Mo-CBP3, an Antifungal Chitin-Binding Protein from Moringa oleifera Seeds, Is a Member of the 2S

24 Pages·2015·1.76 MB·English

RESEARCHARTICLE
Mo-CBP , an Antifungal Chitin-Binding
3
Protein from Moringa oleifera Seeds, Is a
Member of the 2S Albumin Family
JoséE.C.Freire1,IlkaM.Vasconcelos1,FredericoB.M.B.Moreno2,AdelinaB.Batista1,
MarinaD.P.Lobo2,MirellaL.Pereira3,JoãoP.M.S.Lima4,RicardoV.M.Almeida4,
AntônioJ.S.Sousa1,AnaC.O.Monteiro-Moreira2,JoséT.A.Oliveira1,Thalles
B.Grangeiro3*
1 DepartamentodeBioquímicaeBiologiaMolecular,CentrodeCiências,UniversidadeFederaldoCeará,
Fortaleza,Ceará,Brazil,2 NúcleodeBiologiaExperimental,UniversidadedeFortaleza,Fortaleza,Ceará,
Brazil,3 DepartamentodeBiologia,CentrodeCiências,UniversidadeFederaldoCeará,Fortaleza,Ceará,
a11111
Brazil,4 InstitutodeMedicinaTropical(IMT-RN),UniversidadeFederaldoRioGrandedoNorte,Natal,Rio
GrandedoNorte,Brazil
* [email protected]
Abstract
OPENACCESS
Mo-CBP isachitin-bindingproteinfromM.oleiferaseedsthatinhibitsthegerminationand
Citation:FreireJEC,VasconcelosIM,Moreno 3
FBMB,BatistaAB,LoboMDP,PereiraML,etal. mycelialgrowthofphytopathogenicfungi.Thisproteinishighlythermostableandresistant
(2015)Mo-CBP,anAntifungalChitin-BindingProtein topHchanges,andthereforemaybeusefulinthedevelopmentofnewantifungaldrugs.
3
fromMoringaoleiferaSeeds,IsaMemberofthe2S
However,therelationshipofMoCBP3withtheknownfamiliesofcarbohydrate-bindingdo-
AlbuminFamily.PLoSONE10(3):e0119871.
mainshasnotbeenestablished.Inthepresentstudy,full-lengthcDNAsencoding4iso-
doi:10.1371/journal.pone.0119871
formsofMo-CBP (Mo-CBP -1,Mo-CBP -2,Mo-CBP -3andMo-CBP -4)werecloned
3 3 3 3 3
AcademicEditor:WeiWang,HenanAgricultural
fromdevelopingseeds.ThepolypeptidesencodedbytheMo-CBP cDNAswerepredicted
Univerisity,CHINA 3
tocontain160(Mo-CBP -3)and163aminoacidresidues(Mo-CBP -1,Mo-CBP -2and
3 3 3
Received:November26,2014
Mo-CBP -4)withasignalpeptideof20-residuesattheN-terminalregion.Acomparative
3
Accepted:February3,2015
analysisofthededucedaminoacidsequencesrevealedthatMo-CBP isatypicalmember
3
Published:March19,2015 ofthe2Salbuminfamily,asshownbythepresenceofaneight-cysteinemotif,whichisa
Copyright:©2015Freireetal.Thisisanopen characteristicfeatureoftheprolaminsuperfamily.Furthermore,massspectrometryanalysis
accessarticledistributedunderthetermsofthe demonstratedthatMo-CBP isamixtureofisoformsthatcorrespondtodifferentmRNA
3
CreativeCommonsAttributionLicense,whichpermits
products.TheidentificationofMo-CBP asagenuinememberofthe2Salbuminfamilyrein-
3
unrestricteduse,distribution,andreproductioninany
forcesthehypothesisthattheseseedstorageproteinsareinvolvedinplantdefense.More-
medium,providedtheoriginalauthorandsourceare
credited. over,thechitin-bindingabilityofMo-CBP3revealsanovelfunctionalityforatypical
2Salbumin.
