analysis of structures, functions, and epitopes of cysteine protease

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 198250 7 pageshttpdxdoiorg1011552013198250

Research ArticleAnalysis of Structures Functions and Epitopes of CysteineProtease from Spirometra erinaceieuropaei Spargana

Li Na Liu Jing Cui Xi Zhang Tong Wei Peng Jiang and Zhong Quan Wang

Department of Parasitology Medical College Zhengzhou University 40 Daxue Road Zhengzhou 450052 China

Correspondence should be addressed to Jing Cui cuijzzueducn and Zhong Quan Wang wangzqzzueducn

Received 14 September 2013 Accepted 23 October 2013

Academic Editor Fabio Ribeiro Braga

Copyright copy 2013 Li Na Liu et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Spirometra erinaceieuropaei cysteine protease (SeCP) in sparganum ES proteins recognized by early infection sera was identifiedby MALDI-TOFTOF-MS The aim of this study was to predict the structures and functions of SeCP protein by using the fulllength cDNA sequence of SeCP gene with online sites and software programsThe SeCP gene sequence was of 1 053 bp length witha 1011 bp biggest ORF encoding 336-amino acid protein with a complete cathepsin propeptide inhibitor domain and a peptidaseC1A conserved domain The predicted molecular weight and isoelectric point of SeCP were 3787 kDa and 647 respectively TheSeCP has a signal peptide site and no transmembrane domain located outside the membrane The secondary structure of SeCPcontained 8 120572-helixes 7 120573-strands and 20 coils The SeCP had 15 potential antigenic epitopes and 19 HLA-I restricted epitopesBased on the phylogenetic analysis of SeCP S erinaceieuropaei has the closest evolutionary status with S mansonoides SeCP wasa kind of proteolytic enzyme with a variety of biological functions and its antigenic epitopes could provide important insights onthe diagnostic antigens and target molecular of antisparganum drugs

1 IntroductionSparganosis is a serious parasitic zoonosis caused by infectionwith spargana the plerocercoid larvae of some Diphylloboth-rium tapeworms that belong to the genus Spirometra [1] Themost important species of the genus Spirometra tapewormswith plerocercoids that can produce sparganosis in humaninclude Spirometra erinaceieuropaei (syn Spirometra erinaceior Spirometra mansoni) which is the most common in Asiaand Spirometra mansonoides which is mainly distributed inNorth America [2] The adults are intestinal parasites ofsome species of Canidae and Felidae the first intermediatehosts are freshwater copepods (cyclops) whereas the secondintermediate or paratenic hosts belong to different species ofvertebrates (frogs snakes pigs etc) [3 4] Human is an acci-dental host Human infection results mainly from drinkingraw water contaminated with cyclops harboring procercoidingesting raw fleshes of frogs and snakes infected withplerocercoids or placing frog or snake flesh on open woundfor treatment of skin ulcers or eye inflammations [5 6]

Human sparganosis is reported in many countries ofthe world but is most common in Eastern Asia and the

Far East [7] Sparganosis poses a serious threat to humanhealth the plerocercoids usually lodge in the subcutaneoustissues and muscles but sometimes invade the abdominalcavity eye and central nervous system causing blindnessseizures headache epilepsy paralysis and even death [8]Ocular sparganosis is especially prevalent in China andVietnam [9] The clinical diagnosis of sparganosis is ratherdifficult and often misdiagnosed because the larvae haveno predilection site in humans and the specific signs orsymptoms are lacking A definite diagnosis of subcutaneoussparganosis can be achieved by detection of the larvae ina biopsy specimen from the lesion but the confirmativediagnosis is very difficult for visceral and cerebral sparganosissince the larva is found only by surgical removal [10] TheELISA using the crude or excretory-secretory (ES) antiagensof plerocercoids has high sensitivity for the detection ofsparganum infection in humans but the main disadvantageis the false negative results during the early stage of infectionand the cross-reactions with serum samples from patientswith other parasitic diseases (cysticercosis paragonimiasisclonorchiasis etc) [11 12]

