p Leptopilina boulardi targets the Drosophila phenoloxidase cascade

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

DevelopmentalandComparativeImmunology33(2009)681–689

ContentslistsavailableatScienceDirect

DevelopmentalandComparativeImmunology

journalhomepage:http://wendang.chazidian.com/locate/dci

AserpinfromtheparasitoidwaspLeptopilinaboularditargetstheDrosophilaphenoloxidasecascade

´bastienMoreaud,DominiqueColineta,b,c,1,AuroreDubuffetd,1,2,DominiqueCazesa,b,c,Se

`nePoirie´a,b,c,*Jean-MichelDrezend,Maryle

a

InstitutNationaldelaRechercheAgronomique,INRA,UMR1301,France

CentreNationaldelaRechercheScienti?que,CNRS,UMR6243,Francec

´NiceSophiaAntipolis,UFRSciences,FranceUniversite

d

´deTours,FranceInstitutdeRecherchesurlaBiologiedel’Insecte,UMRCNRS6035-Universite

b

ARTICLEINFOABSTRACT

Articlehistory:

Received26October2008

Receivedinrevisedform28November2008Accepted28November2008

Availableonline25December2008Keywords:ParasiteDrosophilaInsectImmunityVirulence

PhenoloxidasecascadeSerineproteaseSerpin

Theinsectphenoloxidase(PO)cascadeisknowntobetightlyregulatedbyserineproteasesandserineproteaseinhibitorsoftheserpinfamily.Asakeycomponentoftheinsectimmunesystem,itisalsosuspectedtobeinhibitedbyseveralendoparasitoidwasps,insectsthatdevelopinsideotherarthropodsashosts.However,theunderlyingmechanismsofthisinhibitionarelargelyundescribed.Here,wereportthecharacterizationofageneencodingaserpin,LbSPNy,highlyexpressedinthevenomofthewaspLeptopilinaboulardi(ISytype),andweshowthateitherthevenomortherecombinantLbSPNyinhibitthePOcascadeinthehemolymphofDrosophilayakubahostlarva.Altogether,ourresultsidentifythe?rstserpinusedasavirulencefactorbyaparasitoidwaspandshowthatitdisruptstheactivationpathwayofthePOintheDrosophilahost.

ß2008ElsevierLtd.Allrightsreserved.

1.Introduction

Endoparasitoidwaspsareinsectsthatdevelopattheexpenseofarthropods,generallyotherinsects,eventuallykillingthem.Onceintroducedintothebodycavityoftheirhosts,endoparasitoidshavetofacetheencapsulationimmuneresponse[1–3].Thisdefensereactionwheneffectiveconsistsintheformationofamulticellularmelanizedcapsulearoundtheeggandrequiresthecoordinationofcellularandhumoralcomponents.Followingparasiterecognition,hemocytesproliferate,becomeadhesive,attachandspreadatthesurfaceoftheforeignbody[3,4].Inthemeantime,acascadeinvolvingseveralserineproteasesistriggered,leadingtotheactivationofprophenoloxidase-activatingproteases(PAPs).Theseenzymesareserineproteasesthatconvertinactiveprophenoloxidase(proPO)into

*Correspondingauthor.InstitutAgrobiotech,UMRInteractionsBiotiqueset´Ve´ge´tale,400RoutedesChappes,06903SophiaAntipolis,France.Sante

Tel.:+33492386409;fax:+33492386401.

´).E-mailaddress:poirie@sophia.inra.fr(M.Poirie

1

Bothauthorsequallycontributedtothiswork.2

Presentaddress:InstituteofIntegrativeandComparativeBiology,FacultyofBiologicalSciences,UniversityofLeeds,UK.

Abbreviations:PAP,prophenoloxidase-activatingprotease;PO,phenoloxidase;proPO,prophenoloxidase;RCL,reactivecenterloop.

0145-305X/$–seefrontmatterß2008ElsevierLtd.Allrightsreserved.doi:10.1016/j.dci.2008.11.013

activephenoloxidase(PO)whichthencatalysesoxidationofphenoliccompoundsthatpolymerisetoformmelanin[5].Cytotoxicmolecules,includingquinonesandreactiveoxygenintermediatesgeneratedduringthemelanizationprocess,arepresumedtoberesponsiblefortheparasitoiddeath[6].Thesemoleculesbeingharmfulforthehosttissuesaswell,theactivationofthePOcascade(andsubsequentmelanization)isthustightlyregulatedbyanarrayofserineproteasesinhibitorsthatbelongtotheserpinsuperfamily[7].

