Molecular cloning and structural modelling of gamma-phospholipase A2 inhibitors from Bothrops atrox and Micrurus lemniscatus snakes

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ContentslistsavailableatScienceDirect

International

Journal

of

Biological

Macromolecules

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / i j b i o m a c

Molecular

cloning

and

structural

modelling

of

gamma-phospholipase

A

2

inhibitors

from

Bothrops

atrox

and

Micrurus

lemniscatus

snakes

Carina

G. Picelli

a

_,

_Rafael

_J.

_Borges

b

_,

_Carlos

_A.H.

_Fernandes

b

_,

_Fabio

_M.

_Matioli

b

_,

Carla

F.C.

Fernandes

a

_,

_Juliana

_C.

_Sobrinho

a

_,

_Rudson

_J.

_Holanda

a

_,

_Luiz

_S.

_Ozaki

a,c,d

_,

Anderson

M. Kayano

a

_,

_Leonardo

_A.

_Calderon

a

_,

_Marcos

_R.M.

_Fontes

b

_,

_Rodrigo

_G.

_Stábeli

a

_,

Andreimar

M. Soares

a,e,∗

a_Centro_de_Estudos_de_{Biomoléculas}_Aplicadas_a_Saúde,_CEBio,_Fundac¸_ão_Oswaldo_Cruz,_FIOCRUZ,_Fiocruz_Rondônia_and_Departamento_de_Medicina,

UniversidadeFederaldeRondônia,UNIR,76812-245PortoVelho,RO,Brazil

b_Departamento_de_Física_e_Biofísica,_Instituto_de_{Biociências,}_Universidade_Estadual_Paulista,_UNESP,_Botucatu,_SP,_Brazil

c_Center_for_the_Study_of_Biological_Complexity,_Life_Sciences,_Virginia_Commonwealth_University,_23284,_Richmond,_USA

d_PVE-CNPq,_Brazil

e_Centro_{Universitário}_São_Lucas,_UNISL,_Porto_Velho,_RO,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30January2017

Receivedinrevisedform3May2017

Accepted15May2017

Availableonline18May2017

Keywords: Snakes

Bothropsatrox

Micruruslemniscatus

InhibitorsofphospholipasesA2

GammatypephospholipaseA2inhibitors

a

b

s

t

r

a

c

t

PhospholipasesA2inhibitors(PLIs)producedbyvenomousandnon-venomoussnakesplayessential roleinthisresistance.TheseendogenousinhibitorsmaybeclassifiedbytheirfoldinPLI␣,PLI␤and PLI␥.PhospholipasesA2 (PLA2s)developmyonecrosisinsnakeenvenomation,aconsequencethatis notefficientlyneutralizedbyantivenomtreatment.Thisworkaimedtoidentifyandcharacterizetwo PLIsfromAmazoniansnakespecies,BothropsatroxandMicruruslemniscatus.LivertissuesRNAof speci-mensfromeachspecieswereisolatedandamplifiedbyRT-PCRusingPCRprimersbasedonknownPLI␥ genesequences,followedbycloningandsequencingofamplifiedfragments.Sequencesimilaritystudies showedelevatedidentitywithinhibitorPLI␥genesequencesfromothersnakespecies.Molecularmodels oftranslatedinhibitors’genesequencesresemblecanonicalthreefingerfoldfromPLI␥andsupportthe hypothesisthatthedecapeptide(residues107–116)mayberesponsibleforPLA2inhibition.Structural studiesandactionmechanismofthesePLIsmayprovidenecessaryinformationtoevaluatetheirpotential asantivenomorascomplementofthecurrentophidianaccidenttreatment.

1. Introduction

Snakevenomcausephysiologicaldisorderintheirpreyorbitten

victims.Thedisturbancesaremainlyrelatedtotheactionoftoxins

thatpossessafﬁnitytoreceptorsorionchannelsandofenzymes

withcatalyticactivity,whichpromotehomeostaticdeviationofthe

centralandperipheralnervoussystems,andcardiovascular,

neuro-motor,andbloodcoagulationsystems.Furthermore,tissuenecrosis

aroundthebite,mainlycommoninBothropsaccidents,mayresult

inpermanentsequelae[1,2].

∗ Correspondingauthorat:CentrodeEstudosdeBiomoléculasAplicadasaSaúde,

CEBio,Fundac¸ãoOswaldoCruz,FIOCRUZ,FiocruzRondôniaandDepartamentode

Medicina,UniversidadeFederaldeRondônia,UNIR,76812-245PortoVelho,RO,

Brazil.

E-mailaddress:andreimar.soares@ﬁocruz.br(A.M.Soares).

Animportantpartoftheeffectsdescribedaboveisduetothe

actionofphospholipasesA2 (PLA2s),whichareasuperfamilyof

enzymes that speciﬁcallycatalyze the hydrolysis of the 2-acyl

ester bond of glycero-phospholipids in cell membranes,

result-inginlysophospholipidsandfattyacidssuchasarachidonicacid.