2 BioMed Research International

1 agaatagg

9 atgaagttcgtaatatacgttgccttcttgttccttcttttgacg

M K F V I Y V A F L F L L L T54 gtctgcagaggctcgactgaaagtgagacgtacgtccggcgggaa

V C R G S T E S E T Y V R R E99 ttgtggaaggcctggaaattggccttcaagaaggagtacttcagt

L W K A W K L A F K K E Y F S

144 agtgaagaagaactccaccgaaagcgtgcattctttaacaatctc

S E E E L H R K R A F F N N L189 gacttcatcatccgacataatcaacgctattatcaacagctcgag

D F I I R H N Q R Y Y Q Q L E234 tcctatgcagtgcgattaaatgattttagtgacctgacgccgggt

S Y A V R L N D F S D L T P G279 gaatttgccgaaagatacctttgcttacggggaattgttttgacg

E F A E R Y L C L R G I V L T324 aagttaagacggaaggaagcagtaagcgtgccactcaaagaaaat

K L R R K E A V S V P L K E N369 cttcccgacagcgtaaactggcgcgagagaggtgccgttacatcg

L P D S V N W R E R G A V T S414 gtcaaaaatcagggtcaatgcggatcctgctggtctttttccgca

V K N Q G Q C G S C W S F S A459 aacggtgcaatagaaggcgcaatccagataaagaccggtgcattg

N G A I E G A I Q I K T G A L504 cgcagcctgtcagaacagcagttgatggactgcagctgggactac

R S L S E Q Q L M D C S W D Y

549 ggcaatcaaggctgcaacgggggactcatgccacaggccttccag

G N Q G C N G G L M P Q A F Q594 tatgcccaaaggtatggcgtcgaagctgaagttgactacagatat

Y A Q R Y G V E A E V D Y R Y639 actgaaagggatggggtttgcagatatcgtcaggacctggttgtt

T E R D G V C R Y R Q D L V V684 gccaatgttactggatatgcggaactgccagaaggcgatgaggga

A N V T G Y A E L P E G D E G729 ggtctacaaagggctgttgcaaccataggcccaatatctgtcgga

G L Q R A V A T I G P I S V G774 atcgatgctgccgatcctgggtttatgtcttacagccacggtgtt

I D A A D P G F M S Y S H G V819 ttcgtcagtaaaacatgctctccatacgccattgaccacggagtt

F V S K T C S P Y A I D H G V864 ctggttgttggttatggcgcggaaaatggtgacgcttactggtta

L Y V G Y G A E N G D A Y W L909 gtgaaaaacagctggggaagctcctggggtgaggatggatacctc

V K N S W G S S W G E D G Y L954 aaaatggcccgcaacagaaacaacatgtgcgggattgccagcatg

K M A R N R N N M C G I A S M999 1019gcaagctatccaaccgtgtaa

A S Y P T V1021 tcacctgtggagtaaataaacatcttttggtatc

lowast

Figure 1 Sequences and amino acid residues of SeCPThe SeCP sequence was of 1 053 bp length with a 1011 bp biggest OFR from 9 bp (ATG)to 1019 bp (TAA) which encoded 336-amino acid protein with 31015840UTR locating at the positions 1020ndash1053 bp

In order to separate the early specific diagnostic antiag-ens the ES proteins of S erinaceieuropaei sparganum wereanalyzed by two-dimensional electrophoresis (2DE) andWestern blot probed with early sera from infected mice at14 days after infection Three immunoreactive protein spotswere successfully identified by MALDI-TOFTOF-MS andcharacterized as the S erinaceieuropaei cysteine protease(SeCP) [13] In this paper the full-length cDNA sequence ofSeCP (GenBank accession no 1834307)was analyzed its stru-cture and function were predicted by using bioinformaticstechniques

2 Materials and Methods

The full-length cDNA sequence of SeCP (GenBank acce-ssion no 1834307) was used in this study The struct-ure domain and function domain were predicted by onlineanalysis httpsmartembl-heidelbergde The amino acidsequence was submitted to httpwwwexpasyorgtoolsprotparamhtml and its physical and chemical propertieswere predicted Signal peptide was predicted by a web-basedtool (httpwwwcbsdtudkservicesSignalP) and subcell-ular localization was predicted using httppsortnibbacjpform2html Hydrophilic prediction was predicted athttpwwwexpasyorgcgi-binprotscalepl Transmembranedomain was predicted through httpwwwcbsdtudkser-vicesTMHMM-20 The secondary structures were constr-ucted using the software PSIPRED v30 httpbioinfcsuclacukpsipred [14 15] The 3D models of proteins wereconstructed by I-TASSER a protein structure server on the

website httpzhanglabccmbmedumicheduI-TASSERwhich is considered to predict protein 3D structures thathave more than 100 amino acids [16ndash18] Visual molecu-lar dynamics (VMD) was used to read standard ProteinData Bank (PDB) files and display the contained struct-ure [19ndash21] VMD is a molecular visualization software fordisplaying animating and analyzing large biomolecularsystems using 3D graphics and built-in scripts httpwwwksuiuceduResearchvmd Amino acid sequence wassubmitted to httpwwwcbsdtudkservicesBepiPred inorder to predict its antigen epitopes Conserved HLA-res-tricted CD8+ T cells epitopes were also predicted using thesoftware from IEDB httpwwwimmuneepitopeorg whichcould identify novel HLA-class I restricted CD8+T cell epi-topes Other cysteine protease amino sequences of modelorganisms of other parasites used in this study were obtainedfrom GenBank (httpwwwncbinlmnihgovGenbankindexhtml) and listed as follows Clonorchis sinensis (AAD-291301) Homo sapiens (CAB428831) Spirometra manso-noides (AAB170511) Taenia solium (BAH033951) Parago-nimus westermani (AAF214572) Schistosoma japonicum(CAX7157810) Schistosoma mansoni (P257921) Taenia pisi-formis (AEE690341) Haemonchus contortus (ACS360901)Entamoeba histolytica (CAA628361) Trichinella spiralis(XP 0033772401) Plasmodium vivax (AAA603681) Brugiamalayi (XP 0018968231) Echinococcus multilocularis (BAF-025161) Arabidopsis thaliana (AAB676261) Mus musculus(AAA374451)Drosophilamelanogaster (AAB183451)Caen-orhabditis elegans (AAA987851) Haemaphysalis longicornis(BAH860621) andAedes aegypti (ABE729701)Themultiple

