Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut

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ContentslistsavailableatSciVerseScienceDirect

Veterinary

Parasitology

j our na l h o me p ag e:w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Expression

and

functional

characterization

of

boophilin,

a

thrombin

inhibitor

from

Rhipicephalus

(

Boophilus

)

microplus

midgut

Tatiane

Sanches

Soares

a

_,

_Renata

_Midori

_Okuta

_Watanabe

a

_,

_Anita

_Mitico

_{Tanaka-Azevedo}

b

_,

Ricardo

José

Soares

Torquato

a

_,

_Stephen

_Lu

a

_,

_Ana

_Carvalho

_Figueiredo

c

_,

Pedro

José

Barbosa

Pereira

c

_,

_Aparecida

_S.

_Tanaka

a,∗

a_Departamento_de_Bioquímica,_Escola_Paulista_de_Medicina,_Universidade_Federal_de_São_Paulo,_Rua₃_de_Maio_100,_04044-020_São_Paulo,_SP,_Brazil

b_Laboratório_de_{Fisiopatologia,}_Instituto_Butantan,_São_Paulo,_SP,_Brazil

c_IBMC_-_Instituto_de_Biologia_Molecular_e_Celular,_Universidade_do_Porto,_4150-180_Porto,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17November2011

Receivedinrevisedform17January2012 Accepted22January2012

Keywords:

Kunitz-typeinhibitor Tick

Anticoagulant Elastaseinhibitor RNAisilencing

a

b

s

t

r

a

c

t

Rhipicephalus(Boophilus)microplusisanectoparasiteresponsibleforanimportantdecrease inmeat,milkandleatherproduction,causedbothbycattlebloodlossandbythe trans-missionofanaplasmosisandbabesiosis.R.microplusisarichsourceofserineprotease inhibitors,includingthetrypsininhibitorsBmTI-AandBmTI-6,thesubtilisininhibitorBmSI, andtherecentlydescribedthrombininhibitor,boophilin.Boophilinisadouble Kunitz-typethrombininhibitor,withtheunusualabilitytoformaternarycomplexwithasecond (non-thrombin)serineproteinasemolecule.Thelarge-scaleexpressionandpurificationof boophilinandofitsisolatedN-terminal(D1)domaininPichiapastoris,itsexpression pro-file,andtheeffectofRNAi-mediatedgenesilencingintickeggproductionarereported. Full-lengthboophilinandD1wereexpressedat21and37.5mg/Lofculture,respectively. Purifiedboophilininhibitedtrypsin(Ki0.65nM),neutrophilelastase(Ki21nM)andbovine

thrombin(Ki57pM),whileD1inhibitedtrypsinandneutrophilelastase(Ki of2.0and

129nM,respectively),butnotthrombin.BoophilingenesilencingusingRNAiresultedin 20%reductionineggweightproduction,suggestingthattheexpressionofboophilininthis lifestagewouldbeimportantbutnotvital,probablyduetofunctionaloverlapwithother serineproteinaseinhibitorsinthemidgutofR.microplus.Consideringourdata,Boophilin couldbecombiningwithotherantigeninavaccineproductionfortickcontrol.

1. Introduction

The cattle tick Rhipicephalus (Boophilus)microplus is widely distributed in tropical and subtropical regions, being responsible for the transmission of thecausative agentsofbabesiosisandanaplasmosis,withasignificant

Abbreviations: D1,boophilindomain1;D2,boophilindomain2; Boophilin,full-lengthboophilin(domains1–2).

∗ Correspondingauthor.Tel.:+551155764445;fax:+551155723006. E-mailaddress:[email protected](A.S.Tanaka).

economicimpactincattleproductionbyreducingweight gainandmilkproduction(Sonenshine,1991).

Bloodsuckinganimalsproduceaconsiderablenumber ofactivemoleculesintheirsalivaryglands(e.g. anticoagu-lants,vasodilatorsandplateletaggregationinhibitors)that interferewithhomeostasisintheirvertebratehosts.In par-ticular,thesehaematophagousparasitesvitallydependon blockingthebloodcoagulationcascadeinordertofacilitate theacquisitionanddigestionoftheirbloodmeal(Ribeiro, 1995).Thrombin(orcoagulationfactorIIa)playsavitalrole inbloodclottingbypromotingplateletaggregationandby convertingfibrinogentofibrinattheendofthepathway (Davieetal.,1991).Thrombinisaserineprotease,which

Open access under the Elsevier OA license.

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contains two functionallyimportant structural features, besidestheactivesite:thesurfaceareasenrichedinbasic residuesknownasexositeI(Bodeetal.,1992)andexosite II(Arnietal.,1994;Sheehanetal.,1993).Asthrombinhas keyrolesintheintrinsicandextrinsicpathwaysofblood coagulation,thrombininhibitorsarethemostoften iden-tifiedanticoagulantmoleculesinbloodsuckingorganisms (Francischettietal.,2008).

