Boophilus microplus cathepsin L-like (BmCL1) cysteine protease: Specificity study using a peptide phage display library

VeterinaryParasitology181 (2011) 291–300

ContentslistsavailableatScienceDirect

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

Boophilus

microplus

cathepsin

L-like

(BmCL1)

cysteine

protease:

Specificity

study

using

a

peptide

phage

display

library

Renan

O.

Clara

_,

_Tatiane

_S.

_Soares

_,

_Ricardo

_J.S.

_Torquato

_,

_Cássia

_A.

_Lima

_,

Renata

O.M.

Watanabe

_,

_Nilana

_M.T.

_Barros

_,

_Adriana

_K.

_Carmona

_,

_Aoi

_Masuda

_,

Itabajara

S.

Vaz

Junior

_,

_Aparecida

_S.

_Tanaka

a_{DepartamentodeBioquímica,UniversidadeFederaldeSãoPaulo,RuaTrêsdeMaio,100,04044-020SãoPaulo,SP,Brazil} b_{DepartamentodeBiofísica,UniversidadeFederaldeSãoPaulo,SãoPaulo,Brazil}

c_{CentrodeBiotecnologiadoEstadodoRioGrandedoSul,UFRGS,PortoAlegre,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5November2010

Receivedinrevisedform24March2011 Accepted1April2011

Keywords: Cysteineproteases

Rhipicephalus(Boophilus)microplus Proteinexpression

Enzymekinetic Phagedisplaylibrary

a

b

s

t

r

a

c

t

ThetickRhipicephalus(Boophilus)microplusisoneofthemostimportantbovine ectopar-asites,adiseasevectorresponsibleforlossesinmeatandmilkproductions.Acysteine proteasesimilartocathepsinL,namedBmCL1,waspreviouslyidentifiedinR.microplus gut,suggestingaroleoftheenzymeinmealdigestion.Inthiswork,BmCL1was success-fullyexpressedinPichiapastorissystem,yielding54.8mg/Lofcultureanditsactivitywas analyzedbysyntheticsubstratesandagainstaR.micropluscysteineproteaseinhibitor, Bmcystatin.AfterrBmCl1biochemicalcharacterizationitwasusedinaselectionofa pep-tidephagelibrarytodeterminerBmCL1substratepreference.Obtainedsequencedclones showedthatrBmCL1haspreferenceforLeuorArgatP1position.ThepreferenceforLeuat positionP1andtheactivationofBmCL1afteraLeuaminoacidresiduesuggestpossibleself activation.

© 2011 Elsevier B.V.

1. Introduction

Thetick Rhipicephalus(Boophilus)microplus isone of themostimportantbovineectoparasites,adiseasevector heldresponsibleforthetransmissionofdiseasessuchas babesiosisandanaplasmosis,whichcausemassivelosses inlivestockproduction(Saueretal.,1995).Onceattached toabovine,R.microplusisabletoingestalargevolumeof blood,inaprocessthatincreasesitsbodyweightbymore than50 times,when comparedwithitsoriginalweight, mostintenselyduringthelast24hofengorgement.Blood digestioninticksisanintracellularprocessvia

phagocy-Abbreviations: rBmCL1, recombinantBoophilusmicroplus cathep-sin L-Like; Kcat, turnover number; Ki, inhibition constant; Km,

Michaelis–Mentenconstant;Vmax,maximumvelocity.

∗ _{Correspondingauthor.Tel.:+551155764445;fax:+551155723006.} E-mailaddress:tanaka.bioq@epm.br(A.S.Tanaka).

tosisbydesquamatedepithelialcellsinthemidgut(Koh etal.,1991;Sonenshine,1991).Recently,Laraetal.(2005) showedthatthemidgutdigestivecellsendocytoseblood componentsand release largeamounts of heme during hemoglobindigestion.Proteolyticenzymessecretedinthe tick midgutmay berequired for various functionsthat areessential toticksurvival via bloodfeedingbehavior (Ribeiro,1987),andplaycriticalrolesinpathogen trans-mission(Tsujietal.,2008).Thus,proteolyticenzymesmay becomeinterestingcandidatesasdrugtargetsanda com-ponentof vaccines for both tick andtick-borne disease controls(Randetal.,1989;Renardetal.,2002;Willadsen andKemp,1988).Amongthetickproteolyticenzymes, sev-eralcysteineproteasesbelongingtothepapainsuperfamily havebeenidentified(Estrelaetal.,2007;Renardetal.,2000, 2002;Seixasetal.,2003;Tsujietal.,2008).Cysteine pro-teasesareexpressedinorganismsfrombacteriatohumans. Inparasitesithasbeensuggestedthatcysteineproteases areinvolvedintheinvasionofhosttissuesandevasionof

