Contentslistsavailableat SciVerseScienceDirect
Molecular
&
Biochemical
Parasitology
Leishmania
(L.)
amazonensis
peptidase
activities
inside
the
living
cells
and
in
their
lysates
Elide
E.
Caroselli
a,
Diego
M.
Assis
a,
Clara
L.
Barbiéri
b,
Wagner
A.S.
Júdice
c,
Maria
A.
Juliano
a,
Marcos
L.
Gazarini
d,
Luiz
Juliano
a,∗aDepartmentofBiophysics,EscolaPaulistadeMedicina,UniversidadeFederaldeSãoPaulo,SP,Brazil
bDepartmentofMicrobiology,ImmunologyandParasitology,EscolaPaulistadeMedicina,UniversidadeFederaldeSãoPaulo,SP,Brazil
cCentroInterdisciplinardeInvestigac¸ãoBioquímica,UniversidadedeMogidasCruzes,Av.Dr.CândidoXavierdeAlmeidaSouza200,08780-911MogidasCruzes,Brazil
dDepartmentofBiosciences,UniversidadeFederaldeSãoPaulo,Santos,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received24November2011
Receivedinrevisedform13March2012 Accepted27April2012
Available online 6 May 2012
Keywords: Cysteineprotease Peptidase OligopeptidaseB Fluorescentpeptides
a
b
s
t
r
a
c
t
InthisstudyweinvestigatedthepeptidaseactivityinLeishmania(L.)amazonensisliveamastigoteby con-focalmicroscopyusingpeptidyl-MCAassubstrates,thehydrolysisofwhichreleasestheMCAfluorophore insidethecells.Cellpre-treatmentwithpeptidaseinhibitorsindicatedthepresenceofcysteineand ser-inepeptidases.ItwasnoteworthythatLeishmaniaamastigotesincorporateonlysubstrates(Z-FR-MCA, Z-RR-MCA)orinhibitors(E64,TLCK)containingpositivelychargedgroups.Thepeptidaseactivitiesinthe supernatantsofamastigotesandpromastigoteslysateswerealsoevaluatedwiththesamepeptidyl-MCA substratesandinhibitorsinthepHrange4.5–9.0.Theeffectsoftemperatureanddifferentsaltswerealso includedinthisstudy.ThehydrolyticactivitiesofsupernatantsonZ-FR-MCAclearlyindicatethe pres-enceofdifferentcysteinepeptidasesthatadaptedtoworkindifferentenvironmentconditions.Intact LeishmaniacellsincorporatedZ-RR-MCA,thehydrolysisofwhichwasinhibitedonlybyTLCKindicating thepresenceofatleastoneserinepeptidase.ThepHprofileofZ-RR-MCAhydrolysisbyamastigotesand promastigoteslysatesupernatants,andthehydrolysistimecourseoftheFRETpeptide Abz-AGRRRAQ-EDDnpatR Abond,followedbyremovalofthetwoC-terminiRtoyieldAbz-AGR-OHthatisaunique characteristicofoligopeptidaseB,indicateitspresenceintheparasite.
© 2012 Elsevier B.V.
1. Introduction
Peptidasesarecriticalforthesurvivalandpathogenicityof par-asites,playingrolesintherecyclingandmetabolismofproteins
[1–6], invasion of host cells and tissues [7,8], parasite nutri-tion,modificationofhostproteins[8,9],parasiteimmuneevasion and differentiation [10–12]. The set of peptidases described in
Leishmaniaspeciesincludesaspartic,cysteine,metallo,serineand threoninepeptidases,andrepresentaround1.8%ofthegenome
Abbreviations: DTT,1,4-dithiothreitol;MCA,[7-amino-4-methyl]coumarin; E64, 1-[l-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane; Abz, ortho -aminobenzoic acid; EDDnp[, N-(2,4-dinitrophenyl) ethylenediamine]; TLCK, tosyl-lysinechloromethylketone;FRET,fluorescenceresonanceenergytransfer; PMSF,phenylmethylsulfonylfluoride;CPB,cysteinepeptidaseB;pFF-MCA,herep meansprolineasd-stereoisomer,-NH2-caproyl-C(SBzl)-C(SBzl)-MCA,hereSBzl meansbenzylboundtosulfydrylgroup;CPB2.8CTE,clonedcysteinepeptidaseB withdeletedC-terminalextension.
∗Correspondingauthorat:DepartmentofBiophysics,UniversidadeFederalde
SãoPaulo,100RuaTresdeMaio,SP,Brazil.Tel.:+551155764450; fax:+551155759617.
E-mailaddress:ljuliano@terra.com.br(L.Juliano).
sequence[13]http://merops.sanger.ac.uk/.Thebestcharacterized peptidases in thegenusLeishmania are thecysteine peptidases (CPs) designatedas CPA,CPB and CPC[14] that belongto clan CA. The cysteine peptidases present stage-regulated levels,are importantvirulencefactors,modulatemammalianhostimmune cellsandfacilitatetissuehostinvasionandconstitutean attrac-tivepotentialtargetforchemotherapy[12,14].Leishmaniaparasites havetwoformsintheirlifecycle:thepromastigote,aflagellate extracellularorganismlivinginthegutofphlebotominaeinsect andanobligateintracellularovalamastigote.Amastigotesare aci-dophilesandadaptedtoliveinsidetheparasitophorousvacuoleof vertebratehostmacrophages[7,15].Theactivitiesofcysteine pep-tidasesareconsiderablyhigherinamastigotethanpromastigote form[12],particularlyCPB-likecysteinepeptidaseactivitiesthat increase during thedifferentiationof internalized promastigote intoamastigotewithinmacrophages[7].Otheridentifiedcysteine peptidases thatdifferfundamentallyfromtheclanCAenzymes havebeenassignedtotheclanCDthatincludesmetacaspase, sep-araseandGPI6[16].Thebeststudiedmetallopeptidaseinseveral
Leishmaniaspeciesisleishmanolysin(gp63,MSP),asurfacezinc endopeptidase,thatmaximizespromastigoteinvasionand intra-macrophagesurvivalofamastigoteform[17,18].Leishmaniasp.has
0166-6851© 2012 Elsevier B.V.
http://dx.doi.org/10.1016/j.molbiopara.2012.04.012
Open access under the Elsevier OA license.