Parasitoidshaveevolvedvariousstrategiestoescapetheencapsulationprocess,rangingfromdisplayingsurfacefeaturesthatpreventtheirrecognitiontoalteringspeci?ccomponentsofthehostimmunesystem[1,8].ReductionofPOactivityisoneofthemostlargelydescribedstrategy,althoughsofarreportedalmostexclusivelyinbraconidandichneumonidspecies(withtheexceptionoftheEulophidaeEulophuspennicornis)[9–17].How-ever,itisgenerallyunknownwhetheritisthePOitselforitsactivationcascadewhichisinhibitedbyparasitoids.Besides,onlytwovirulencefactorsresponsibleforreductionofhostPOactivityhavebeenidenti?edsofarinparasitoids:Vn50,aserineproteasehomolog,inCotesiarubecula[14],andEgf1.0,aserineproteaseinhibitorofthesmapinfamily,expressedbythepolydnavirusofMicroplitisdemolitor[16].Interestingly,bothofthesefactorsinhibitactivationofproPOintoactivePO[16,17].

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ThecynipoidwaspLeptopilinaboulardiparasitizesthesecondlarvalstageofsomeDrosophilaspeciesbelongingtothemelanogastersubgroup,includingD.melanogasterandD.yakuba[18].WerecentlydemonstratedthattheISmlineofthisparasitoidinjectsamajorimmunesuppressivefactor,aRacGAP(RacGTPaseActivatingProtein;LbGAP)topreventencapsulationofitseggsbyD.melanogaster[19].LbGAPaltersthelamellocytemorphologybyenteringthesecellsandtargetingDrosophilaRac1andRac2[20,21].TheISylineofL.boulardidiffersfromtheISmlineinitsabilitytosuccessfullyparasitebothD.melanogasterandD.yakubahosts:whileISmsucceedsitsparasitismonlyonD.melanogaster,ISycansucceedonbothspecies,butdependingontheirresistant/susceptiblegenotype[22].AnotherstrikingdifferenceisthatISyparasitoidssuccessisassociatedneitherwithchangesinhostlamellocytemorphologynorwiththepresenceofhighLbGAPquantitiesinthevenom.However,parasitismbyISyfemalesorinjectionofISyfemalevenombothdelayencapsulationofforeignbodiesinD.yakuba,suggestingthatimmunesuppressivefactorsotherthanLbGAParepresentinISyvenom[23].

Inthispaper,weshowthatthevenomofISyL.boulardifemalesinhibitsproPOactivationinthehemolymphofD.yakubalarvaeandthatitcontainsaserineproteaseinhibitoroftheserpinsuperfamily,namedLbSPNy.TheLbSPNy-encodinggeneisspeci?callyoverexpressedinvenomglandsandarecombinantLbSPNyproteinreproducestheinhibitionofthePOcascadeobservedwiththevenom.Altogether,theseresultsdemonstratethatLbSPNyactsasavirulencefactorbytargetingthehostmelanizationprocess.2.Materialsandmethods2.1.Insects

TheISylineofL.boulardi(Gifstock,no.486)hasbeendescribedpreviously[22,23].Brie?y,itderivesfromasinglefemaleoriginatingfromamasscultureofaBrazzaville(Congo)populationandwasrearedonasusceptibleD.melanogasterstrain(Gifstockno.1333)at258C.TheD.yakubareferencestrainusedduringtheexperimentsistheisofemaleline1880-D[22],whichwasraisedat258C.

2.2.Venomglandextracts

L.boulardifemalevenomglands,correspondingtovenomglandsandtheirassociatedreservoirs,weredissectedeitherinsodiumcacodylatebuffer(cacodylicacid10mM;CaCl2,2H2O5mM;pH7)forPOactivityassays,orinRinger’ssaline(KCl182mM;NaCl46mM;CaCl23mM;Tris–HCl10mM)forproteinanalysis.Sampleswerethencentrifugatedbrie?yat12,000ÂgtoremovecellulardebrisandthesupernatantwaskeptoniceuntilbeingusedforPOactivityassaysorkeptatÀ208Cforproteinanalysis.Proteinconcentrationwasdeterminedspectrophotome-tricallyusingtheBradfordmethod.