DataAvailabilityStatement:Ourdataareall
containedwithinthepaper.
Funding:Thisworkwassupportedbyresearch
grantsfromConselhoNacionaldeDesenvolvimento
CientíficoeTecnológico(CNPq),Coordenaçãode
AperfeiçoamentodePessoaldeNívelSuperior Introduction
(CAPES)andFundaçãoCearensedeApoioao
Moringa,theonlygenusofthefloweringplantfamilyMoringaceae,comprises13speciesrang-
DesenvolvimentoCientíficoeTecnológico
ingfromsmallherbstolargetreesdistributedintropicalandsubtropicalregions.Thedrum-
(FUNCAP).TBG,JTAO,JPMSLandIMVaresenior
investigatorsofCNPq. sticktree(M.oleiferaLam.),alsoknownasthehorseradishtree,isadrought-resistantspecies
PLOSONE|DOI:10.1371/journal.pone.0119871 March19,2015 1/24
CloningofcDNAsEncodingaChitin-BindingProteinofMoringaoleifera
CompetingInterests:Theauthorshavedeclared thatisnativetonorthwesternIndiaandisnowcultivatedinmanyareas.Thisspecieshasbeen
thatnocompetinginterestsexist. describedasamultipurposetreebecauseofitsmanyusesandpotentialapplications.Theseeds
ofM.oleifera,forexample,possesscoagulantandantimicrobialagentsthathavebeenexplored
fortheirabilitytotreatwaterandwastewater[1].Theactivecomponentorcomponentsre-
sponsibleforthesecoagulantandantimicrobialeffectsofM.oleiferaseedextractshavebeen
underinvestigationsincethe1990s.Mostpreviousstudiessupportthehypothesisthatcationic
peptidesandsmallbasicproteinsaretheactivemolecules[2–6],althoughtheinvolvementof
anorganic3-kDapolyelectrolyteofunknownstructure[7]orotheras-yetunrevealedactive
agentsisapossibility.
Recently,anovelchitin-bindingprotein(CBP)waspurifiedfromtheseedsofM.oleifera
andnamedMo-CBP [8].Mo-CBP isa14-kDathermostableantifungalproteinthatinhibits
3 3
thesporegerminationandmycelialgrowthoftheascomyceteFusariumsolaniandotherfungi
[8],[9].Thisproteinmaybeusefulinthedevelopmentofnewantifungaldrugsortransgenic
cropswithenhancedresistancetophytopathogenicfungi.Althoughthemechanismofaction
ofMo-CBP andmanyotherchitin-bindingproteinsisnotfullyunderstood,theantifungalac-
3
tivityoftheseCBPsislikelytheresultofproteinbindingtonascentfungalcellwallchitin,as
demonstratedforAFP1,achitin-bindingproteinfromStreptomycestendae[10].
Carbohydrate-bindingmodules(CBMs)andlectinsarethemainclassesofcarbohydrate-
recognizingproteinsdescribedtodate.CBMsarenon-catalyticpolysaccharide-recognizingdo-
mainsthattypicallyoccurwithinmulti-modularcarbohydrate-activeenzymes[11],although
insomerarecases,theyarefoundasindependentunits.Forexample,Olee10isa10-kDapol-
lenproteinfoundintheolivetree(Oleaeuropaea)thatpreferentiallybinds1,3-β-glucanand
comprisesanindependentCBMnotlinkedtoacatalyticallyactivemodule[12].Sixty-nine
familiesofstructurally-relatedCBMsarecurrentlydefinedinthecarbohydrate-activeenzymes
database(CAZy)[13],and12outofthe69CBMfamiliesareknowntoincludememberswith
chitin-bindingactivity.Lectinsareaheterogeneousgroupofproteinsthatpossessone(mero-
lectins)ortwoormore(hololectins)non-catalyticdomainsthatbindspecificallytoamonosac-
charideoroligosaccharide[14].Thechimerolectinsconstituteathirdtypeoflectininwhich
oneormorecarbohydrate-bindingdomains(CBDs)arefusedtounrelateddomains(notnec-
essarilyacarbohydrate-activecatalyticdomain).Lectinsoccurasfamiliesofstructurallyand
evolutionaryrelatedproteins,andsomeofthesefamiliescharacteristicallypossessasugar-
bindingspecificityforoligomersofN-acetylglucosamineandchitin,suchasthosecontaining
theNictabaorheveindomain[15].