These metabolites act primarily as precursors of inﬂammatory

mediators(eicosanoids),which areallimportantinintracellular

signalingpathwayssuchasneuronaltransmission,mitogenesis,

smoothmusclecontraction,andplateletactivation[3,4].Because

oftheirimportanceininﬂammatoryevents,thisfamilyofproteins

isalsowellstudiedinautoimmuneandinﬂammatorydiseases.

PLA2sandothersnakevenomtoxinscanbeinhibitedby

differ-entmoleculesthatvaryinnature,includingsyntheticornatural

chemical agents of animal and plant origin. For example,

syn-theticlowmolecularweightheparin,p-bromophenacylbromide,

tetracycline, mono/polyclonalantibodies,and naturalﬂavonoids

andalkaloidsextractedfromplantsandmoleculesstemmingfrom

marineorganisms,areinhibitorsofPLA2fromsnakevenoms[5].

http://dx.doi.org/10.1016/j.ijbiomac.2017.05.076

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Molecularstructureswithinhibitoryactivitiesofphospholipase

A2andmetalloproteinasearealsoknowntobefoundinsnakes.

Thesemolecules arerelated totheself-envenomingprevention

mechanism[6].Besidessnakes,otheranimals,mammals,ﬁshand

someinvertebrateshavebeenstudiedfornaturalresistanceagainst

snakevenoms[7],fungiandbacteria[8].

PLA2 inhibitors(PLIs)isolatedfromsnakebloodmaypresent

differencesamongdifferentsnakespeciesPLIs[9].Thesemolecules

areoligomeric,globular,acidicglycoproteinswithmolecularmass

between75and180kDa.ThemechanismofactionofsecretedPLA2

inhibitorsisthroughtheformationofasolublecomplexbetween

theinhibitorandthetargetenzyme[10].Theseinhibitorsare

clas-siﬁed into three groups, ␣, ␤ and ␥, based ontheir structural

characteristicsandmaybepresentinasinglesnake,whether

poi-sonousornot [11].Inhibitors fromthethreegroups havebeen

isolatedfromdiversesnakespeciesandfamilies[10,12].

ThePLI␥spossessthehighestinhibitoryactionamongthethree

groups [13].They haveinhibitory activitiesonacidic and basic

groupIIaswellasonthoseofgroupIIIPLA2s[14]andareacidic

gly-coproteinsoligomeric,withmolecularweightsofeachmonomer

between20and31kDa.ThePLI␥shavebeenclassiﬁedintotwo

subgroups,PLI_␥IandII,accordingtotheiraminoacidsequence,

biochemicalcharacteristicsandinhibitionproﬁle.Membersofthe

PLI␥Isubgrouphaveaheteromericcompositionandtwosubunits

(AandB)withlessthan 33%identitytoeachotherand inhibit

groupsI,II,andIIIPLA2s.MembersofthePLI␥IIsubgroup

pref-erentiallyinhibitsthosePLA2sofgroupIIandareconstitutedofa

singlesubunit[7,13,15,16].However,recentlythepossibilityofa

secondsubunitinaPLI␥IIfromCrotalusdurissusterriﬁcushasbeen

raised[17].

PLIsareinterestingcandidatesfordesigningdrugsorevenasa

toolinenvenomingtreatment,sincetheyinhibitPLA2activityand

consequentlymanyoftheimportantsymptomsofsnakebite

acci-dents[13,18].Furthermore,thestudyofPLA2inhibitorsisacritical

areaofresearchbecauseofthepharmacologicalpotentialofthese

componentsinthetreatmentofinﬂammationandasatoolto

inves-tigatetheroleofPLA2sinphysiologicalfunctionsofcelldeathand

injury[19].Inrecentyears,syntheticderivedpeptidesfromPLI␥s

wereshowedtohave therapeuticpotentialinPLA2-related

dis-easesandasantivenom-likebioactivemoleculesforsnakevenom

neutralization[20,21].

Inthisstudy,weperformedmolecularcloningofPLI␥softhe

Amazonianoccurringsnakespecies,BothropsatroxandMicrurus

lemninscatus,andbuildinsilicomodelsoftheirstructures.

2. Materialandmethods

2.1. Ethicalcompliance

Thisstudy was approved by theCouncil of Management of

GeneticPatrimony(ConselhodeGestãodoPatrimônioGenético)by

reviewsfromCNPqnumber010627/2011andfromIBAMAnumber

27131-1.Snakesspecimensrequiredforthisstudyweredonatedby

thewildliferescueteamoftheSantoAntônioHydroelectricPlant

project.

