/lM. The reactions were performed in triplicate and presented as mean ± S.D. 4-MC, 4-methylcatechol; L-DOPA, 3,4-dihydroxy-L-phenylalanine; dopamine, 3,4-dihydroxyphenethylamine; 2-MHQ, 2-methylhydroquinone; HQ, hydroquinone. (B) Effect of pH on the diphenolaseactivity of recCsTYR3 toward catechol and L-DOPA. The activity was spectrophotometrically assessed by changing the pH of the reaction buffer (5, 6 and 7). (C) The steady-state kinetic parameters of recCsTYR3 were determined by measuring diphenolase activity against varying concentrations of catechol (1–60 mM) and L-DOPA (1–15 mM).

Fig. 5. Scanning electron microscopic images of Clonorchis sinensis eggs obtainedfrom ex-host incubation of the worms. Ten fresh, viable adults were incubated inRPMI-1640 medium containing FCS (10%), L-glutamine (2 mM) and antibiotics(100 lg/ml) for 24 h at 37 �C in a 5% CO2 atmosphere in the presence/absence ofdiethyldithiocarbamic acid (DDC) (100 lg/ml). Incubated worms were fixed in 2.5%glutaraldehyde. The worms were dehydrated and dried in a critical point dryer. Theworms were teased and the eggs liberated from the uterus were coated with gold.(A) The worms were incubated in RPMI-1640 medium alone. (B) The worms wereincubated with RPMI-1640 medium supplemented with 100 lg/ml DDC.Bar = 5 lm.

Y.-A. Bae et al. / International Journal for Parasitology 43 (2013) 891–900 897

genital primodium containing undifferentiated germ cells (bluecircle in Fig. 6A). The sexual organs could be detected in 1 weekold worms, whereas the worms seemed to initiate egg production,at the earliest, between 1 and 2 weeks p.i. However, consideringthe light-colored eggshells and empty seminal receptacles in2 week old worms, genuine sexual reproduction of C. sinensiswould start sometime between 2 and 2.5 weeks p.i. Expression ofCsTYRs was significantly increased in worms over 2 weeks of ageat the mRNA transcription level and coincided with sexual matura-tion of the worm (Fig. 6B). EPPs such as CsEPP1 and CsEPP2, are inti-mately involved in eggshell formation of C. sinensis by provision ofmajor eggshell components (Ebersberger et al., 2005; Bae et al.,2007). CsEPPs also showed induction patterns similar to those ofCsTYRs, whereas that of a housekeeping tropomyosin gene(CsTROP) remained constant through the developmental stagesexamined (Fig. 6B).

Since we were not able to purify recCsTYR4, probably due to ahigh degree of intermolecular disulfide bonds, we comparedimmunological cross reactivity of CsTYR2 and CsTYR3 employingantibodies generated against respective recombinant proteins.These proteins showed negligible cross-activity with each of theother paralogous proteins (Supplementary Fig. S2). We observedexpression patterns of CsTYRs by immunohistochemical stainingemploying these antibodies. In the adult worms, proteins immuno-logically homologous to recCsTYR2 and recTYR3 were detectedexclusively within the vitellaria and the intrauterine prematureeggs (only the positive sites against anti-recTYR3 antibody are pre-sented in Fig. 7). These collective results indicate that CsTYRs arespecifically expressed in reproductive systems in an upregulatedfashion according to the development of the worm in the definitivehost.

898 Y.-A. Bae et al. / International Journal for Parasitology 43 (2013) 891–900

4. Discussion

Vitelline cells play a key role during sexual reproduction oftrematode parasites by supplying the nutrients for fertilised ovaas well as by provision of resources involved in the eggshell forma-tion (Smyth and Halton, 1983). The eggshell proteins exhibit un-ique primary structures; homologs have not yet been identifiedin other animal taxa. These molecules are particularly enrichedwith specific aa residues including glycine and tyrosine, each ofwhich is intimately associated with the cross-linking processes toharden the eggshell (Kawanaka and Sugiyama, 1992; Bae et al.,2007). The tyrosine residues are converted into DOPA quinonesand are then condensed with tyrosine or lysine on adjacent pro-teins (Cordingley, 1987). Previous experimental evidence has indi-cated the pivotal roles of TYR in the tanning process mediated bytyrosine hydroxylation/oxidation (Fitzpatrick et al., 2007 and refer-ences therein). In this study, we determined the enzymatic param-eters of a recombinant CsTYR protein against mono- anddiphenolic compounds, which is, to our knowledge, the first knownreport analysing actual biochemical property of trematode TYRs atthe individual molecular level.