BioMed Research International 3

SMART domain pept_C1 (Probably catalytically inactive)E value 539eminus114Start 121End 335

SMART domain Inhibitor_I29 E value 223eminus17Start 32End 92

Signal peptide

InhibitorI29 Pept_C1

Figure 2 Prediction of structure domains of SeCP by SMARTservers The confidently predicted SeCP structure domains con-tained a complete cathepsin propeptide inhibitor domain (I29)located at 32aandash92aa and a peptidase C1A with an active site locatedat 39aandash303aa and a S2 subsite 189aandash330aa which has the functionof cysteine-type peptidase activity

sequence alignment of SeCP and the above-mentionedsequences were carried out by Clustal X then molecularevolutionary tree was constructed by MEGA41 [22] Phylo-genies were estimated under the neighbor-joining (N-J)method [23]

3 Results

31 The Basic Properties of SeCP Sequence The SeCP seque-nce was of 1 053 bp length with a 1011 bp biggest OFR from9 bp (ATG) to 1019 bp (TAA) which encoded 336-amino acidprotein with 31015840UTR locating at the positions 1020ndash1053 bpNucleotide sequence and deduced amino acid sequence wereshown in Figure 1

32 Physical and Chemical Properties of SeCP The SeCP hadthe molecular weight of 3787 kDa and theoretical isoelectricpoint (pI) of 647 Extinction coefficients are 74300Mminus1 cmminus1at 280 nm measured in water assuming all pairs of Cysresidues form cysteines The half-life was 30 h gt20 h andgt10 h in mammalian reticulocytes (in vitro) yeast (in vivo)and Escherichia coli (in vivo) respectively The instabilityindex (II) was computed to be 3211 This classifies theprotein as stable Aliphatic index is 7574 Grand average ofhydropathicity (GRAVY) is minus0321

33 Structural Domain Hydrophobicity Signal Peptide Sub-cellular Localization and Transmembrane Domain The con-fidently predicted SeCP structure domains contained a com-plete cathepsin propeptide inhibitor domain (I29) located at32aandash92aa and a peptidase C1A with an active site locatedat 39aandash303aa and a S2 subsite 189aandash330aa which has thefunction of cysteine-type peptidase activity (Figure 2) Usingthe scale HphobKyte and Doolittle the SeCP protein has anobvious hydrophobic regions at 51015840 (Figure 3)

The prediction results of SeCP signal by Signal P-41showed that there was a peak fraction at 19aa residue position(Figure 4) The score was 387 which was high enough withsplit site So the SeCP protein had a cleavable signal peptide(from 1 to 19) andwith possible cleavage site between 19aa and20aa

minus4

minus3

minus2

minus1

0

1

2

3

50 100 150 200 250 300

Scor

e

Position

Protscale output for user sequence

HphobKyte and Doolittle

Figure 3 Hydrophobicity of SeCPThe SeCP protein has an obvioushydrophobic regions at 51015840 predicted by using the scale HphobKyteand Doolittle

00

02

04

06

08

10

0 10 20 30 40 50 60 70

Scor

e

Position

Signal P-41 prediction (euk networks) 1

C-score S-score Y-score

MKFVIYVAFLFLLLTVCRGSTESETYVRRELWKAWKLAFKKEYFSSEEELHRKRAFFNNLDFIIRHNQRY

Figure 4 Prediction of SeCP signal peptide There was a peakfraction at 19aa residue position and the score was 387 which washigh enough with split site The SeCP protein had a cleavable signalpeptide (1 to 19) with possible cleavage site between 19aa and 20aa

Results of the k-NN prediction of SeCP suggested thatthe peptide chain was located in the extracellular (includingcell wall) vacuolar mitochondrial and endoplasmic reticu-lum with the possibility of 556 222 111 and 111respectively The maximum possible location was in theextracellular (119896 = 23)

Prediction of transmembrane domain of SeCP withTMHMMServer v 20 suggested that the SeCP had no trans-membrane domain located outside the membrane

34 2D Structure Alignment for SeCP PSIPRED v 33 wasused to predict the secondary structures of SeCP which had8 120572-helixes 7 120573-strands and 20 coils (Figure 5)