184 (2012) 82–89 83
atleasttwoasparticpeptidases,asolublecathepsinD-simileand onemembrane associatedpresenelin-simile (PS1)[13].Another peptidasecalledsignalpeptidase(SPP)hasbeenidentified[19,20]. Genesthatencodeserinepeptidases inLeishmania include pro-lyloligopeptidase(POP),andoligopeptidaseB,which arerelated toparasitevirulence[21,22].OligopeptidaseBpertainstoprolyl oligopeptidasefamily(clanSC,familyS9)butishighlyspecificfor basicaminoacids[23,24].
Theaimofthisstudywastoinvestigatethepeptidase activ-ity in live Leishmania (L.) amazonensis amastigotes by confocal microscopy.Z-FR-MCAand Z-RR-MCA wereused assubstrates, whosehydrolysisinsidethecellsreleasestheMCAfluorophore, resultinginintensefluorescencethatenabledquantizationofthe peptidaseactivities.Z-FR-MCAisapeptidesubstratethatis effi-cientlyhydrolyzedbyserineandcysteinepeptidases[25] while Z-RR-MCAismainlyhydrolyzedbyserinepeptidasesthatprefer orevenrequireapairofbasicresiduesasreportedfor oligopepti-daseB[24,26],dengueandyellowfevervirusNS2B-NS3proteases
[27,28].Thepeptidaseactivitiesinthesupernatantsof amastig-otesandpromastigoteslysateswerealsoevaluatedwiththesame peptidyl-MCAsubstratesinthepresenceandtheabsenceofthe inhibitorsinthepHrange4.5–9.0.Theeffectsoftemperatureand differentsaltsonthepeptidaseactivitiesofamastigotesand pro-mastigoteslysateswerealsoinvestigatedinordertoexploreifthe enzymesaredifferentiallyadaptedtoenvironmentalconditions.
The significant hydrolysis of Z-RR-MCA inside the parasites and in thesupernatant of amastigote and promastigote lysates mainlyatalkalinepHsuggestedtheexpression ofan oligopep-tidaseB,whichwaspreviouslycloned[29].Theseresultsledus toassayassubstratethefluorescenceresonanceenergytransfer (FRET) peptide Abz-AGRRRAQ-EDDnp that characteristically is hydrolyzedbyTrypanosomacruziandTrypanosomabrucei oligopep-tidase B at the R A bond (Abz-AGRRR↓AQ-EDDnp) followed by removal of the two C-terminus R from the Abz-AGRRR-OH product(Abz-AGRR↓R-OH and Abz-AGR↓R-OH)resulting in Abz-AGR-OH [24]. In FRET peptides Abz (ortho-aminobenzoic acid)isthefluorescencedonorattheC-terminusandQ-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine])isthe fluo-rescence acceptor at the N-terminus. While this work was in progress,McKerrow’sgroupidentified, cloned,knocked-outthe geneandcharacterizedanoligopeptidaseBinLeishmania(L.) dono-vani[30].
2. Materialsandmethods
2.1. Preparationofamastigotes,promastigotesandtheirlysates
The L. (L.) amazonensis strain (MHOM/BR/1973/M2269) was characterizedandkindlyprovidedbyDr.JeffreyJ.Shaw,Instituto EvandroChagas,Belém,Pará,Brazil,andmaintainedasamastigotes byinoculationintofootpadofgoldenhamstersevery4–6weeks asearlierreported[31].Briefly,amastigotesuspensionswere pre-paredbyhomogenization of excisedlesions,disruptionbyfour passagesthrough22-gaugeneedlesandcentrifugationat250×g
for10min;theresultingsupernatantwascentrifugedat2000×g
for10min,andthepelletwassuspendedinmediumRPMI1640 (Invitrogen,RoswellParkMemorialInstitute1640).The suspen-sionwaskeptunder agitationfor4hatroomtemperature and centrifugedat250×gfor10min.Thefinalpelletcontained puri-fiedamastigotes,whichwereessentiallyfreeofcontaminationby othercells[32].
L. (L.) amazonensis strain promastigotes were kept by the firstpassageat26◦Cin199medium(Gibco-BRL)supplemented
with40mMHEPES,0.1mMadenine,2mMl-glutamine,5gml−1
hemin(in50%triethanolamine),100Uml−1penicillin,100gml−1
streptomycin,and10%heatinactivatedfetalbovineserum (Gibco-BRL). The promastigotes were isolated from stationary growth phase(5to6-day-old)cultures[33].
Promastigotesandamastigoteslysateswerepreparedby cen-trifugationoftheparasitesat2000×gfor10min,separatedinto smallflaskscontaining1×109cells/mlandfrozenat−20◦C.The
pelletsweresuspendedin 100lofNaCl(0.9%)containing0.1%
TritonX100andkeptonice,sonicated(UltrasonicSonicatorXL 2020,MisonicInc.,NY,USA)for3cyclesduring20swith5s inter-valsbetweenthecycles.Thesolutionwasdilutedto200land
centrifugedat6000×gfor10min.Thesupernatantwasseparated fromthepelletandusedinthepeptidaseactivityassays.