Amongblood suckinganimals, ticksarerich sources of serine protease inhibitors, many of them belonging to the BPTI-Kunitz family (Azzolini et al., 2003; Mans etal.,2008;Sasakietal.,2004),suchasBmTIs(Boophilus

microplustrypsininhibitor)fromlarvaeand eggs,which

targettrypsin,chymotrypsin,neutrophilelastase,plasma kallikreinandplasmin(Andreottietal.,2001,2002;Sasaki etal.,2004;SasakiandTanaka,2008;Tanakaetal.,1999); thetickanticoagulantpeptide(TAP),afactorXainhibitor

fromOrnithodoros moubata (Waxman et al., 1990); and

thethrombin inhibitors ornithodorin (from O. moubata

(vandeetal.,1996)),savignin(fromOrnithodorossavignyi

(Mansetal.,2002b)),monobin(fromArgasmonolakensis

(MansandRibeiro,2008))andboophilin(fromR.microplus

(Macedo-Ribeiro et al., 2008)). Boophilin is a doubled-headedKunitzinhibitordisplayingaP1Lysresidueatthe canonicalreactiveloopofitsN-terminalKunitzdomain. However,boophilininhibitsthrombininanon-canonical manner,insertingitsN-terminalsegmentintothrombin’s activesite,whileitsC-terminalKunitzdomainbindstothe exositeIoftheprotease(Macedo-Ribeiroetal.,2008).

GiventheimportantroleofKunitz-typeinhibitorsinthe

R.micropluslifecycleandthehighspecificityofboophilin

forthrombin,weexpressedandpurifiedfull-lengthmature boophilinanditsN-terminalKunitzdomaininlargescale usingaPichiapastorissystem.Wealsoprofiledboophilin gene expression and evaluated theeffect of RNAi gene silencingintickeggproduction.

2. Materialsandmethods

2.1. Animals

R.(Boophilus)microplus(Babesiaspp.-free)tickswere

suppliedby Dr.Itabajarada SilvaVazJunior(Centrode Biotecnologia,UniversidadeFederaldoRioGrandedoSul, RS,Brazil).

2.2. Materials

ThepPICZ␣BvectorandP.pastorisstrainKM71Hwere purchasedfromInvitrogen(Carlsbad,CA,USA)andused following the supplier’s instructions. DNA sequencing wasperformedusingtheBigDye TerminatorV3.1Cycle Sequencing Kit on an ABI 377 or ABI 3130 sequencer (AppliedBiosystems,FosterCity,CA,USA).Thesubstrates S2484 (Pyro-Glu-Pro-Val-pNa) and S2238 (HD-Phe-Pip-Arg-pNa),werepurchased from Chromogenix(Molndal, Sweden)and tosyl-Gly-Pro-Arg-pNAfromSigma (Darm-stadt,Germany).Bovinetrypsin(EC3.4.21.4)andbovine thrombin(EC3.4.21.5)wereobtainedfromSigma(St.Louis,

MO, USA)and humanneutrophilelastase(EC3.4.21.37) fromCalbiochem(SanDiego,CA,USA).

2.3. QuantitativeReal-TimePCR

RNAfromovary,fatbody,salivarygland,gut,and hemo-cytesofengorgedR.microplusadultfemaleswasextracted usingTryzolReagent(Invitrogen,Carlsbad,CA,USA) fol-lowingthemanufacturer’sinstructions.ThecDNAswere synthesizedusingtheImProm-IITM_Reverse_{Transcription} System(Promega,Madison,WI,USA).QuantitativePCRwas performedusingtwospecificprimersdesignedbasedon theboophilinsequencewithGenBankaccessionnumber AJ304446:Boophilinfw(5′_-CAG_AGA_AAT_GGA_TTC_TGC_CGA CTGCCGGCA-3′₎_and_Boophilinrev₍₅′_-ACA_CTC_CTC_TAT GGT CTCGAA-3′_)._R._microplus_elongation_factor_1-alpha (ELF1a)specificprimers–ELF1afw(5′_-CGT_CTA_CAA_GAT TGGTGGCATT-3′₎_and_ELF1arv₍₅′_-CTC_AGT_GGT_CAG_GTT GGC AG-3′₎_– _were_used _for _DNA _{amplification}_control. Boophilin andELF1a (endogenouscontrol)were quanti-fiedusing SYBR® _Green_PCR _Master_Mix _in_a ₇₅₀₀_Fast Real-Time PCR System(AppliedBiosystems, FosterCity, CA,USA),accordingtothefollowingconditions:40cycles of95◦_C_for₁_min,₆₀◦_C_for₁_min_and₇₂◦_C_for₁_min._For eachtissue,50ngofcDNAwereusedastemplate.AllqPCR runswereconductedin triplicate,in threeindependent experiments.Theamountof eachmRNAwascalculated accordingtothe2-DDCtmethod(Livakand Schmittgen, 2001).ANOVA(p<0.05)andtheTukeytestwereusedin thestatisticalanalysis.