2.2. ExtractionofRNA

RNA-later® solution(ThermoFisherScientiﬁc)wereaddedto

thedissectedlivertissuesfromthesnakespecimens andstored

inliquid nitrogenat theOswaldoCruzFoundation −

FIOCRUZ-Rondônia facilities until use. RNAwas isolated from200mg of

frozenlivertissuehomogenizedinliquidnitrogenusingtheTRIzol®

Plus RNA Puriﬁcation Kit (Thermo Fisher Scientiﬁc), according

Table1

OligonucleotideprimerssequencesusedfortheampliﬁcationofB.atrox.

PLI(ATG)A−SENSE 5-ATGAAATCYCTACACACCATCTGCC-3 PLI(ATG)B−SENSE 5ATGAAATCYCTACAGATCATCTGTCTTC-3 PLI(STOP)A−ANTISENSE 5_{-ATCAGAGGCTTGCCAATCTGATG-3} PLI(STOP)B−ANTISENSE 5_{-TTATTGTTTTTCAACTTGGATGGC-3} PLI(STOP)C−ANTISENSE 5_{-GGTGACGGAATTATTCRGAAGGTG-3}

tothemanufacturer’sprotocols.ThepuriﬁedRNAsampleswere storedat−80◦_C_until_use.

2.3. CloningofthePLA2inhibitorgenesandsequencing

Oligonucleotide primers (Table 1) for the polymerase chain

reaction(PCR)weredesignedbasedonknownsequencesofPLA2

genes,correspondingto␥typeinhibitorsanddepositedatNCBI

(The National Center for Biotechnology Information) GenBank

(AB462512,AF211168,AF211166,AF211165,AF211163,AJ249829,

AB021425,AB559509, EU155177, EU155169e AB003472). First

strandedcDNA wassynthesized from thepuriﬁed RNAs ofthe

snakespecimenslivertissuesusingtheSuperScript®IIIFirst-strand

SynthesisSystem(ThermoFisherScientiﬁc)andthekit’soligo(dT)

primer.ThePLIgenesweresubsequentlyampliﬁedusingthe“pool”

ofPLI␥designedprimers(Table1)ataﬁnalconcentrationof0.2␮M,

ina volumeofcontaining5␮Lof theﬁrststrandedcDNA

reac-tionmixture,200␮MdNTPsandoneunitofHotMasterTM_Taq_DNA

polymerase(5Prime).PCRconditionswere:95◦Cfor3minand30

cyclesat95◦Cfor30s,55◦Cfor30sand70◦Cforoneminute.

TheampliﬁedfragmentswereclonedintopGEM®-TEasyVector

(PromegaCo,Madison,WI,USA).Theclonedinsertswerepuriﬁed

bycolonyPCRusingthevectorprimersT7andSP6andthe

ampli-ﬁedfragments sent for sequencing.Sequencing wasperformed

inanABIPrism® 377automaticsequencer(ThermoFisher

Scien-tiﬁc)atthecompanyGenomic−EngenhariaMolecularinSãoPaulo,

SP,Brazil.

2.4. Alignmentandphylogeneticanalysis

DifferentPLI␥wereobtainedfromNCBIGenBankdatabaseusing

BLASTPalgorithm[22]andBLOSUM62substitutionmatrix,withB.

atroxPLIsequenceasquery.Theminimume-valuepresentedbythe

selectedsequenceswas1e-74_._Multiple_sequence_alignment_was

executedusingtheonlineprogramMuscle[23]andphylogenetic

treewasbuildbasedontheBayesianmethodMonteCarlo-Markov

chain(MCMC)usingtheprogramMrBayes3.2.6[24].Two

concur-rentMCMCrunsof500,000generationswereperformedusingfour

progressivelyheatedchainswithaheatingparameterof0.2,tree

samplingevery100generationsandaburn-insettingof250trees.

After,thephylogenetictreewasvisualizedusingMesquitev.3.04

program[25].

2.5. Structuralmodellingandcomparison

ThededucedPLIaminoacidsequencesexcludingpeptide

sig-nalwereusedtogeneratestructuralmodelsfor bothPLIsusing

Phyre2_[26]_based_on_Domains_II_and_III_of_the_models_of_Urokinase

plasminogenactivatorsurfacereceptor(uPAR)fromMusmusculus

(PDBid/chain:3LAQ/V)[27]andfromHomosapiens(PDBid/chain:

1YWH/A)[28].Sinceidentitydifferenceamongisoformswereless

than1%,onlyonesequenceofeachsnakePLIsweremodeled:B.

atrox(At.PLI2)andM.lemniscatusmodels(mi.PLI1).Morethan94%

ofresiduesweremodeledwithconﬁdencehigherthan90%based

ontemplatehomologousproteins(100%chance).Theother10%of

theresiduesarelocatedinC-terminalregion,last9and7residues

fromB.atroxandM.lemniscatusmodels,respectively,andwere

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Fig.1. Agarosegel(1%)electrophoresisofthePCRampliﬁedfromthesnakesM.

lemniscatusandB.atroxlivertissuecDNAs.PCRprimersaredescribedinTable1.

100bp,sizemarkers.