2.2. Detectionofintracellularproteolysisactivitywithconfocal microscopy
L. (L.) amazonensis amastigotas (1×109cells/ml) were sus-pendedinbuffercontaining75mMTRIS,25mMglycine,25mM MES and 25mM acetic acid, with 10mM CaCl2, 116mM NaCl, 0.8mMMgSO4,0.8mMKCland55mMglucose,pH7.0,atroom temperatureandplacedinaglassbottomdishformicroscopy (Mat-TekCorp.,USA)pre-treatedfor1hwithpoly-lysinesolutionforcell adhesion.DynamicimagingwasperformedwithaLSM510 laser-scanningmicroscope(CarlZeiss)usingLSM510software,version 2.5.TheAxiovert100Mmicroscopeisequippedwitha63×water immersionobjective.Parasiteswereplatedontomicroscopycover slips(MatTekCorp.)pretreatedfor1hwithl-polylysinesolution andexcitedat351nm(AbzorMCA).Emittedlightwascollected througha bandpassfilterat387–470nm(Abzor MCA). Trans-mittedlightobservationswereperformedduringtheexperiments inordertofollowtheintegrityofthecells.Fluorescencearbitrary unitswereacquiredfromanaverageofselectedwholeparasite areas.Theresultsarerepresentativeofatleastthreeexperiments adding10Mofpeptidesubstrates.
2.3. Hydrolysisofpeptidyl-MCAandAbz-peptidyl-Q-EDDnp substratesbyamastigotesandpromastigoteslysates
ThesubstratespFF-MCA,Z-FR-MCA,Z-RR-MCA,
-NH2-caproyl-C(SBzl)-C(SBzl)-MCAwere synthesizedas earlier described [34]
and assayed in a Shimadzu 1501 spectrofluorimeter, at 37◦C.
The fluorescence measurements were monitored continuously at ex=380nm and em=460nm. The fluorescent rates were convertedintoenzymaticactivityunits(nanomolesofsubstrate hydrolyzedpersecondpernanomoloftotalprotein)basedona calibrationcurveobtainedfromthecompletehydrolysisofeach peptide. The FRETpeptide Abz-AGRRRAQ-EDDnp was obtained bysolid-phasepeptidesynthesisaspreviouslydescribed[35,36]
usingtheFmoc-procedureinanautomatedbench-top simultane-ousmultiplesolid-phasepeptidesynthesizer(PSSM8systemfrom Shimadzu,Tokyo,Japan). Theassayconditionfor this substrate wasex=320nmandem=420nm.Thekineticparameterswith respectivestandarderrorswerefitusingGraFit®software
(Eritha-cusSoftware,Horley,Surrey,UK),andalldatawerecollectedin triplicate.
2.4. EffectsofpH,salts,temperatureandinhibitorsonhydrolytic activityoftheamastigoteandpromastigotelysates
ThepHdependenceofthehydrolyticactivitiesofthe amastig-oteandpromastigotelysatesupernatantsweremeasuredat37◦C
84 184 (2012) 82–89
cysteinepeptidaseactivityifpresent. Theeffectoftemperature onthepeptidaseactivitieswasmonitoredatpH5.0andpH8.0, andrangingfrom27to60◦C.TheinfluenceofNaCl,sodiumsulfate
andsodiumcitrate,wasinvestigatedoveraconcentrationrangeof 50–1000mM,andperformedatpH5.0andpH8.0,inaShimadzu 1501spectrofluorimeterasdescribedabove.Thefollowing pepti-daseinhibitorswereassayedinordertoidentifytheclassofenzyme presentinthelysates:1MpepstatinA,10MPMSF,100MTLCK
and10ME64.Alltheinhibitorswerepre-incubatedfor10min
withthesupernatantsbeforetheadditionofthesubstrateat35◦C
inthebufferdescribedabove.
2.5. HydrolysisofAbz-AGRRRAQ-EDDnpbylysatesmonitoredby HPLC
Thehydrolysisof50MAbz-AGRRRAQ-EDDnpbythelysate
supernatantsofpromastigotesandamastigotesin50mMTris/HCl bufferwith1mM DTT,at 37◦C andpH8.0, weremonitoredby
HPLC.Aliquotsof100lofthereactionmixturewereanalyzedin
anUltrasphereC18column(5m,4.6mm×250mm),whichwas elutedwiththesolventsystem(water/TFA1000:1)andB (acetoni-trile/water/TFA,900:100:1)withflowof1ml/minandagradient 0–80%ofBfor40min.TheproductsweredetectedbyUVat220nm and the molecularweight of each product wasdetermined by LC/MSusingLCMS-2010EVequippedwithESI-probe(Shimadzu), connectedtothecircuitaftertheUVdetector.