0304-4017© 2011 Elsevier B.V.

doi:10.1016/j.vetpar.2011.04.003

Open access under the Elsevier OA license.

thehostimmunesystem(reviewedinMcKerrow(1989) and Rocheet al. (1999)).In hematophagousorganisms, cysteineproteaseshavebeendescribedasresponsiblefor hemoglobindigestionin thegutofSchistosomamansoni (Brady et al., 1999)and in thevacuole of the Plasmod-ium falciparum (Salas et al., 1995). In the R. microplus tick, cathepsin L-like enzymes have been found in the midgutandeggs,whichsuggeststheinvolvementofthese enzymesin meal digestion(Renardet al.,2002)and in vitellindegradation(Estrelaetal.,2007;Seixasetal.,2003), respectively.

Previously,Renardetal.(2000)clonedandexpressedan R.micropluscathepsinL-likeenzymenamedBmCL1.The recombinantenzymewasabletohydrolyzenatural sub-stratessuchastickvitellin,bovinehemoglobinandgelatin atacidicpH.Moreover,itwasshownthattheBmCL1 tran-scriptisexpressedinpartiallyengorgedtickfemales,and thattheenzymeseemstobelocalizedinsecretorygutcells. Takingalltheseobservationstogether,theauthors postu-latedthatBmCL1maybeinvolvedinbovinehemoglobin degradationintheR.microplusgut(Renardetal.,2002). Recently,itwasshownthatbovinehemoglobin degrada-tionbyBmCL1generatespotentialantimicrobialpeptides basedonhumanandbovinehemocidins(Cruzetal.,2010). Variousmethodshavebeenusedtoidentifydrug tar-getsforparasitecontrol(Canalesetal.,2009),amongwhich combinatorialmutatedpeptidesorproteinsdisplayedon filamentous bacteriophage surface, which is known as phagedisplaysystem,haveevolvedasanimportanttool tostudyprotein–orpeptide–proteininteractions(Smith, 1985).Phagedisplaylibrarieshavebeenusedtoselect spe-cificprotease inhibitors(Campos etal., 2004;Markland etal., 1996a,1996b; Robertset al.,1992; Tanakaetal., 1999), toincrease theanticoagulant activityof proteins (Maunetal.,2003;Yangetal.,2002),andtoselect spe-cificantibodies(Barbasetal., 1991;Marksetal.,1991). In addition, peptide phage display libraries have also beensuccessfullyemployedtodefineenzymesubstrates (Deperthes,2002;MatthewsandWells,1993).

Inthepresentwork,weclonedandhighlyexpressed theactiveBmCL1(rBmCL1)enzymeusingthePichia pas-torissystem.The purified rBmCL1 enzyme wasused in biochemicalcharacterizationand insubstratespecificity investigationusingapeptidephagedisplaylibrary.

2. Materialsandmethods

2.1. Materials

ThepPIC9vectorandP.pastorisstrainGS115were pur-chased fromInvitrogen Corporation(Carlsbad,CA,USA) andusedfollowingthesupplier’sinstructions.Media com-ponents werefromDifco (Detroit, MI,USA). Restriction endonucleasesandT4DNAligasewereobtainedfrom Fer-mentas(Beverly,Vilnius,Lithuania).TaqDNApolymerase wasobtained fromPromega Corporation(Madison, WI, USA); Oligonucleotides were synthesized by Invitrogen (Carlsbad,CA,USA).Allotherreagentswereobtainedfrom Sigma (St. Louis, MO, USA). DNA sequencing was per-formedusing DYEnamicTM _{ET Dye Sequencing kit from} AmershamLittleChalfont(Buckinghamshire,England,UK)

onan ABI377sequencer from AppliedBiosystems (Fos-terCity,CA,USA).AmiconUltra5000MWCOmembrane wasfromMillipore(Billerica,MA,USA), andtheHiTrap SPcolumnwasfromAmershamBiosciences(Piscataway, NJ,USA). Thesubstrates:Bz-Phe-Arg-pNa,Z-FR-MCA, Z-RR-MCA(MCA=methyl-7-aminocoumarinamide)andthe inhibitor E-64 ([trans-epoxy-succinyl-l -leucylamido-(4-guanidino)butane])werepurchasedfromSigma(St.Louis, MO,USA).ThesubstrateZ-LR-MCAwasfromNovabiochem (Darmstadt,Germany).