2.4. CloningofboophilinandD1intopPICZ˛B

The DNA fragments encoding boophilin or D1 were amplifiedbyPCRusing amidgutcDNApreparation and theprimersetBoophifw1std(5′_-GTA_TCT_CTC_GAG_AAA AGACAGAGAAATGGATTCTGCCGACTGCCGG-3′₎_and Boophirv2ndd(5′_-CGA_ATT_AAT _TCG _CGG_CCG_CCT_ACA TGTTCTTGCAGACGAGTTCACAC-3′₎_for_boophilin_and Boophifw1stdandBoophirv1std(5′_-CGA_ATT_AAT_TCG_CGG CCGCCTAAGCTCCGCACGCCTTTTGACAATC-3′₎_for_D1. PCRreactionswereconductedinafinalvolumeof50␮Lin 100mMTris–HClpH8.8,500mMKCl,0.8%(v/v)Nonidet P40,1.5mMMgCl2,100␮MdNTPs,10pMofeachprimer, 5UTaqDNApolymerasewiththefollowingparameters: 94◦_C_for₂_min,_prior_to₃₀_cycles_of₉₄◦_C_for₄₅_s,₅₅◦_C for 45s and72◦_C _for ₁_min_followed _by ₅_min_at ₇₂◦_C. Boophilin and D1 DNA fragmentamplificationproducts wereseparatedbyagarosegelelectrophoresisandpurified usingtheQIAEXIIgelextractionsystem(QIAGEN,Hilden, Germany) accordingto the manufacturer’s instructions. PurifiedDNAfragmentsweredigestedwithXhoIandNotI restrictionenzymes,andligatedintothepPICZ␣Bvector, previouslydigestedwiththesameenzymes,generatingthe constructionsBoophilin-pPICZ␣BandD1-pPICZ␣B,which wereverifiedbyautomatedDNAsequencing.

2.5. PreparationandtransformationofP.pastorisyeast

P.pastorisKM71Hstrainwastransformedwith10␮g

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electroporationina Gene Pulser(Bio-Rad, Hercules,CA, USA)followingthemanufacturer’sinstructions.The eletro-poratedcells wereimmediatelysuspended in 1.0mL of ice-cold1.0MsorbitolandspreadonMDagarplates(1.34% yeastnitrogenbase(YNB),2%dextrose,4×10−5_%_biotin) without histidine. The target gene wasdetected in the recombinant P.pastoris by PCR using 3′_AOX _and ₅′_AOX primers(Invitrogen,Carlsbad,CA,USA).Clonesthatwere homologousrecombinantswiththeAOXIsequencewere selected.

2.6. ExpressionofrecombinantBoophilinandD1

To identify positive yeast clones expressing each of theinhibitors,sixisolatedP.pastorisKM71Hstrains car-rying the boophilin or D1 gene fragment, identified by PCR,wereindividuallyinoculatedin2.5mLBMGYmedium (1.0%(w/v)yeastextract,2.0%(w/v)peptonein100mM potassium phosphate buffer pH 6.0, 1.34% (w/v) YNB, 4×10−5_%_(w/v)_biotin_and _1%_(v/v)_glycerol)_in_a ₅₀_mL steriletube,andgrownat30◦_C_for₂₈_h_at₂₅₀_rpm._The yeastcells wereharvested bycentrifugationat3000×g

for5minat4◦_C_and_resuspended_in_BMMY_(BMGY_with glycerolreplacedby0.5%(v/v)methanol)mediumtoan absorbanceof1.0at600nm.Expressiontookplaceat30◦_C withshakingat250rpmfor4days,withadditionof0.5% (v/v) methanol every24h. After removing the cells by centrifugation(4000×gfor20minat4◦_C),_the_inhibitory activityof thesupernatantsagainst bovinethrombin or bovine trypsin was assessed in activity assays using a chromogenicsubstrate(S2238ortosyl-Gly-Pro-Arg-pNA). Individualcloneswithhighexpressionlevelsforboophilin orD1wereselected(datanotshown).

AsingleP.pastoriscolony(Mut+)expressinghigh lev-elsofboophilinorD1wasselectedandusedtoinoculate 120mLBMGYmediumina1Lsterileflask,andincubated at30◦_C_and₂₅₀_rpm_for₂₄_h._Expression_was_performed_as describedaboveandtheculturesupernatantwasstoredat 4◦_C_prior_to_{purification.}

2.7. PurificationofrecombinantBoophilinandD1

RecombinantboophilinorD1-containingyeastculture supernatantwasloadedontoanaffinitytrypsin-Sepharose column previously equilibrated with 50mM Tris–HCl buffer pH 8.0 (buffer A). Weakly bound proteins were washedoutwithbufferAsupplementedwith0.15MNaCl. Theboundmaterialwaselutedwith0.5MKClpH2.0and thecollectedfractionswereimmediatelyneutralizedwith 1MTris–HClbufferpH8.0.Absorbanceat280nmwasalso monitored.Theinhibitoryactivityofthefractionswas ana-lyzedinproteaseactivityassays(seebelow).Thefractions containinginhibitoryactivityanddisplayingonemain pro-teinbandinSDS-PAGEwerepooledandconcentratedusing a5000MWCOmembrane(Millipore,Billerica,MA,USA).