ThePLI␥modelsweresubmittedtomoleculardynamic(MD)

simulationsusingtheprogramGROMACS(GroningenMachinefor

ChemicalSimulation)v.5.0.5[29,30]withGROMOS9654A7force

ﬁeld[31].Theproteinchargedgroupsusedwerethe

correspon-dentofpH7.TheMDsimulationswereexecutedinthepresenceof

explicitwatermoleculesneutralizedbyadditionofcounterions,in

aperiodictruncatedcubicbox,underconstanttemperature(298K)

andpressure (1.0bar) heldbycouplingtoanisotropicpressure

andexternalheatbath[32,33].Inthecubicbox,5%ofthewater

moleculeswerereplacedbypositiveions,Na+_,_and_negative_ions,

Cl−,inequalamounts.TheMDsimulationswererunwithenergy

minimizationusingthesteepestdescentalgorithm, followedby

2nsofrestrainedproteinpositionandby100nsofunrestrained

proteinposition.TheoptimizedPLIinsilicomodelsfromB.atroxand

fromM.lemniscatuswerefurtherselectedevaluatingthestability

inMDbyaveragerootmeansquaredeviation(RMSD)/timegraph

oftheproteinbackboneatomsandbyoverallstereochemicaland

energyqualityinRAMPAGE[34]andProSA-web[35],respectively.

The ﬁnal models obtainedafter MDwere analyzed in VMD

[36].ThestructuralcomparisonwereevaluatedwithRMSDs

calcu-latedusingshortfragmentclusteringbyGESAMP(GeneralEfﬁcient

StructuralAlignmentofMacromolecularTargets)approach[37].

Similarfold wasevaluatedby TM-Scoregenerated byTM-align

algorithm[38]andbyPDBeFold[39].Themodelresidues

acces-siblesolventareawascalculatedbyAREAIMOLavailableinCCP4

suite[40].Theﬁguresandsurfaceillustrationsweregeneratedin

CHIMERA[41]andPymol(ThePyMOLMolecularGraphicsSystem,

Version1.3Schrödinger,LLC.).

3. Resultsanddiscussion

Usingoligonucleotidesprimersbasedonsequencesof␥type

phospholipase A2 inhibitors from several families of snakes

depositedatNCBIGenBankandRNAextractedfromtheliver

tis-sueofspecimens of thesnakesB. atroxand M.lemniscatus,we

obtainedbyRT-PCRfragments ofapproximately600basepairs

(Fig.1).ThesefragmentswereclonedinthevectorpGEM®-TEasy

(PromegaCo,Madison,WI),resultingin5clonesforB.atroxand8

Table2

ComparativeanalysisofAt.PLI1andAt.PLI2sequenceswiththeprogramBLASTn.

Identiﬁcation Species Class Score Similarity Gaps E-value

EU155175 B.moojeni ␥ 1057 98% 0% 0.0

EU155173 B.jararacussu ␥ 1051 98% 0% 0.0

EU155166 B.alternatus ␥ 1046 98% 0% 0.0

AY425346 Lachesismutamuta ␥ 1046 98% 0% 0.0

EU155170 B.jararaca ␥ 1018 97% 0% 0.0

CDU08289 Crotalusd.terriﬁcus ␥ 963 95% 0% 0.0

EU155168 B.erythromelas ␥ 957 95% 2% 0.0

EU155177 B.neuwiedi ␥ 946 95% 2% 0.0

AB018372 Gloydiusb.siniticus ␥ 935 95% 0% 0.0

AB003472 T.ﬂavoviridis ␥ 857 93% 0% 0.0

AB559508 P.elegans ␥ 852 93% 0% 0.0

*Dataabout thephospholipaseA2inhibitorswereselectedby species,witha sequenceofeachbeingchosenforanalysis.Theprogramcomparedthesequences fromthisstudywithothersdepositedinGenBank.

clonesforM.lemniscatus(Fig.2),fromwhichnucleotidesequences

wereobtained.

Two consensus sequences were obtained from B. atrox and

namedAt.PLI1andAt.PLI2andtwofromM.lemniscatus,Mi.PLI1

andMi.PLI2.Weanalyzedseparatelythesequencesofeachsnake

speciesasthesearedifferentatfamilylevel,Viperidaeand

Elapi-dae,respectively.Bythesequencealignment,itwasobservedthat

thenucleotidesequencesofAt.PLI1and2aresimilartothosefrom

B.moojeniandB.jararacussu[42],B.alternatus[43],Lachesismuta

[16],with98%identity(Table2).Onesequencefromeachspecies

withidentityhigherthan90%andanE-valueof0.0wasselected

foranalysis.Thesearchdataforhomologoussequencesegments

(Score)showthatthesequenceofonePLI␥foundinB.moojeniis

themostsimilartothoseobtainedinthisstudy(Table2).Multiple

nucleotidealignmentconﬁrmedthesimilaritybetweensequences.

Deducedaminoacidsequenceswereobtainedusingtheprogram

ORFFinder.