3. Results
3.1. Confocalmicroscopymeasurementsofintracellular proteolysisinL.(L.)amazonensisamastigotes
Livecellassayswereperformedwithisolatedparasitesplated ontomicroscopycoverslips,andthetransmittedlightobservations wereperformedduringtheexperimentsinordertofollowthecell integrity.Fluorescencearbitraryunitswereacquiredfroman aver-ageofselectedwholeparasiteareasusingassubstratesZ-FR-MCA atpH5.0inthepresenceandtheabsenceofpeptidaseinhibitorsas showedinFig.1.E-64wasthemosteffectiveinhibitorfollowed byTLCK,demonstrating significantcysteineprotease activityin
L.(L.)amazonensis amastigote livecells. Amastigoteswerealso assayedwithpeptidyl-MCAsubstratesthatarewellhydrolyzedby cysteinepeptidases(pFF-MCAand
-NH2-caproyl-C(SBzl)-C(SBzl)-MCA)[34,37]butnohydrolysisinsidethecellswasdetectedwith them.Theintegrityofcellswasconfirmedwithfurtheradditionof Z-FR-MCA(10M),withwhichintracellularhydrolysiswasclearly
observed(datanotshowed).However,allthesepeptideswerewell hydrolyzedbytheamastigotesupernatantlysateascomparedto Z-FR-MCAandtheirhydrolysiswasmorethan90%inhibitedbyE64 (Fig.2).Theseresultsindicatethatatleastonepositivechargeis requiredforthepeptidepermeationintotheamastigotelivingcell asthepositivechargedgroupspresentinthearginine,lysineand agmatine([4-aminobutyl]-guanidine)ofZ-FR-MCA,TLCKandE64, respectively.Inaccordancewiththeseobservations,Z-RR-MCAwas incorporatedandhydrolyzedinsidetheamastigotecells, andin contrasttothehydrolysisofZ-FR-MCA,theZ-RR-MCAhydrolysis wasinhibitedbyTLCKbutnotbyE64(Fig.3).Theseresultsindicate thatZ-RR-MCApeptidewashydrolyzedbyaserinepeptidasethat appearstobeanoligopeptidaseBasdemonstratedbelowwiththe peptidaseactivitiesoftheparasitesupernatantlysates.
3.2. PeptidaseactivityinL.(L.)amazonensisamastigotesand promastigotelysates
ThehydrolysisofZ-FR-MCAbyL.(L.)amazonensisamastigote lysateswassubstantiallyactivatedbyDTTinthepHrange4.5–9.0
Fig.1. DetectioninlivecellsofLeishmania(L.)amazonensisamastigotesof intracel-lularhydrolysisofZ-FR-MCAusingconfocalmicroscopy.Theassayswerecarried outinthepresenceandtheabsenceofpepstatin,TLCKandE64.Thebarsrepresent thefluorescenceincreaseafterZ-FR-MCAcleavage.DetailsaredescribedinSection 2.
asshowedin Fig.4Aand theinhibitionbyE64 ofthis cysteine peptidaseactivityonZ-FR-MCAaswellasthat onthepeptides pFF-MCAand-NH2-caproyl-C(SBzl)-C(SBzl)-MCAhydrolysisare
showedinFig.2.Itisnoteworthythatthiscysteinepeptidase activ-ityismaintainedinbothacidandbasicrangeofpHthatcontrasts withthenarrowpHprofileofmammaliancathepsincysteine pep-tidases,which are quiteinstable inalkaline environments[38]. ThepHprofileofhydrolyticactivitiesofthelysatesupernatants fromamastigotesandpromastigotesshowstwooptimumpHs,one aroundpH5andanotheratpH8,indicatingthatmorethanone cysteinepeptidaseispresentinthelysatesupernatants.The pres-enceoftwoor morecysteinepeptidases couldalsoexplain the broadpHprofileinamastigotelysatesupernatants(Fig.4A).Abroad pH-profilewasalsoobservedforthehydrolysisofZ-FR-MCAby CPB2.8CTE(Fig.4B)thatisapurifiedrecombinantcysteine pep-tidaseofLeishmaniamexicana[39].Thehighactivityandstability inalkalinepHofcysteinepeptidasesfromparasitesseemstobea generalcharacteristicoftheseenzymesaspreviouslyreported[5]. ThepHprofileofL.(L.)amazonensisamastigoteslysate super-natantshydrolyticactivityonZ-RR-MCAisshowninFig.4C.The pHoptimumisaround8.0withitsactivitycompletelylostatpH 4.5.SlightactivationbyDTTwasdetectedonlyinthepHrange 7–9andisnotinhibitedbyE64,whichisincontrastto hydrol-ysis of Z-FR-MCA. Different peptidase inhibitors were used to characterizethesupernatanthydrolyticactivityonZ-RR-MCAas substrate,andalmosttotalinhibition wasobservedinthe pres-enceofTLCKwhereaspepstatin,PMSF,E64andEDTAdidnotaffect thehydrolyticactivity(Fig.5).Thesameexperimentswerealso performedwiththepromastigotelysatesupernatantandsimilar resultswereobtained,exceptthattheremaininghydrolyticactivity onZ-FR-MCAinthepresenceofE64andthehydrolyticactivityon Z-RR-MCAwerehighercomparedwiththatobservedwith amastig-otelysatesupernatant(Fig.6).
184 (2012) 82–89 85
pH
9 8.5 8 7.5 7 6.5 6 5.5 5 4.5
Enzymatic Activity Units
0 50 100 150 200
pH
9.0 8.5 8.0 7.5 7.0 6.0 5.5 5.0 4.5
Enzymatic Activity Units
0 5 10 15 20
pH
9 8.5 8 7.5 7 6.5 6 5.5 5 4.5
Enzymatic Activity Units
0 100 200 300
400
(A)
(B)
(C)
2-Cap-Cys(SBzl)-Cys(SBzl)-MCA (D)Pro-Phe-Phe-MCA
Z-Phe-Arg-MCA ε
Fig.2. HydrolyticactivityoftheLeishmania(L.)amazonensisamastigoteslysatesinpHrange4.5–9.0,inthepresenceof1mMDTTusingassubstrates:Z-FR-MCA(A), -NH2-Caproyl-C(SBzl)-C(SBzl)-MCA(B)andpFF-MCA(C)assubstratesintheabsence(fullbars)andthepresence(emptybars)ofE64.