2.3.POactivitymeasurementsandmelanizationassays

Phenoloxidaseactivitywasassayedusingamethodmodi?edfromMoreauetal.[11].Foreachreplicate,thewholehemolymph(i.e.,plasmaandhemocytes)of?veD.yakubalarvaeagedof96hÆ2hwascollecteddirectlyinamicroplatewell(Nunc-ImmunoPlate1FP96Maxisorp,NUNC)in12mlcacodylatebuffercontainingvenomorrecombinantproteinstobetestedattheappropriateconcentration.After45minofincubationat378C,duringwhichproPOisconvertedintoPO,thesubstrate(50mlof5mML-DOPApreparedincacodylatebuffer)wasadded.Theplatewasthenincubated1hat218C,andthePOactivitylevelwasestimated

spectrophotometricallybyrecordingat490nmthequantityofdopachromesformedfromtheconversionofL-DOPAbythePOenzyme.Atleastthreereplicatesweremadeforeachtreatment.Todeterminewhetherabsorbancevaluesdifferedsigni?cantlyaccord-ingtothetreatment,ANOVAanalyseswereperformed,followedbypairwisecomparisonsusingFisher’sLeastSigni?cantDifference(LSD)test(Software:Systast10,SPSSInc.,Chicago,IL).

TheeffectofvenomonPOactivitywasassayedbycomparingtheabsorbancevaluesobtainedwithhemolymphcollectedincacodylatebufferaloneorinpresenceofISyvenom(0.1g/lor0.3g/l),BSA(0.3g/l)orPTU(0.01%1-phenyl-2-thiourea),aninhibitorofPOandperoxidase.TodeterminewhetherthevenomaffectsthePOenzymeitselforitsactivation(byserineproteases),theabsorbancevalueswerecomparedbetweenhemolymphsamplescollectedinpresenceofISyvenom(?nalconcentration0.1g/l)orinwhichthevenomwasaddedafterthe45minincubationperiodthatfollowshemolymphcollection.HemolymphsamplescollectedinPTU(?nalconcentration0.01%),inwhichPTUwasaddedafterthe45minincubationperiod,orcollectedincacodylatebufferalone,wereusedascontrols.Hemolymphwascollectedin6mlvolumeand6mlwereaddedaftertheincubationperiodtoensuresimilar?nalhemolymph,venomandPTUdilutionsinthedifferenttreatments.

TheeffectoftherecombinantserpinLbSPNyproducedasafusionwithglutathioneS-transferase(GST-LbSPNy;seebelow)onPOactivitywastestedbycomparingtheabsorbancevaluesobtainedwithhemolymphofthreelarvaecollectedincacodylatebufferaloneorinpresenceoftheGST-LbSPNyfusionprotein(0.3g/l),GST(0.3g/l),orPTU(0.01%).

TherecombinantLbSPNywitha6xHis-tagattheN-terminus(His-LbSPNy;seebelow)beingproducedmoreeasilythanGST-LbSPNy,theeffectsofLbSPNyonmelanizationweretestedbyinjecting20nlof0.5g/lsolutionofHis-LbSPNyinRingersolutioninthreedaysoldlarvae.ControlswerelarvaeinjectedwithvenomorBSAatthesameconcentration.InjectionswereperformedusingaNanojectIIinjector(DrummondScienti?cCompany,Broomall,PA).ThehemolymphfromBSA,His-LbSPNyorvenominjectedlarvae(48,48and24larvaepersample,respectively)wascollectedindividually30minlaterin10mlofRingersolutioncontaining0.2g/loflaminarin,anactivatorofthePOcascade,andincubatedfor2hatroomtemperature.Sampleswerethencategorizedbasedonobservationofthemelanizationlevel:nomelanization,weakmelanizationorstrongmelanization.2.4.Proteinicanalysisofvenomglands

Theproteincontentof130L.boulardivenomglands(80mgofproteins),wasanalysedbynative-PAGEelectrophoresis(7%polyacrylamidegel,8h,48C,25mA)andstainedwithCoomassieblueG-250.Thetwomostintensebandswereexcised,incubatedinLaemmlibufferfor10minat558C,andthenanalysedbySDS-PAGE(10%polyacrylamidegel,4h,40mA).AfterstainingwithCoomassieblueR-250,thebandsobservedwerecomparedwiththemigrationpatternofextractsof40venomglands(24mgofproteins).