Conf

Pred

CCEEEEEEECCCCCCCEEEEEECCCCCCCCCCCEEEEEECPred

IDHGVLVVGYGAENGDAYWLVKNSWGSSWGEDGYLKMARNAA290 300 310 320

Conf

Pred

Pred

AA

CCCCCCCCCCCCCCCC

RNNMCGIASMASYPTV

330

HelixStrand Coil

Conf = confidence of predictionminus +

Pred predicted secondary structureAA target sequence

ConfPred

CHHHHHHHHHHHHHHHHHHCCCCCCHHHHHHHHHHHHHCCPredMKFVIYVAFLFLLLTVCRGSTESETYVRRELWKAWKLAFKAA

10 20 30 40

ConfPred

CCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCEEECPredKEYFSSEEELHRKRAFFNNLDFIIRHNQRYYQQLESYAVRAA

50 60 70 80

ConfPredPredAA

CCCCCCCCHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCC

LNDFSDLTPGEFAERYLCLRGIVLTKLRRKEAVSVPLKEN

90 100 110 120

ConfPredPredAA

CCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHH

LPDSVNWRERGAVTSVKNQGQCGSCWSFSANGAIEGAIQI

130 140 150 160

ConfPredPredAA

HHCCCCCCCCHHHHCCCCCCCCCCCCCCCHHHHHHHHHHC

KTGALRSLSEQQLMDCSWDYGNQGCNGGLMPQAFQYAQRY

170 180 190 200

ConfPredPredAA

CCCCCCCCCCCCCCCCCCCCCCCCCEEECEEEECCCCCHH

GVEAEVDYRYTERDGVCRYRQDLVVANVTGYAELPEGDEG

210 220 230 240

ConfPredPredAA

HHHHHHCCCCCCEEEEECCCCCCCCCCCCCCCCCCCCCCC

GLQRAVATIGPISVGIDAADPGFMSYSHGVFVSKTCSPYA

250 260 270 280

Figure 5 The predicted secondary structure of SeCP by using PSIPRED There were 8 120572-helixes 7 120573-strands and 20 coils of the predictedsecondary structure of SeCP

35 Construction of 3DModel and Enzyme Activity PredictingFive models were set up for each protein by Dr Zhangrsquoslab [16] We selected the model with the highest confidenceC-score (Figure 6) which estimates the quality of predictedmodels by I-TASSER It was calculated based on the sig-nificance of threading template alignments and the conver-gence parameters of the structure assembly simulations C-score is typically in the range of [minus5 2] and a model witha C-score above 2 suggested a high confidence Enzymehomologs in PDB predicted by I-TASSER showed that themost reliable enzyme classification (EC) number predictionwas 342243 with the highest CscoreEC (0664) CscoreEC isthe confidence score for the EC number prediction CscoreECvalues range in between [0-1] where a higher score indicatesa more reliable EC number prediction The recombinant

enzyme hydrolyzes proteins (serum albumin collagen) andCA synthetic substrates (Z-Phe-Arg-NHMec gt Z-Leu-Arg-NHMec gt Z-Val-Arg-NHMec) The maximum activity ofthe enzyme was at pH 57 and was unstable at neutralpH Compound E-64 leupeptin and chicken cystatin areinhibitors of cysteine protease which belongs to peptidasefamily C1 (httpenzymeexpasyorgEC342243)

36 Antigenic Epitope of SeCP The sequence of SeCP wascomparedwith the hostrsquos homologous sequences correspond-ing regional sequence by BepiPred 10b Server The SeCPhad 15 potential antigen epitopes (19aandash26aa 44aandash50aa86aandash92aa 118aandash128aa 130aandash143aa 151aandash155aa 179aandash189aa 199aandash205aa 207aandash214aa 231aandash243aa 254aandash264aa276aandash280aa 291aandash296aa 305aandash312aa and 332aandash336aa)


Table 1 The predicted HLA restricted CD8+ T cell epitopes for SeCP

Allele Start End Peptide Method Percentile rankHLA-Alowast0201 3 16 FVIYVAFLFLLLTV Ann 03HLA-Alowast0201 263 272 FMSYSHGVFV Consensus (annsmm) 04HLA-Alowast0201 194 206 FQYAQRYGVEAEV Ann 04HLA-Alowast0201 194 202 FQYAQRYGV Consensus (annsmmcomblib sidney 2008) 05HLA-Alowast0201 188 197 GLMPQAFQYA Consensus (annsmm) 055HLA-Alowast0201 6 16 YVAFLFLLLTV Consensus (annsmm) 065HLA-Alowast0201 3 14 FVIYVAFLFLLL Ann 08HLA-Alowast0201 9 16 FLFLLLTV Consensus (annsmm) 09HLA-Alowast1101 124 137 SVNWRERGAVTSVK Ann 03HLA-Alowast1101 33 40 KAWKLAFK Consensus (annsmm) 045HLA-Alowast1101 307 316 SSWGEDGYLK Consensus (annsmm) 05HLA-Alowast1101 262 274 GFMSYSHGVFVSK Ann 06HLA-Alowast1101 33 41 KAWKLAFKK Consensus (annsmm) 06HLA-Alowast1101 266 274 YSHGVFVSK Consensus (annsmm) 06HLA-Alowast1101 264 274 MSYSHGVFVSK Consensus (annsmm) 07HLA-Blowast0702 277 286 SPYAIDHGVL Consensus (annsmm) 035HLA-Blowast0702 317 330 MARNRNNMCGIASM Ann 05HLA-Blowast0702 277 288 SPYAIDHGVLVV Ann 06HLA-Blowast0702 106 117 KLRRKEAVSVPL Ann 07