Fig.3.DetectionofintracellularhydrolysisofthepeptideZ-RR-MCAinLeishmania(L.)amazonensisamastigotelivecellsusingconfocalmicroscopy.Theassayswerecarried outintheabsence(A)andthepresenceofTLCK(B)andE64(C).
peptideAbz-AGRRRAQ-EDDnpthatwascleavedatR Abond (Abz-AGRRR↓AQ-EDDnp)followedbyremovalofthetwoC-terminus RfromtheAbz-AGRRR-OH product(Abz-AGRR↓R-OHand Abz-AGR↓R-OH)resultinginAbz-AGR-OH(Fig.7).Thiscleavagepattern ofAbz-AGRRRAQ-EDDnpwassimilartothatpreviouslyobserved withT.cruziandT.bruceioligopeptidaseB[24].Allthesedata sug-gestthatthehydrolyticactivitiesofamastigoteandpromastigote lysatesonZ-FR-MCAand Z-RR-MCAare essentiallydueto cys-teinepeptidasesandoligopeptidaseB,respectively.Inaddition,it shouldbenotedthatZ-RR-MCAwasnotcleavedbyCPB2.8CTE that similar tomost cysteine proteases from clanCA hashigh specificity for substrates with hydrophobic amino acids at P2 position[39,40].
3.3. Effectsoftemperatureandsaltsonhydrolyticactivityof amastigotesandpromastigotelysates
Theeffectoftemperatureonthehydrolyticactivityof amastig-otes and promastigote lysates was assayed using substrates Z-FR-MCAatpH5.0and8.0andZ-RR-MCAatpH8.0(Fig.8).The cysteinepeptidaseactivityofamastigotelysateonZ-FR-MCAwas maintainedatahighertemperaturerangethanthatof promastig-ote lysates,and this occurredmainlyat pH5.0. Incontrast, no differencewasobservedfortheoligopeptidaseBhydrolytic activ-itiesofthetwolysatesonZ-RR-MCA.Theseresultsstrengthenthe differenceofcysteineproteaseactivitybetweenamastigotesand promastigotesofL.(L.)amazonensisasfirstlydemonstratedinL.
86 184 (2012) 82–89
Fig.5.pHprofileofLeishmania(L.)amazonensisamastigoteslysatesinthepresence ofdifferentpeptidaseinhibitorsonZ-RR-MCAsubstrate.Theinhibitorassayswere performedusingpepstatin,EDTA,PMSFandTLCK.
(L.)mexicana[1].In ordertofurtherexplore thedependenceof theenvironmentalconditionsonthecysteinepeptidaseactivity weexploredtheeffectofsodiumcitrateandNa2SO4(kosmotropic salts)andNaClonthehydrolyticactivityofamastigotelysateon Z-FR-MCA.AsshowninFig.9,atpH5.0bothkosmotropicsalts increasedtheparasitehydrolyticactivityatconcentrationshigher than0.5M,whereasatpH8.0thisactivityremainsconstantwith sodiumcitrateandwasreducedwithsodiumsulfate.NaClhadno effectonthehydrolyticactivityatpH8.0butdecreaseditatpH 5.0.Theeffectsofthesesaltswerealsoexaminedontheisolated CPB2.8CTEandonlysodiumcitratewasabletoinducesignificant increaseinthehydrolysisofZ-FR-MCA(Fig.9D).
4. Discussion
ThedetectionofpeptidaseactivityinsideLeishmaniacellswas limitedtohydrolysisofpeptidescontainingatleastonepositively chargedresidue(Z-FR-MCAandZ-RR-MCA)andtheinhibitionof theseactivitiesalsooccurredwithinhibitorscontaining a posi-tivechargegroup(E64andTLCK).ThisselectivityofLeishmania
amastigotestoincorporatepositivelychargedpeptidescouldbe related toLeishmania dependence ontheexogenous sources of R for protein synthesis and as precursor for polyamine syn-thesis[41]. Inaddition, cysteinepeptidases areup-regulatedin amastigotes [7] and these peptidases have efficient hydrolysis on substrates at the C-terminus of arginine [39,42] increasing theavailabilityofargininetotheparasite.Ametabolic perspec-tive of Leishmania–macrophage interaction was reviewed [43], wheretheimportanceoftheroleofaminoacidsandpolyamines are discussed as carbon sources and growth-limiting nutrients, respectively.
TheeffectsofpH,temperatureandsaltsonZ-FR-MCA hydrol-ysisbyamastigotesandpromastigotelysatesclearlyindicatethe expression of different cysteine peptidases adapted to workin differentenvironmentalconditionsoftheparasitelifecycle.The hydrolysisofZ-RR-MCAbytheintactLeishmaniacellsand amastig-otesorpromastigotelysates,itsinhibitionbyTLCKandparticularly thetime-course hydrolysis of theFRET peptide Abz-AGRRRAQ-EDDnp clearly indicated the expression of oligopeptidase B in bothparasiteforms,possiblysimilartothatrecentlyreported[30]
with83.1kDa. In addition,thehydrolyticactivityofamastigote supernatantonAbz-AGRRRAQ-EDDnpco-elutedwiththeactivity ofrecombinant80kDaoligopeptidaseBofT.brucei[23]inagel
pH
4.5 5.0 5.5 6.0 7.0 7.5 8.0 8.5 9.0
En
z
y
m
a
ti
c
A
c
ti
v
ity
U
n
it
s
0 4 8 12 16 20
(B)
pH
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
Enz
ym
a
ti
c
A
c
ti
v
ity
U
n
it
s
0 100 200 300 400 500
(A)
pH
4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
En
z
y
m
a
ti
c
A
c
ti
v
ity
U
n
it
s
0 40 80 120 160 200
(C)
pH
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
Enzymatic Activity Units
0 40 80 120
(D)
2-Cap-Cys(SBzl)-Cys(SBzl)-MCA
(D) Pro-Phe-Phe-MCA Z-Phe-Arg-MCA
Z-Arg-Arg-MCA ε
184 (2012) 82–89 87
Fig.7.HPLCprofileofthehydrolysisofAbz-AGRRRAQ-EDDnpbyLeishmania(L.)amazonensispromastigotes(A)andamastigotelysates(B).TheHPLCprofileafterincubation ofthesubstrate(50M)withLeishmania(L.)amazonensispromastigotesandamastigotelysatesat37◦Cin50mMTris/HCl,pH8.0,followingpre-activationwith5mMDTT. Theelutionprofilesweredeterminedat220nmatdifferenttimeintervals,asindicated.Thenumbersindicatethefollowingproducts,whichwereidentifiedby MALDI-TOFMSafterisolation:(1)Abz-AGRRRAQ-EDDnp;(2)Abz-AGRRR-OH;(3)Abz-AGRR-OH(firstproductofcarboxypeptidaseactivity);(4)Abz-AGR-OH(secondproductof carboxypeptidaseactivity);and(5)AQ-EDDnp.
filtration chromatography (data not showed). Serine oligopep-tidase activity was earlier detected from L (L.) amazonensis
promastigotes[44]anditsgeneexpressionanalysisshowedthat thisoligopeptidaseisconservedduringtheparasitelifecycle[29]. Experimentsinprogressinourlaboratoryshowasignificant increase of Z-FR-MCAhydrolysis inside Leishmania amastigotes aftertreatmentwiththapsigarginwhichincreasescytosolicCa2+ ions (data not shown). Thus, in addition to the cysteine pep-tidases and oligopeptidase B, part of the observed hydrolysis
inside the cells and in theparasite lysates could berelated to a calcium-dependent-cysteine peptidases as the metacaspases. Theseenzymes areexpressedbyLeishmania [45] and cleave Z-FR-MCA or Z-RR-MCA since metacaspase of T. brucei has high selectivityforarginine[46]andalsocleavesthesepeptidyl-MCA substrates(datanotpublished).Calpainorcalpain-likepeptidase isalsopresentinLeishmania[47]andrequiresCa2+,butthe prefer-enceofcalpainsforthehydrolysisofsubstratesatbasicresiduesis verylimited[48].
88 184 (2012) 82–89
Fig.9. EffectofsaltsonhydrolyticactivityofLeishmania(L.)amazonensisamastigoteslysatesupernatantsonZ-FR-MCA.Sodiumcitrate(A),sodiumsulfate(B)andsodium chloride(C).ThesamesaltsweretestedonCPB2.8CTE(D)atpH6.5.
Leishmania are exposed to dramatic alterations in tempera-tureandpHduringitslife cycle,acquiringthermotoleranceas previouslyreviewed[49].Theenvironmentosmolarityisanother variablethatshouldbetakenintoaccountasindicatedbytheeffect ofthekosmotropicsaltssodiumcitrateand Na2SO4 onthe cys-teinepeptidaseactivityofamastigotelysatesatpH5.0aswellas onCPB2.8CTE.Themacromolecularcrowdingconceptappears adequatetointerprettheobservedincreaseofZ-FR-MCA hydroly-sisbyhighsodiumcitrate[50,51].Themacromolecularcrowding obtainedbyhighsaltconcentrationcompactsthepeptidase(s)with reductionofsurface–volumeratio,whichresultsinactive confor-mationoftheenzyme.
In conclusion, the serine peptidase activity observed inside
LeishmanialivecellsisessentiallyduetooligopeptidaseB,whereas thecysteinepeptidaseactivitiesareduetomorethanoneenzyme andtheoptimumactivitiesdisplayedbytheseparasitesare depen-dentonpH,temperatureandsalinityconditions.
Acknowledgments
ThisworkwassupportedbyFundac¸ãodeAmparoàPesquisado EstadodeSãoPaulo(FAPESP),ConselhoNacionalde Desenvolvi-mentoCientíficoeTecnológico(CNPq)andInstitutoNacionalde CienciaeTecnologiaemFluidosComplexos(INCT-Fx).Wethank Prof.MichaelBlaberforthecommentsandeditionofthispaper.
References
[1]CoombsGH.ProteinasesofLeishmaniamexicanaandotherflagellateprotozoa. Parasitology1982;84:149–55.
[2]McKerrowJH,SunE,RosenthalPJ,BouvierJ.Theproteasesandpathogenicity ofparasiticprotozoa.AnnualReviewofMicrobiology1993;47:821–53. [3] Rosenthal PJ. Proteasesof protozoan parasites. Advances in Parasitology
1999;43:105–59.
[4]LecailleF,KaletaJ,BrommeD.Humanandparasiticpapain-likecysteine pro-teases:theirroleinphysiologyandpathologyandrecentdevelopmentsin inhibitordesign.ChemicalReview2002;102:4458–88.
[5]SadijM,McKerrowJH.Cysteineproteaseofparasiticorganisms.Molecular BiochemistryParasitology2002;120:1–21.
[6]McKerrowJH, CaffreyC, KellyB, Loke P, SajidM. Proteasesinparasitic diseases. Annual Review of Pathology: Mechanisms of Disease 2006;1: 497–536.
[7]BesteiroS,WilliamsRAM,CoombsGH,MottranJC.Proteinturnoverand differ-entiationinLeishmania.InternationalJournalofParasitology2007;37:1063–75. [8] TroebergL,PikeRN,MortyRE,BerryRK,CoetzerTHT,Londsdale-EcclesJD. ProteasesfromTrypanosomabruceibrucei:purification,characterizationand interactionswithhostregulatorymolecules.EuropeanJournalofBiochemistry 1996;238:728–36.