BasedontheabilityofMo-CBP tobindchitin,theprimarymotivationofthepresentstudy
3
wastoinvestigatethepossiblerelationshipofthisproteinwithanyclassifiedCBMorlectin
familyortodeterminewhetherthisnovelCBPdefinesanewCBMorlectinfamily.Cloningof
full-lengthcDNAsandanalysisofthededucedaminoacidsequencesshowedthat,contraryto
anypreviousexpectations,Mo-CBP isatypicalmemberofthe2Salbuminfamily,whichis
3
oneofthemainclassesofseedstorageproteins.
MaterialsandMethods
Plantmaterial
DevelopingseedsofM.oleiferaat65daysafteranthesiswereharvestedfromtreesnaturally
growingattheCampusdoPici,Fortaleza,Ceará,Brazil.Voucherspecimens(EAC54112)were
depositedattheHerbárioPriscoBezerra,UniversidadeFederaldoCeará.BecauseM.oleiferais
anintroducedspeciesthatisnotnativetoBrazil,specificpermissionsfromlocalauthoritiesto
obtainthesamplesusedinthepresentworkwerenotrequired.Thefieldstudiesdidnotinvolve
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CloningofcDNAsEncodingaChitin-BindingProteinofMoringaoleifera
endangeredorprotectedspecies.Onceharvested,immatureseedswerefrozeninliquidnitro-
genandstoredat−80°Cuntiluse.
Plasmids,bacterialstrainsandreagents
TheplasmidpGEM-TEasywaspurchasedfromPromega(Madison,WI,USA),whereasthe
0
EscherichiacolicloningstrainTOP10F wasfromInvitrogen(Carlsbad,CA,USA).Allother
reagentswereofanalyticalgrade.
Nucleicacidpurification
TotalRNAwasisolatedusingtheConcertPlantRNAReagent(Invitrogen)accordingtothe
manufacturer’sinstructions.TheintegrityoftheRNAsampleswasdeterminedby1%agarose
gelelectrophoresis,andtheyieldwasestimatedbymeasuringtheabsorbanceat260nm[16].
PriortocDNAsynthesis,totalRNAwastreatedwithRQ1RNase-freeDNaseI(Promega)at
37°Cfor30min(1UofDNaseIperμgofRNA)andcleanedupusingtheRNeasyMinikit
(Qiagen,Hilden,Germany).TreatedRNAwasrecoveredin30μLofnuclease-freewaterand
usedforcDNAsynthesis.
0
3 RACE
TotalRNAwasreverse-transcribedtoDNAusingtheImProm-IIReverseTranscriptionSys-
tem(Promega)andoligo(dT) primer(Promega)accordingtotheprotocolsuppliedbythe
18
manufacturer.Thefirst-strandcDNAproductswerethensubmittedtoamplification
0 0 0
(3 RACE)usingagene-specificprimer(5-CCGTGYCCGGCNATHCAGCGTTGCT-3)and
oligo(dT) .Thegene-specificprimerwasdesignedtakingintoaccounttheN-terminalamino
18
acidsequencedeterminedfromthematureMo-CBP [8].Amplificationswereperformedina
3
finalvolumeof20μL,whichcontainedfirst-strandcDNA(640ng),1×GoTaqreactionbuffer
(Promega),1.5mMMgCl ,200μMeachdNTP,0.5μMeachprimer,and1.25UGoTaqDNA
2
Polymerase(Promega).ThereactionswereperformedinaPTC-200thermocycler(MJRe-
search,USA)usingthefollowingcyclingparameters:aninitialdenaturationstep(2minat
95°C)followedby27cyclesof1minat95°C,40sat52°C,and30sat72°C.Afterthelastcycle,
thereactionswerefurtherincubatedfor5minat72°Candstoredat−20°Cuntiluse.