Thesedatawereused fora similaritysearchin theGenBank

databaseandconserveddomainswereinvestigatedusingBLASTp.

AsshowninFig.3A,At.PLI1andAt.PLI2aminoacidsequencesshow

homologytothePLI␥s’three-ﬁngerfold,whicharealsopresentin

urokinase-typeplasminogenactivatorreceptors(u-PAR)andincell

surfaceantigensfromthesuperfamilyLy-6andCD59(Fig.3).These

resultscorroboratepreviousworkwithaPLA2inhibitorofNajanaja

kaouthia[44].Furthermoretheoreticalphysico-chemical

parame-tersofthePLIPLIofB.atrox(At.PLI1andAt.PLI2)weredetermined

usingPROTParam(isoelectricpointandmolecularmass)and

com-paredtothesameparametersofPLI␥sofothersnakesspecies.As

showninTable3,closesimilaritiesexistbetweentheB.atroxPLIs

andthelistedPLI␥s.

The Mi.PLI1 and 2 sequences present divergence as to the

inhibitorclassinthesearchforsimilarity.Interestingly,theﬁve

sequences that presented the highest identities (89–91%) were

inhibitorsrecordedinGenbankasPLI␣andonlytwoshowed

sim-ilaritieswiththe␥-type(Table4).Thededucedtranslationofthe

nucleotidecrackswasusedforthemultiplealignmentand

con-serveddomainsearch,accordingtothemethodologydescribedfor

B.atrox.Again,thesimilaritysearchresultedinPLIsequencesof

␣and␥types(Table5).Therefore,thePLI␥sequencesofBrazilian

snakesthatpresentedthelowestidentity(65–68%)wereincluded

intheanalyses.

The searchfor conserved domainsshowed that Mi.PLI1and

Mi.PLI2possessrepeatedcysteineresiduesinthethree-ﬁngerfold

(PLI␥)andnotthoseofcarbohydraterecognition(CRD)ofCa2

+-dependent lectins (CTLD family) characteristic of PLI␣. Similar

sequencesrecordedasPLI␣wereanalyzedandalsoshowed

simi-laritieswithPLI_␥domains.

Thus,At.PLI1and2,Mi.PLI1and2,aswellasthecrotoxin

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Fig.2.Agarosegel(1%)ofPLI␥clonedgenesfromB.atrox(bands1–5inA);andM.lemniscatus(inB).B)2,4-10bandscorrespondtoampliﬁedDNAfragmentsfromselected

coloniesusingthepGEM®-TEasy(Promega,Madison,WI,USA)primersT7andSP6.100bp,sizemarkers.

Table3

Predicted/deductedbiochemicalcharacteristicsofthephospholipaseA2inhibitorfromB.atroxcomparedwiththoseofothersnakes.

Inhibitor Identiﬁcation sN◦_of_Amino_acids _Molecular_mass_(Da) _Isoelectric_Point_(pI)

At.PLI1 – 200 22196.2 5.97 At.PLI2 – 200 22180.2 5.97 B.moojeni ABV91335.1 200 22181.1 5.97 B.jararacussu ABV91332.1 200 22178.1 5.79 B.alternatus ABV91326.1 200 22208.1 5.79 L.mutamuta P60592.1 200 22207.1 5.97 B.jararaca ABV91331.1 200 22197.1 6.04 C.d.terriﬁcus Q90358.1 200 22267.4 6.07 B.erythromelas ABV91328.1 200 22281.1 5.24 B.neuwiedi ABV91336.1 200 22266.1 5.50 Table4

AnalysisofMi.PLI1andMi.PLI2sequences.

Identiﬁcation Species Class Identity Score Gaps E-value

AF211165 O.scutellatus ␣ 91% 813 0% 0.0 AF211161 Notechisater ␣ 91% 808 0% 0.0 AJ249829 Notechisscutatus ␣ 90% 797 0% 0.0 AF211167 O.microlepidotus ␣ 90% 785 0% 0.0 AF211166 P.textilis ␣ 89% 769 0% 0.0 AB462512 E.climacophora ␥ 89% 756 0% 0.0 AB021425 E.quadrivirgata ␥ 88% 745 0% 0.0

terriﬁcus,areorthologoustoplasminogenactivatorfactorfromH. sapiens,characterizedbythree-ﬁngerdomains[17],withe-values

betweene-26_/e-30_as_calculated_by_HHPred_server_[45]₍_Table₆_)._In

contrast,PLI␣sareorthologoustolungsurfactantproteinD,which

presentsacarbohydraterecognitiondomain[43,46].Interestingly,

these sequences from Elapidae snakes previously deposited as

PLI␣sinNCBI(GIs:6685092,6685094,5931772,6685090)present

similare-values toplasminogenactivatorfactorsuggesting that

thesesequences are in fact PLI␥s. Furthermore,these Elapidae

sequencesshowanE-valuearounde-39fortheplasminogen

acti-vatorfactor domainobservedinCNF,asseeninPFAMsoftware

[47].