2.8. Determinationofdissociationconstantfordifferent

serineproteases

The concentration of active trypsin was determined by activesite titrationwithp-nitrophenyl-p′ -guanidino-benzoate as previously described (Chase and Shaw,

1969). The equilibrium dissociation constants of com-plexesformedbyboophilinorD1withbovinetrypsinor neutrophilelastase were determinedusing the method described by Bieth (1980). Briefly, the serine proteases were incubated at 37◦_C _with _different _{concentrations} of inhibitors in 0.1M Tris–HCl buffer pH 8.0 contain-ing 0.15M NaCl and 0.1% Triton X-100. The residual enzymeactivitywasmeasuredaftertheadditionof the chromogenicsubstratetosyl-Gly-Pro-Arg-pNAorelastase substrateI(MeOSuc-Ala-Ala-Pro-Val-pNA)fortrypsinand neutrophilelastase,respectively.ApparentKivalueswere calculatedbyfittingthesteady-statevelocitiestothe equa-tion (Vi/Vo=1−{Et+It+Ki−[(Et+It+Ki)2−4EtIt]1/2}/2Et) fortight-bindinginhibitorsandusinganon-linear regres-sionanalysis(Morrison,1969).

2.9. Thrombininhibitionassays

Boophilin(1.2and2.4␮M)waspre-incubatedwith␣ -thrombin (0.025U) or ␥-thrombin (1␮g) for 10min at 37◦_C_in₁₀₀_mM_Tris–HCl_buffer_pH_8.0_containing₁₅₀_mM NaCland0.1%TritonX-100.Theresidualthrombinactivity against the fluorogenic substrate Benzoyl-Phe-Val-Arg-AMC(200␮M)wasmeasured,afterincubationinthesame conditionsfor20min.Thefluorescencewasmonitoredat em=460nmandex=380nminaSynergyHTmicroplate reader(BioTek,Winooski,VT,USA)for20min.Asacontrol, thesameassaywasperformedintheabsenceofboophilin.

2.10. RNAinterference

AboophilinfragmentconjugatedwiththeT7promoter regionwasamplifiedbyPCRusingprimersBoophRNAifw (5′_-GGA_TCC_TAA _TAC_GAC _TCA_CTA_TAG_GCA_GAG_AAA TGGATTCTGCCGAC-3′₎_and_BoophRNAirv₍₅′_-GGA_TCC TAA TAC GAC TCA CTA TAG TCA TGT TCT TGC AGA CGA GTTCAC-3′_)._PCR _products _were_purified _with _the QIAEXIIkit(QIAGEN,Hilden,Germany)andusedas tem-platefor double-strandedRNA (dsRNA) synthesis using theT7RibomaxTM_Express_RNAi_system_(Promega, Madi-son,WI,USA).ThedsRNAwasdigestedwithDNAseand RNAse,precipitatedwithisopropanol,resuspendedin ster-ile PBS, and quantifiedby measuringits absorbance at 260nm. Engorged R. microplus females (35 individuals) wereinjectedwith2␮LofdsRNA-boophilin(3.5␮g),using aninsulinsyringe.Anidenticalcontrolgroupwasinjected with2␮LofPBSbuffer,andathirdgroupwasnotinjected. AfterdsRNAinjection, allgroups werekeptat 22–25◦_C and95%humidityfor24h,afterwhichtenticksofeach groupweredissectedandtheirgutsplacedinTrizolreagent (Invitrogen,Carlsbad,CA,USA)forsubsequentRNA extrac-tion. Eggs of 25 tickswere collected 24 and 48h after injectionandweighed.

2.11. Transcriptionanalysisofboophilingeneexpression

intickgutbyPCR

cDNAfromR.microplusengorgedadultfemalegutwas

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Fig.1. AminoacidsequencealignmentofboophilinwithseveralKunitz-typeinhibitors.FulllengthboophilinandD1(thiswork)arecomparedtoboophilin variantsG2(UniProtentryQ8WPI3)andH2(UniProtentryQ8WPI2),thethrombininhibitorshaemalin(UniProtentryB6ZIW0)fromHaemaphysalis longicornis(Liaoetal.,2009),monobin(UniProtentryQ09JW6)fromArgasmonolakensis(Mansetal.,2008),amblin(UniProtentryQ5VJG2)fromAmblyomma hebraeum(Laietal.,2004),ornithodorin(UniProtentryP56409)fromOrnithodorosmoubata(vandeetal.,1996),andsavignin(UniProtentryQ8WPG5) fromOrnithodorossavignyi(Mansetal.,2002a).Forcomparisonpurposes,twomoleculesofBPTI(UniProtentryP00974),thebovinebasictrypsininhibitor, arealsoincluded.Invariantresiduesaredisplayedwithablackbackground,whileconservedaminoacidsareshadedgray.Theresiduesofboophilindistinct frombothG2andH2variantsarehighlightedinredandthosewhereitisidenticalonlytoH2aremarkedinblue.ThesinglepositionwheretheD1sequence differsfromthatofboophilinishighlightedingreen.Thenumberingabovethealignmentcorrespondstothatofmatureboophilin(Macedo-Ribeiroetal., 2008).FigurepreparedwithALINE(BondandSchuttelkopf,2009).(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferred tothewebversionofthisarticle.)

boophilinwasthenamplifiedbyPCRusingcDNAsas tem-plateandthespecificprimersBoophilinfw(5′_-CAG_AGA_AAT GGATTCTGCCGACTGCCGGCA-3′₎_and_Boophilinrev₍₅′ -ACACTCCTCTATGGTCTCGAA-3′_).