2.2. TranscriptionanalysisofBmCL1byPCR

The cDNA sequence encoding BmCL1 was amplified using cDNA prepared of mRNA from ovary, fat body, salivary gland, gut, and haemocytes of R. microplus engorged adult females. The PCR was performed using two specific primers, the sense primer BmCL1fw (5′

-GTATCTCTCGAGAAAAGATCTCAAGAAATCCTACGCACC-3′₎

and the antisense primer BmCL1rv (5′

-CCCGTGCGGCCGCTTAGACGAGGGGGTAGC-3′_{). The PCR}

wasperformedin50␮Lreactionvolumecontaining1␮L ofcDNAsample,25pmolofeachprimer,100␮MdNTPs, 1.5mMMgCl2,and2.5UTaqDNApolymerase(Fermentas, Vilnius,Lithuania).ThecontrolofDNAamplificationwas madeusing25pmolofR.microplusactinspecificprimers: Actf (5′_{-TCCTCGTCCCTGGAGAAGTCGTAC-3}′_{) and Actr}

(5′_{-CCACCGATCCAGACCGAGTACTTC-3}′_{). PCR conditions}

were: 94◦_{C for 5min; 25 cycles(94}◦_{C–40s, 62}◦_C–40s,

72◦_C–1min),72◦_Cfor5min.

2.3. BmCL1DNAfragmentamplificationandcloninginto pPIC9vector

TheDNAfragmentencodingtheBmCL1genewas ampli-fiedbyPCRusingaplasmidconstructcontainingtheBmCL1 coding sequence in vectorpMAL-pas template (Renard etal.,2000).ThePCRwasperformedusingaprimerset constructedbasedontheBmCL1codingregionsequence, introducingrestrictionsitesforXhoIandNotIenzymesat 5′_and3′_{ends,respectively.Thesenseoligonucleotidewas:}

5′_{-CTCGAGGTATCTCTCGAGAAAAGATCTCAAGAAATC}

CTACGCACC-3′_{,whiletheantisensewas:5}′_-GCGGCCGC

CCC GTGCGGCCGCTTAGACGA GGGGGT AGT-3′_.PCR

reactionconditionswereconductedina finalvolumeof 50␮L,1.5mMMgCl2,100␮MdNTPs,10pMofeachprimer, 5UofTaqDNApolymerase,anditscorrespondingbuffer (100mMTris–HClpH8.8,500mMKCl,and0.8%(v/v) Non-idetP40).ThePCRparameterswere:pre-denaturationat 94◦_{Cfor2min;30cyclesof(94}◦_Cfor30s,55◦_Cfor45s,

72◦_{Cfor1min);followedby5minat72}◦_C.BmCL1DNA

insertionofPCRfragmentwasverifiedbyDNA sequenc-ing.Onecorrectconstruction(BmCL1-pPIC9)wasusedina midi-plasmidDNApreparationusingPlasmidMidiKitfrom QIAGEN(Hilden,Germany). BmCL1deducedaminoacid sequencewasalignedwithothercathepsinL-likecysteine proteasesusingClustalWprogram.

2.4. TransformationofP.pastorisyeast

P. pastoris GS115 strain was grown in a yeast extract-peptone-dextrose(YPD)mediumandpreparedfor transformation by electroporation following the manu-facturer’sinstructions.Afterlinearizationoftheplasmid BmCL1-pPIC9(10␮g)withNsiIrestrictionenzyme, compe-tentP.pastorisGS115wastransformedbyelectroporation. Yeasttransformationwasperformedina0.2-cmcuvette inaGenePulser(Bio-Rad,Hercules,CA)usingthe follow-ingparameters:1.5kV,25␮Fand400.Theeletroporated cells were immediatelysuspended in1.0mLofice-cold 1.0MsorbitolandspreadonMDagarplate(1.34%YNB,2% dextrose,4×10−5_{%biotin)withouthistidine.Clonesthat}

werehomologousrecombinantswiththeAOXIsequence wereselected.Afterincubationat30◦_{Cfor48h,theclones}

weretransferredtoanMDagarplatewithouthistidineto screenformethanolutilizationplus(Mut+).Thetargetgene intherecombinantswasdetectedbyPCRreactionsusing 3′_and5′_{AOXprimersfromInvitrogen(Carlsbad,CA,USA).}

2.5. Selectionofyeastclonesfunctionalexpressing BmCL1

Toidentifypositiveyeastclones,fiveisolatedP.pastoris GS115strainscarryingBmCL1genefragment,confirmedby PCRreaction,wereindividuallyinoculatedin5mLBMGY medium (1.0% yeast extract, 2.0% peptone in 100mM potassiumphosphatebufferpH6.0,1.34%YNB4×10−5%

(w/v) D-biotinand 1%(v/v)glycerol) in a 50-mLsterile tube,andcultivatedat30◦_{Cfor28hat250rpm.Theyeast}

cellswereharvestedbycentrifugationat3000gfor5min at4◦_{C,andcellswereresuspendedinBMMY(1.0%yeast}

extract, 2.0% peptone in 100mM potassium phosphate bufferatpH6.0,1.34%YNB4×10−5%(w/v)D-biotinand

0.5% (v/v) methanol) medium toabsorbance at 600nm of 1.0,following expressionfor4 daysat30◦_{C, shaking}

at 250rpm. The protein expression was induced every 24hbyadding0.5%methanoltotheyeastcultures.After fermentation,yeastcellswereremovedbycentrifugation (4000×gfor 20minat 4◦C)and thesupernatantswere

used in enzymatic activity detection by chromogenic substrate (HD-Pro-Phe-Arg-pNa) hydrolysis. Analysis of individualclonesshowedthesameexpressionlevelforall checkedclones(datanotshown).

