tein from an endoparasitoid wasp inhibits melanization of the host hemolymph

阿斯顿飞阿斯顿飞阿斯顿飞阿斯顿飞阿斯顿飞阿斯顿发第三方

InsectBiochemistryandMolecularBiology33(2003)

1017–1024

http://wendang.chazidian.com/locate/ibmb

Aserineproteinasehomologvenomproteinfromanendoparasitoid

waspinhibitsmelanizationofthehosthemolymph

SassanAsgaria, ,GuangmeiZhanga,RezaZareieb,OttoSchmidta

a

InsectMolecularBiologyLab.,PlantandPestScience,WaiteCampus,UniversityofAdelaide,GlenOsmond,SA5064,Australia

b

ProteinPharmaceuticalDivision,BresaGenLtd.,POBox259,RundleMall,Adelaide,SA5000,Australia

Received6March2003;accepted20June2003

Abstract

Activationofprophenoloxidase(proPO)ininsectsisadefensemechanismagainstintrudingmicroorganismsandparasites.Patternrecognitionmoleculesinduceactivationofanenzymaticcascadeinvolvingserineproteinases,whichleadstotheconversionofproPOtoactivephenoloxidase(PO).PhenoliccompoundsproducedbypPO-activationaretoxictoinvaders.Here,wedescribetheisolationofavenomproteinfromtheparasitoid,Cotesiarubecula,injectedintothehost,Pierisrapae,whichishomologoustoserineproteinasehomologs(SPH).Thedatapresentedhereindicatethattheproteininterfereswiththeproteolyticcascade,whichundernormalcircumstancesleadstotheactivationofproPOandmelaninformation. 2003ElsevierLtd.Allrightsreserved.

Keywords:Cotesiarubecula;Parasitoid;Venom;Prophenoloxidase;Serineproteinasehomolog;Melanization

1.Introduction

Endoparasitoidwaspsintroducematernalfactorsintothebodyoftheirhostuponparasitizationtomanipulatehostphysiology.Theseincludecalyx uidcontainingvirusorvirus-likeparticles,suchaspolydnaviruses(PDVs),andproteinsecretionsfromvenomglands.PDVsareproducedintheovariesofcertainhymenop-teranparasitoidwaspsbelongingtotwoichneumonoidfamilies,BraconidaeandIchneumonidae(StoltzandVinson,1979).ThesewaspsusePDVstoovercomethehostimmunedefensesandhavebeenshowntobeessen-tialforsuccessfulparasitismanddevelopmentoftheparasitoidinsidethehost(Edsonetal.,1981;FlemingandSummers,1991).

Inmosthost-parasitoidsystems,PDVsareonlyeffec-tivewhenaccompaniedbyvenomproteins.InCotesiamelanoscela,venomfacilitatestheuncoatingofPDVsinvitroandviruspersistenceinvivo(Stoltzetal.,1988).

Correspondingauthor.Tel.:+61-8-8303-6565;fax:+61-8-8379-4095.

E-mailaddress:sassan.asgari@adelaide.edu.au(S.Asgari).

Genbankaccessionnumber:theNCBI/GenbankaccessionnumberforthesequencereportedhereisAY293826.

0965-1748/$-seefrontmatter 2003ElsevierLtd.Allrightsreserved.doi:10.1016/S0965-1748(03)00116-4

InCotesiaglomeratus(Kitano,1986)andApanteleskariyai(Tanaka,1987),venomisanessentialrequire-mentforsuccessfulparasitism.ItisalsoproposedthatduringtheperiodbetweenovipositionandtheexpressionofPDVgenes,venomproteinsmightprotecttheeggfromthehostimmunereaction(WebbandDahlman,1985).InadditiontotheirsynergisticeffectstogetherwithPDVs,venomproteinsaffecthostphysiologyanddevelopment(Digilioetal.,2000;GuptaandFerkovich,1998).InendoparasitoidsthatlackPDVs,venomseemstoplayamajorroleinhostimmunesuppressionandhostregulation.Forexample,inPimplahypochondriaca(Braconidae),venomadverselyaffectsthemorphology,viability,andimmunefunctionofhemocytesofthetom-atomoth,Lacanobiaoleracea(RichardsandEdwards,1999;RichardsandParkinson,2000).Inthissystemvenomcontainsaprophenoloxidase,aprophenoloxidaseinhibitorandacytotoxicfactor(ParkinsonandWeaver,1999).

