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articleinfoabstract346837Articlehistory:Inthisstudywehaveinvestigatedtheanatomicsitesofexpressionanddevelopmentalexpressionpat-38Received11December201339Receivedinrevisedform28April2014ternsoftwocathepsinB-likecysteineproteases(AC-cathB-1,-2)ofAngiostrongyluscantonensis.The40Accepted3June2014immunolocalizationresultsrevealedthatnativeAC-cathBswerefoundpresentintheL1andL3larvae,41Availableonlinexxxxfemaleandmaleadults,andtheAC-cathBswerelocalizedmainlyonthedigestivetractofA.cantonensisandexpressedatvariedlevelsandindifferentpatternsintheinternaltissuesaccordingtotheirdevelop-42Keywords:mentalstage.ConsistentwiththeinfectivestageofL3isamuchmoreintensestainingofAC-cathBsinthe43Angiostrongyluscantonensisesophaguscomparedwiththeintestine.IncontrasttoL3,moreabundantsignalswerelocatedtothe44CathepsinBcysteineproteaseintestineofadults,suggestingthatnutritiondigestionlikelytobethemainfunctionoftheproteaseat45Developmentalexpressionpatternthispoint.AC-cathBsfluorescentsignalswerepresentinexcretorypore,excretorytubeinlateralcords,4647Immunolocalizationandmuscularesophagusoflarvae,furthersupportedtheAC-cathB-1,-2likelytobereleasedbyA.can-tonensisasexcretory/secretoryproducts.Additionally,onlytheproteinAC-cathB-2wasdetectedinthereproductivesystem,especiallyinthewallofvasdeferens,uterus,andoviductoftheparasites,whethertheAC-cathB-2hassomefunctioningermcellsdevelopmentandmaturationneedtobefurthercharac-terized.Althoughtheanatomicsitesandexpressionpatternsweredifferentinlarvaeandadultsandthecorrespondingfunctionmightnotthesame,AC-cathB-1and-2involvedinthehost-parasiteinteractioninadditiontodigestivefunction.Ó2014PublishedbyElsevierInc.⇑Correspondingauthorat:SchoolofLifeSciences,XiamenUniversity,Xiamen,Fujian361005,China.E-mailaddress:dmluo@xmu.edu.cn(D.Luo).http://dx.doi.org/10.1016/j.exppara.2014.06.0080014-4894/Ó2014PublishedbyElsevierInc.Pleasecitethisarticleinpressas:Yu,C.,etal.ImmunolocalizationanddevelopmentalexpressionpatternsoftwocathepsinBproteases(AC-cathB-1,-2)ofAngiostrongyluscantonensis.Exp.Parasitol.(2014),http://dx.doi.org/10.1016/j.exppara.2014.06.0084950515253545556575859606162636465666768
YEXPR689214June20142697071727374757677787980C.Yuetal./ExperimentalParasitologyxxx(2014)xxx–xxxNo.ofPages7,Model5G1.IntroductionCysteineproteaseshavebeenshowntobeoneofthemostabundantlyexpressedproteasefamiliesinthegastrointestinaltractsofparasitichelminths.Theseproteasesareoftendevelop-mentallyregulatedthroughoutthecomplexparasiticlifecycles,arefrequentlyup-regulatedinactivelyfeedingstagesandhavebeenshowntobecommonlyexpressedinorgansororganellesinvolvedinfeeding(Jasmeretal.,2004).Haematophagousnema-todesexpressproteasesofdifferentmechanisticclassesintheirintestines,manyofwhichhaveprovenorputativerolesindegra-dationofhaemoglobinandotherproteinsinvolvedinnutrition,however,mostoftheproteasesdescribedpreviouslyareexpressedmanufacturer’sprotocols;RNAintegritywasverifiedbyelectro-phoresison1%formaldehyde-agarosegels.OnemicrogramofRNAwasthenconvertedtothefirststrandcDNAinareactionvol-umeof20lLusingthe1stStrandcDNASynthesisKit(Takara,Dalian,China)witholigo-d(T)primerfollowingtheinstructiondescribedbythemanufacturer.2.3.ConstructionofexpressionplasmidofcathepsinBprotease(AC-cathB-1,-2)cDNAThefirststrandcDNAderivedfromadultA.cantonensiswasservedasatemplatetoamplifyacDNAofcathepsinBproteinase(AC-cathB-1,-2)genebyRT-PCR.PCRexperimentwasperformed12812913013113213313413513613713881intheintestineandmanyofthemdonotappearinexcretory/inafinalreactionvolumeof50lLthatconsistedof2lLofthe82secretory(ES)products,implyingthattheyactlocallyintheintes-productofreversetranscription,10ÂExTaqBuffer5lL(TaKaRa,83tine(Williamsonetal.,2003).TheESproductsandthecharacter-Dalian,China),8lLdNTPsmixture(2.5mMeach),0.5lLTaKaRa84izationofcathepsinBproteaseindifferentstagesofdevelopmentExTaqpolymerase(5U/lL),6llMgCl2(25mM),Primerpairs85inAngiostrongyluscantonensishasbeendescribedusingSignal-PMEcathB-1FandMEcathB-1R(4lLeach),andautoclavedddH2O.86(Fangetal.,2010),RT-PCR,andwesternblotanalyses(Nietal.,Amplificationwasperformedunderthefollowingconditions:872012),however,therearenodatathatdirectlysupportthecathep-pre-denaturingat95°Cfor5min;38cyclesof95°Cfor30s,88sinBprotease(AC-cathB-1,-2)werereleasedbytheparasiteasES60°Cfor30sand72°Cfor1min;andafinalextensionat72°C89productsandtheanatomiclocationoftheproteasesinA.cantonen-for7min.ThesamesystemwerecarriedoutinthePCRreaction90sisremainsunclear.HerewelookattheanatomicallocalizationofAC-cathB-2asadoptedforAC-cathB-1,inadditiontothedifferent91andexpressioncharacterizationoftwocysteineproteases(AC-primers(MEcathB-2FandMEcathB-2R)andtheannealingtemper-92cathB-1,-2)inordertoassistusinunderstandingbettertheroleatures(56°C).PCRproductswerecheckedon1.0%(w/v)agarose93ofcathepsinBintheA.cantonensis.gelsandvisualizedusingethidiumbromideandUVilluminationtoconfirmproductsize.942.MaterialsandmethodsThePCRproductswerepurifiedusingEZ-10SpinColumnDNAGelExtractionKit(Sangon,Shanghai,China).ThepurifiedDNAwas952.1.NematodeandratmaterialssubclonedintoapMD18-Tvector(TaKaRa,China).AfterPCRscreeningtheinsertsandplasmidsdigestion,positiveplasmids96ThelifecycleofA.cantonensiswasmaintainedinthelaboratoryweresenttoInvitrogen(Invitrogen,Shanghai,China)for97byroutinepassagethroughmiceandtheintermediatesnailhostsequencing.98Pomaceacanaliculata.TheA.cantonensismaintainedinSprague–ForconstructionoftheexpressionvectorofcathepsinBprote-99Dawley(SD)ratsattheParasitologicalResearchLaboratoryofXia-asepET32a-Ac-CathB-1andpET32a-Ac-CathB-2,therightplasmid100menUniversitywasused.AdultA.cantonensiswerecollectedfromwasdigestedbyKpnIandSalIandclonedintotheexpressionvec-101thepulmonaryarteryofratsaftermercykilling.Thefirststagelar-torofpET-32a(Invitrogen,Shanghai,China)betweenthesetwo102vae(L1)ofA.cantonensiswerecollectedfromfaecesbyusinga500siteswithT4DNAligase(TransGenBiotech,Beijing,China),then103meshsieveat45daftertheratinfection.Freshpositiveratfeces,transformedintocompetentEscherichiacoliBL21.Theidentityof104addingwaterintoapaste,wereappliedinthesurfaceoflettuce,theindividualcolonieswereconfirmedbyPCRscreeningthe105theapplesnailswerethenfedonadietoflettuceandinfectedcon-insertsusingsameprimersasemployedfortheiramplification,106sequently.After3weeksofbeinginfected,thesnailswerecutintoandrestrictionenzymesdigestionaswellastheplasmids107smallpiecesanddigestedovernightindigestivefluid(0.7%pepsinsequencing.108in0.5%HCl).Thirdstagelarvae(L3)werecollectedunderadissect-109ingmicroscope.Thenematodesofeachstagewerethenpreserved2.4.Expressionofrecombinantproteinfragmentsandpolyclonal110inaRNAstoresolutionandkeptunderÀ80°Cuntiluse.SDratsantiseraproduction111andimmunizedmouseweremaintainedintheLaboratoryAnimal112Center,XiamenUniversityandthestudywasconductedadheringThetransformantE.coliBL21clonesweregrownat37°Ctoan113totheguidelinesforanimalhusbandryandalsoapprovedbyEthicOD600valueof0.7inLB/ampicillin(100lg/mL),inducedwith114CommitteefromXiamenUniversity.1mMisopropylthio-b-D-galactoside(IPTG)andincubatedfor5h.TheinducedcellsweredisruptedbysonicationandrecombinantproteinwaspurifiedusingHisTrap™HPColumns(GEHealthcare,1152.2.RNAextractionandreversetranscriptionShanghai,China)accordingtothemanufacturer’sprotocol.Threemouseeachwasinjectedintramuscularlywith60lgof116Twoprimerpairsweresynthesized(Sangon,Shanghai,China)recombinantproteinmixedwithanequalvolumeofFreud’scom-117accordingtoGenBankaccessionNo.forcathepsinB-likecysteinepleteadjuvant,followedbythreeadditionalboostinjectionsof118proteasegenes1,2(AC-cathB-1,AC-cathB-2)ofA.cantonensis(Ni60lgofrecombinantproteininthesameFreud’sincompleteadju-119etal.,2012).ForwardprimerMEcathB-1F(50-CGGGGTACCTCAGvantat1weekintervals.Bloodwastakenfromthemouseafterthe120AAGACAACGACAAT-30)andreverseprimer(MEcathB-1R50-ACGClastboost10dbeforetheimmunization,thepolyclonalmouse121GTCGACACAGACAATGAAGTGGAA-30)forAC-cathB-1(HQ110099);anti-A.cantonensisantiserawerestoredatÀ20°Cuntilused.122ForwardprimerMEcathB-2F(50-CGGGGTACCGTCTCGGCAGCATCT123TGGC-30)andreverseprimer(MEcathB-2R50-ACGCGTCGACATTT2.5.Westernblotanalysis124CGGTTCTCCGGCAA-30)forAC-cathB-2(HQ110100)withrestriction125enzymeofKpnIandSalI(underlined),respectively.ForWesternblots,5lLofrecombinantpolypeptideweresepa-126TotalRNAwereextractedfromadultA.cantonensisusingratedthrough12%SDS–PAGE,andelectro-transferredtonitrocel-127RNApreppureTissueKit(Tiangen,China)accordingtothelulosemembraneforwesternblottingaccordingtostandardPleasecitethisarticleinpressas:Yu,C.,etal.ImmunolocalizationanddevelopmentalexpressionpatternsoftwocathepsinBproteases(AC-cathB-1,-2)ofAngiostrongyluscantonensis.Exp.Parasitol.(2014),http://dx.doi.org/10.1016/j.exppara.2014.06.008139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180181182183184185186187YEXPR689214June2014C.Yuetal./ExperimentalParasitologyxxx(2014)xxx–xxx188189190191192193194195196197No.ofPages7,Model5G3230231232233234235236237238239procedures.WesternblotdetectionwasperformedusingaMini-ProteanIIapparatus(Bio-Rad,China).Themembraneswereblockedandincubatedwithprimaryantiseraat1:1000dilutionsovernightat4°C.Themembraneswerewashed3times(10mineach)inTBSTbufferandincubatedwithgoatanti-mouseIgG(H+L)conjugatedtohorseradishperoxidase(HRP)(Boster,Wuhan,China)at1:2000dilutionfor1hatroomtemperature.DetectionwaswithClarity™WesternECLSubstratereagent(Bio-Rad,China).Blotsprobedwithpre-immunizationmouseseraservedasnegativecontrols.2.6.ImmunolocalizationofcathepsinBprotease(AC-cathB-1,-2)inA.cantonensis3.Results3.1.CloningofthecDNAencodingcathepsinBproteasegenes(AC-cathB-1,-2)ThecDNAfragmentofcathepsinBprotease(AC-cathB-1,-2)wasamplifiedbyRT-PCRfromadultworm(Fig.1A,C),andclonedintothepET-32avector(Fig.1B,D),thensequenced.BLASTsearchesconfirmedthatthecDNAwaspartialsequenceofcathep-sinBprotease(AC-cathB-1,-2)geneofA.cantonensis.3.2.ExpressionandpurificationofthecathepsinBprotease(AC-cathB-1,-2)inE.coli198199200L1andL3LarvaewereplacedintheboxmadebyPAPpenonToobtainlargequantitiesofcathepsinBprotease(AC-cathB-1,201poly-lysine(300lL,0.25mg/mL)coatedglassslideandcovered-2)forimmunologicandfunctionalstudies,thecDNAofcathepsin202withcoverslips,removingexcesswaterbyvacuumfiltration,thenBprotease(AC-cathB-1,-2)wereclonedintopET-32avectors,203frozeninliquidnitrogenandimmersedinfixativesolution(4%resultingintheexpressionvectorpET-AC-cathB-1,-2.Theconstructs204formaldehyde30min,cold100%methanol10min).Afterwashedwereconfirmedbyrestrictionenzymeanalysis(Fig.1B,D)and205inPBSfor2times(10mineach),theslideswereincubatedinblocknucleotidesequencing.ThecellextractsfromE.coliBL21cultures206solution(PBScontaining5%skimmilk)1.5hat37°C,thenwashedinducedwithIPTGshowedamajorproteinbandofabout60and207inPBScontaining0.1%TritonX-100for10min.Theslideswere55kDainaSDS–PAGEanalysis,respectively(Fig.2A,D).Then,the208incubatedinprimaryantibody(1:500dilutedinblocksolution)recombinantAC-cathBswerepurified(Fig.2A,D)andusedtoraise209overnightat4°C,followedby3timeswashinPBST(15mineach).specificantibodiesinmouseandfunctionalassayforevaluating210Theslideswerestainedwithsecondaryantibodyfor2h(cy3-theirputativeproteaseactivity.Afterobtainedtheanti-AC-cathB-211conjugatedgoatanti-mouseIgG1:1000inblocksolution)atroom1,-2serums,recombinantproteinswereblottedwiththeircorre-212temperature,followedby4timeswashesinPBST(10mineach).spondingantibodiesandthecross-specificitybetweenrAC-cathB-213Afterward,adding0.1%dyeHoechst(dilutedwith3%BSA)for1andrAC-cathB-2wastested;thepositiveserumsrecognizedthe21415min,washedinPBSfor2times(10mineach).70%glycerolcorrespondingrecombinantproteins