2.6. ExpressionofrBmCL1

One isolated P. pastoris colony (Mut+) expressing BmCL1 in high level was inoculated in 50mL BMGY medium in 1L sterile flask, and cultivated at 30◦_{C for}

24hat250rpm.Theyeastcellswereharvested by cen-trifugation at 3000×g for 5min at4◦C, and cells were

resuspendedinBMMYmediumtoabsorbanceat600nm

of1.0,followingexpressionfor4daysat30◦_C,shakingat

250rpm.Theproteinexpressionwasinducedafterevery 24hbyadding0.5%methanoltotheyeastculture.After fermentation,yeastcellswereremovedbycentrifugation (4000×g for 20min at 4◦C) and the supernatant was

storedat4◦_{Cforpurificationprocedure.}

2.7. PurificationofrecombinantBmCL1

RecombinantBmCL1secretedtotheyeastsupernatant waspre-activated by dialysisin 40mM sodiumacetate buffer,pH5.5,for 12hat4◦_{C. Afterdialysis,the}

super-natantwasfilteredin0.45-␮mfilterandBmCL1purified byionexchangechromatographyinaHiTrapSP(5mL) col-umnconnectedtoanÄKTApurifierchromatograph. The supernatant(400mL)wasappliedtoaHiTrapSPcolumn pre-equilibratedwith50mMsodiumacetatebuffer,pH5.5. Afterthewashingstep,theadsorbedproteinswereeluted byalinearNaClgradient(0–1M)atflowrate1mL/min.The proteinselutionwasmonitoredbyabsorbanceat280nm.A majorproteinpeakwasdetectedatapproximately400mM NaCl.Fractionswereanalyzedbychromogenicsubstrate hydrolysisandbySDS-PAGE(12%).Thefractionscontaining proteolyticactivityandpresentedonemainproteinband werepooledandconcentratedusingamembraneAmicon Ultra5000MWCOfromMillipore(Billerica,MA,USA).

2.8. SDS-PAGE

SDS-PAGEwasperformedaccordingtoLaemmli(1970). 12%SDS-PAGEwith5%stackinggelwasusedtoanalyze rBmCL1.ProteinswerestainedbyCoomassieBrilliantBlue R-250solution.

2.9. Enzymaticactivityassay

The rBmCL1 activity was measured using the chro-mogenic substrate Bz-Phe-Arg-pNa (Sigma) or HD-Pro-Phe-Arg-pNa (Chromogenix) (Erlanger et al., 1961). The sampleswerepre-incubatedat37◦_{Cfor10minin0.1M}

sodiumacetatebufferpH5.5,containing1mMDDT.Then thesubstrate(0.2mM)wasaddedandtheenzyme activ-itywasmonitoredbymeasuringtheabsorbanceat405nm in a platereader Synergy HT model (Biotek, Winooski, USA). The specific cysteine protease inhibitor E-64 (L-trans-epoxy-succinyl-leucylamido-[4-guanidino] butane) was used to determine the rBmCL1 active concentra-tion by active site titration. The enzyme was activated by pre-incubation with 1% DTT in acid medium, fol-lowingincubationwithdifferentconcentrationsofE-64, and finally theactivitywas monitoredby adding chro-mogenicsubstrate,asdescribedabove.Theexperiments withhumancathepsinLandcathepsinBwereperformed usingthesamemethod.

2.10. DeterminationofkineticparametersofrBmCL1

Fig.1.Virionparticleschemeusedforpeptidephagelibraryconstruction.Itisrepresentedbythevirionparticleanditsproteincomposition,withthe singlestrandDNAinside.Asshown,themonovalentsystemwasusedandtheN-terminalofproteingIIIwasfusedto6randomaminoacidsfollowedbya histidinetag.

Table1

YieldandqualityofrBmCl1expressedinthePichiapastorissystem.