Figure 6 The 3D model of SeCP with highest confidence C-scorewhich estimates the quality of predicted models by I-TASSER

Epitope prediction algorithm consensus was used to predictpeptides that could stimulate human to induce effectiveand protective immune response against S erinaceieuropaeiwhen the conserved HLA-restricted CD8+ T cells epitopesof SeCP were predictedThe SeCP had 19 conserved peptidesbased on a high HLA allele binding score (percentile rank lt1) (Table 1)

37 Multiple Sequence Alignment and Molecular Evolution ofSeCP Multiple sequence alignment and phylogenetic ana-lysis of SeCP with the cysteine protease of other specieswere displayed in Figure 7 Based on the phylogenetic ana-lysis of SeCP Spirometra erinaceieuropaei has the closestevolutionary status with Spirometra mansonoides

Taenia pisiformis Taenia solium Spirometra erinaceieuropaei

Spirometra mansonoides Mus musculus

Haemaphysalis longicornis Drosophila melanogaster Aedes aegypti

Echinococcus multilocularis Arabidopsis thaliana

Homo sapiens Clonorchis sinensis

Paragonimus westermani Brugia malayi

Entamoeba histolytica Plasmodium vivax

Trichinella spiralis Schistosoma japonicum Schistosoma mansoni

Haemonchus contortus Caenorhabditis elegans

100

6498

100

100

97100

100

10098

100

6634

28

25

53

43

54

02

Figure 7Neighbor-joining phylogenetic tree referred from cysteineprotease amino acid sequence of Spirometra erinacei Bootstrapvalues are indicated on branches

4 Discussion

Cysteine protease is a kind of proteolytic enzyme whichcontains cysteine residues in the center of enzyme activityIt has been shown that the cysteine protease of manyparasites acts extracellularly to help invade tissues and cellsto uptake nutrient to hatch or to evade the host immunesystem [24ndash26] Cysteine protease is the key factor in theparasitic pathogenicity either by inducing tissue damage and


facilitating invasion or by empowering the parasites to salvagemetabolites from host proteins [27 28] Cysteine proteasehas been detected in S erinacei [29 30] The plerocercoidsof S erinacei is also known to secrete a large amount ofcysteine proteases [31] The cysteine protease from S erinaceican hydrolyze collagen hemoglobin and immunoglobulinG (IgG) in vitro and may be concerned with digestion ofhost tissue in pathogenesis [32 33] Our previous study on2DE analysis showed that the ES proteins of S erinaceiplerocercoids had a total of approximately 149 proteins spotswith molecular weight varying from 20 to 115 kDa andisoelectric point (pI) from 3 to 75 When probed with serafrom infected mice at 14 days after infection seven proteinspots with molecular weight of 23ndash31 kDa were recognizedand analyzed byMALDI-TOFTOF-MSThree of seven spotswere successfully identified and characterized as the sameprotein SeCP [13] The SeCP might come from the excretoryand secretory products and the cuticles (membrane proteins)and are directly exposed to the hostrsquos immune system andare the main target antiagens which induce the immuneresponses

Based on the construction of full-length cDNA libraryof SeCP the sequence of SeCP gene was of 1 053 bp lengthwith a 1011 bp biggest ORF encoding 336-amino acid proteinwith a complete cathepsin propeptide inhibitor domain anda peptidase C1A conserved domainThe predictedmolecularweight and isoelectric point of the deduced SeCP proteinwere 3787Da and 647 respectively Based on the phyloge-netic analysis of SeCP Spirometra erinaceieuropaei has theclosest evolutionary status with Spirometra mansonoides Thesecondary structure of SeCP contained has 8 120572-helixes 7 120573-strands and 20 coils The SeCP had 15 potential antigenicepitopes and 19 HLA-I restricted epitopes These predictedantigenic epitopes could provide important insights on thediagnostic antiagens and target molecular of antiparasiticdrugs for sparganosis

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (no 81172612) and the ZhengzhouUniversity Scientific Research Grant for the Postgraduates(no 12Y03001)

References

[1] B J Bogitsch C E Carter and T N Oeltmann Human Par-asitology Academic Press New York NY USA 3rd edition2005

[2] L S Roberts G D Schmidt and J Janovy Foundations ofParasitology McGraw-Hill New York NY USA 8th edition2009

[3] S Nithiuthai M T Anantaphruti JWaikagul and A GajadharldquoWaterborne zoonotic helminthiasesrdquo Veterinary Parasitologyvol 126 no 1-2 pp 167ndash193 2004

[4] E H Shin S M Guk H J Kim S H Lee and J Y ChaildquoTrends in parasitic diseases in the Republic of Koreardquo Trendsin Parasitology vol 24 no 3 pp 143ndash150 2008

[5] T Fukushima and Y Yamane ldquoHow does the sparganosisoccurrdquo Parasitology Today vol 15 no 3 p 124 1999