(Fig.2B,E)andnocross-spec-215wasmountedandinspectedimmediatelyunderfluorescenceificitywasobservedinrAC-cathB-1/anti-AC-cathB-2serum(Fig.2C)216microscope.Mousepre-immuneserawereusedasnegativeandrAC-cathB-2/anti-AC-cathB-1serum(Fig.2F),separately.Since217control.nocrossreactionwasfound,andtherewasasinglebandandthe218Sections(4lm)offormalin-fixedandparaffin-embeddedadultmolecularsizewasconsistentwiththeexpectedone,thespecificity219andfemaleA.cantonensiswerecutandmountedonglassslides.oftheantibodiesweredetermined.220Thewormsectionswereincubatedovernightat4°Cinahumidity221chamberwithmouseanti-recombinantpolypeptideserumata3.3.Immuno-stainingofcathepsinBproteaseindifferentstagesofA.222dilutionof1:1000in3%BSA.AfterwashinginTBSTfor3timescantonensis223(5mineach),thesectionswereincubatedincy3-conjugatedgoat224anti-mouseIgG,diluted1:1000in3%BSA,for2hat37°C.TheyTheexpressionofcathepsinBproteasesinadultandlarvalstage225werethenwashedasbeforebut10mineachandcounterstainedofA.cantonensisweredetectedbyimmuno-fluorescenceantibod-226with0.1%Hoechstinahumidifiedatmospherefor0.5hat37°C,iesstainingusinganti-rAC-cathB-1,-2antibodies.OverallL1,227thenwashed4timesinTBST(10mineach).Negativecontrolsec-cross-sectionsofL3andadultofA.cantonensiswereincubated228tionswereexposedtoserum(diluted1:3000)takenfromthewithanti-rAC-cathB-1,-2antibodies.Asshownbyimmuno-stain-229mousebeforeimmunization.ing,AC-cathB-1expressionthroughoutthealimentarycanalFig.1.cDNAamplificationofcathepsinBproteasegenesbyRT-PCR.(A,C)singlebandof1100bpand900bpwereobtainedafteramplificationofcysteineproteasemRNAsfromA.cantonensisadultcDNA,separately;M:DNAladdermixturemarker;lane1:AC-cathB-1inpanel(A)andAC-cathB-2inpanel(C).(B),(D)DoubleenzymedigestionofAC-cathB-1,-2expressionvectors,respectively.M:DNAladdermixturemarker;lane1:AC-cathB-1inpanel(B)andAC-cathB-2inpanel(D).Pleasecitethisarticleinpressas:Yu,C.,etal.ImmunolocalizationanddevelopmentalexpressionpatternsoftwocathepsinBproteases(AC-cathB-1,-2)ofAngiostrongyluscantonensis.Exp.Parasitol.(2014),http://dx.doi.org/10.1016/j.exppara.2014.06.008240241242243244245246247248249250251252253254255256257258259260261262263264265266267YEXPR6892No.ofPages7,Model5G14June20144C.Yuetal./ExperimentalParasitologyxxx(2014)xxx–xxxFig.2.PurificationoftherecombinantcathepsinBprotease.(A,D)SDS–PAGEanalysisofpurificationofrAC-cathB-1andrAC-cathB-2proteininducedwith1mMIPTGat37°C.LaneM:standardproteinmarker;lane1:E.colilysateswithrecombinantplasmidthatproducedrAC-cathB-1andrAC-cathB-2inducedbyIPTG,respectivelyinpanels(A)and(D);lane2:elutedpurifiedrecombinantprotein.(B,C,E,F)Westernblotsanalysisofrecombinantproteinwithanti-AC-cathB-1,2mouseserum,andtestsforcross-specificitybetweenAC-cathB-1andAC-cathB-2,respectively.TherecombinantAC-cathB-1andAC-cathB-2reactedwithratserumimmunizedwithAC-cathB-1inpanel(B)andAC-cathB-2inpanel(E),whilenocrossreactionwasobservedinrAC-cathB-1/anti-AC-cathB-2serum(Fig.2C)andrAC-cathB-2/anti-AC-cathB-1serum(Fig.2F),separately.AC-cathBsdidnotreactwithserumfromnaïverat(notshow).268(esophagusplusintestine)ofL1,exceptforstyletregion(Fig.3A,B,L1wasbasicallysimilartoAC-cathB-1,buttheexpressionlevel269C).Thereisasmallamountofexpressioninlarvaebodywall,andinintestinewasabundantthanthatofesophagus(Fig.3D).No270theexpressionproductswerevisibleatexcretoryporeandanus.fluorescencewasdetectedinthenegativecontrolbyusingpre-271Therewasnoexpressioninnervering.AC-cathB-2expressioninserumincubated.Fig.3.ImmunolocalizationofAC-cathB-1,-2inL1ofA.cantonensis.(A,B)L1wasstainedwithanti-AC-cathB-1antibodyandHoechst,respectively;(C)Combinedmicrographof(A)and(B).