PhylogenetictreeusingthesesequencesfromElapidae,At.PLI1

andAt.PLI2,Mi.PLI1and2andallotherPLI␥savailableonNCBI

databank shows that Elapidae sequences nest in a clade with

PLI␥sfromnon-venomousColubridaesnakes(Fig.3B).

Interest-ingly,sequencesfromvenomousViperidaefamilyalsonestina

cladewithsequencesfromsnakesfromanon-venomousfamily

(Phytonidae)(Fig.3B).Aminoacidsequencesalignmentofthese

sequencesshowswellconservedregionsinallPLI␥s(Fig.3A).

ThesimilaritiesofthetranscriptstoGenBankwereveriﬁedin

themultiplealignment,andconserveddomainregionswere

iden-tiﬁed.Itwasobservedthatthereissimilaritybetweentherecorded

sequences,suchasPLI␣sandPLI␥s.Moreover,itisnotedthat

Elapi-daesequencespossesswell-conservedregionsdifferentfromthose

ofBothrops.Insomeoftheseplaces,Mi.PLI sequencesare

simi-laronlytoMicrurus,andinotherstoBothrops(Fig.3).Finally,a

sequenceoftheB.jararacussuCTLDchosenrandomlyfromGenbank

wasalignedtoMi.PLIandtherewasnosimilarity.Thesequences

thatarerecordedinGenbankasPLI␣s(Table5)maypossiblybe

thosereferredtoasElapidaePLI␥sinTable3,sincethereareno

publicationsofPLI␣sinthesespeciesyet.ThestudiesaboutPLI␥in

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Fig.3. Aminoacidsequencesalignment(A)andphylogeneticanalyses(B)ofgammaphospholipasesA2inhibitors(PLI␥s)ofsnakesfromdifferentfamilies.A)Thedecapeptide

(87–96)ishighlightedintheredbox.B)Phlylogenetictreeresultedfromthealignedsequences.Eachbranchcolorcorrespondstoadifferentsnakefamily.PLI␥ssequences

usedforthealignmentwere:Malayopythonreticulates(GI:8163992);Protobothropselegans(302698904);Gloydiusbrevicaudussiniticus(26454644);Crotalusneutralization

factorfromCrotalusdurissusterriﬁcus(45477157);Lachesismutamuta(45477023);Bothropsneuwiedi(157885078);Bothropserythromelas(157885064);Bothropsjararaca

(157885068);Bothropsalternates(157885058);Bothropsjararacussu(157885072);Bothropsmoojeni(157885076);At.PLI1(thiswork);At.PLI2(thiswork);Elapheclimacophora

(226235139);Sinonatrixannularis(385843188);Najakaouthia(47117137);Mi.PLI1(thiswork);Mi.PLI12(thiswork);Notechisater(6685090);Notechisscutatus(5931772);

Oxyuranusscutellatus(6685092);Pseudonajatextilis(6685096)andOxyuranusmicrolepidotus(6685094).(Forinterpretationofthereferencestocolourinthisﬁgurelegend,

thereaderisreferredtothewebversionofthisarticle.)

PLIsweremodeledusingdomains2and3ofmammalianuPARs

templates and were submitted to 100ns classical MD. Models

werestabilizedwithintheﬁrst20nswithaRMSDof5Å.At.PLI2

(m.At.PLI2)andMi.PLI1(m.Mi.PLI1)modelshave94.4%and92.2%

ofresiduesinfavoredandallowedregionsandZ-scoreof−4.48

and−5.08,respectively,whosevaluesarewithinexpected

crystal-lographicstructuresofsimilarproteinsize.

M.At.PLI2andm.i.PLI1keptinitialfoldaftermoleculardynamics

stabilizationasshownbyalowRMSDdeviationofapproximately

6Å.ThisfoldisalsoseeninuPARasconﬁrmedbyT-scoresabove

0.6when insilicomodelsare comparedtotheuPARsmodeling

templates(Fig.4A1,B1andC).Thisfeatureisconﬁrmedwhen

com-paringthestudiedmodelstoallavailablestructuresintheprotein

databankbyPDBeFold,whereuPARsaretopinthelistwithZ-scores

between4and5.Asamatteroffact,uPARisanextracellularprotein

containing283residuesdividedintothreedomains(approximate

90residueseach)thatadoptathree-ﬁngerfoldandthatare

clas-siﬁedasLy-6/uPAR/␣-neurotoxinproteindomainfamily[27,28].

M.At.PLI2andm.Mi.PLI1homologytouPARconﬁrmsthemasPLI␥s,

astheseinhibitorsarecomposedoftwostructuralunitsofhighly

conservedtandemrepeatsofhalfcysteinesknownasthree-ﬁnger

motifs.