Inhibitionofmelanizationafterparasitismhasbeenshowninseveralsystems(reviewedinLavineandBeckage,1995).Sincetheprophenoloxidase(proPO)activationcascade,leadingtotheformationofmelaninandothertoxicphenoliccompounds,isavitaldefensemechanismagainstintrudingorganisms(Ashidaand

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Brey,1998;VassandNappi,2000),itispossiblethatparasitoidsinterferewiththeregulationofthatcascade.Arecentstudyindicatesthatserineproteinasehomologs(SPH)inthehemolymph,thatdonothaveproteolyticactivity,mediateactivationofproPOinconjunctionwithothercomponentsfromthehemolymph(Yuetal.,2003).Here,wedescribepuri cationandcharacterizationofavenomprotein(Vn50)fromtheendoparasitoidCotesiarubeculathatinterfereswiththeproteolyticcascadeleadingtotheactivationofproPOandformationofmel-anininthehosthemolymph.Sequencehomologyanaly-sisindicatesthatVn50isaSPHwithoutproteolyticactivity.

2.Methodsandmaterials2.1.ReversephaseHPLC(rpHPLC)

Venomsacsfrom20femaleC.rubeculawaspsweredissectedanddisruptedinsterilewaterbymicro-scissorstoreleasevenomproteinsfollowedbycentrifugationfor1minat16,000×gtoremovemembranedebris.RpHPLCofthesamplewascarriedoutinaHewlettPackard1090LiquidChromatograph.ThecrudevenomsamplewasloadedontoaVydacreversephase(RP)C18columnandelutedatthe owrateof0.2ml/minusingagradientof5–100%ofbufferB(0.04%tri uoroaceticacid,TFA,in70%acetonitrile)againstbufferA(0.05%TFAinwater)over82min.Proteinsweredetectedbyabsorbanceat214nmandproteinpeakswerecol-lectedmanually.

2.2.N-terminalandpeptidesequencing

ToobtaintheN-terminalsequenceforVn50,approxi-mately100pmolofthepuri edproteinfromrpHPLCwasfreeze-dried,reconstitutedin8Mureacontaining0.1MNH4HCO3and4mMDTTand nallyalkylatedbyadditionofsodiumiodoacetatetothe nalconcen-trationof10mM.Thesamplewasacidi edwithTFAtostopthereaction.TheproteinwassequencedusingaHewletPackardG1000AProteinSequencer.

InordertoobtainmorepeptidesequencedatatodesigndegenerateprimerstoVn50,theproteinwasexcisedfromaSDS-PAGEgelanddigestedingelusingtrypsin.DigestedandelutedpeptideswereseparatedonarpHPLCfromwhichonewassequencedasabove.2.3.RNAisolationandRT-PCR

VenomglandsweredissectedfromfemaleC.rubec-ulawaspsfromwhichtotalRNAwasisolatedasdescribed(Ausubeletal.,1993).ToconstructcDNAmolecules,apoly-dT(17mer)primerwasusedinareversetranscriptionreactionusingAMVreversetran-

scriptase.Inatotalvolumeof11µl,2µgextractedRNAplus0.1µgprimerwasheatedat95°Cfor5minandchilledimmediatelyonice.Then3µlAMVreversetran-scriptionbuffer(5×),0.5µlAMVreversetranscriptase(9U/µl),0.5µlRNasin(40U/µl)and0.5µldNTPs(15mM)wereadded(Promega).Thereactionwascarriedoutat42°Cfor1hfollowedby95°Cfor5mintoinactivatethereversetranscriptase.