0
5 RACE
0
5 RACEwasperformedusingtheFirstChoiceRLM-RACEKit(AmbionLifeTechnologies,
Carlsbad,CA,USA)followingthemanufacturer’sprotocolwithminormodifications.Briefly,
totalRNA(10–15μg)wastreatedinitiallywithcalfintestinalalkalinephosphatase(CIP)and
subsequentlywithtobaccoacidpyrophosphatase(TAP);bothreactionswereperformedat
0 0
37°Cfor1h.The5 RACEadapter(5-GCUGAUGGCGAUGAAUGAACACUGCGUUUG-
0
CUGGCUUUGAUGAAA-3)wasthenligatedtotheCIP/TAP-treatedRNAusingT4RNAli-
gase(37°Cfor1h)andusedinreversetranscription.cDNAsynthesiswasperformedusing
0
M-MLVreversetranscriptaseandrandomdecamersat50°Cfor1h.Next,the5 endofthe
0
transcriptencodingMo-CBP wasamplifiedbyPCRusingthe5 RACEouterprimer
3
0 0
(5-GCTGATGGCGATGAATGAACACTG-3)andagene-specificreverseprimer.Threedis-
tinctreverseprimerswereused,andtheseprimersweredesignedbasedonthesequenceinfor-
0
mationobtainedfromthe3 RACEproducts.Thesequencesoftheseprimerswereasfollows:
0 0 0
5-CACGGGGTACATTTGAGCAACTAGC-3 (gene-specificreverseprimer1,GSRP1),5-
0 0
AGCTTCGAGCTCTACGAACACACAC-3 (GSRP2),and5-GTTACACCGC-
0
TAGTGGCTCTCGTCT-3 (GSRP3).Theamplificationswereperformedinafinalvolumeof
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CloningofcDNAsEncodingaChitin-BindingProteinofMoringaoleifera
50μL,whichcontainedfirst-strandcDNA(640ng),1×GreenGoTaqreactionbuffer(Pro-
mega),1.5mMMgCl ,200μMeachdNTP,0.5μMeachprimer,and1.25UGoTaqDNAPoly-
2
merase(Promega).ThereactionswerecarriedoutinaMastercyclergradientthermocycler
(Eppendorf,Hamburg,Germany)usingthefollowingcyclingparameters:aninitialdenatur-
ationstep(5minat95°C)followedby33cyclesof1minat95°C,1.5minat60°C,and1.5min
at72°C.Afterthelastcycle,thereactionswerefurtherincubatedfor15minat72°Candstored
at−20°C.
CloningofPCRproducts
0 0
ThespecificityofthePCRamplifications(5 and3 RACE)andthesizesoftheampliconswere
determinedby1%agarosegelelectrophoresis[16].Analiquotoftheremainingamplified
productswasligatedintothepGEM-TEasyvectorusingT4DNAligase(Promega)at4°Cfor
0
16h.ProductsfromtheligationreactionswereintroducedintoE.coliTOP10F cellsbyelectro-
poration,andthetransformantswereselectedonLBagarcontaining100μg.mL-1carbenicillin
and30μg.mL-1streptomycin.PlasmidDNAwasisolatedfromantibiotic-resistantcolonies
usingthealkalinelysismethod[16],andthepresenceoftheinsertswasconfirmedbyrestric-
tiondigestionwithEcoRI(FermentasLifeSciences,Ontario,Canada).