M.At.PLI2andm.Mi.PLI1comparisonconﬁrmsconservationof

thefoldwithsomelocaldifferencesalreadyexpectedforproteins

fromdifferentsnakefamilies(Fig.4A2andB2),witha

superposi-tionRMSDof2.3Å(75%ofmatchingresidues)andT-scoreof0.54.

␤-sheetsanddisulphidebridges(yellowsticksinFig.4A2andB2)

arethemostsimilarregionsinthestudiedPLIs(colouredinbluein

Fig.4A2andB2);ontheotherhand,extremitiesof␤-sheets,loops

andinmoreexposedregionsofmodelswerecolouredwhiteras

theydeviatesmorethanbluishregions.Fewregionscolouredin

redwereexcludedbysuperimpositionalgorithmsincethatthey

present highstructural deviation.Despite of thefew structural

studiesonsnakePLIsstructuresavailable,CNFisoneofthemost

studiedPLI␥anditsinsilicostructurehasbeenmodelled[17].The

superpositionofm.At.PLI2andm.Mi.PLI1withtheinsilicomodel

ofCNF[17]generatedRMSDof2.7and2.9Åfrommatching85and

73%ofresidues,respectively(Fig.4D).PLI␥weredescribedas

pos-sessinghigh-orderquaternarystructure, suchastrimers[49]or

(6)

Table5

SimilarityofthesequencesusedtoanalyzetheM.lemniscatusinhibitor.

Class Family Species Identiﬁcation MaximumIdentity(%) Reference

A Elapidae Notechisater AAF23778 85 [54]

Oxyuranusscutellatus AAF23781 83 [54]

Pseudonajatextilis AAF23783 82 [54]

Notechisscutatus CAB56617 82 [8]

Oxyuranusmicrolepidotus AAF23785 81 [54]

Elapidae Elapheclimacophora BAH47550 82 [48]

Viperidae B.moojeni ABV91335 68 [42]

B.jararaca ABV91331 68 [42]

B.alternatus ABV91326 68 [42]

B.jararacussu ABV91333 67 [42]

B.neuwiedi ABV91336 66 [42]

B.erythromelas ABV91328 65 [42]

Fig.4. FoldsimilaritiesbetweeninsilicomodelsofAtPLI2andMiPLI1andthecrystallographicstructureofahumanUrokinaseplasminogenactivatorsurfacereceptor(uPAR).

StructuresincartoonrepresentationofA1-A2BothropsatroxPLI(AtPL2),B1-B2M.lemniscatusPLI(MiPLI1),C)HomosapiensuPAR(1YWH/chainA)andD)C.d.terriﬁcusCNF.

Theywereorientatedaftersuperimpositionoffourlong␤-sheetscoloredincyan.MoleculesinA1,B1andCareina90◦rotationinthehorizontalaxisinrelationshiptoA2,B2

andD.MoleculesinA1,B1andCweregeneratedinChimeraandwerecoloredaccordingtoHomosapiensuPARdomains:D1(yellow),D2(cyan)andD3(purple);moreover

thePLIdecapeptide,theputativePLA2sinteractionsite,iscoloredingreenandiscorrespondenttotheendofuPARD2.MoleculesinA2,B2andDweregeneratedinPymol

andwerecoloredbasedondistancegeneratedbysuperimpositioninarangeof2.5Åinwhiteto0Åinblue,whereastheredareresiduesexcludedinsuperimposition.While

thedistancesinAtPLI2inA2andMiPLI1inB2werecalculatedusingonlythesetwomodels,CNFdistancesinDwerecalculatedbythemeanofthedistanceswithboth

previousmodels.TheinsilicoPLImodelssharesconservancyof␤-sheets(bluish)anddisulﬁdebridges(yellowsticksinA2,B2,andD).(Forinterpretationofthereferences

(7)

Table6

StructuralhomologyevaluationofgammaphospholipasesA2inhibitorssequences

incomparisonwithcrystalstructureofhumanurokinaseplasminogenactivator.

CrystalStructureofUrokinasePlasminogenActivatorfrom H.sapiens(ChainU;PDBID2FD6)

Score E-value

At.PLI2 187.17 4.3e-26

Mi.PLI1 184.85 4.4e-26

CNF 212.63 1.3e-30

O.scutellatusPLI* 187.21 2.5e-26

O.microlepidotusPLI* 188.38 1.6e-26

N.scutatusPLI* 189.51 5.8e-27

N.aterPLI* 192.5 1.8e-27

P.textilisPLI* 187.72 1.4e-26

Scoreande-valueswereobtainedbyHHPredsoftware[45].Thesequencesmarked

with*werepreviouslynotedasalphaphospholipasesA2inhibitors.At.PLI2and

Mi.PLI1weredescribedinthiswork.CNFisCrotalusneutralizationfactor,a␥PLIfrom

Crotalusdurissusterriﬁcus(GI:45477157).ThePLIsfromOxyuranusscutellatus,

Oxyu-ranusmicrolepidotus,Nothechisscutatus,NotechisaterandPseudonajatextiliswere

foundonNCBIdatabasewiththefollowingGIcodes:6685092,6685094,5931772,

6685090.

structurestoCNF.Therefore,thisprobablyindicatesthestudied

proteinsarrangeinhigherquaternarystructure.Thestructural

sim-ilaritiesanddifferencesinstudiedPLIsmodelsarealsoseeninCNF

insilicomodel(Fig.4D).