Ampli cationfollowedthecDNAsynthesisusingdegenerateprimersdesignedbasedonVn50peptidesequences(Vn50-F,5 -CCNCCNCARCARGCNGCNCC-3 ;Vn50-R,5 -AANGCYTCNARYTGYTGRTC-3 ).Thereactionwascarriedoutin50µlbyaninitialheatingat96°Cfor5minfollowedby35cyclesof94°Cfor30s,48°Cfor70s,72°Cfor90sanda nalextensionat72°Cfor15min.ThePCRproductwasexcisedfromanagarosegel,puri edusingWizardPCRPuri cationkit(Promega)andclonedintopGEM-T-Easyvector(Promega).2.4.ScreeningacDNAlibrary

Toobtainafull-lengthcDNAcodingforVn50,acDNAlibrarymadefromvenomglandandovariesofC.rubeculawaspswasscreenedusingthepartialsequenceobtainedfromRT-PCRusingdegenerateprimersasaprobe(seeabove).Positivecloneswerere-screenedandthelongestcDNAwassequencedinbothdirections.2.5.Productionandpuri cationofVn50expressedinE.coli

ApartialVn50proteinwasexpressedinbacteriacon-tainingmostpartsoftheproteinfromaminoacid28to385.Thecodingregionwasampli edbydesigningspe-ci cprimerscontainingrestrictionsites(Vn50-BamHI-F,5 -GCGCGGATCCCCCCAGCAAGCCGCTCCG-3 ;Vn50-KpnI-R,5 -GCGCGGTACCTTACGCCTCTAGTTGCTGGTC-3 ,restrictionsitesareunderlined)tofacilitatedirectcloningintotheexpressionvector.ThePCRampli edproductwasdigestedwiththerestrictionenzymesandclonedintothecorrespondingsitesinpQE30expressionvector(Qiagen).Theproteinexpressedinthissystemisafusionproteincontainingsixhistidineresidues.Trans-formedbacteriawereinducedwith1mMIPTGfor2handanalyzedona12%SDS-PAGEgelasdescribed(Laemmli,1970).Theidentityoftheproteinwascon- rmedbyWesternblottingusingamonoclonalanti-polyhistidinealkalinephosphataseconjugate(1:5000,Sigma)andapolyclonalantiserumraisedagainsttotalvenomproteins(1:5000).Alkalinephosphatase-conju-gatedanti-rabbitIgGantibodieswereusedassecondaryantibodies(1:5000,Sigma).

Sincetheexpressedproteinwasfoundintheinsolublefraction,itwaspuri edunderdenaturingconditions

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accordingtothemanufacturer’sinstructions(Qiagen).Thefusionproteinwaspuri edbyaf nitychromato-graphyusingNi-NTAtechnology(Qiagen)andelutedwith8Murea,0.1MNa-phosphate,0.01MTrisatpH4.5.Torefoldtheprotein,itwas rstdialyzedagainst1l1Murea,150mMNaCl,10mMTris(pH7.5)andthen150mMNaCl,10mMTris(pH7.5),30heachat4°C.

2.6.Detectionofglycosylation

Venomproteinsfromtwowaspswererunona12%SDS-PAGEgel,transferredontoanitrocellulosemem-brane.Todetectcarbohydrateportionsofvenompro-teins,ECLGlycoproteinDetectionModulewasusedaccordingtothemanufacturersinstructions(Amersham).2.7.Enzymaticassays