DNAsequencingandassembly
PlasmidsamplesforDNAsequencingwerepurifiedusingtheAxyPrepplasmidminiprepkit
(AxygenScientific,UnionCity,CA,USA).TheinsertsweresequencedusingtheDYEnamic
ETDyeterminatorcyclesequencingkit(GEHealthcare,Buckinghamshire,UK)followingthe
protocolsuppliedbythemanufacturer.Bothstrandsweresequencedusingtheuniversalprim-
0 0
ersM13(-40)forward(5-GTTTTCCCAGTCACGACGTTGTA-3)andM13(-46)reverse
0 0
(5-GAGCGGATAACAATTTCACACAGG-3).Thesequencingproductswereresuspended
in10μLof70%formamide/1mMEDTA,andpriortocapillaryelectrophoresis,10μLofaga-
rosewasadded(0.06%finalconcentration)assuggestedpreviously[17],[18].Thesequencing
reactionswereanalyzedinaMegaBACE1000automaticsequencer(GEHealthcare)usingthe
followingparameters:injectionat3kVfor50sandelectrophoresisat6kVfor200min.Auto-
matedbase-callingwasperformedusingCimarron3.12software,andcompletesequences
wereassembledusingthePhred/Phrap/Consedpackage[19–21].Beforefurtheranalysis,the
endsoftheassembledcontigsweretrimmedtoremovelow-quality(phred<20)sequences.
Sequenceanalysis
MultiplealignmentsofDNAandaminoacidsequenceswereusuallyperformedusingthepro-
gramClustalW[22]implementedwiththeBioEdit7.2.5softwarepackage[23],whichwas
routinelyusedforsequencemanipulation,editingandcomparisons.Ontheotherhand,the
3ʹUTRsequenceswerealignedusingtheprogramClustalOmega[24]atthewebserverwww.
ebi.ac.uk/Tools/msa/clustalo/.ThedefaultalignmentparametersofClustalOmegawereem-
ployed,althoughthenumberofcombinediterationsandthemaximumnumberofHMMitera-
tionswerebothsettofive.Codon-basedalignmentswereperformedusingtheprogram
PAL2NAL[25]attheprogram’swebserver(www.bork.embl.de/pal2nal/).Theidentitybe-
tweentwoalignedsequenceswascalculatedasthenumberofpositionscontainingidenticalnu-
cleotidesoraminoacidresiduesdividedbythenumberofalignedpositions,excludingthesites
withgaps,andexpressedasapercentage.RNAsecondarystructureswerepredictedusingVi-
ennaRNASecondaryStructurePredictionversion2.1.6(http://rna.tbi.univie.ac.at/cgi-bin/
RNAfold.cgi.)[26].Thepresenceofsignalpeptidesandtheirputativecleavagesiteswerepre-
dictedusingthealgorithmSignalP4.1(www.cbs.dtu.dk/services/SignalP/)[27].Searchesfor
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CloningofcDNAsEncodingaChitin-BindingProteinofMoringaoleifera
homologousproteinsinpublicsequencedatabaseswereperformedusingBLASTp[28].The
presenceanddelimitationofproteindomainswasaccomplishedusingtheConservedDomain
Database(CDD)[29].
Phylogeneticanalysis
PhylogeneticanalysiswasperformedusingMolecularEvolutionaryGeneticsAnalysis(MEGA)
softwareversion6.0[30].TheaminoacidsequencesoftheproteinswerealignedusingClustal
Omega,andthepairwiseevolutionarydistanceswerethencomputedusingthePoissoncorrec-
tionmethod[31].Thetreesweregeneratedusingtheneighbor-joiningmethod[32],andthe
stabilityofthecladeswasassessedusingthebootstrapmethod[33].