ThenatureoftheproteinicbandsintheSDSpro?lesofthetwomajornativebandswasassessedusingmatrixassistedlaserdesorption/ionizationtime-of-?ight(MALDI-TOF)massspectro-metry.Brie?y,thebandswereexcisedandsubmittedtoin-geltrypticdigestion,followingtheprotocolofBelghazietal.[24].Priortomassspectrometryanalysis,proteolyticdigestsweretreatedonC18ZipTips(Millipore)fordesaltingandremovalofresidualacrylamide.Partialamino-acidsequenceoftrypticpeptideswasthenobtainedusingmassspectrometryandadatabasesearchwasperformedtoidentifythecorrespondingproteins(http://dove.embl-heidelberg.de/Blast2/).

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2.5.CloningandmolecularanalysisofLbSPNy

TotalRNAwasextractedfrom100L.boulardiISyvenomglandsusingTRIzolReagent(Invitrogen)accordingtomanufacturer’sinstructions.AcDNAlibrarywasthenconstructedusing1mgoftotalRNAandtheCreatorSMARTcDNALibraryConstructionKit(Clontech),accordingtomanufacturer’sinstructions.Atotalof120librarycloneswasrandomlyanalysedtoobtaintheentiresequenceofLbSPNy.

DatabasesearcheswerethenperformedusingBLASTPatNCBI(http://www.ncbi.nlm.nih.gov/blast/).Occurrenceandpositionofsignalpeptidecleavagesites,N-glycosylationsitesandphosphor-ylationsitesintheLbSPNycompletecDNAsequencewerepredictedusingtheCBSpredictionservers(http://www.cbs.dtu.dk/services/).Pairwisesequencecomparisonswereper-formedusingtheEMBOSSprogramNeedleatEMBL-EBI(http://www.ebi.ac.uk/emboss/align/).MultipleaminoacidsequencealignmentswereperformedusingMUSCLE[25],andphylogeneticanalysiswasconductedusingPhyML[26]atLIRMM(http://www.phylogeny.fr/).

2.6.MolecularmodelingofLbSPNy

TheserpindomainofLbSPNywasmodeledbyhomologyusingSwissModel[27]withnativeproteinCinhibitorstructureastemplate[28].ThemodelqualitywasassessedbyWhatcheck[29]andProcheck[30].AssignmentofsecondarystructureandpredictionofsolventaccessibilitywereperformedusingDSSPprogram[31].Visualizationofstructures,structuralalignmentsandroot-mean-squaredeviations(RMSD)weredoneusingPymol(http://wendang.chazidian.com/).

2.7.ProductionofrecombinantproteinsinEscherichiacoli

ForproductionofGST-LbSPNy,acDNAfragmentcorrespondingtothematureLbSPNyproteinwasampli?edbyPCR(948C,2min;5cycles:948C15s,558C30s,728C90s;25cycles:948C15s,658C30s,728C90s;728C2min)usingthePwoSuperYieldDNAPolymerase(Roche)andthespeci?cprimers50-CCAGGAATTCTG-TATGGTAACGTAAATTACTTTAG-30and50-GCCGCTCGAGTTACTG-TATATGTTTGACGTTACCA-30.Theampli?edfragmentwasclonedintothepGEX-4T-2vector(AmershamBiosciences)http://wendang.chazidian.competentBL21E.colicellsweresubsequentlytransformedwiththerecombinantplasmid.TheproductionoftheGST-LbSPNyfusionproteinandGSTalonewasperformedaccordingtotheGSTGeneFusionSystemHandbook(AmershamBiosciences).Thefusionproteinwasthenpuri?edusingglutathione-Sepharosebeads(AmershamBiosciences).ForPOactivityassays,GST-LbSPNyfusionproteinandGSTaloneweretransferredincacodylatebufferusingaMicroconYM-10membrane(Millipore).