ThePCRreaction(25␮L)contained1␮LofcDNA sam-ple,25pmolofeachprimer,100␮MdNTPs,1.5mMMgCl2, and2.5UTaqDNApolymerase(Fermentas,Vilnius, Lithua-nia)andwasperformedwiththefollowing parameters: 94◦_C_for₅_min,₂₅_cycles_of₉₄◦_C_for₄₀_s,₅₅◦_C_for₄₀_s_and 72◦_C_for₁_min,_followed_by₇₂◦_C_for₅_min._For_DNA ampli-ficationcontrol a similar reactionwas performedusing

25pmolofR.micropluselongationfactor1-alpha(ELF1a)

specificprimers:ELF1afw(5′_-CGT_CTA_CAA_GAT_TGG_TGG CATT-3′₎_and_ELF1arv₍₅′_-CTC_AGT_GGT_CAG_GTT_GGC AG-3′_).

3. Results

3.1. Cloning,expressioninP.pastorisand

characterizationofrecombinantboophilin

Aspecific tandemKunitzdomainthrombin inhibitor

from R. microplus, named boophilin, was previously

described(Macedo-Ribeiroetal.,2008).Inanattemptto producelargeamountsofrecombinantboophilin,theDNA fragment coding for the full-length inhibitor or for its N-terminaldomain(D1)wereamplifiedbyPCRusing spe-cificoligonucleotidesbasedonthesequenceofboophilin variantG2(EMBLaccessioncodeAJ304446.1)andcloned

into the P. pastoris pPICZ␣B expression vector. Positive

clonesforboophilinandD1wereconfirmedbyautomated DNAsequencingandusedtotransformP.pastorisyeast. The sequence of cloned boophilin differedfrom that of boophilin variantsG2 (EMBL accessioncode AJ304446.1)

andH2(EMBLaccessioncodeAJ304447.1),beingclosestto

theformer(Fig.1).Theclonedmatureboophilinwas dis-tinctfrombothpreviouslydescribedvariantsatpositions 27(GlninsteadofGlu)and92(ThrinsteadofAla),and iden-ticaltovariantH2atposition66,whileD1wasidenticalto variantG2atthisposition.Alltheseaminoacidexchanges occuronthesolvent-exposedfaceoftheinhibitoronits complexwiththrombin(Macedo-Ribeiroetal.,2008)and arethereforeunlikelytoaffectitsanticoagulantactivity.

Full-lengthboophilinandD1wereexpressedinP.

pas-torisat highlevels(21 and 37.5mg/L,respectively) and

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Fig.2. PurificationofboophilinandD1expressedinP.pastorisyeast.(A)AffinitychromatographyofD1onatrypsin-Sepharosecolumn.Yeastsupernatant containingrecombinantD1wasappliedontoatrypsin-Sepharosecolumnpreviouslyequilibratedwith0.05MTris–HClbufferpH8.0.Boundmaterial waselutedwith0.5MKClpH2.0.(B)Affinitychromatographyofboophilinonatrypsin-Sepharosecolumn.Yeastsupernatantcontainingrecombinant boophilinwaspurifiedasdescribedforD1.(C)SDS-PAGE(15%)analysisofpurifiedrecombinantproteins.M,proteinmolecularmassstandard(Bio-Rad); Booph,full-lengthboophilin(2␮g);D1,N-terminaldomainofboophilin(2␮g).BoophilinandD1displayapparentmolecularmassesofapproximately20

and11kDa,respectively.

(Fig.2AandB).OnSDS-PAGE,purifiedboophilindisplayed anapparentmolecularmassof20kDaandpurifiedD1of 11kDa(Fig.2C).Theinhibitoryactivityofboophilinagainst thrombin, trypsinandneutrophilelastase wasassessed, and the corresponding inhibition constants (Ki) deter-mined(Table1).Purifiedboophilinshowedhighselectivity to thrombin with a Ki of 57pM, a value significantly lowerthanthe1.80nMreportedforboophilinproducedin

Escherichiacoli(Macedo-Ribeiroetal.,2008).Thesecond

Table1

Inhibitionconstants(Ki)fordifferentserineproteases(nM).

Bovine trypsin

Bovine thrombin

Human neutrophil elastase

D1 2.00 n.i. 128.90 Boophilin 0.65 0.057 21.22 Boophilina _n.d. _1.80 _n.d.

n.i.,noinhibition;n.d.,notdetermined. a_{Macedo-Ribeiro}_et_al.₍₂₀₀₈₎_.

Kunitzdomainofboophilindisplaysanalanineresidueat thereactiveloopP1position(SchechterandBerger,1967), suggestingitcouldinhibitelastase.BothboophilinandD1 inhibitedhumanneutrophilelastaseinvitrowithKi val-uesof21nMand129nM,respectively.Boophilininhibits thrombinbybindingsimultaneouslytotheactivesiteand theexosite1oftheprotease(Macedo-Ribeiroetal.,2008). Thecontributionoftheinteractionwiththeexosite1tothe inhibitoryactivityofboophilinwasprobedbycomparing itsactivitytowards␣-thrombinandtheexosite1-lessform, ␥-thrombin(Fig.3).Recombinantboophilinrevealedno activitytowards␥-thrombin,inamountsthatcompletely abolishedtheamydolyticactivityof␣-thrombin,therefore underscoringtheimportanceoftheinteractionwiththe exosite1.