In situ zymography with phenolic compounds allowed us tolocalise TYR activity in adult C. sinensis toto specimens. The CsTYRtranscripts were not detected in the metacercarial stage; however,expression levels of CsTYRs appeared to be upregulated in accor-dance with the growth and sexual development of the liver flukein the definitive host. The CsTYR proteins were exclusively local-ised in the vitelline follicles and intrauterine eggs proximal to oo-type, where the tanning process occurs (Figs. 3 and 7). Clonorchissinensis underwent rapid sexual maturation between 1 and

Fig. 6. Expression regulation of Clonorchis sinensis tyrosinase (CsTYR) genes during sexuexperimentally infected rats at different developmental stages as indicated. The wormswere adjusted similarly during the image processing, although there were substantial dgenital primodium; IE, intrauterine eggs; IN, intestine; OS, oral sucker; OV, ovary; SR, semTotal RNAs were extracted from C. sinensis worms at various developmental stages, as indtranscription-PCR. The amplified products were separated on 1.2% agarose gels and stainand tropomyosin (CsTROP) genes were included as comparison and standard controls foDNA was confirmed by preparing reactions without reverse transcriptase during the fir

2 weeks p.i. in experimental animals and the process was accom-panied by the appearance of male and female sex organs includingovary, testis and vitelline follicles. During this period, genes essen-tial for egg production are induced and eggs with tanned eggshellwere observable at least 2 weeks after the experimental infection(Fig. 6). The specific spatiotemporal induction patterns weresimilarly observed in other trematode parasites. Fasciola hepaticainitiated the development of vitellaria and expression of the sexorgan-related genes, such as eggshell precursor and tyrosinasegenes, only after migrating into the host bile ducts, where theparasite performs sexual reproduction (Robinson et al., 2001).TYR transcripts could not be observed in the cercarial and earlyjuvenile stages of S. japonicum and S. mansoni (Fitzpatrick et al.,2007; Cai et al., 2009). Taken together, our results indicate thatCsTYRs isolated in this study represent genuine enzymatic activityfor the biological conversion of tyrosine to DOPA quinone ineggshell proteins.

Trematode TYRs harbored biphasic activities, of which mono-phenolase activity is critical to initiate the tanning process duringeggshell formation. However, native CsTYRs and recCsTYR3showed negligible activity toward monohydroxylated compounds,compared with those against dihydroxy phenols (Figs. 3 and 4).The diphenolase-biased biochemical feature was also observedwith schistosome orthologs (Fitzpatrick et al., 2004, 2007; Caiet al., 2009), which strongly suggests that the reduced monophe-nolase/diphenolase ratio is general to trematode TYRs, rather thanspecific to Clonorchis TYRs. Therefore, a step for the conversion oftyrosine into DOPA would be a rate-limiting reaction during theenzymatic process, similar to the cases of other TYR-mediated bio-chemical pathways found in soil-transmitted pathogenic bacteria

al maturation. (A) Clonorchis sinensis worms were harvested from the bile ducts ofwere observed with a light microscope after carmine staining. The sizes of wormsifferences in their actual sizes (bars indicate �100 lm). Ab: MC, metacercaria; GP,inal receptacle; TE, testis; UT, uterus; VS, ventral sucker; VT, vitellaria; wk, week. (B)icated. The mRNA transcripts of CsTYRs were amplified by a semiquantitative reverseed with ethidium bromide. Reactions to amplify eggshell precursor protein (CsEPP)r each RNA sample, respectively. RTase (�), absence of any contaminated genomic

st reverse transcription step. M, 100-bp DNA size standards.

Vitellaria

Intrauterine eggs proximal to ovary

Fig. 7. Localisation of tyrosinases (CsTYR) in adult Clonorchis sinensis sections. The native CsTYR proteins expressed in adult worms were reacted with the recCsTYR-specificantibodies. The sites of positive reaction were stained by subsequent incubation with a rhodamine-conjugated secondary antibody and the immunohistochemical stainingpatterns were observed under a fluorescent microscope (red). The sections were also observed under the laser-scanning microscope. Representative images around thevitelline glands and uterine eggs, where the positive signals were exclusively detected against anti-recCsTYR3, are presented in this figure.