(D)L1wasstainedwithanti-AC-cathB-2antibodyandHoechst.AC-cathB-1expressedthroughoutthealimentarycanal,exceptforthestylet.Thereisapositiveexpressioninbodywall,excretoryporeandanus;noexpressioninnervering.Asshowninpanel(D),AC-cathB-2expressedsimilarly,buttheexpressionlevelinintestinewasabundantthanthatofesophagus.Intestine(i);bodywall(w);anus(a);excretorypore(e);nervering(n).Pleasecitethisarticleinpressas:Yu,C.,etal.ImmunolocalizationanddevelopmentalexpressionpatternsoftwocathepsinBproteases(AC-cathB-1,-2)ofAngiostrongyluscantonensis.Exp.Parasitol.(2014),http://dx.doi.org/10.1016/j.exppara.2014.06.008272273274275YEXPR689214June2014C.Yuetal./ExperimentalParasitologyxxx(2014)xxx–xxxNo.ofPages7,Model5G5Fig.4.ImmunolocalizationofAC-cathB-1,-2incross-sectionsofL3A.cantonensis.(A,B)Sectionswerestainedwithanti-AC-cathB-1antibodyandHoechst,respectively;(C)Combinedmicrographof(A)and(B).(D,E)Sectionswerestainedwithanti-AC-cathB-2antibodiesandHoechst,separately;(F)Mergedfigureof(D)and(E).Thepositivesignalswerepresentindigestivetract,whereastheexpressionlevelinmuscularesophaguswasstrongerthanthatofintestineforbothAC-cathB-1,-2.Also,thebodywallincludingthelongitudinalalae,excretorytubesinlateralcordsshowedintensestaining.Longitudinalala(a);lateralcord(orexcretorytube)(l);esophagus(e);intestine(i);bodywall(w).Fig.5.ImmunolocalizationoftheAC-cathB-1,-2geneproductsofA.cantonensisintransversesectionsofmaleadult.(A,D)Micrographsofcrosssectionsthroughtesticle;(B,E)Crosssectionsthroughseminalvesicle;(C,F)Crosssectionsthroughvasdeferens.Sectionswerestainedwithanti-AC-cathB-1antibodyandHoechstintheupperpanel(A–C).Sectionswerestainedwithanti-AC-cathB-2antibodyandHoechstintheunderpanel(D–F).Specificstainingwasonlyinthealimentarytracts,especiallyinthemicrovillisurfaceofdigestivetubeforAC-cathB-1.However,strongexpressionofAC-cathB-2wasobservedinthesectionsthroughthegenitaltractincludingtesticle,seminalvesicle,andvasdeferensinadditiontoalimentarytract.Despitebeingmaskedbynucleusofthesperms,clearpunctiformstainingisvisibleinthedeferentduct.Therewasaremarkablyintensestainingonthewallofdeferentduct.Intestine(i);microvillilayer(m);testicle(t),seminalvesicle(s),vasdeferens(v);wallofdeferentduct(w).Pleasecitethisarticleinpressas:Yu,C.,etal.ImmunolocalizationanddevelopmentalexpressionpatternsoftwocathepsinBproteases(AC-cathB-1,-2)ofAngiostrongyluscantonensis.Exp.Parasitol.(2014),http://dx.doi.org/10.1016/j.exppara.2014.06.008YEXPR689214June20146276277278279280281282283284285286287288C.Yuetal./ExperimentalParasitologyxxx(2014)xxx–xxxNo.ofPages7,Model5GWhencross-sectionsofL3wereincubatedwithanti-rAC-cathB-1,-2antibodies,positivefluorescentsignalswerepresentinthedigestivetractforbothAC-cathB-1,-2(Fig.4A,B,C;D,E,F),whereastheexpressionlevelinmuscularesophaguswasstrongerthanthatofintestineasbeingcontrastedtothedigestivetractofL1.Also,thebodywallincludingthelongitudinalalaeandexcre-torytubesinlateralcordsshowedintensestaining.Atadultstage,control(pre-immunization)mouseserumdidnotreactwithanyinternalstructuresofadultA.cantonensis(notshown).Bycontrast,cross-sectionsincubatedwiththemouseanti-Ac-CathB-1serumdemonstratedspecificstainingonlyinthealimentarytracts(Fig.5A,B,C),especiallyinmicrovillilayeroftheesophagusandintestine.Contrastingthenuclearlocalizationdeferens(Fig.5F).Itcanbealsoobservedthatthesignalstrengthinthedigestivetractwasalittlebitstrongerthanthereproductiveorgans,withtheexceptionofthewallofvasdeferens,redfluores-centsignalsinlongitudinalmusclesofthebodywallweretheweakest.ComparisonsofadultintestineandesophagusindicatedtheAC-cathB-1,and-2tobemoreabundantintheformer,atthispoint,themainfunctionoftheproteaselikelytobedigestionoferythrocytefromthehost.TheanatomicsitesofAC-cathBsexpressionweresimilarinfemaleadult.AC-cathB-1waslocalizedmainlytoesophagusandintestine.Therewasnonspecificstainingofreproductiveorgansincludingtheeggsintheuterus(Fig.6A,B).Inthesameexpressionpatterns,AC-cathB-2wasdistributedinbothdigestivesystemand305306307308309310311312313314315316317289stain(blue),afewspecificstainingoccurredinparietallongitudi-genitalsystemoffemaleadult.Eventhoughthespecificstainingof290nalmusclesintreatedsections.Othersectionsthroughthegenitaloocytesinovariesandeggsinuteruswasweakerascomparedto291tractsincludingtesticle,seminalvesicle,andvasdeferensdidnottheesophagusorintestine,AC-cathB-2washighlyexpressedin292showanyspecificreactions.theuterinewallandoviductwall(notshown).293Inlikefashion,redfluorescencewasobviouslyobservedinthe294alimentarytractwhensectionsincubatedwiththemouse295anti-AC-cathB-2serumand,bycontrast,thesignalstrengthof4.Discussion296AC-cathB-2wasmoreabundantthanthatofAC-cathB-1(Fig.5D,297E,F).However,thereactionsonmalereproductiveorganswereThemajorroleofparasitecysteineproteasesinhost-parasite298obviouslydissimilarwiththemouseanti-AC-cathB-1antibody.Ininteractions,includinglarvaemigration,tissueinvasion,299cross-sectionsthroughthetestical,seminalvesiclesandvasdefer-immuno-evasion,nutrientacquisitionaswellascuticlerenewal,300ens,AC-cathB-2distributedincytoplasmofthespermatocytesandhavebeendescribedbroadly(ShompoleandJasmer,2001;Que301spermsseparately(Fig.5D,E,F),althoughwithlessintensityetal.,2002;Ranjitetal.,2008;Malagonetal.,2010).Althoughvar-302comparedwithesophagusorintestinecells;anothersignificantiedinphysicochemicalpropertiesanddistributions(Sajidand303differencebetweenAC-cathB-2andAC-cathB-1indistributionMckerrow,2002),theexpressionofcysteineproteaseinparasite304wasthattheAC-cathB-2expressedstronglyinthewallofvasiscorrespondingtotheirfunctions.Forexample,cysteineproteaseFig.6.ImmunolocalizationofAC-cathB-1,-2infemaleadultA.cantonensis.(A,C)Micrographsofcrosssectionthroughovaries;(B,D)Crosssectionsthroughuterus.Sectionswerestainedwithanti-AC-cathB-1antibodyandHoechstintheupperpanel(A,B).Sectionswerestainedwithanti-AC-cathB-2antibodyandHoechstintheunderpanel(C,D).Exceptforintestine,sectionsincubatedwithmouseanti-AC-cathB-1serumdidnotshowanyspecificreactionsingenitaltract.Bycontrast,sectionsincubatedwithmouseanti-AC-cathB-2antibodydemonstratedspecificstaininginbothdigestivesystemandgenitalsystemoffemaleadult.Eventhoughthespecificstainingofoocytesinovariesandeggsinuteruswasweakerascomparedtotheesophagusorintestine,theexpressionofAC-cathB-2intheuterinewallandoviductwall(notshown)wasthestrongest.Intestine(i);microvillilayer(m);ovary(o);uterus(u);wallofuterus(w).Pleasecitethisarticleinpressas:Yu,C.,etal.ImmunolocalizationanddevelopmentalexpressionpatternsoftwocathepsinBproteases(AC-cathB-1,-2)ofAngiostrongyluscantonensis.Exp.Parasitol.(2014),http://dx.doi.org/10.1016/j.exppara.2014.06.008318319320321322323324325326327328329330YEXPR689214June2014C.Yuetal./ExperimentalParasitologyxxx(2014)xxx–xxx331332333334335336337338339340341342343No.ofPages7,Model5G7396397398399400401402403404405406407falcipainwhichexpressedintrophozoitesandconcentratedinfoodvacuolesisrequiredformalarialglobinhydrolysis(Rosenthaletal.,1988;Salasetal.,1995;Shenaietal.,2000).Likewise,schistosomecathepsinL,termedSmCL1wascapableofdegradinghumanhemoglobinwithinthegutoftheschistosome(Bradyetal.,1999).Inaddition,immunohistologicalexperimentsrevealedthattheproteinasecathepsinLislocalizedinstructuresassociatedwiththereproductivesystemoffemalesandwiththesubtegumentalregionofthegynecophoriccanalofmales(Micheletal.,1995),sug-gestingitmayoperateareproductive-relatedfunction.Fortheblood-feedingnematodes,fourkindsofproteasewerethoughttomediatepeptidehydrolysis,namelyserine,aspartic,cysteine,andmetalloproteases,amongwhichcysteineproteaseAcknowledgmentsThisworkwassupportedbytheNationalNatureScienceFoun-dationofChina(GrantNo.81171595).ReferencesBrady,C.P.,Dowd,A.J.,Brindley,P.J.,Ryan,T.,Day,S.R.,Dalton,J.P.,1999.RecombinantexpressionandlocalizationofSchistosomamansonicathepsinL1supportitsroleinthedegradationofhosthemoglobin.Infect.Immun.67(1),368–374.Ding,B.L.,Xu,S.E.,Shen,H.X.,Lu,X.J.,Zhang,D.P.,1990.ScanningelectronmicroscopicobservationsonlarvaeandyoungadultsofAngiostrongycantonensis.Chin.J.Parasitol.Parasit.Dis.8(4),53–56.Dzik,J.M.,2006.Moleculesreleasedbyhelminthparasitesinvolvedinhost344wasthemostfrequentlyreported,whosemainroleistodegradecolonization.ActaBiochim.Pol.53,33–64.345hosthemoglobin,providenutritionfortheparasiticnematodeFang,W.,Xu,S.,Wang,Y.,Ni,F.,Zhang,S.,Liu,J.,Chen,X.,Luo,D.,2010.ESproteins346(Williamsonetal.,2003).Therefore,intheseblood-feedingnema-analysisofAngiostrongyluscantonensis:productsofthepotentialparasitismgenes?Parasitol.Res.106(5),1027–1032.347todesthecysteineproteasesaregenerallylocatedinthedigestiveHarrop,S.A.,Sawangjaroen,N.,Prociv,P.,Brindley,P.J.,1995.Characterizationand348systemsuchasintestinesandpharynx(Harropetal.,1995;JasmerlocalizationofcathepsinBproteinasesexpressedbyadultAncylostomacaninum349etal.,2001;Williamsonetal.,2002;Yangetal.,2011).Similarly,A.hookworms.Mol.Biochem.Parasitol.71(2),163–171.Jasmer,D.P.,Roth,J.,Myler,P.J.,2001.CathepsinB-likecysteineproteasesand350cantonensiscysteineproteaseAC-cathB-1,and-2werelocatedonCaenorhabditiseleganshomologuesdominategeneproductsexpressedinadult351thedigestivetractorgutmicrovilli.Haemonchuscontortusintestine.Mol.Biochem.Parasitol.116(2),159–169.352ESproductsareconstantlyincontactwithhostimmunecells.Jasmer,D.P.,Mitreva,M.D.,McCarter,J.P.,2004.mRNAsequencesforHaemonchuscontortusintestinalcathepsinB-likecysteineproteasesdisplayanextremein353Parasitescontinuouslyreleasemoleculesnecessaryfortissuepen-abundanceanddiversitycomparedwithotheradultmammalianparasitic354etration,immunesystemevasion,oxidativestressandnutrientnematodes.Mol.Biochem.Parasitol.137(2),297–305.355acquisition(Dzik,2006).CysteineproteasesinparasiticnematodesLee,J.D.,Yen,C.M.,2005.ProteasesecretedbytheinfectivelarvaeofAngiostrongyluscantonensisanditsroleinthepenetrationofmouseintestine.Am.J.Trop.Med.356alsoexistasESproteins,suchashookwormproteinaseAcCP-1Hyg.72(6),831–836.357localizedimmunohistochemicallytoesophageal,amphidialandMalagon,D.,Diaz-Lopez,M.,Benitez,R.,Adroher,F.J.,2010.CathepsinB-andL-like358excretoryglands,indicatedthattheAcCP-1proteinaseisexpressedcysteineproteaseactivitiesduringtheinvitrodevelopmentofHysterothylacium359and/orstoredintheseglands(Harropetal.,1995).TheESproteinsaduncum(Nematoda:Anisakidae),aworldwidefishparasite.Parasitol.Int.59,89–92.360fromthethirdstagelarvaeofA.cantonensishaveshowntopossessMichel,A.,Ghoneim,H.,Resto,M.,Klinkert,M.Q.,Kunz,W.,1995.Sequence,361serineproteaseandmetalloproteaseactivitieslikelyassociatedcharacterizationandlocalizationofacysteineproteinasecathepsinLin362withduodenalpenetration(LeeandYen,2005).Astudyinvestigat-Schistosomamansoni.Mol.Biochem.Parasitol.73(1–2),7–18.Morassutti,A.L.,Levert,K.,Pinto,P.M.,daSilva,A.J.,Wilkins,P.,Graeff-Teixeira,C.,363ingtheantioxidantenzymeprofileofadultA.cantonensisworms2012.CharacterizationofAngiostrongyluscantonensisexcretory–secretory364demonstratedthatsuperoxidedismutaseandcatalasewerehighlyproteinsaspotentialdiagnostictargets.Exp.Parasitol.130(1),26–31.365activeESproductslikelyinvolvedinmediatingparasitesurvivalMorassutti,A.L.,Pinto,P.M.,Dutra,B.K.,Oliveira,G.T.,Ferreira,H.B.,Graeff-Teixeira,C.,2011.Detectionofanti-oxidantenzymaticactivitiesandpurificationof366againstoxidativestressesgeneratedbyhostimmuneresponsesglutathionetransferasesfromAngiostrongyluscantonensis.Exp.Parasitol.127,367(Morassuttietal.,2011).Asshownbyimmunolocalizationsinthis365–369.368study,thepositivesignalswerepresentintheexcretorypore,Ni,F.,Wang,Y.,Zhang,J.,Yu,L.,Fang,W.,Luo,D.,2012.CathepsinB-likeandhemoglobin-typecysteineproteases:stage-specificgeneexpressionin369excretorytubeinlateralcord,muscularesophagus,andanusofAngiostrongycantonensis.Exp.Parasitol.131(4),433–441.370larvaeforbothAC-cathB-1and-2,indicatedthattheAC-cathBsQue,X.,Ngo,H.,Lawton,J.,Gray,M.,Liu,Q.,Engel,J.,Brinen,L.,Ghosh,P.,Joiner,371likelytobereleasedasESproductsofA.cantonensis.Obviously,K.A.,Reed,S.L.,2002.ThecathepsinBofToxoplasmagondii,toxopain-1,iscritical372consistentwiththeinfectivestageofL3isanintensestainingofforparasiteinvasionandrhoptryproteinprocessing.J.Biol.Chem.277,25791–25797.373AC-cathBsintheesophagusandexcretory.So,theresultsinthisRanjit,N.,Zhan,B.,Stenzel,D.J.,Mulvenna,J.,Fujiwara,R.,Hotez,P.J.,Loukas,A.,374reportfurthersupportedourpreviousobservationsresultingfrom2008.AfamilyofcathepsinBcysteineproteasesexpressedinthegutofthe375Signal-P,RT-PCR,andwesternblotanalyses(Fangetal.,2010;Nihumanhookworm,Necatoramericanus.Mol.Biochem.Parasitol.160,90–99.Rosenthal,P.J.,McKerrow,J.H.,Aikawa,M.,Nagasawa,H.,Leech,J.H.,1988.A376etal.,2012),andfromelectrosprayionizationmassspectrometrymalarialcysteineproteinaseisnecessaryforhemoglobindegradationby377(Morassuttietal.,2012).ThereisabigdifferenceinthestructurePlasmodiumfalciparum.J.Clin.Invest.82,1560–1566.378betweenlarvaeandadults,nolateralglandsandlateralalaecouldSajid,M.,Mckerrow,J.H.,2002.Cysteineproteasesofparasiticorganisms.Mol.Biochem.Parasitol.120(1),1–21.379beseeninthebodywallofadultA.cantonensis,whiletheywereSalas,F.,Fichmann,J.,Lee,G.K.,Scott,M.D.,Rosenthal,P.J.,1995.Functional380moreobviousinL1andL3(Xuetal.,1989;Dingetal.,1990).expressionoffalcipain,aPlasmodiumfalciparumcysteineproteinase,supports381Althoughthedistributionsitesandexpressionpatternswereveryitsroleasamalarialhemoglobinase.Infect.Immun.63(6),2120–2125.Shenai,B.R.,Sijwali,P.S.,Singh,A.,Rosenthal,P.J.,2000.Characterizationofnative382differentinthelarvaeandadultsandthecorrespondingfunctionsandrecombinantfalcipain-2,aprincipaltrophozoitecysteineproteaseand383mightnotbethesame,AC-cathB-1and-2,especiallylocatedinESessentialhemoglobinaseofPlasmodiumfalciparum.J.Biol.Chem.275(37),384proteinsoflarvae,involvedinthehost-parasiteinteractioninaddi-29000–29010.Shompole,S.,Jasmer,D.P.,2001.CathepsinB-likecysteineproteasesconfer385tiontodigestivefunction.intestinalcysteineproteaseactivityinHaemonchuscontortus.J.Biol.Chem.386TheimmunolocalizationofAC-cathBsinthetegumentoflarvae,276(4),2928–2934.387butnotobviousinadults,presumablyasimilarfunctionmaybeWilliamson,A.L.,Brindley,P.J.,Abbenante,G.,Prociv,P.,Berry,C.,Girdwood,K.,Pritchard,D.I.,Fairlie,D.P.,Hotez,P.J.,Dalton,J.P.,Loukas,A.,2002.Cleavageof388attributedtotheseproteaseswhichinvolvedintheprocessoflar-hemoglobinbyhookwormcathepsinDasparticproteasesanditspotential389valmolting.TheAC-cathB-2locatedinthetesticle,andstronglycontributiontohostspecificity.FASEBJ.16(11),1458–1460.390expressedinthewallofdeferentduct,whichmaybe,similartoWilliamson,A.L.,Brindley,P.J.,Knox,D.P.,Hotez,P.J.,Loukas,A.,2003.Digestive391Q3thecysteineproteasesinFasciola(Wijffelsetal.,1994),asthetar-proteasesofblood-feedingnematodes.TrendsParasitol.19(9),417–423.Xu,S.,Ding,B.,Lu,X.,Shen,H.,Zhang,D.,Xiao,Y.,1989.Scanningelectron392getoftheimmuneresponse,therebyprotecttheeggsandsperm.microscopicobservationsonadultsofAngiostrongyluscantonensis.Chin.J.393DifferencesinthematrixliningthevasdeferensledustoconcludeParasitol.Parasit.Dis.7(1),32–34.394thatAC-cathB-2mayalsoaltertheviscosityoftheseminalfluidorYang,Y.,Qin,W.,Wei,H.,Zhen,J.,2011.CharacterizationofcathepsinBproteinase(AcCP-2)ineggsandlarvaestagesofhookwormAncylostomacaninum.Exp.395itmaybejustparticipatedinmaturationofgermcells.Parasitol.129(3),215–220.Pleasecitethisarticleinpressas:Yu,C.,etal.ImmunolocalizationanddevelopmentalexpressionpatternsoftwocathepsinBproteases(AC-cathB-1,-2)ofAngiostrongyluscantonensis.Exp.Parasitol.(2014),http://dx.doi.org/10.1016/j.exppara.2014.06.008408409410411412413414415416417418419420421422423424425426427428429430431432433434435436437438439440441442443444445446447448449450451452453454455456457458459460461462463464465466467468469470471472473474475476
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