PurificationofMo-CBP
3
Mo-CBP waspurifiedfromcrudeextractsofmatureM.oleiferaseedsusingaffinitychroma-
3
tographyonachitinmatrixfollowedbycationexchangechromatographyonaResourceSma-
trix(GEHealthcare)asdescribedpreviously[8].Thepurityoftheproteinsampleswas
determinedbytricine-SDS-polyacrylamidegelelectrophoresis(tricine-SDS-PAGE)according
toapreviouslydescribedmethod[34].Proteinbandswerestainedwith0.1%(w/v)Coomassie
BrilliantBlueR250in40%methanol/1%aceticacid.Destainingwascarriedoutwith50%
(v/v)methanol.
N-terminalaminoacidsequencing
N-terminalsequencingwasperformedonaShimadzuPPSQ-10AutomatedProteinSequencer
(Kyoto,Japan).Proteinsampleswereblottedontoapolyvinylidenefluoride(PVDF)mem-
braneaftertricine-SDS-PAGEandsubmittedtoEdmandegradation[35].Thephenylthiohy-
dantoin(PTH)aminoacidsweredetectedat269nmafterseparationonareverse-phaseC18
column(4.6mmx2.5mm)underisocraticconditionsaccordingtothe
manufacturer’sinstructions.
Capillaryliquidchromatography/nanoelectrosprayionizationtandem
massspectrometry(LC-ESI-MS/MS)
In-geltrypticdigestionsofproteinsbandsresolvedbytricine-SDS-PAGEwereperformedac-
cordingtoaprotocoldescribedpreviously[36].Proteinsampleswerealsosubmittedtoin-
solutiondigestions.Tothisend,thesampleswerereducedwith5mMDTTat60°Cfor30min,
treatedwith15mMiodoacetamideatroomtemperaturefor30mininthedark,anddigested
withsequencing-gradetrypsin(Promega)at37°Cfor16h.Thetrypticpeptidesfromin-gel
andin-solutiondigestionswereanalyzedbyLC-ESI-MS/MSusingaSynaptG1HDMSQ-ToF
massspectrometer(WatersCo.,Milford,MA,USA)coupledtoaWatersultra-high-perfor-
manceliquidchromatography(UPLC)unit.Thedigestedsampleswereinjectedusingthe
nanoACQUITYUPLCsamplemanager,andthechromatographywasperformedusinga
UPLCC18nanoACQUITYcolumn(75μmx10cm,1.7μmparticlesize)ataflowrateof
0.35μL/min.Themassspectrawereacquiredusingthedata-dependentacquisition(DDA)
mode,inwhichthetopthreepeaksweresubjectedtoMS/MS.MobilephasesAandBconsisted
of0.1%formicacidinwaterand0.1%formicacidinacetonitrile,respectively.Thepeptides
wereelutedusingthefollowingstepgradient:3–40%Bfor30minand40–85%Bfor5min.
ThedatawereprocessedusingProteinLynxGlobalServersoftware(WatersCo.)andsubjected
toadatabasesearchusingtheMascotsearchengine[37].Thesearcheswereperformedwith
theassumptionsthattherewasamaximumofonemissedtrypsincleavageandthe
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CloningofcDNAsEncodingaChitin-BindingProteinofMoringaoleifera
experimentalmassesofthepeptidesweremonoisotopic.Furthermore,carbamidomethylation
ofcysteinewasincludedasafixedmodification,whereasoxidationofmethionineandcycliza-
tionofN-terminalglutaminetopyroglatamicacid(pyro-Glu)wereincludedaspossiblevari-
ablemodifications.MS/MSionssearcheswereperformedagainsttheNCBInon-redundant
database(lastaccessedonJanuary21st,2015)usingasignificancethresholdofp<0.05.The
peptidemasstoleranceandfragmentmasstolerancewerebothinitiallysetto±0.1DaforMS/
MSionsearching.However,candidatepeptideIDswereonlyacceptedifthem/zvalueswere
within0.1Da(typicallylessthan0.05Da)ofthetheoreticalmassofthecandidateID,asdeter-
minedwhenmanuallyreviewingtheMASCOTsearchresults.Themassspectrometryproteo-
micsdatahavebeendepositedtotheProteomeXchangeConsortium[38]viathePRIDE
partnerrepositorywiththedatasetidentifierPXD001762andnull.