Y.-A. Bae et al. / International Journal for Parasitology 43 (2013) 891–900 899

(Hernández-Romero et al., 2006). Since the precursor molecules in-volved in the eggshell formation of the trematodes including egg-shell proteins and TYRs are synthesised and co-packaged intosecretory vesicles in the vitelline cells, the lower monophenolaseactivity would have a biological implication to block untimely ini-tiation of the essential process in the vitelline droplets. Activationof trematode TYRs might depend on a phenol compound(s) se-creted by the Mehlis’ gland in the ootype of these digenean para-sites (Smyth and Halton, 1983). The physicochemical changessuch as pH and concentration of calcium ion were found to partic-ipate in the induction of the monophenolase activity (Wells andCordingley, 1991). Alternatively, catechol (or other dihydroxy phe-nols) may act as a cofactor to trigger the monophenolase activity ofTYR (Land et al., 2003) as DOPA did during hydroxylation reactionof a monophenol (Körner and Pawelek, 1982). Further studies de-signed to elucidate the chemicals and/or physiological factors,which increase the first-step reaction rate, might be necessary toaddress the controversial enzymatic properties of CsTYRs.

In addition to catechol oxidases, TYR binds oxygen molecules ata binuclear copper active site, known as the type-3 copper center(Gerdemann et al., 2002), to incorporate those into the phenol ringof tyrosine. The characteristic active site is also conserved in hae-mocyanins, which are principally responsible for the precise deliv-ery of oxygen in the haemolymph of various molluscs andarthropods (Decker and Tuczek, 2000; Guo et al., 2009). The funda-mental roles of type-3 copper proteins are associated with the pro-tection of organisms by sequestrating harmful oxygen during theearly evolutionary phase (Burmester, 2001; Jaenicke and Decker,2004). After amplification or during multiplication, a certain

lineage of the common ancestral gene would have evolved intothe contemporary TYR subfamily. A previous cladistic analysisdemonstrated that the intermediate gene underwent an additionalduplication event during an early phase of Chordate evolution,each of which has led to TYR or TYRP (TYRP1 and TYRP2) genes (Bra-asch et al., 2007). In our analysis, it was apparent that a TYRP-line-age gene(s) had already emerged before separation of Tunicata andChordata, although divergence between duplicated TYRP genesseemed to have occurred in vertebrates (Fig. 2). The genomes oftrematode species examined in this study encoded multiple para-logous TYR genes. Considering the close phylogenetic relationshipsamong trematode orthologs, these genes are likely to have not yetundergone the structural/functional diversification leading to theemergence of TYRP-related gene. The duplication events might berelated to an increase in genic dosage for amplified enzymaticactivity. Interestingly, tightly conserved primary structures andfunctional domains were observed between trematode and verte-brate homologs (Fig. 1, Supplementary Fig. S1). This result sug-gested either that these genes have been subjected to commonlyoriented evolutionary forces or that they undertook a horizontalgene transfer(s). Further investigations are warranted to clarify thisintriguing issue.

When we observed an in vitro effect of TYR inhibitor on C. sin-ensis eggshell formation, DDC introverted the production of pheno-typically intact eggs. In addition to irregular general shape,underdevelopment of muskmelon-like wrinkling with several de-fects found in the eggshell surface strongly suggested that inhibi-tion of diphenol oxidase activity subsequently prevented normaleggshell formation. When S. mansoni eggs were incubated with

900 Y.-A. Bae et al. / International Journal for Parasitology 43 (2013) 891–900

kojic acid for 48 h in vitro, several morphological defects, such asloss of the lateral spine and invaginations of the eggshell surface,were observed (Fitzpatrick et al., 2007). Such profound effects ofthe TYR inhibitor were not observed in this study. We thought thatincubation for a short period (24 h) might invoke less prominentchanges of C. sinensis eggs. Our ongoing in vivo experiments dem-onstrate that treatment of the TYR inhibitor in the C. sinensis-in-fected rats significantly reduced egg laying capacity of the liverfluke (unpublished observation). We are currently analysingwhether a single or multiple species of CsTYR is involved in egg-shell maturation by employing anti-CsTYR antibodies.

Eggs of trematode parasites are central etiological agents fortransmission of the parasites in maintenance and propagation oftheir lifecycle. They also participate in pathobiological changesduring the course of parasitic diseases, thereby eliciting severalclinical symptoms and immunological alterations in patients(Cho et al., 2000; Hoffmann et al., 2002; Fairfax et al., 2012). Con-trol of parasite fecundity may contribute to blocking transmissionof parasitic diseases. The sexual reproductivity of schistosomes ismarkedly impaired either by treatment of TYR-specific inhibitorsin ex- and in-host environments (Fitzpatrick et al., 2007; Caiet al., 2009) or by down-regulation of TYR activity employing smallinterfering RNA-mediated RNA interference (He et al., 2012). Clo-norchiasis remains one of the major infectious diseases in severalAsian countries and approximately 600 million people are at riskof infection worldwide (Keiser and Utzinger, 2009). The CsTYRgenes characterised in this study might provide target candidatesto develop novel, and potentially control, modalities. It may beespecially effective for treatment of clonorchiasis in reservoirhosts. The modular evolutionary pathways of the tyrosinase-re-lated protein family across animal taxa would also be informativein understanding the diverse evolutionary mechanisms of eukary-otic genes, which have been accompanied with functionaldiversification.