[6] S Magnino P Colin E Dei-Cas et al ldquoBiological risks asso-ciated with consumption of reptile productsrdquo InternationalJournal of Food Microbiology vol 134 no 3 pp 163ndash175 2009

[7] M T Anantaphruti Y Nawa and Y Vanvanitchai ldquoHumansparganosis inThailand an overviewrdquoActa Tropica vol 118 no3 pp 171ndash176 2011

[8] K Shirakawa H Yamasaki A Ito and H Miyajima ldquoCerebralsparganosis the wandering lesionrdquo Neurology vol 74 no 2 p180 2010

[9] J Cui X M Lin H W Zhang B L Xu and Z Q Wang ldquoSpa-rganosis Henan province central Chinardquo Emerging InfectiousDiseases vol 17 no 1 pp 146ndash147 2011

[10] K Murata T Abe M Gohda et al ldquoDifficulty in diagnosing acase with apparent sequel cerebral sparganosisrdquo Surgical Neu-rology vol 67 no 4 pp 409ndash411 2007

[11] T Nishiyama T Ide S R Himes Jr S Ishizaka and TAraki ldquoImmunodiagnosis of human sparganosis mansoniby micro-chemiluminescence enzyme-linked immunosorbentassayrdquo Transactions of the Royal Society of Tropical Medicine andHygiene vol 88 no 6 pp 663ndash665 1994

[12] J Cui N Li Z QWang P Jiang and X M Lin ldquoSerodiagnosisof experimental sparganum infections of mice and humansparganosis by ELISA using ES antigens of Spirometra mansonisparganardquo Parasitology Research vol 108 no 6 pp 1551ndash15562011

[13] D D Hu J Cui L Wang L N Liu T Wei and Z Q WangldquoImmunoproteomic analysis of the excretory-secretory pro-teins from Spirometra mansoni Sparganumrdquo Iranian Journal ofParasitology vol 8 no 3 pp 408ndash416 2013

[14] M I Sadowski and D T Jones ldquoThe sequence-structure rela-tionship and protein function predictionrdquo Current Opinion inStructural Biology vol 19 no 3 pp 357ndash362 2009

[15] D W Buchan S M Ward A E Lobley T C O Nugent KBryson and D T Jones ldquoProtein annotation and modellingservers at University College Londonrdquo Nucleic Acids Researchvol 38 pp W563ndashW568 2010

[16] Y Zhang ldquoI-TASSER server for protein 3D structure predic-tionrdquo BMC Bioinformatics vol 9 article 40 2008

[17] A Roy A Kucukural and Y Zhang ldquoI-TASSER a unified plat-form for automated protein structure and function predictionrdquoNature Protocols vol 5 no 4 pp 725ndash738 2010

[18] A Roy J Yang and Y Zhang ldquoCOFACTOR an accurate com-parative algorithm for structure-based protein function anno-tationrdquo Nucleic Acids Research vol 40 pp W471ndashW477 2012

[19] W Humphrey A Dalke and K Schulten ldquoVMD visualmolecular dynamicsrdquo Journal of Molecular Graphics vol 14 no1 pp 33ndash38 1996

[20] T Schwede J Kopp N Guex and M C Peitsch ldquoSWISS-MODEL an automated protein homology-modeling serverrdquoNucleic Acids Research vol 31 no 13 pp 3381ndash3385 2003

[21] N Guex M C Peitsch and T Schwede ldquoAutomated compara-tive protein structure modeling with SWISS-MODEL andSwiss-PdbViewer a historical perspectiverdquo Electrophoresis vol30 supplement 1 pp S162ndashS173 2009

[22] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 molec-ular evolutionary genetics analysis (MEGA) software version40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007


[23] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[24] F Salas J Fichmann G K Lee M D Scott and P JRosenthal ldquoFunctional expression of falcipain a Plasmodiumfalciparum cysteine proteinase supports its role as a malarialhemoglobinaserdquo Infection and Immunity vol 63 no 6 pp2120ndash2125 1995

[25] M M Wasilewski K C Lim J Phillips and J H McKerrowldquoCysteine protease inhibitors block schistosome hemoglobindegradation in vitro and decrease worm burden and eggproduction in vivordquo Molecular and Biochemical Parasitologyvol 81 no 2 pp 179ndash189 1996

[26] J E Olson G K Lee A Semenov and P J Rosenthal ldquoAnti-malarial effects in mice of orally administered peptidyl cysteineprotease inhibitorsrdquo Bioorganic andMedicinal Chemistry vol 7no 4 pp 633ndash638 1999

[27] Y B Chung Y Kong I J Joo S Y Cho and S Y Kang ldquoExcyst-ment of Paragonimus westermanimetacercariae by endogenouscysteine proteaserdquo Journal of Parasitology vol 81 no 2 pp 137ndash142 1995

[28] J Tort P J BrindleyDKnox KHWolfe and J PDalton ldquoPro-teinases and associated genes of parasitic helminthsrdquo Advancesin Parasitology vol 43 pp 161ndash266 1999