ForproductionofHis-LbSPNy,thefragmentcorrespondingtothematuresequencewasampli?edusingthespeci?cprimers50-CGTCAGGAATTCGCCTTGGAAAAATTCAATAACGAC-30and50-GCCGCTCGAGTTACTGTATATGTTTGACGTTACCA-30andclonedinthepET-28a(+)vector(Novagen).TheproductionofHis-LbSPNywasperformedaccordingtothepETSystemManual(Novagen).Therecombinantproteinwasthenpuri?edusingNiSepharoseFastFlowbeads(GEHealthcare)andtransferredinRingersolutionusingaMicroconYM-10membrane(Millipore).2.8.Quantitativereal-timePCR

TotalRNAwasisolatedeitherfrom50dissectedvenomglandsorfrom50residualfemalebodies(withoutvenomglands)andreverse-transcribedusingtheiScriptcDNASynthesisKit(BioRad).

qPCRreactionswerethencarriedoutonanOpticonmonitor2(BioRad)usingtheAbsoluteqPCRSYBRMasterMixPlusforSYBRGreenINoROX(Eurogentec)andthespeci?cprimers50-CGCATTACAGATGGAAATCTTTC-30and50-GATTTCATCGATCT-GAACCCT-30.PCRconditionswereasfollows:508C,2min;958C,10min;40cycles:958C30s,608C30s,688C30s.Eachreactionwasperformedintriplicateandthemeanofthreeindependentbiologicalreplicateswascalculated.AlldatawerenormalizedusingtheITS2(InternalTranscribedSpacer2)ribosomalgeneasacontrolandresultswerecalculatedusingtheDCtmethod.3.Results

3.1.ThevenomofL.boulardiinhibitsPOactivation

TheeffectofL.boulardivenomonPOactivityofD.yakubalarvaewasassessedusingspectrophotometricassaysonhosthemo-lymphsamples.Highabsorbancevalueswereobservedinsamplescollectedincacodylatebufferalone,indicatingthatDrosophilalarvaehemolymphcollectedusingstandardconditionshasanelevatedspontaneousPOactivitylevel.CollectionofhemolymphincacodylatebuffersupplementedwithPTU,ledtoa80to98%reductionofabsorbance(Fig.1A).Thissuggeststhat

the

Fig.1.(A)EffectofISyvenomonPOactivityofD.yakubalarvaehemolymph.Hemolymphwascollectedincacodylatebufferalone(–),inpresenceofvenomextracts(concentration0.1g/lor0.3g/l),inpresenceofaPOinhibitor(0.01%PTU),orinpresenceofBSA(concentration0.3g/l).Absorbancewasmeasuredat490nm1hafteradditionofL-DOPA.Meanvalues(ÆSE)aregivenforeachcategoryandnumberswithinbracketsindicatenumbersofreplicates.(B)EffectofISyvenomonPOactivation.HemolymphofD.yakubalarvaewascollectedincacodylatebuffer(CB)alone(–,VafterandPTUafter),inpresenceofvenomextracts(Vbefore),orinpresenceofaPOinhibitor(PTUbefore).After45minofincubationat378C,cacodylatebuffer(–,VbeforeandPTUbefore),venom(Vafter)orPTU(PTUafter)wasaddedandabsorbancemeasuredat490nm1hafteradditionofL-DOPA.Meanvalues(ÆSE)aregivenforeachcategoryandnumberswithinbracketsindicatenumbersofreplicates.

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Fig.2.(A)NativePAGEpro?leofvenomextractsfromtheISylineofL.boulardi.Venomglandproteins(80mg)wereseparatedona8%native-PAGEandstainedwithCoomassieblueG-250(migration4h,40mA,48C).Fivemajorbandswereidenti?ed(N1toN3–2).BandsN2–1andN2–2weresoclosethattheywereconsideredassinglebandforfurtheranalyses.(B)SDS-PAGEpro?leofN1andN2bandscutandelutedfromthenativegelshowninA.Proteinswereseparatedona10%SDS-PAGEandcomparedwithproteinsofvenomglandextracts(24mg).GelwasstainedwithCoomassieblueR-250(migration4h,40mA).Py1aandPy1bindicatetheproteinicbandsresultingfromthemigrationoftheN1nativeband.Py2aandPy2bresultfromtheN2band.MW:Molecularweightmarker(NewEnglandBiolabs).

productionofdopachromesrecordedbymeasuringabsorbanceismainlyduetoPOactivity.