Acknowledgement

This work was supported by a grant from Ministry of Health andWelfare (Health & Medical Technology R&D Program, Korea, 2008–2009, A084596).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ijpara.2013.05.006.

References

Bae, Y.A., Kim, S.H., Cai, G.B., Lee, E.G., Kim, T.S., Agatsuma, T., Kong, Y., 2007.Differential expression of Paragonimus westermani eggshell proteins during thedevelopmental stages. Int. J. Parasitol. 37, 295–305.

Bae, Y.A., Kim, S.H., Lee, E.G., Sohn, W.M., Kong, Y., 2011. Identification andbiochemical characterization of two novel peroxiredoxins in a liver fluke,Clonorchis sinensis. Parasitology 138, 1143–1153.

Bouvard, V., Baan, R., Straif, K., Grosse, Y., Secretan, B., El Ghissassi, F., Benbrahim-Tallaa, L., Guha, N., Freeman, C., Galichet, L., Cogliano, V., WHO InternationalAgency for Research on Cancer Monograph Working Group, 2009. A review ofhuman carcinogens - Part B: biological agents. Lancet Oncol. 10, 321–322.

Braasch, I., Schartl, M., Volff, J.N., 2007. Evolution of pigment synthesis pathways bygene and genome duplication in fish. BMC Evol. Biol. 7, 74.

Burmester, T., 2001. Molecular evolution of the arthropod hemocyanin superfamily.Mol. Biol. Evol. 18, 184–195.

Cai, G.B., Bae, Y.A., Kim, S.H., Sohn, W.M., Lee, Y.S., Jiang, M.S., Kim, T.S., Kong, Y.,2008. Vitellocyte-specific expression of phospholipid hydroperoxideglutathione peroxidases in Clonorchis sinensis. Int. J. Parasitol. 38, 1613–1623.

Cai, G.B., Bae, Y.A., Zhang, Y., He, Y., Jiang, M.S., He, L., 2009. Expression andcharacterization of two tyrosinase from the trematode Schistosoma japonicum.Parasitol. Res. 104, 601–609.

Cho, S.Y., Kong, Y., Yun, D.H., Kang, S.Y., Kim, L.S., Chung, Y.B., Yang, H.J., 2000.Persisting antibody reaction in paragonimiasis after praziquantel treatment iselicited mainly by egg antigens. Korean J. Parasitol. 38, 75–84.

Colhoun, L.M., Fairweather, I., Brennan, G.P., 1998. Observation on the mechanismsof eggshell formation in the liver fluke, Fasciola hepatica. Parasitology 116, 555–567.

Cordingley, J.S., 1987. Trematode eggshells: novel protein biopolymers. Parasitol.Today 3, 341–344.

Decker, H., Tuczek, F., 2000. Tyrosinase/catecholoxidase activity of hemocyanins:structural basis and molecular mechanism. Trends Biochem. Sci. 25, 392–397.

Ebersberger, I., Knobloch, J., Kunz, W., 2005. Cracks in the shell – zooming in oneggshell formation in the human parasites Schistosoma mansoni. Dev. GenesEvol. 215, 261–267.

Fairfax, K.C., Amiel, E., King, I.L., Freitas, T.C., Mohrs, M., Pearce, E.J., 2012. IL-10Rblockade during chronic schistosomiasis mansoni results in the loss of B cellsfrom the liver and the development of severe pulmonary disease. PLoS Pathog.8, e1002490.

Fitzpatrick, J.M., Hirai, Y., Hirai, H., Hoffmann, K.F., 2007. Schistosome eggproduction is dependent upon the activities of two developmentally regulatedtyrosinases. FASEB J. 21, 823–835.

Fitzpatrick, J.M., Johansen, M.V., Johnston, D.A., Dunne, D.W., Hoffmann, K.F., 2004.Gender-associated gene expression in two related strains of Schistosomajaponicum. Mol. Biochem. Parasitol. 136, 191–209.

Garcia-Borron, J.C., Solano, F., 2002. Molecular anatomy of tyrosinase and its relatedproteins: beyond the histidine-bound metal catalytic center. Pigment Cell Res.15, 162–173.

Gerdemann, C., Eicken, C., Krebs, B., 2002. The crystal structure of catechol oxidase.New insights into the functions of type-3 copper proteins. Acc. Chem. Res. 35,183–191.