Sample volume(L)

Proteina (mg/mL)

Total protein (mg)

Active protease (mg/mL)

Totalactive protease (mg)

Specificactivity (activemg/ proteinmg)

Recovery(%) Yield (fold)

Yeastsupernatant 1 1.33 1330.0 0.0548 54.80 0.041 100.0 1

HiPrepSPP1 8.0 2.40 19.2 0.639 5.11 0.266 9.3 6.5

HiPrepSPP2 12.0 3.20 38.4 1.630 19.56 0.509 35.7 12.4

a_{ProteinquantificationusingtheBradfordmethod(Bradford,1976).}

5.0,at37◦_{C.BmCL1waspre-activatedinthepresenceof}

2mMDTT for 5minat 37◦_{C beforethe additionof the}

substrates.Thefluorescence changes werecontinuously monitoredatex=380nmandem=460nm.The appar-entsecondorder rateconstant kcat/Km wasdetermined underpseudofirst-orderconditions,where[S]≪Kmand performedattwodifferentsubstrateconcentrationsand calculatedbyanon-linearregressionGrafitprogram.Inall determinations,theerrorswerelessthan5%.

2.11. pHactivityprofile

TherBmCL1optimumpHcurveactivitywasperformed at 37◦_{C using Z-FR-MCA assubstrate. We useda}

four-componentbuffercontaining25mMglycine,25mMacetic acid,25mMMes,and75mMTris(3.0<pH<8.0).rBmCL1 was pre-activated with 2mM DTT for 5min at 37◦_C

beforeadding the substrate.For each pH,the apparent second-orderrateconstant (kcat/Km)wasdeterminedat lowsubstrateconcentrations,wherethereactionfollowed thefirst-orderconditions([S]≪K_m).

Fig.2.BmCL1transcriptionanalysis.RNApreparationsofsalivarygland (SG),fatbody(FB),gut,ovary(OV)andhaemocytes(H)ofR.microplus engorgedadultfemalesandspecificprimersofBmCL1wereusedinPCR analysis.Actinspecificprimerswereusedascontrol.

2.12. Determinationofinhibitionconstant(Ki)

TheinhibitionconstantofBmcystatin(Limaetal.,2006) for rBmCL1 was determined usingthe fluorogenic sub-strate Z-FR-MCA. Briefly, theenzyme waspre-activated in0.1Msodiumacetatebuffer, pH5.0,containing1mM DTTfor5minat37◦_{Candthenpre-incubatedwith}

Bmcys-tatin (0.2–0.7␮M) for 10min before adding Z-FR-MCA (10␮M). The residual enzyme activity was continu-ouslymonitoredbyfluorescencechangesatex=380nm and em=460nm. The inhibition constant (Ki) was calculatedbyfittingthesteady-statevelocitiestothe equa-tion (V_i/Vo=1−{Et+It+Ki−[(E_t+I_t+K_i)2−4E_tI_t]1/2}/2Et) fortight-bindinginhibitorsusinganon-linearregression analysis(Morrison,1969).

2.13. Peptidemutantphagedisplaylibraryconstruction

The library was designed with random mutation in six positions. Initially, a PCR reaction was performed witha mutatedoligonucleotideas template(PEPTN: 5′

- CTTTCTATGCGGCCCAGCCGGCCAGCCATCATCATCATCATC- ATCATGGAGCAGCNNSNNSNNSNNSNNSNNSGCGGCCGC-TTTTCCTTG-3′_{–N=A/T/C/G;S=G/C)toachieve}

Fig.3. AlignmentofBmCL1(accessnumberAF227957)aminoacidsequencewithothercathepsinL-likeproteasesfromtickDermacentorvariabilis(DvCL) (accessnumberEU025855),Rhipicephalusappendiculatus(RaCL)(accessnumberAY208824),Ixodesricinus(IrCL)(accessnumberEF428205),andhuman Homosapiens(CathL1)(accessnumberNM001912).Signalpeptideaminoacidsequencesareboxed.AminoacidresiduesofputativeBmCL1pro-peptideare underlined.Theaminoacidresidueswhichappearatleastinthreeenzymesequencesareshadowed.Thecatalytictriadaminoacidresiduesareidentified byablackbox.

2.14. PeptidelibraryselectionforrecombinantBmCL1

After the titration of peptide phage display library (9.0×106CFUtotal), E.coli TG1 transformed cells were

grown in 2YTmedium containing200␮g/mLampicillin

and2%glucoseuntilanopticaldensityofA550=0.5–0.7was reached,afterwhichhelperphage(M13K07)witha mul-tiplicityofinfectionof50wasaddedinordertoproduce fusionphages,asdescribedinthemanufacturer’sguide. Theculturewascentrifugedandthemediumwasreplaced

Table2

KineticparametersforthehydrolysisoffluorogenicsubstratesbyBmCL1andothercysteineproteases.