ThepresenceofISyfemalevenomextractssigni?cantlydecreasedtheabsorbancemesuredinD.yakubahemolymphsamples(Fig.1A):50%inhibitionwasobservedwith0.1g/lofvenom(P<0.001)and75%inhibitionwith0.3g/lofvenom(P<0.001).Therewasnostatisticaldifferencebetweenhemo-lymphsamplescollectedinpresenceofvenomattheconcentra-tionof0.3g/landsamplescollectedinpresenceofPTU(P=0.216).Noinhibitioncouldbedetectedwhencollectinghemolymphinbuffercontaining0.3g/lBSA(P=0.109),thusdemonstratingthatinhibitioneffectsareduetovenomcomponentsandnottothesimpleadditionofproteins.

Toinvestigatewhetherthereductionofabsorbancere?ectedtheinhibitionofthePOitselfortheinhibitionofitsactivation,weperformedparallelexperimentsinwhichcacodylatebufferwassupplementedwithparasitoidvenom,cacodylatebufferorPTU,eitherbeforehemolymphcollectionorafterPOactivationbyincubationofhemolymphfor45minat378C(Fig.1B).Asexpected,therewasnodifferencebetweensamplescontainingPTU(P=0.594),thePOactivitybeingstronglyreducedinbothsamples.Bycontrast,ahighlysigni?cantdifferencewasfoundbetweenvenom-containingsamples(P<0.001).Incomparisontothecacodylatecontrol,a2-foldreductionofPOactivitywasobservedforhemolymphsamplescollectedinvenom(P=0.002)butnotwhenvenomwasaddedaftertheincubationperiod(P=0.153).Thisindicatesthatvenomcomponentsaffectoneor

severalstepsleadingtotheactivationofthePOenzyme,butnotthePOactivityitself.

3.2.ThevenomofL.boulardicontainsprotein(s)oftheserpinfamilyAsmallnumberofbandswereobservedfollowingnativeproteinelectrophoresisofL.boulardiISyvenomglands,aspreviouslyreported[19].Amongthe?vebandsvisualized,N1,N2–1andN2–2wereintenselycoloredcomparedtoN3–1andN3–2(Fig.2A).TheN2–1andN2–2bandscouldnotbeeasilyseparated,evenatalowergelconcentration(datanotshown)andwerethuslaterconsideredasasingleband,N2.SDS-PAGEelectrophoresiswasthenperformedforthetwomostintensenativebands,N1andN2,excisedfromthenativegel.Eachbandwasresolvedintotwobandsofclosemolecularweight:Py1aandPy1b(53and52kDa,respectively)forN1,andPy2aandPy2b(51and50kDa,respectively)forN2(Fig.2B).

Followingexcisionfromthegel,Py1a,Py1b,Py2a,andPy2bweredigestedwithtrypsin,andapeptidespectrumwasgeneratedbyMALDI-TOF.Ofthe14peptidesequencesobtained,sevenwereobservedinatleasttwobands,andoneofthem,TPLPTV[SD][RK]LNGK,wasfoundinthefourproteinicbands.Thissuggestedthatthefourbandscontainedeitherthesameproteinorrelatedproteins.Databasesearchesidenti?edtwopeptides,NGAEAAAASVAVLGFRandVVLTNAIYFKGEWK,asbelongingtoproteinsoftheserpinfamily.Py1aandPy2bbandseachcontainedoneofthesetwo

peptides.

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Fig.3.MolecularanalysisandmodelingofLbSPNy.(A)StructureandorganizationofLbSPNyprotein.Signalpeptide,serpindomainandreactivecenterloop(RCL)areshowningrey,whiteandmagentarectangles,respectively.(B)TertiarystructureofLbSPNyserpindomainintheclassicorientationwiththeRCLontopandb-sheetAfacing,andcomparisonwiththenativestructureoftheprototypicalserpina1-antitrypsin[57].b-sheetsA,BandCarecoloredinred,greenandyellow,respectively.Theninea-helicesarecoloredinblue.TheRCLiscoloredinmagenta,withtheP1residueshownasasphere.(Forinterpretationofthereferencestocolorinthis?gurelegend,thereaderisreferredtothewebversionofthearticle.)