3.2. BoophilingeneexpressionanalysisbyqPCR

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Fig.3.Inhibitionofalphaandgamma-thrombinbypurifiedrecombinant boophilin.(A)Theresidualactivityofalpha(A)orgamma-thrombin(B) againstthefluorogenicsubstrateTosyl-Gly-Pro-Arg-AMCwasdetermined inthepresenceofdifferentamountsofrecombinantboophilin.

synthesis(Fig.4).Boophilingeneexpressionwasmostly detectedinthemidgut(25,000foldaboveothertissues) withminorexpressionlevelsinhemocytes,althougha con-taminationwithmidgutcellsduringdissectioncannotbe discarded.

SG FB Gut OV _Hem

0 10000 20000 30000 40000

Fold expression

**

Fig.4. Boophilingeneexpressionprofileindifferenttissuesofengorged adultR.microplusfemalesbyquantitativePCR.Gut(Gut),ovary(OV), hemocyte(Hem),salivarygland(SG)andfatbody(FB)cDNAoffedticks wereusedinqPCRexperiments.Alldatawerenormalizedtothe expres-sionlevelofELF1a(elongationfactor1-alpha),usedasaninternalcontrol. Theexpressionlevelofboophiliningutshowedasignificantincrease whencomparedtoothertissues(**p<0.05inTukey’sMultiple Compari-sonTest).Errorbarscorrespondtothestandarderrorofthemeanofthree independentexperiments.

3.3. BoophilingenesilencingusingRNAinterference

Inanattempttounveilboophilin’sphysiologicalrole,a RNAi-mediatedgenesilencingexperimentwasperformed. Threegroupsofticks,eachcomposedof25animals,were injected witheitherboophilindsRNA,PBS bufferorleft untreated.Incomparisontothecontrolanimals,an effi-cientsilencingofboophilinexpressionwasachievedafter boophilin dsRNA treatment (Fig. 5A). Boophilin down-regulationresultedinadecrease(∼20%after24and48h) ineggproduction(Fig.5B).

4. Discussion

ConsideringtheimportantroleofKunitz-typeinhibitors inthelifecycleofR.microplusandthehighspecificityof thetandemKunitzinhibitorboophilinforthrombin, full-lengthboophilinanditsN-terminalKunitzdomain(D1) wereexpressed,purifiedandcharacterized.Furthermore, boophilingeneexpressionanalysisandevaluationofthe effectofRNAisilencingineggproductionwerealso per-formed.

ActiveboophilinandD1wereefficientlyexpressedinP.

pastorisandpurifiedinasinglestepbyaffinity

chromatog-raphy.Purifiedrecombinantboophilinstronglyinhibited thrombin,withadissociation constantinthepMrange. Moreover,italsodisplayedconsiderableactivityagainst trypsin (Ki 0.65nM) andneutrophil elastase (Ki 21nM). AsforpurifiedrecombinantD1,itdisplayedaninhibitory activityagainsttrypsinsimilartothat ofthefull-length inhibitor (Ki 2nM), and also inhibited neutrophil elas-tase,althoughwithasignificantlydecreasedefficiency(Ki 0.129␮M),suggestingasignificantcontributionfromthe C-terminalKunitzdomaintothisinteraction,compatible withthe presenceof an alanineresidue in thereactive loopP1position.Thethree-dimensionalstructureofthe thrombin-boophilin complexrevealeda bidentate inter-action of boophilinwiththe activesite and theexosite I of␣-thrombin. The N-terminal regionof theinhibitor bindstoandblockstheactivesiteofthrombinwhilethe negativelychargedC-terminalKunitzdomainofboophilin docksintothebasicexositeI(Macedo-Ribeiroetal.,2008). Asexpectedfromthethrombin-boophilincomplex archi-tecture, isolated D1does not display inhibitoryactivity againstthrombin,confirmingthefundamental contribu-tion of the C-terminaldomain-mediated interaction for thrombininhibition.Furtherhighlightingtheimportance oftheexositeIforthrombininhibition,boophilininhibited strongly␣-thrombininvitrobutwasunabletoinhibitthe exositeI-disruptedformoftheenzyme,␥-thrombin.

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Fig.5.(A)BoophilinexpressioninR.microplusgutaftergenesilencing.(a)TicksinjectedwithboophilindsRNA,eachlinerepresentsthegutofonetick, showingthatthesilencingwasefficient.Resultsoftwoindependentexperiments(onlyonewasshown);(b)ticksinjectedwithPBSbufferand(c) non-injectedticks.(B)ReductioninovipositionafterRNAi-silencingofboophilin.TickinjectedwithPBSshowed5%ofovipositionreductionwhileticksinjected withds-boophilinshowed20%ofovipositionreductioncomparedtonon-injectedticks.Theresultsarethemeanoftwoindependentexperiments±standard deviation.

engorgementprocess,anditisthereforeconceivablethat boophilinmightbeusedtocontrolanyplasminor elas-taseactivityarisinginthemidgutduringthisperiod,even whencomplexedwiththrombin,avoidingunwantedtissue damage.