[9]Alexander J, Russel DG. The interaction of Leishmania species with macrophages.AdvancesinParasitology1992;31:175–254.
[10]BrettonCB,BlisnickT,JouinH,BaraleJC,RabilloudT,LangsleyG,etal. Plasmod-iumchabaudip68serineproteaseactivityrequiredformerozoiteentryinto mouseerythrocytes.ProceedingsoftheNationalAcademyofSciencesofthe UnitedStatesofAmerica1992;20:9647–51.
[11]AlvesCR,Corte-RealS,BourguignonSC,ChavesCS,SaraivaEM.Leishmania amazonensis:earlyproteinaseactivitiesduringpromastigote–amastigote dif-ferentiationinvitro.ExperimentalParasitology2005;112:63–5.
[12] MottramJC,CoombsGH,AlexanderJ.Cysteinepeptidasesasvirulencefactors ofLeishmania.CurrentOpinioninMicrobiology2004;7:375–81.
[13]Rawlings ND, Barrett AJ, Bateman A. MEROPS: the database of prote-olytic enzymes, their substrates and inhibitors. Nucleic Acids Research 2012;40:D343–50.
[14]SelzerPM,Pingel S,HsiehI, UgeleB, ChanVJ, BogyoM,et al.Cysteine proteaseinhibitorsaschemotherapy:lessonsfromaparasitetarget. Proceed-ingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica 1999;96:11015–22.
184 (2012) 82–89 89
[16]MottranJC,HelmsMJ,CoombsGH,SajidM.ClanCDcysteinepeptidasesof parasiticprotozoa.TrendsinParasitology2003;19:182–7.
[17]HalléM,GomezMA,StuibleM,ShimizuH,McMasterWR,OlivierM,etal. TheLeishmaniasurfaceproteaseGP63cleavesmultipleintracellularproteins andactivelyparticipatesinp38mitogen-activatedproteinkinaseinactivation. JournalofBiologicalChemistry2009;284:6893–908.
[18]GomezMA,ContrerasI,HalléM,TremblayML,McMasterRW,OlivierM. Leish-maniaGP63altershostsignalingthroughcleavage-activatedproteintyrosine phosphatases.ScienceSignalling2009;90:ra58.
[19] RafatiS,SalmanianAH,TaheriT,MasinaS,SchaffC,TaslimiY,etal.TypeI signalpeptidasefromLeishmaniaisatargetoftheimmuneresponseinhuman cutaneousandvisceralleishmaniasis.MolecularandBiochemicalParasitology 2004;135:13–20.
[20] TaheriT,SalmanianAH,GholamiE,DoustdariF,ZahedifardF,RafatiS. Leish-maniamajor:disruptionofsignalpeptidasetypeIanditsconsequenceson survival,growthandinfectivity.ExperimentalParasitology2010;126:135–45. [21]PolgarL.Theprolyloligopeptidasefamily.CellularandMolecularLifeSciences
2002;59:349–62.
[22]Silva-LopesRE,Giovanni-De-SimoneS.Leishmania(Leishmania)amazonensis: purificationandcharacterizationofapromastigoteserineprotease. Experi-mentalParasitology2004;107:173–82.
[23] CoetzerTH,GoldringJP,HusonLE.OligopeptidaseB:aprocessingpeptidase involvedinpathogenesis.Biochimie2008;90:336–44.
[24] HemerlyJP,OliveiraV,DelNeryE,MortyRE,AndrewsNW,JulianoMA,etal. Subsitespecificity(S3,S2,S1′,S2′andS3′)ofoligopeptidaseBfromTrypanosoma cruziandTrypanosomabruceiusingfluorescentquenchedpeptides: compara-tivestudyandidentificationofspecificcarboxypeptidaseactivity.Biochemical Journal2003;373:933–9.
[25]MeloRL,AlvesLC,DelNeryE,JulianoL,JulianoMA.Synthesisand hydroly-sisbycysteineandserineproteasesofshortinternallyquenchedfluorogenic peptides.AnalyticalBiochemistry2001;293:71–7.
[26] MortyRE,Londsdale-EcclesJD,MoreheadJ,CallerEV,MenteleR,Auerswald EA,etal.OligopeptidaseBfromTrypanosomabrucei,anewmemberofan emergingsubgroupofserineoligopeptidases.JournalofBiologicalChemistry 1999;274:26149–56.
[27] GouveaIE,Izidoro MA, JudiceWA, CezariMH, CaliendoG, SantagadaV, etal.Substrate specificityofrecombinantdengue2virusNS2B-NS3 pro-tease:influenceofnaturalandunnaturalbasicaminoacidsonhydrolysis ofsyntheticfluorescentsubstrates.ArchivesofBiochemistryandBiophysics 2007;457:187–96.
[28] KondoMY,OliveiraLC,OkamotoDN,deAraujoMR,DuartedosSantosCN, JulianoMA,etal.YellowfevervirusNS2B/NS3protease:hydrolyticproperties andsubstratespecificity.BiochemicalandBiophysicalResearch Communica-tions2011:640–4.
[29] GuedesHLM,CarneiroMPD,GomesDCO,Giovanni-De-SimoneS. Oligopepti-daseBfromL.amazonensis:molecularcloning,geneexpressionanalysisand molecularmodel.ParasitologyResearch2007;101:853–63.
[30] SwenertonRK,ZhangS,SajidM,MedzihradszkyKF,CraikCS,KellyBL,etal.The oligopeptidaseBofLeishmaniaregulatesparasiteenolaseandimmuneevasion. JournalofBiologicalChemistry2011;286:429–40.
