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

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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

_,

_Renata

_Midori

_Okuta

_Watanabe

_,

_Anita

_Mitico

_{Tanaka-Azevedo}

_,

Ricardo

José

Soares

Torquato

_,

_Stephen

_Lu

_,

_Ana

_Carvalho

_Figueiredo

_,

Pedro

José

Barbosa

Pereira

_,

_Aparecida

_S.

_Tanaka

a_{DepartamentodeBioquímica,EscolaPaulistadeMedicina,UniversidadeFederaldeSãoPaulo,Rua3deMaio100,04044-020SãoPaulo,SP,Brazil} b_{LaboratóriodeFisiopatologia,InstitutoButantan,SãoPaulo,SP,Brazil}

c_{IBMC-InstitutodeBiologiaMoleculareCelular,UniversidadedoPorto,4150-180Porto,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

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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.

© 2012 Elsevier B.V.

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:tanaka.bioq@epm.br(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

0304-4017/© 2012 Elsevier B.V. doi:10.1016/j.vetpar.2012.01.027

Open access under the Elsevier OA license.

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(Babesia spp.-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_{ReverseTranscription}

System(Promega,Madison,WI,USA).QuantitativePCRwas

performedusingtwospecificprimersdesignedbasedon

theboophilinsequencewithGenBankaccessionnumber

AJ304446:Boophilinfw(5-CAGAGAAATGGATTCTGCCGA

CTGCCGGCA-3)andBoophilinrev(5-ACACTCCTCTAT

GGT CTCGAA-3).R.micropluselongationfactor1-alpha

(ELF1a)specificprimers–ELF1afw(5-CGTCTACAAGAT

TGGTGGCATT-3)andELF1arv(5-CTCAGTGGTCAGGTT

GGC AG-3)– wereused for DNA amplificationcontrol.

Boophilin andELF1a (endogenouscontrol)were

quanti-fiedusing SYBR® _{GreenPCR MasterMix ina 7500Fast}

Real-Time PCR System(AppliedBiosystems, FosterCity,

CA,USA),accordingtothefollowingconditions:40cycles

of95◦Cfor1min,60◦Cfor1minand72◦Cfor1min.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-GTATCTCTCGAGAAA

AGACAGAGAAATGGATTCTGCCGACTGCCGG-3)and

Boophirv2ndd(5-CGAATTAAT TCG CGGCCGCCTACA

TGTTCTTGCAGACGAGTTCACAC-3)forboophilinand

Boophifw1stdandBoophirv1std(5-CGAATTAATTCGCGG

CCGCCTAAGCTCCGCACGCCTTTTGACAATC-3)forD1.

PCRreactionswereconductedinafinalvolumeof50␮Lin

100mMTris–HClpH8.8,500mMKCl,0.8%(v/v)Nonidet

P40,1.5mMMgCl2,100␮MdNTPs,10pMofeachprimer,

5UTaqDNApolymerasewiththefollowingparameters:

94◦Cfor2min,priorto30cyclesof94◦Cfor45s,55◦C

for 45s and72◦C for 1minfollowed by 5minat 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

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 3AOX and 5AOX

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)biotinand 1%(v/v)glycerol)ina 50mL}

steriletube,andgrownat30◦Cfor28hat250rpm.The

yeastcells wereharvested bycentrifugationat3000×g

for5minat4◦CandresuspendedinBMMY(BMGYwith

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),theinhibitory

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◦Cand250rpmfor24h.Expressionwasperformedas

describedaboveandtheculturesupernatantwasstoredat

4◦Cpriortopurification.

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◦Cin100mMTris–HClbufferpH8.0containing150mM

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-GGATCCTAA TACGAC TCACTATAGGCAGAGAAA

TGGATTCTGCCGAC-3)andBoophRNAirv(5-GGATCC

TAA TAC GAC TCA CTA TAG TCA TGT TCT TGC AGA

CGA GTTCAC-3).PCR products werepurified with the

QIAEXIIkit(QIAGEN,Hilden,Germany)andusedas

tem-platefor double-strandedRNA (dsRNA) synthesis using

theT7RibomaxTM_{ExpressRNAisystem(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

preparedfromallsilencinggeneexpressionexperimental

groupsusingtheImProm-IITM_{ReverseTranscription}

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-CAGAGAAAT

GGATTCTGCCGACTGCCGGCA-3)andBoophilinrev(5

-ACACTCCTCTATGGTCTCGAA-3).

ThePCRreaction(25␮L)contained1␮LofcDNA

sam-ple,25pmolofeachprimer,100␮MdNTPs,1.5mMMgCl2,

and2.5UTaqDNApolymerase(Fermentas,Vilnius,

Lithua-nia)andwasperformedwiththefollowing parameters:

94◦Cfor5min,25cyclesof94◦Cfor40s,55◦Cfor40sand

72◦Cfor1min,followedby72◦Cfor5min.ForDNA

ampli-ficationcontrol a similar reactionwas performedusing

25pmolofR.micropluselongationfactor1-alpha(ELF1a)

specificprimers:ELF1afw(5-CGTCTACAAGATTGGTGG

CATT-3)andELF1arv(5-CTCAGTGGTCAGGTTGGC

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

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-Ribeiroetal.(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

DifferenttissuesofengorgedR.microplusfemaleswere

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.

Incontrasttootherpreviouslydescribednatural

throm-bininhibitorsfromblood-suckinganimals,boophilinmay

alsotargetadditionalserineproteasessuchastrypsinand

plasmin(Macedo-Ribeiroetal.,2008).Theobserved

activ-ityofboophilinagainstneutrophilelastasecorroborated

thishypothesis,suggestingaroleotherthancounteracting

bloodcoagulationinthemidgutofR.microplus.Bloodis

acomplexmixtureofnumeroussolubleproteins,

includ-ingplasminprecursorplasminogen,andofdifferentcells,

amongwhichtheelastase-producingneutrophils.Inticks,

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

boophilindecreaseinthedsRNA-boophilininjectedticks,

leadingtoalowerimpactineggproduction.Similarresults

were obtainedwhen other Kunitz type inhibitors were

silencedoranti-BmTIswereinjectedinengorgedfemales

(A.S.Tanaka,unpublisheddata).Thepresentfindings,as

wellasthepresenceofanotherthrombininhibitorinthe

midgutofR.microplus(Riccietal.,2007),highlightthehigh

redundancyofR.microplusKunitz-basedserineprotease

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.

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