Boophilin amino acid sequence is 37% identical to that ofhemalin (Liao etal., 2009),a thrombin inhibitor describedinthetickHaemaphysalislongicornis.However, whilehemalinwasexpressedinallmajortissues(including salivaryglands,midgut,hemocytesandfatbody)ofadult femaleticks,boophilinwasexclusivelyexpressedinthe midgut,suggestinganimportantroleinthisorgan. Con-sideringitslimitedexpressionanditsmultipleinhibitory activities,silencingofboophilinexpressionwasexpected toimpactserineproteaseactivitylevelsinthemidgutand consequentlythedigestionprocess,withavisibleeffectin eggproduction.InjectionofdsRNA-boophilininengorged

R.microplusfemalesalmostabolishedboophilin

transcrip-tioninthemidgutandresultedin20%reduction inegg production,aresultsimilartothatobservedforhemalin

genesilencing(Liaoetal.,2009).Apossibleexplanation fortheapparentlysmallimpactofboophilingene silenc-ingineggproduction mightbethevast arrayofKunitz type inhibitorspresent in R. microplus, which werenot observedintherelatedRhipicephalussanguineus(Azzolini etal.,2003).Besidesboophilin,R.microplusproducesother Kunitztypeinhibitorswithactivityagainsttrypsin, plas-min,plasmakallikreinandneutrophilelastase(Sasakietal., 2004;SasakiandTanaka,2008;Tanakaetal.,1999). Inter-estingly,someoftheseinhibitorgenesarehighlyexpressed

inR. microplusmidgut, and couldpartiallymake upfor

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efficiencyasabovineectoparasite.Consideringourdata, andtheredundancyofR.microplusKunitzinhibitorswe believethatboophilinmaybeusefulasanantigentogether withothertickproteininavaccineproductionfortick con-trol.

Acknowledgements

Thisworkwassupportedby Fundac¸ãode Amparoà PesquisadoEstadodeSãoPaulo(FAPESP;grants 05/03514-9and09/05405-3),ConselhoNacionaldeDesenvolvimento CientíficoeTecnológico(CNPq;grant490574/2006-8),and INCT-EntomologiaMolecular.A.S.T.wastherecipientofa CNPqfellowship.ThisworkwasfundedinpartbyFundac¸ão para a Ciência ea Tecnologia,Portugal, through grants PTDC/BIA-PRO/70627/2006andREEQ/564/B10/2005 (EU-FEDERandPOCI2010) andthepost-doctoralfellowship SFR/BPD/46722/2008toA.C.F.We thankCassia A. Lima, RenatoSakaiandRafaelMarchesanoforhelpingincloning andexpressionexperiments.

References

Andreotti,R.,Gomes,A.,Malavazi-Piza,K.C.,Sasaki,S.D.,Sampaio,C.A., Tanaka,A.S.,2002.BmTIantigensinduceabovineprotectiveimmune responseagainstBoophilusmicroplustick.Int.Immunopharmacol.2, 557–563.

Andreotti,R.,Malavazi-Piza,K.C.,Sasaki,S.D.,Torquato,R.J.,Gomes,A., Tanaka,A.S.,2001.Serineproteinaseinhibitorsfromeggsandlarvae oftickBoophilusmicroplus:purificationandbiochemical characteri-zation.J.Protein.Chem.20,337–343.

Arni,R.K.,Padmanabhan,K.,Padmanabhan,K.P.,Wu,T.P.,Tulinsky,A., 1994.Structureofthenon-covalentcomplexofprothrombinkringle 2withPPACK-thrombin.Chem.Phys.Lipids67–68,59–66. Azzolini,S.S.,Sasaki,S.D.,Torquato,R.J.,Andreotti,R.,Andreotti,E.,Tanaka,

A.S.,2003.Rhipicephalussanguineustrypsininhibitorspresentinthe ticklarvae:isolation,characterization,andpartialprimarystructure determination.Arch.Biochem.Biophys.417,176–182.

Bieth,J.G.,1980.Pathophysiologicalinterpretationofkineticconstants ofproteaseinhibitors.Bull.Eur.Physiopathol.Respir.16(Suppl.), 183–197.

Bode,W.,Turk,D.,Karshikov,A.,1992.Therefined1.9-AX-ray crys-talstructureofd-Phe-Pro-Argchloromethylketone-inhibitedhuman alpha-thrombin:structureanalysis,overall structure,electrostatic properties,detailed active-site geometry, and structure–function relationships.ProteinSci.1,426–471.

Bond,C.S.,Schuttelkopf,A.W.,2009.ALINE:aWYSIWYGprotein-sequence alignmenteditorforpublication-qualityalignments.ActaCrystallogr. D:Biol.Crystallogr.65,510–512.

ChaseJr.,T.,Shaw,E.,1969.Comparisonoftheesteraseactivitiesoftrypsin, plasmin,andthrombinonguanidinobenzoateesters.Titrationofthe enzymes.Biochemistry8,2212–2224.