[29] T M Fukase Y Akihama and H Itagaki ldquoPurification andsome properties of cysteine protease of Spirometra erinaceiplerocercoid (Cestoda Diphyllobothriidae)rdquo Japanese Journalof Parasitology vol 35 no 5 pp 351ndash360 1985

[30] C Y Song D H Choi T S Kim and S H Lee ldquoIsolationand partial characterization of cysteine proteinase from spar-ganumrdquo Kisaengchunghak Chapchi vol 30 no 3 pp 191ndash1991992

[31] Y B Chung and H J Yang ldquoPartial purification and character-ization of a cysteine protease Inhibitor from the plerocercoid ofSpirometra erinaceirdquo Korean Journal of Parasitology vol 46 no3 pp 183ndash186 2008

[32] C Y Song and C L Chappell ldquoPurification and partial char-acterization of cysteine proteinase from Spirometra mansoniplerocercoidsrdquo Journal of Parasitology vol 79 no 4 pp 517ndash524 1993

[33] Y Kong Y B Chung S Y Cho and S Y Kang ldquoCleavageof immunoglobulin G by excretory-secretory cathepsin S-likeprotease of Spirometra mansoni plerocercoidrdquo Parasitology vol109 part 5 pp 611ndash621 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

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Zoology


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GenomicsInternational Journal of


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BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


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Stem CellsInternational



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Microbiology


1 agaatagg

9 atgaagttcgtaatatacgttgccttcttgttccttcttttgacg

M K F V I Y V A F L F L L L T54 gtctgcagaggctcgactgaaagtgagacgtacgtccggcgggaa

V C R G S T E S E T Y V R R E99 ttgtggaaggcctggaaattggccttcaagaaggagtacttcagt

L W K A W K L A F K K E Y F S

144 agtgaagaagaactccaccgaaagcgtgcattctttaacaatctc

S E E E L H R K R A F F N N L189 gacttcatcatccgacataatcaacgctattatcaacagctcgag

D F I I R H N Q R Y Y Q Q L E234 tcctatgcagtgcgattaaatgattttagtgacctgacgccgggt

S Y A V R L N D F S D L T P G279 gaatttgccgaaagatacctttgcttacggggaattgttttgacg

E F A E R Y L C L R G I V L T324 aagttaagacggaaggaagcagtaagcgtgccactcaaagaaaat

K L R R K E A V S V P L K E N369 cttcccgacagcgtaaactggcgcgagagaggtgccgttacatcg

L P D S V N W R E R G A V T S414 gtcaaaaatcagggtcaatgcggatcctgctggtctttttccgca

V K N Q G Q C G S C W S F S A459 aacggtgcaatagaaggcgcaatccagataaagaccggtgcattg

N G A I E G A I Q I K T G A L504 cgcagcctgtcagaacagcagttgatggactgcagctgggactac

R S L S E Q Q L M D C S W D Y

549 ggcaatcaaggctgcaacgggggactcatgccacaggccttccag

G N Q G C N G G L M P Q A F Q594 tatgcccaaaggtatggcgtcgaagctgaagttgactacagatat

Y A Q R Y G V E A E V D Y R Y639 actgaaagggatggggtttgcagatatcgtcaggacctggttgtt

T E R D G V C R Y R Q D L V V684 gccaatgttactggatatgcggaactgccagaaggcgatgaggga

A N V T G Y A E L P E G D E G729 ggtctacaaagggctgttgcaaccataggcccaatatctgtcgga

G L Q R A V A T I G P I S V G774 atcgatgctgccgatcctgggtttatgtcttacagccacggtgtt

I D A A D P G F M S Y S H G V819 ttcgtcagtaaaacatgctctccatacgccattgaccacggagtt

F V S K T C S P Y A I D H G V864 ctggttgttggttatggcgcggaaaatggtgacgcttactggtta

L Y V G Y G A E N G D A Y W L909 gtgaaaaacagctggggaagctcctggggtgaggatggatacctc

V K N S W G S S W G E D G Y L954 aaaatggcccgcaacagaaacaacatgtgcgggattgccagcatg

K M A R N R N N M C G I A S M999 1019gcaagctatccaaccgtgtaa

A S Y P T V1021 tcacctgtggagtaaataaacatcttttggtatc

lowast

Figure 1 Sequences and amino acid residues of SeCPThe SeCP sequence was of 1 053 bp length with a 1011 bp biggest OFR from 9 bp (ATG)to 1019 bp (TAA) which encoded 336-amino acid protein with 31015840UTR locating at the positions 1020ndash1053 bp

In order to separate the early specific diagnostic antiag-ens the ES proteins of S erinaceieuropaei sparganum wereanalyzed by two-dimensional electrophoresis (2DE) andWestern blot probed with early sera from infected mice at14 days after infection Three immunoreactive protein spotswere successfully identified by MALDI-TOFTOF-MS andcharacterized as the S erinaceieuropaei cysteine protease(SeCP) [13] In this paper the full-length cDNA sequence ofSeCP (GenBank accession no 1834307)was analyzed its stru-cture and function were predicted by using bioinformaticstechniques