ToinvestigatepossibleproteaseactivityofVn50,trypsinandα-chymotrypsinassayswerecarriedout.Enzymeassayswereperformedasdescribedpreviouslywithminormodi cations(Muharsinietal.,2001).Brie y,4mMNα-benzoyl-dl-Arg-p-nitroanilide(BAPNA,Sigma)andsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide(SAPNA,Sigma)werepreparedin100mMTris,pH8.0,containing20mMCaCl2assubstratesol-utionsfortrypsinandα-chymotrypsinenzymes,respect-ively.Reactionswerecarriedoutinmicrotitreplatesina nalvolumeof100µlcontaining40µlsubstratesol-ution,10µlenzyme(100U/mltrypsinand15U/mlchy-motrypsin,Sigma)or5µlVn50(fourwaspsequivalent,ca.0.5µg).Afterincubationat37°Cfor2h,absorptionwasmeasuredat405nminaBioRADmicroplatereader.2.8.HemolymphphenoloxidaseenzymeactivityTheassaywascarriedoutasdescribedpreviouslywithminormodi cations(Becketal.,2000).Hemo-lymphwascollectedfromtwofourthinstarP.rapaelar-vaebysurfacesterilizingin70%ethanolandbleedingfromaproleginto100µlice-coldPBS.Toobtainacell-freeserum,collectedhemolymphwascentrifugedat800×gfor5min.Cell-freehemolymphwasaddedto900µlsubstratesolution(20mMdl-3,4-dihydroxy-phenylalanineinPBS,dl-DOPA)andmixed.Absorb-encyat490nmwasmonitoredatroomtemperaturefor100minusingaDMS100spectrophotometer(VarianTechtron).Forinhibitionassays,totalvenomfromfourwaspsinPBS(10µl),fourwaspsequivalentVn50(ca.0.5µg)inPBSpuri edonHPLC,or0.5µgpuri edrecombinantVn50wasaddedtotheabovemixtureandabsorbencywasmeasuredasabove.Theassayswererepeatedthreetimesforeachtreatment.

3.Results

3.1.IsolationandcharacterizationofVn50

Whencrudevenompreparationsareseparatedonareversephasehighpressureliquidchromatography(rpHPLC)oneofthemajorproteinselutesat55s(Fig.1A).Whentheelutedproteinwasanalyzedona12%SDS-PAGEgel,a50kDaproteinwasdetected(Vn50)withCoomassiestainingastheonlymajorprotein(Fig.1B).N-terminalsequencingoftheelutedfractionfromrpHPLCresultedin22aminoacids(ENSDVXPPPQQAAPVXTXTNXL).Theproteinwasalsodigestedwithtrypsinandfractionatedon

rpHPLC.

Fig.1.(A)RpHPLCofcrudeC.rubeculavenom.Severalcompo-nentsarepresentinthevenom.Themostabundantproteinwaselutedat28.7%acetonitrile(arrow).(B)SDS-PAGEanalysis(12%)ofthemostabundantpolypeptideinthevenomfrom(A).(C)Detectionofglycoproteinsinthevenom.Venomproteinsfromtwowaspswererunona12%SDS-PAGEgel,transferredontoanitrocellulosemembrane.GlycoproteinsweredetectedusingECLGlycoproteinDetectionMod-ule.TwomajorglycoproteinsweredetectedinthevenomfractionincludingVn50(arrow).

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Oneofthepeptideswassequencedcontaining10aminoacids(EWIDQQLEAF).

DegenerateprimersweredesignedbasedonN-ter-minalandpeptidedigestsequences(Fig.2).TotalRNAisolatedfromvenomglandofC.rubeculawaspswasreversetranscribedusinganoligodTprimerfollowedbyaPCRreactionusingthedegenerateprimers.Aca.1000bpfragmentwasobtainedwhichwasclonedandsequencedinbothdirections(Fig.2).Toobtainacom-pletecDNA,avenom/ovarycDNAlibrarywasscreenedusingtheampli edRT-PCRproductasaprobe.Thelongestclonewassequencedinbothdirections.Ameth-ionineatposition1wasidenti edastheputativeinitiationcodon(ATG;CavenerandRay,1991).Thecompleteopenreadingframe(ORF)contained388aminoacids.SeveralputativeN-andO-glycosylationsiteswerefoundinthesequence(Hansenetal.,1988).Tocon rmthattheproteinisglycosylated,aglycosyl-ationdetectionkitwasused,whichshowedthatVn50isindeedheavilyglycosylated(Fig.1C).InadditiontoVn50,http://wendang.chazidian.comingSignalPV1.1software,aputativecleav-agesitewasdetectedbetweenaminoacids19and20(Fig.2;Nielsenetal.,1997),consistentwiththeN-ter-minalsequenceofthesecretedproteinfromthevenomstoragesac(see

above).