Davie,E.W.,Fujikawa,K.,Kisiel,W.,1991.Thecoagulationcascade: initi-ation,maintenance,andregulation.Biochemistry30,10363–10370. Francischetti,I.M.,Meng,Z.,Mans,B.J.,Gudderra,N.,Hall,M.,Veenstra,

T.D.,Pham,V.M.,Kotsyfakis,M.,Ribeiro,J.M.,2008.Aninsightinto thesalivarytranscriptomeandproteomeofthesofttickandvector

ofepizooticbovineabortion,Ornithodoroscoriaceus.J.Proteomics71, 493–512.

Lai,R.,Takeuchi,H.,Jonczy,J.,Rees,H.H.,Turner,P.C.,2004.A throm-bininhibitorfromtheixodidtick,Amblyommahebraeum.Gene342, 243–249.

Liao,M.,Zhou,J.,Gong,H.,Boldbaatar,D.,Shirafuji,R.,Battur,B.,Nishikawa, Y.,Fujisaki,K.,2009.Hemalin,athrombininhibitorisolatedfroma midgutcDNAlibraryfromthehardtickHaemaphysalislongicornis.J. InsectPhysiol.55,164–173.

Livak,K.J.,Schmittgen,T.D.,2001.Analysisofrelativegeneexpressiondata usingreal-timequantitativePCRandthe2(-deltadeltaC(T))method. Methods25,402–408.

Macedo-Ribeiro,S.,Almeida,C.,Calisto,B.M.,Friedrich,T.,Mentele,R., Sturzebecher,J.,Fuentes-Prior,P.,Pereira,P.J.,2008.Isolation,cloning andstructuralcharacterisationofboophilin,amultifunctional Kunitz-typeproteinaseinhibitorfromthecattletick.PLoSOne3,e1624. Mans,B.J.,Andersen,J.F.,Schwan,T.G.,Ribeiro,J.M.,2008.Characterization

ofanti-hemostaticfactorsintheargasid,Argasmonolakensis: implica-tionsfortheevolutionofblood-feedinginthesofttickfamily.Insect Biochem.Mol.Biol.38,22–41.

Mans, B.J.,Louw, A.I.,Neitz,A.W., 2002a. Aminoacid sequenceand structuremodelingofsavignin,athrombininhibitorfromthetick, Ornithodorossavignyi.32,821–828.

Mans,B.J.,Louw,A.I.,Neitz,A.W.H.,2002b.Aminoacidsequenceand structuremodelingofsavignin,athrombininhibitorfromthetick, Ornithodorossavignyi.InsectBiochem.Mol.Biol.32,821–828. Mans,B.J.,Ribeiro,J.M.,2008.Function,mechanismandevolutionofthe

moubatin-cladeofsoftticklipocalins.InsectBiochem.Mol.Biol.38, 841–852.

Morrison, J.F.,1969. Kineticsofthereversibleinhibition of enzyme-catalysedreactionsbytight-bindinginhibitors.Biochim.Biophys.Acta 185,269–286.

Ribeiro,J.M.,1995.Blood-feedingarthropods:livesyringesorinvertebrate pharmacologists?Infect.AgentsDis.4,143–152.

Ricci, C.G.,Pinto, A.F.,Berger, M., Termignoni,C., 2007.A thrombin inhibitorfromthegutofBoophilusmicroplusticks.Exp.Appl.Acarol. 42,291–300.

Sasaki,S.D.,Azzolini,S.S.,Hirata,I.Y.,Andreotti,R.,Tanaka,A.S.,2004. Boophilusmicroplusticklarvae,arichsourceofKunitztypeserine proteinaseinhibitors.Biochimie86,643–649.

Sasaki,S.D.,Tanaka,A.S.,2008.rBmTI-6,aKunitz-BPTIdomainprotease inhibitorfromthetickBoophilusmicroplus,itscloning,expressionand biochemicalcharacterization.Vet.Parasitol.155,133–141. Schechter,I.,Berger,A.,1967.Onthesizeoftheactivesiteinproteases.I.

Papain.Biochem.Biophys.Res.Commun.61,157–162.

Sheehan, J.P.,Wu,Q.,Tollefsen,D.M.,Sadler, J.E.,1993.Mutagenesis of thrombin selectively modulates inhibition by serpins heparin cofactorIIandantithrombinIII.Interactionwiththeanion-binding exositedeterminesheparincofactorIIspecificity.J.Biol.Chem.268, 3639–3645.

Sonenshine,D.E.,1991.Evolutionandsystematicrelationshipofticks.In: Biologyofticks.Oxforduniversitypress,NewYork,Oxford,pp.13–50. Tanaka,A.S.,Andreotti,R.,Gomes,A.,Torquato,R.J.,Sampaio,M.U., Sam-paio,C.A.,1999.Adoubleheadedserineproteinaseinhibitor– human plasmakallikreinandelastaseinhibitor–fromBoophilusmicroplus larvae.45,171–177.

vande,L.A.,Stubbs,M.T.,Bode,W.,Friedrich,T.,Bollschweiler,C.,Hoffken, W.,Huber,R.,1996.Theornithodorin-thrombincrystalstructure,a keytotheTAPenigma?Immunopharmacology15,6011–6017. Waxman,L.,Smith,D.E.,Arcuri,K.E.,Vlasuk,G.P.,1990.Tickanticoagulant