AcDNAlibrarywasthenconstructedfromtotalRNAextractedfromISyvenomglands.Ofthe120bacterialclonessequenced,?veoverlappingclonesencodedproteinssharingsimilaritieswithserpins.Insertswere487bpupto1627bplong,withonlyfewnucleotidesandnoaminoaciddifferences.ThelongestcontainedalargeORFencodinga411aminoacidsprotein(GenBankaccessionnumber1118746),withapredictedsizeof46kDaandanisoelectricpointof7(Fig.3A).ThisproteinwasnamedLbSPNy.AllbutthreepeptidesgeneratedbyMALDI-TOFMSfromPy1a,Py1b,Py2aandPy2bproteinicbandsmatchedwiththeLbSPNyaminoacidsequencepredictedfromthe1627bpcloneandthepeptidescoveredallpartsofthesequence(23.35%coverage).Thiscon?rmedthatN1andN2nativebandsbothcorrespondtoLbSPNy.3.3.AnalysisofLbSPNy

TheLbSPNyproteincontainsapredictedN-terminalsignalpeptideof22aminoacids,followedbyaputativeserpindomainrangingfromaminoacids42to407(Fig.3A).FourN-glycosylationsitesand13phosphorylationsiteswereidenti?edasputativepost-translationalmodi?cationsandpredictedtobeaccessibletothesolvent.Thepresenceofthesepost-translationalmodi?cationsitesmightexplainwhydifferentbandsareobservedforLbSPNyonnativegels.

Serpins(serineproteaseinhibitors)arealargefamilyoffunctionallydiverseproteaseinhibitors.Theyshareaconservedstructuralarchitectureanduseaconformationalchangetoinhibittargetserineproteases[http://wendang.chazidian.comingtheSwissModelFirstApproachModeandsimilaritieswithproteinCinhibitor(thecloseststructurallycharacterizedserpin)asasupport,weconstructeda3DmodelfortheserpindomainofLbSPNy(Fig.3B).Theoverallstructuralorganizationwasthatofatypicalserpinwithnineahelicesandthreebsheetsconstitutingtheconservedserpinarchitecture[32].Thesuperpositionoftheprototypicalserpin,a1-?for291a-carbonatoms,antitrypsin,resultedinaRMSDof1.891A

revealingstrongsimilaritiesbetweenbothstructures.Incontrast,signi?cantdifferenceswereobservedinthepredictedC-terminalextendedreactivecenterloop(RCL),whichisresponsibleforinteractionwiththeactivesiteoftargetserineproteases[32].P1,P2andP3residues,locatedinthishypervariableloop,areknowntobeimportantforproteasespeci?cityofserpins[32].TheLbSPNyproteinhasanArgatthepredictedP1site,suggestingthatitmayinhibitserineproteaseswithtrypsin-likespeci?city[32].

Phylogeneticanalyseswereperformedusing(i)insectserpinsknowntobeinvolvedinthemelanizationprocessand(ii)theclosestserpinsisolatedorpredictedininsectspecieswhosecompletegenomehadbeenobtained(Anophelesgambiae,Bombyxmori,D.melanogaster,Triboliumcastaneum).AlltheserpinspredictedinthetwosequencedgenomesofHymenoptera(ApismelliferaandtheparasitoidNasoniavitripennis)werealsoincludedintheanalysis.Lookingattheentireserpindomain,LbSPNyisnotcloselyrelatedtoanyotherserpin(Fig.4A)butitgroupswith11insectserpinsincluding5sequencesofHymenopteraandonesequenceofaserpinknowntobeinvolvedinmelanization,serpin-1JfromM.sexta[33].Interestingly,alignmentofRCLsequencesofLbSPNyandinsectserpinsthatinhibitthePOcascadeshowsaperfectconservationofP3-P10residuesbetweenLbSPNyandserpin-6ofM.sexta(Fig.4B),suggestingthattheseproteinsmightsharesimilarfunctions.Incontrast,P2andP3residues,aswellasP10,arenotconservedbetweenLbSPNyandserpin-1J(Fig.4

B).