Fig.2.NucleotidesequenceanddeducedaminoacidsequenceofacDNAcodingforVn50.Thenumbersofnucleotidesandaminoacidresiduesareshownattheendofeachline.AminoacidsequencesobtainedfromN-terminalandpeptidemicrosequencingareunderlined.Aputativepolyadenylationsiteisunderlined.Thelocationofdegener-ateprimersusedforinitialisolationofthegeneisdotunderlined.PutativeN-andO-glycosylationsitesareunderlinedandshowninbold,respectively.

SequencehomologysearchesatGenBankshowedthatVn50hassimilaritytoSPHfromManducasextaSPH1(44%),Tenebriomolitor(50%),Drosophilamelanogas-ter(48%),andLimulusfactorD(34%).Vn50sequencecontainsallthecysteineresiduesconservedinamino-terminalclipandserineproteinasedomainsofSPHs(Fig.3;JiangandKanost,2000).However,SPHsinclud-ingVn50donothaveproteolyticactivity(seebelow)sincetheconservedserineattheactivesiteofthepro-teinase-likedomainischangedtoglycine(Fig.3,arrowhead).3.2.Enzymaticassays

ToexperimentallyshowthatVn50doesnothavepro-teolyticactivity,syntheticpeptideswereusedassub-strates.Whenfourwaspequivalentsofpuri edVn50(ca.0.5µg)weretestedintrypsinandα-chymotrypsinassaysusingBAPNAandSAPNAassubstrates,respect-ively,noactivitywasdetected(Fig.4AandB).Inbothcontrols,trypsinandα-chymotrypsincleavedthecorre-spondingsubstrates.ThisindicatesthatVn50lacksenzy-maticactivity,atleastwiththelimitednumberofsub-stratestested.

3.3.Inhibitionofmelaninformation

Serineproteinasehomologsisolatedsofarfromvari-ousinsectshavebeenshowntobenecessaryforacti-vationofproPOzymogen(e.g.Yuetal.,2003).TodeterminewhetherVn50fromC.rubeculavenomhasasimilareffectonthehostP.rapaehemolymph,melaniz-ationassayswerecarriedout.Whenhemolymphalonewastestedinthepresenceofdl-DOPA,asubstrateforthePOenzyme,melanizationwasdetectedat490nmwavelength(Fig.5;Hem).However,whentotalvenom(fourwaspsequivalent)orpuri edVn50(fourwaspsequivalent,ca.0.5µg)wereaddedtothereaction,mel-anizationwasinhibited(Fig.5;Hem+VenandHem+Vn50).Tofurthercon rmthisinhibitionusingrecom-binantprotein,thecodingregionforthesecretedVn50wasexpressedinE.coli,whichresultedinthepro-ductionofaca.42kDaprotein(Fig.6A)closetothepredictedsize(40.7kDa).Theproteinwaspuri edanditsidentitywascon rmedbyitscrossreactionwithanti-venomantibodies(Fig.6B).WhentherecombinantVn50wasusedinmelanizationassays,italsoinhibitedmelanization(Fig.5;Hem+Rec).Thereducedinhi-bitionofmelanizationobservedwithrecombinantVn50,althoughstillsigni cantcomparedtothecontrol,couldbeduetopost-translationalmodi cationsorthefactthatproteinrenaturationisusuallylessef cientafterpuri -cationoftheproteinunderdenaturingconditionsanddialysis,whereasactiveVn50fromvenomismoreeffec-tiveininhibitinghosthemolymphmelanization.