2 Materials and Methods

The full-length cDNA sequence of SeCP (GenBank acce-ssion no 1834307) was used in this study The struct-ure domain and function domain were predicted by onlineanalysis httpsmartembl-heidelbergde The amino acidsequence was submitted to httpwwwexpasyorgtoolsprotparamhtml and its physical and chemical propertieswere predicted Signal peptide was predicted by a web-basedtool (httpwwwcbsdtudkservicesSignalP) and subcell-ular localization was predicted using httppsortnibbacjpform2html Hydrophilic prediction was predicted athttpwwwexpasyorgcgi-binprotscalepl Transmembranedomain was predicted through httpwwwcbsdtudkser-vicesTMHMM-20 The secondary structures were constr-ucted using the software PSIPRED v30 httpbioinfcsuclacukpsipred [14 15] The 3D models of proteins wereconstructed by I-TASSER a protein structure server on the

website httpzhanglabccmbmedumicheduI-TASSERwhich is considered to predict protein 3D structures thathave more than 100 amino acids [16ndash18] Visual molecu-lar dynamics (VMD) was used to read standard ProteinData Bank (PDB) files and display the contained struct-ure [19ndash21] VMD is a molecular visualization software fordisplaying animating and analyzing large biomolecularsystems using 3D graphics and built-in scripts httpwwwksuiuceduResearchvmd Amino acid sequence wassubmitted to httpwwwcbsdtudkservicesBepiPred inorder to predict its antigen epitopes Conserved HLA-res-tricted CD8+ T cells epitopes were also predicted using thesoftware from IEDB httpwwwimmuneepitopeorg whichcould identify novel HLA-class I restricted CD8+T cell epi-topes Other cysteine protease amino sequences of modelorganisms of other parasites used in this study were obtainedfrom GenBank (httpwwwncbinlmnihgovGenbankindexhtml) and listed as follows Clonorchis sinensis (AAD-291301) Homo sapiens (CAB428831) Spirometra manso-noides (AAB170511) Taenia solium (BAH033951) Parago-nimus westermani (AAF214572) Schistosoma japonicum(CAX7157810) Schistosoma mansoni (P257921) Taenia pisi-formis (AEE690341) Haemonchus contortus (ACS360901)Entamoeba histolytica (CAA628361) Trichinella spiralis(XP 0033772401) Plasmodium vivax (AAA603681) Brugiamalayi (XP 0018968231) Echinococcus multilocularis (BAF-025161) Arabidopsis thaliana (AAB676261) Mus musculus(AAA374451)Drosophilamelanogaster (AAB183451)Caen-orhabditis elegans (AAA987851) Haemaphysalis longicornis(BAH860621) andAedes aegypti (ABE729701)Themultiple




Signal peptide




3 Results





minus4

minus3

minus2

minus1

0

1

2

3

50 100 150 200 250 300

Scor

e

Position




00

02

04

06

08

10

0 10 20 30 40 50 60 70

Scor

e

Position









Conf

Pred



Conf

Pred

Pred

AA

CCCCCCCCCCCCCCCC

RNNMCGIASMASYPTV

330

HelixStrand Coil



ConfPred


10 20 30 40

ConfPred


50 60 70 80

ConfPredPredAA



90 100 110 120

ConfPredPredAA



130 140 150 160

ConfPredPredAA



170 180 190 200

ConfPredPredAA



210 220 230 240

ConfPredPredAA



250 260 270 280




















100

6498

100

100

97100

100

10098

100

6634

28

25

53

43

54

02


4 Discussion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Signal peptide




3 Results





minus4

minus3

minus2

minus1

0

1

2

3

50 100 150 200 250 300

Scor

e

Position




00

02

04

06

08

10

0 10 20 30 40 50 60 70

Scor

e

Position









Conf

Pred



Conf

Pred

Pred

AA

CCCCCCCCCCCCCCCC

RNNMCGIASMASYPTV

330

HelixStrand Coil



ConfPred


10 20 30 40

ConfPred


50 60 70 80

ConfPredPredAA



90 100 110 120

ConfPredPredAA



130 140 150 160

ConfPredPredAA



170 180 190 200

ConfPredPredAA



210 220 230 240

ConfPredPredAA



250 260 270 280




















100

6498

100

100

97100

100

10098

100

6634

28

25

53

43

54

02


4 Discussion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Conf

Pred



Conf

Pred

Pred

AA

CCCCCCCCCCCCCCCC

RNNMCGIASMASYPTV

330

HelixStrand Coil



ConfPred


10 20 30 40

ConfPred


50 60 70 80

ConfPredPredAA



90 100 110 120

ConfPredPredAA



130 140 150 160

ConfPredPredAA



170 180 190 200

ConfPredPredAA



210 220 230 240

ConfPredPredAA



250 260 270 280




















100

6498

100

100

97100

100

10098

100

6634

28

25

53

43

54

02


4 Discussion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















100

6498

100

100

97100

100

10098

100

6634

28

25

53

43

54

02


4 Discussion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

analysis of structures, functions, and epitopes of cysteine protease

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