Snake PLA2s and PLI share a high variability in sequences,

which,in thecaseof theinhibitors,couldbeimportant to

efﬁ-cientlyneutralizedifferentPLA2.Although,aspeciﬁcregionfrom

residue107–116isconservedamongdifferentPLI␥s(Fig.3inred

box,differentnumberingisduetotheabsenceofsignalpeptide

in alignment). The synthetic decapeptide, called P-PB.III, based

on the non venomous snake Python reticulatus PLI␥ sequence

(107_PGLPLSLQNG116₎_was_shown_to_recognize_different_PLA

2sand

inhibittheircatalyticactivity[50].SimilarlytoP-PB.III,At.PLIand

Mi.PLIsequencespossessthedecapeptides107_PGLPLSRPNG116_and

107_PGLPLSHPNG116_,_{respectively,}_which_differs_only_by_two

substi-tutions,inthepositions112and113.Thisdecapeptideinm.At.PLI2

andm.Mi.PLI1areexposedtothesolventforbothproteinswithno

secondarystructure(Fig.4A1 andB1ingreen),withexceptionof

thelastthreeresiduesinm.Mi.PLI1whichpresents␤-sheet

con-formation.Thesumofaccessiblesolventareaforthisdecapeptide

inAt.PLI1andMi.PLI1modelareapproximately620and680Å2_,

respectively.Despitesimilarexposedareasandsequence

conser-vation,thedifferentdecapeptidespossessdifferentconformations,

asitsRMSDofm.At.PLI2andm.Mi.PLI1is3.9Å.ComparingtoCNF

decapeptide,m.At.PLI2sharesmoresimilaritiesthanm.Mi.PLI1,as

theirRMSDis2.7and3.6Å,respectively.ThePLI␥broadinhibiting

spectrumbydifferentsnakevenomPLA2s[50–53]mayberelatedto

theconservationofdecapeptidesequencetogetherwithitssolvent

expositionandloopﬂexibility.Thus,suchobservationsstrength

thehypothesisthatthedecapeptide(107–116)mayparticipatein

PLA2sinteractionandeventualinhibition.

4. Conclusion

Themethodologyappliedtotheampliﬁcationoftheliver

tis-suegeneticmaterialfromthesnakesB.atroxandM.lemniscatus

showedtobeefﬁcientinidentifyingthephospholipaseA2inhibitor

transcripts.

Thepresenceof␥classinhibitorswasprovenforthespecies

B.atrox,whichpresentedelevatedsimilaritytoothersequences

characterizedas such. The sequences fromM. lemniscatus

pre-sentedparticularcharacteristicsofsimilaritywithotherelapidor

bothropicsnakesatpreservedsites.However,thepresenceofa

three-ﬁngerfoldspeciﬁctothePLI␥groupandtheabsenceofa

CRDdomainspeciﬁctoPLI␣sintheinsilicomodels,amongtheir

similartertiarystructuretoCNF,suggestthatthestudiedinhibitors

arePLI␥.Theconservationoftheprimarystructureofdecapeptide

amongdifferentPLIs,whichwashypothesizedtobetheresponsible

regionforPLA2sinhibition,isnotfollowedbyconservationof

ter-tiarystructureseenintheinsilicoevaluatedmodelsandthishigh

deviationmayberelatedtothePLI␥broadinhibitingspectrumof

differentsnakevenomPLA2s.

Conﬂictofinterest

Therearenoconﬂictsofinterest.

Acknowledgements

The authors acknowledge Conselho Nacional de

Desen-volvimento Cientíﬁco e Tecnológico (CNPq), Coordenac¸ão de

Aperfeic¸oamentodePessoaldeNívelSuperior(CAPES),Instituto

Nacionalde CiênciaeTecnologiaemPesquisaTranslacionalem

SaúdeeAmbientenaRegião Amazônica(INCT-INPeTAm),

Insti-tuto Nacional de Ciência e Tecnologia em Toxinas (INCT-Tox),

Fundac¸ãodeAmparoàPesquisadoEstadodeRondônia(FAPERO),

andFundac¸ãodeAmparoàPesquisadoEstadodeSãoPaulofor

ﬁnancialsupport.RJB andCAHF acknowledgetheirgrants from

Fundac¸ãodeAmparoàPesquisadoEstadodeSãoPaulo(FAPESP).

TheauthorsthanktheProgramforTechnologicalDevelopmentin

ToolsforHealth-PDTIS-FIOCRUZforuseofitsfacilities.Amy

Grab-nerprovidedtheEnglisheditingofthemanuscript.

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