[31]Fedeli CE, Ferreira JH, Mussalem JS, Longo-Maugéri IM, Gentil LG, dos Santos MR,etal. Partialprotectiveresponses inducedby arecombinant cysteineproteinasefromLeishmania(Leishmania)amazonensisinamurine model of cutaneous leishmaniasis. Experimental Parasitology 2010;124: 153–8.
[32]BarbiériCL,DoineAI,FreymullerE.Lysosomaldepletioninmacrophagesfrom spleenandfootlesionsofLeishmania-infectedhamster.Experimental Parasitol-ogy1990;71:219–28.
[33]AlberioSO,DiasSS,FariaFP,MortaraRA,BarbiériCL,FreymüllerHaapalainen E.UltrastructuralandcytochemicalidentificationofmegasomeinLeishmania (Leishmania)chagasi.ParasitologyResearch2004;92:246–54.
[34]Alves LC, Almeida PC,Franzoni L, JulianoL, Juliano MA. Synthesisof N alpha-protectedaminoacyl7-amino-4-methyl-coumarinamideby phospho-rousoxychlorideandpreparationofspecificfluorogenicsubstratesforpapain. JournalofPeptideResearch1966;9:92–6.
[35]HirataI,CezariMHC,NakaieCR,BoshcovP,ItoAS,JulianoMA,etal.Internally quenchedfluorogenicproteasesubstrates:solid-phasesynthesisand fluores-cencespectroscopyofpeptidescontainingortho-aminobenzoyl/dinitrophenyl groupsasdonor–acceptorpairs.LettersinPeptideScience1994;1:299–308. [36]KorkmazB,AttucciS,JulianoMA,KalupovT,JourdanML,JulianoL,etal.
Mea-suringelastase,proteinase3andcathepsinGactivitiesatthesurfaceofhuman neutrophilswithfluorescenceresonanceenergytransfersubstrates.Nature Protocols2008;3:991–1000.
[37]CabralH,LeopoldinoAM,TajaraEH,GreeneLJ,Fac¸aVM,MateusRP,etal. Preliminaryfunctionalcharacterization,cloningandprimarysequenceof Fas-tuosain,acysteinepeptidaseisolatedfromfruitsofBromeliafastuosa.Protein andPeptideLetters2006;13:83–9.
[38]MasonRW,GreenGD,BarrettAJ.HumanlivercathepsinL.BiochemicalJournal 1985;226:233–41.
[39]AlvesLC,JudiceWA,StHilairePM,MeldalM,SandersonSJ,MottramJC,etal. Substratespecificityofrecombinantcysteineproteinase,CPB,ofLeishmania mexicana.MolecularandBiochemicalParasitology2001;114:1–9.
[40]AlvesLC,MeloRL,CezariMH,SandersonSJ,MottramJC,CoombsGH,etal. Anal-ysisoftheS(2)subsitespecificitiesoftherecombinantcysteineproteinases CPBofLeishmaniamexicana,andcruzainofTrypanosomacruzi,using fluo-rescentsubstratescontainingnon-naturalbasicaminoacids.Molecularand BiochemicalParasitology2001;117:137–43.
[41] RobertsSC,TancerMJ,PolinskyMR,GibsonKM,HebyO,UllmanB.Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania. Characterizationofgenedeletionmutants.JournalofBiologicalChemistry 2004;279:23668–78.
[42]StHilairePM,AlvesLC,SandersonSJ,MottramJC,JulianoMA,JulianoL,etal.The substratespecificityofarecombinantcysteineproteasefromLeishmania mex-icana:applicationofacombinatorialpeptidelibraryapproach.Chembiochem: AEuropeanJournalofChemicalBiology2000;1:1206–12.
[43]Naderer T, McConville MJ. The Leishmania–macrophage interaction: a metabolicperspective.CellularMicrobiology2008;10:301–8.
[44]Andrade ASR, Santoro MM,Melo MN, Mares-Guia M. Leishmania( Leish-mania) amazonensis: purification and enzymatic characterization of a solubleserineoligopeptidasefrompromastigotes.ExperimentalParasitology 1998;89:153–60.
[45]AmbitA,FaselN,CoombsGH,MottramJC.AnessentialrolefortheLeishmania majormetacaspaseincellcycleprogression.CellDeathandDifferentiation 2008;15:113–22.
[46] MossCX,WestropGD,JulianoL,CoombsGH,MottramJC.Metacaspase2of Trypanosomabruceiisacalcium-dependentcysteinepeptidaseactivewithout processing.FEBSLetters2007;581:5635–9.
[47]Avila-LevyCM,MarinhoFA,SantosLO,MartinsJL,SantosAL,BranquinhaMH. AntileishmanialactivityofMDL28170,apotentcalpaininhibitor.International JournalofAntimicrobialAgents2006;28:138–42.
[48]CuerrierD,MoldoveanuT,DaviesPL.Determinationofpeptidesubstrate speci-ficityformu-calpainbyapeptidelibrary-basedapproach:theimportanceof primedsideinteractions.JournalofBiologicalChemistry2005;280:40623–41. [49] ZilbersteinD,ShapiraM.TheroleofpHandtemperatureinthedevelopment
ofLeishmaniaparasites.AnnualReviewofMicrobiology1994;48:449–70. [50]HuangX,KnoellCT,FreyG,Hazegh-AzamM,TashjianJrAH,HedstromL,
etal.Modulationofrecombinanthumanprostate-specificantigen:activation by Hofmeister saltsand inhibitionby azapeptides. Appendix: thermody-namicinterpretationoftheactivationbyconcentratedsalts.Biochemistry 2001;40:11734–41.