Substrate rBmCL1 CathepsinLa _CathepsinBa

kcat(s−1_{) K}

m(␮M) kcat/Km

(mM−1_s−1₎

kcat(s−1_{) K}

m(␮M) kcat/Km

(mM−1_s−1₎

kcat(s−1_{) K}

m(␮M) kcat/Km

(mM−1_s−1₎

Z-FR-MCA 11.0 0.3 36,700 10.3 2.0 5150 75.9 23.4 3243

Z-LR-MCA 3.0 0.12 25,000 – – – – – –

Z-RR-MCA b b b _{0.22 51.0 4.3 18.0 82.0 220.0}

a_{Meloetal.(2001).}

Fig.4. PurificationandcharacterizationofrecombinantBmCL1.(A)IonexchangechromatographyofsupernatantcontainingrBmCL1onHiTrapSPcolumn. Afterexpression,yeastcellswereharvestedandthesupernatantsubmittedtoanion-exchangechromatographyinHiTrapSPcolumn.Yeastsupernatant containingrBmCL1wasdialyzedagainst50mMsodiumacetatebufferpH5.5anditwasappliedtothecolumnpre-equilibratedwiththesamebuffer.After thewashingstep,theproteinswereelutedwith50mMsodiumacetatebufferpH5.5containing1MNaCl.Theblacklinesshowtheproteolyticactivity fractionsselectedforpoolsP1andP2,respectively.(B)SDS-PAGE(12%)ofpurifiedrBmCL1.MM.Molecularweightstandard.1.PeakP1ofrBmCL1after HiTrapSPcolumn.2.PeakP2ofrBmCL1afterHiTrapSPcolumn.Theblackarrowshowsproteinbandaround27kDa.

by 2YT containing 200␮g/mL ampicillin and 50␮g/mL kanamycinand incubatedfor15h,at37◦_{C.Next,fusion}

phageparticles released in thesupernatant were sepa-ratedfromthebacterialpelletbycentrifugation. Fusion phagesolutionwasusedforfourroundsofselection.The phageparticleconstructionis representedinFig.1.The constructedfusionphageparticlesexpressHisTagat pro-teinIIIN-terminalthataffordedphageaffinitybindingto Ni-NTAmagneticagarosebeads(GE).A500-␮Laliquotof

Table3

Inhibitionconstant(Ki)ofBmcystatinfordifferentcysteineproteases.

Ki(nM)

rBmCL1 CathepsinL CathepsinB

Bmcystatin 370 0.1a ₈₀a

Ca074 n.i. 233,000b _1.94b

n.i.:notinhibiteduntiltheconcentrationof1␮M.

a_{Limaetal.(2006).} b _{Towatarietal.(1991).}

thesupernatantcontainingfusionphageswasincubated with30␮LofagaroseNi-NTAfor6hinatubemixeratroom temperature. Subsequently, the beads were centrifuged andwashedseventimeswith50mMTris–HClbuffer,pH 8.0,containing20mMimidazoleand100mMNaCl.Atthis moment,rBmCL1wasaddedandincubatedfor1hat37◦_C.

rBmCL1cleavedpeptideswerereleasedinthesupernatant and1␮ME64wasaddedtoavoidthecleavageofselected phageparticleproteins.Theelutedphageswereusedto transfectnewE.coliTG1strainatA550=0.5–0.7toamplify theseselectedphagesforanotherroundofselection.Atthis step,ineveryroundofselectionthenumberofselected phageswasdeterminedbytitration.

3. Resultsanddiscussion

3.1. TranscriptionanalysisofBmCL1byPCR

Table4

SelectedpeptidesequencesforrBmCL1usingapeptidephagedisplaylibrary.

Sequence Occurrence

Sequenced clones

G N Q V E L A A A G A

15

S S V W V R# S A A

A P

6

G S S A V R

# G Q G A P

6

H G S S

_{L L*}

Q V S

A A

6

H G S S

_{L L}

_*

Y V S A A

5

H H G S S R#

T

_{L L}

G E

5

S S R E L R#

G G A A A

3

G S S K

_{L L}

_*

K S A A A

3

H H G S S L Q V S N

W

3

C P G V L R# A A A P A

2

S S G A V L G A

A A A

2

Sequencesnot showed**

*

Leu-Leu (appeared in more

:)

6

_{Single Leu (appeared in more:)}

₅

#Arg (appeared in more:)

6

**Selectedpeptidesequenceswithfewrecurrencethatpresentsthesameobserveddatainthemostrepetitiveclones. *SequencescontainingLeu–Leurepresentaround25%ofalltheselectedpeptidesequences.

&_{SequenceswithoneLeuresiduerepresentaround30%ofallselectedpeptidesequences.} #_{SequencescontainingArgrepresentaround28%ofalltheselectedpeptidesequences.}

engorgedfemalesusingtotalRNAastemplate.TheBmCL1 transcriptinovary,fatbody,salivarygland,gutand hemo-cytesofR.microplusengorgedadultfemaleswasanalyzed (Fig.2), andtheBmCL1transcription wasidentifiedonly inthegut,reinforcingitsimportanceindigestion(Renard et al.,2002).The RNAsamples integrity wasconfirmed usingactinspecificprimers,ascontrol.