Guo, D., Zhang, Y., Zeng, D., Wang, H., Li, X., Li, Y., Fan, X., 2009. Functional propertiesof hemocyanin from Oncomelania hupensis, the intermediate host of Schistosomajaponicum. Exp. Parasitol. 123, 277–281.

He, Y., Cai, G., Ni, Y., Li, Y., Zong, H., He, L., 2012. siRNA-mediated knockdown of twotyrosinase genes from Schistosoma japonicum cultured in vitro. Exp. Parasitol.132, 394–402.

Hernández-Romero, D., Sanchez-Amat, A., Solano, F., 2006. A tyrosinase with anabnormally high tyrosine hydroxylase/dopa oxidase ratio. FEBS J. 273, 257–270.

Hoffmann, K.E., Wynn, T.A., Dunne, D.W., 2002. Cytokine-mediated host responsesduring schistosome infection: walking the fine line between immunologicalcontrol and immunopathology. Adv. Parasitol. 52, 265–307.

Jaenicke, E., Decker, H., 2004. Functional changes in the family of type 3 copperproteins during evolution. ChemBioChem 5, 163–169.

Kawanaka, M., Sugiyama, H., 1992. Incorporation of radiolabelled amino acids byadult Schistosoma japonicum: further characterization of a putative eggshellprecursor protein. Int. J. Parasitol. 22, 589–595.

Keiser, J., Utzinger, J., 2009. Food-borne trematodiases. Clin. Microbiol. Rev. 22, 466–483.

Körner, A., Pawelek, J., 1982. Mammalian tyrosinase catalyzes three reactions in thebiosynthesis of melanin. Science 217, 1163–1165.

Land, E.J., Ramsden, C.A., Riley, P.A., 2003. Tyrosinase autoactivation and thechemistry of ortho-quinone amines. Acc. Chem. Res. 36, 300–308.

Lun, Z.R., Gasser, R.B., Lai, D.H., Li, A.X., Zhu, X.Q., Yu, X.B., Fang, Y.Y., 2005.Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect. Dis. 5, 31–41.

Ma, L., 1963. Trace elements and polyphenol oxidase in Clonorchis sinensis. J.Parasitol. 49, 197–203.

Mansour, T.E., 1958. Effect of serotonin on phenol oxidase from the liver flukeFasciola hepatica and from other sources. Biochim. Biophys. Acta 30, 492–500.

Matoba, Y., Kumagai, T., Yamamoto, A., Yoshitsu, H., Sugiyama, M., 2006.Crystallographic evidence that the dinuclear copper center of tyrosinase isflexible during catalysis. J. Biol. Chem. 281, 8981–8990.

Nellaiappan, K., Vinayagam, A., 1986. A rapid method for detection of tyrosinaseactivity in electrophoresis. Satin Technol. 61, 269–272.

Nellaiappan, K., Devasundari, A.F., Dhandayuthapani, S., 1989. Properties of phenoloxidase in Fasciola gigantica. Parasitology 99, 403–407.

Robinson, M.W., Colhoun, L.M., Fairweather, I., Brennan, G.P., Waite, J.H., 2001.Development of the vitellaria of the liver fluke, Fasciola hepatica in the rat host.Parasitology 123, 509–518.

Seed, J.L., Boff, M., Bennett, J.L., 1978. Phenol oxidase activity: induction in femaleschistosomes by in vitro incubation. J. Parasitol. 64, 283–289.

Shin, H.R., Oh, J.K., Masuyer, E., Curado, M.P., Bouvard, V., Fang, Y.Y., Wiangnon, S.,Sripa, B., Hong, S.T., 2010. Epidemiology of cholangiocarcinoma: an updatefocusing on risk factors. Cancer Sci. 101, 579–585.

Smyth, J.D., Halton, D.W., 1983. The biology of the egg. In: Smyth, J.D., Halton, D.W.(Eds.), The Physiology of Trematodes. Cambridge University Press, Cambridge,pp. 86–114.

Solomon, E.I., Sundaram, U.M., Machonkin, T.E., 1996. Multicopper oxidases andoxygenases. Chem. Rev. 96, 2563–2606.

Threadgold, L.T., 1982. Fasciola hepatica: stereological analysis of vitelline celldevelopment. Exp. Parasitol. 54, 352–365.

van Gelder, C.W., Flurkey, W.H., Wichers, H.J., 1997. Sequence and structuralfeatures of plant and fungal tyrosinases. Phytochemistry 45, 1309–1323.

Wells, K.E., Cordingley, J.S., 1991. Schistosoma mansoni: eggshell formation isregulated by pH and calcium. Exp. Parasitol. 73, 295–310.