Melaninformationwascompletelyinhibitedwhen

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Fig.3.SequencealignmentofC.rubeculaVn50withserineproteinasehomologs(SPH).ThematurepolypeptidesequencesofVn50arealignedwithSPHfromM.sexta(Ms-SPH1,AAM69352),T.molitor(Tm,CAC12696),D.melanogaster(Dm,AAF52904),andLimulusfactorD(Lm,BAA13312).GenBankaccessionnumbersareprovidedinthebrackets.IdenticalresidueswithVn50areblackboxed.Conservedcysteineresiduesintheclipdomainandserineproteinasedomainaremarkedbyasterisks.ResiduesinthecatalytictriadofserineproteinasesHis,AspandSerareindicatedbysolidcircles.ThesubstitutionofserinebyglycineintheactivesiteofSPHsisshownbyanarrowhead.

totalvenomorpuri edVn50ortheproteinequivalentoffourwaspswasincludedinmelanizationassays,whereasonewaspequivalenthadnoeffectandtwowaspequivalentsshowedintermediatemelanization(Fig.7)4.Discussion

Maternalfactorsintroducedbyhymenopteranendopa-rasitoidsintohostinsectsinterferewithhostphysiologyanddevelopment.Oneofthemajoreffectsofthesefac-torsissubversionofthehostimmunesystem.Thoseparasitoidsthatdonotproducevirus-likeparticles(suchaspolydnaviruses,PDVs)mainlyrelyonthevenom uidforhostregulation,includingsuppressionofthehostimmunedefense.Waspspecieswithparticlesuseacombinationoffactors,wherevenomusuallyplaysasynergisticrolewithparticles(e.g.Stoltzetal.,1988;Tanaka,1986;WagoandTanaka,1989).Factorsintro-ducedbythewaspsaffectcellular(e.g.preventionofencapsulation)aswellashumoralimmunity(e.g.inhi-bitionofmelanization)ofthehost(Beckage,1998).Calyx uidcontainingPDVshasbeenshowntobeinvolvedininactivationofhosthemocytes(Asgarietal.,1996;Strand,1994;WebbandLuckhart,1996).Inhi-bitionofmelanizationafterparasitismhasbeenreportedfromseveralsystems(Beckageetal.,1990;Shelbyetal.,2000;StoltzandCook,1983;Becketal.,2000),althoughtheexactmechanism(s)isnotwellunderstood.Ininsects,proPOisactivateduponinjuryorinvasion,whichresultsinlocalizedmelanizationofthewoundareaornodules/capsulescapturinginvadingmicro-organismsandparasites.Thisinvolvesactivationofaseriesofserineproteinasesinaso-calledproPOacti-vationcascade(AshidaandBrey,1998).SPHpresentintheplasmaofseveralinsectsfunctionasco-factorsforproPO-activatingproteinase(PAP).Inarecentstudy,Yuetal.(2003)showedthatSPHsfromM.sextaincon-junctionwithothercomponentsfromthehemolymphmediateactivationofproPO(Yuetal.,2003).Intheproposedmodel,immunolectin-2,apatternrecognitionmolecule,initiatesprotein–proteininteractionwithSPHandproPOafterbindingtotheforeignsurface.Then,SPHmediatesrecruitmentofotherplasmaproteinssuchasPAP,whichleadstotheactivationofproPOandmel-anization.ThemediationeffectofSPHisnotwellunder-stoodatthemolecularlevel.ItisassumedthatSPHsmightbringproPOintoacorrectpositionforproteolysisbyPAPorcauseconformationalchangesinproPOtofacilitateitscleavagebyPAP(Yuetal.,2003).

WehaveisolatedavenomproteinfromC.rubecula(Vn50)withsimilaritytoSPHs,whichinhibitsmelaniz-ationofthehosthemolymphP.rapaeinadose-depend-antmanner.Whentotalvenom,puri edorrecombinantVn50wereaddedtoproPOactivationassays,melaniz-ationwascompletelyinhibited(Fig.5).SimilartootherSPHs,Vn50containsthetwostructuraldomains,acar-boxyl-terminalserineproteinasedomainandanamino-