3.2. Cloning,expression,purificationandbiochemical characterizationofrBmCL1

Previously, a cysteine protease similar to cathepsin L from R. microplus, named BmCL1 was cloned and expressed in E. coli (Renard et al., 2000, 2002). The recombinant BmCL1wasabletohydrolyzenatural sub-stratesuchashemoglobinandalsosyntheticsubstrates (Renard et al., 2002). BmCL1 expression was localized in the gut cells of ticks, suggesting that it can be a promising target to be used for R. microplus control (Renardet al., 2002).In an attempt toproduce a large amountofBmCL1,theDNAfragmentencodingforBmCL1 (GenBank:AAF61565) wasclonedintopPIC9expression vector. The construction pPIC9-BmCL1 (10␮g) was lin-earizedandusedinthemethylotrophicyeastP.pastoris transformation. Several clones were tested for rBmCL1 expression.

Several clones of P. pastoris carrying BmCL1 DNA fragment were cultivated for rBmCL1 secretion to the supernatant.Allcell-freeculturemediawereanalyzedby SDS-PAGEandpresentedapredicted28kDaproteinband, whichwasnot presentin themedium beforemethanol induction.AmongthedifferentclonesproducingrBmCL1, theoneexpressingthelargestamountofactiveenzyme was chosen to scale up protein expression. The active rBmCL1expressionlevelwas54.80mg/L,beinghigherthan that expressed by E. coli (Renard et al., 2000). BmCL1 expression was comparable to other cysteine protease expressedintheP.pastorissystem(Aokietal.,2003;Chan etal.,1999;Linneversetal.,1997).

TherBmCL1secretedtotheP.pastorismediumwas puri-fiedbyonechromatographystepinaHiTrapSPcolumn (Fig.4A).ActiverBmCL1 wasfractionatedin two peaks, namedP1andP2,yielding5.11mgand19.56mgpurified enzyme,respectivelythatdisplayedthesameN-terminal. rBmCL1P2showedhigherenzymaticrecoveryand purifi-cation(35.7%and12.4-fold,respectively;Table1).BothP1 andP2showedamajorproteinbandofabout28kDa,but severallowerproteinbandsweredetectedinaSDS-PAGE, whichsuggesteddegradationprocess

PurifiedrBmCL1ofP1fractionpresentedhighspecific activitywasusedinkineticassays.Theapparentsecond orderrateconstant kcat/Km ofrBmCL1for two different substrateswasdeterminedandcomparedtothatobtained for cathepsin L and cathepsin B (Table 2). Z-FR-MCA was cleaved by rBmCL1 (kcat/Km=36,700mM−1s−1), cathepsin L (kcat/Km=5150mM−1s−1) and cathep-sin B (kcat/Km=3243mM−1s−1), while Z-LR-MCA was hydrolyzed only by rBmCL1(kcat/Km=25,000mM−1s−1) and Z-RR-MCA was poorly hydrolyzed by cathep-sin L (kcat/Km=4.3mM−1s−1) and cathepsin B (kcat/Km=220.0mM−1s−1), and not at all by BmCL1. These results indicated substrate selectivity between these enzymes. The BmCL1 kcat/Km relationship for Z-FR-MCAhydrolysiswas1.5-foldhigherthanthatforthe hydrolysistheZ-LR-MCA.

Ininhibitoryassays,BmCL1,cathepsinLandcathepsin BweretestedwithBmcystatin,acystatinfromR.microplus (Limaetal.,2006)andCA-074,airreversiblecathepsinB inhibitor(Table3).BmcystatinwasabletoinhibitBmCL1, cathepsinL and B, withK_i in nM range,indicating that BmCL1 can be one target of the natural cystatin of R. microplustick,controllingitsexcessiveproteolyticactivity. Previously,Towatarietal.(1991)hadshownthatCA-074 inhibited cathepsin L and cathepsin Bwith Ki value of 23.3nMand1.94nM,respectively;however,CA-074was notabletoinhibitBmCL1.

TherBmCL1optimumpHdeterminationwasperformed at37◦_{C usingafour-componentbufferoverapHrange}

of 3.0–7.0 (Fig. 5) and the second-order rate constant wasdetermined.rBmCL1optimumpHwas5.5,confirming itssimilaritytotheexpressedenzymebyE.coli(Renard et al., 2002). These findings are consistent with verte-bratecathepsinsandalsowithBmCL1roleinthedigestion process,whichmayoccurinacidpHoftheintestine envi-ronment(Laraetal.,2005).

3.3. SelectionofsubstrateforrBmCL1usingapeptide phagedisplaylibrary

Therandomsequence(His)6(Gly)1(Ser)2(X)6wasfused toM13protein3geneatthephagemidpCANTAB-5E.The constructedpeptidephagelibrarytitlewas9.0×106CFU.

To analyze the efficiency of input phages coupling on the beads, the library titer was calculated before and after incubation with beads, presenting 4.6×1012CFU

and 1.43×1011CFU, respectively, which indicates that

97% of the fusion phages were bound to the beads. After the 3rd round of selection, the phage enrich-ment for rBmCL1 was 83 times higher than in the 2nd round. Taken together, these results demonstrate

pH

9

8

7

6

5

4

3

2

% of

A

ct

ivi

ty

0

20

40

60

80

100

Fig.5.OptimumpHcurveforrBmCL1.Thehighestproteolyticactivity wasconsideredtobe100%.TheassaywasperformedusingZ-FR-MCAas substrate.

robustnessofphagedisplayasamethodforsubstrate selec-tion.

Theknowledgeofproteasespecificitycanhelpto iden-tifybiologicalsubstratesandguidethedesignofspecific inhibitors(UhlenandMoks,1990).Inordertounderstand BmCL1substratespecificitywesequenced100phagemids selectedforBmCL1andthetranslatedpeptidesequences werealignedinaweblogoanalysisprogram(Crooksetal., 2004)(Fig.6).Analysisofthe100phagemidsindicatesthat chargedamino acidsand non-polar aminoacids appear frequentlyatthesixmutatedpositions,corroborating pre-viousreportsthatrBmCL1hasapreferencefortheseamino acidsatpositionP1andP2(Cruzetal.,2010).Of signif-icancewasthepresenceofLeuatallsixpositions,while Argwasthenextmostfrequentlyobservedamino acid. ThisresultsuggeststhatrBmCl1maycleaveafterLeuor Leu–Leuresidue,andthiscouldbetheprocessingsitefor intestinalproteins,i.e.BmTIs(Sasakietal.,2004;Sasakiand Tanaka,2008).N-terminalsequencingofrBmClconfirmed thatrBmCL1iscleavedat106L/PPA,whichisincontrastto the115S/LPKsequenceproposedbyRenardetal.(2000).

rBmCL1 preferred non-polar residues at position P2 usinghemoglobinassubstrate(Cruzetal.,2010).Thisdata corroboratesourfindingsinwhichifGln,Arg,LeuorGlu arefixedatP1position,non-polarresiduesrepresents60% ofalldeterminedsequences(Table4).Ontheotherhand, whenmerelyArgisfixedatpositionP1,rBmCL1hasa pref-erenceforunchargedaminoacidresidues(mainlyValor Leu)atpositionP2(Table4).Anotherrelevantdataofthis studywasthehighfrequencyofLeu–Leuresidueswhich mayindicateotherroleforBmCL1besidesitsinvolvement inproteindigestion.

Fig.6. Weblogorepresentationofaminoacidrecurrenceatthesixmutatedpositionof100randomsequencedclonesforrBmCL1.Weblogowasgenerated usingWebLogo3:PublicBeta.Intherepresentation,thecorrespondingfrequencyplotisreported.

hypothesisthatBmCL1isadigestiveenzyme.Hydrolysisof hemoglobinbygutcellsgenerateshydroxylradicalsinthe gut(Citellietal.,2007),andinthisenvironmentthe plas-maticproteinscouldbedenaturedfavoringtheirdigestion byrBmCL1.

In conclusion, weshoweda highrBmCL1expression level by yeast P.pastoris. Also, it wasobserved that its purificationwillaffordtheproductionoflargeamountof purifiedrBmCL1tobeusedinbovineimmunizationand crystallization experiments.Moreover,we describedthe firstsubstratephagedisplayselectionforacysteine pro-tease,determiningitscleavagesitespreference,reinforcing thehypothesisthatrBmCL1isapotentdigestiveenzyme ofR.microplusbutalsocanplayanotherimportantrolein thetickphysiology.

Acknowledgments

Thisworkwassupportedby:Fundac¸ãodeAmparoa PesquisadoEstadodeSãoPaulo(FAPESP)(N◦

05/03514-9, N◦ _{05/03339-2, N}◦ _{09/50434-1); Conselho Nacional}

deDesenvolvimentoCientíficoeTecnológico(CNPq)(N◦

470297/2006-9); INCT-Entomologia Molecular. We are gratefultoDr.IzauraY.HirataofDepartamentodeBiofisica, UNIFESP-EPMforperformingaminoacidsequencingand Fernando Sakon for helping in figurelayout. A.S.T. was recipient of CNPq fellowship. R.O.C. was supported by CNPq.

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