Results
RACEandcDNAassembly
0 0
Usingacombinationof5 RACEand3 RACE,full-lengthcDNAsencodingchitin-binding
0
protein3fromM.oleifera(Mo-CBP )wereobtainedfromdevelopingseeds.The3 endofthe
3
Mo-CBP mRNAwasamplifiedusingoligo(dT)asanantisenseprimerandagene-specificde-
3
generateoligonucleotideasasenseprimer;thisprimerwasdesignedbyreferencingthe
0
N-terminalsequenceofthepurifiedprotein[8].Agarosegelelectrophoresisofthe3 RACE
productsrevealedasingleDNAbandofapproximately420bp(Fig.1A).TheamplifiedcDNA
fragmentwascloned,andtheinsertsof20cloneswerecompletelysequenced.Whenthesese-
quenceswerealignedandcompared,itwaspossibletoclustertheminto3groupsaccordingto
theiroverallsimilarity.Therefore,gene-specificoligonucleotidestargetingeachoneofthese
0 0
3distinct3 untranslatedregions(UTRs)weredesignedandusedasreverseprimersinthe5
0
RACEexperiment.AnoligonucleotidetargetingtheRNAadapter,whichwasligatedtothe5
0
endsofthetotalmRNAs,wasusedasaforwardprimer.Agarosegelelectrophoresisofthe5
RACEproductsshowedthat3specificampliconswereproduced,withestimatedsizesofap-
proximately790,800and780bp(Fig.1B).ThesePCRproductswerecloned,andthecomplete
sequencesoftheinsertsfrom42clonesweredetermined.
0
TheoverlappingDNAsequencesobtainedfromallclonedPCRproductsfromboththe3
0
RACEand5 RACEreactionswereassembled.Asaresult,4uniquecDNAcontigspresump-
tivelyencodingMo-CBP weregenerated.Herein,thesecDNAsequences(GenBankaccession
3
numbersKF616830-KF616833)arereferredtoasMo-CBP –1(695bp),Mo-CBP –2(797bp),
3 3
Mo-CBP –3(819bp),andMo-CBP –4(827bp)accordingtotheirlengths.Threeoftheseas-
3 3
sembledfragments,Mo-CBP –2,Mo-CBP –3,andMo-CBP –4,correspondedtofull-length
3 3 3
0 0
cDNAs,aseachonecontainedcomplete5 and3 UTRsandasinglecodingsequence(CDS).
Thefourthcontig(Mo-CBP –1)containedanear-full-lengthcDNA,withacomplete50UTR
3
andCDSbutwithapartial30UTR(Table1).Thefull-lengthcDNAsequenceofMo-CBP –3
3
anditsdeducedaminoacid(aa)sequenceareshowninFig.2asarepresentativesequenceof
the4assembledcDNAs.
Pairwisecomparisonsofthe4cDNAsequencesrevealedanoverallmeansequenceidentity
ofapproximately82.8%(excludingallsiteswithinsertions/deletions).ThecDNAsequencesof
Mo-CBP –1andMo-CBP –4wereverycloselyrelatedtoeachother(99.3%pairwisesequence
3 3
identity),whereasthesequenceofMo-CBP –3wasthemostdivergentfromtheotherse-
3
quences(averagepairwisesequenceidentityofapproximately78.1%).
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CloningofcDNAsEncodingaChitin-BindingProteinofMoringaoleifera
Fig1.AgarosegelelectrophoresisofMo-CBP cDNAfragmentsamplifiedbyPCR.A.30RACEproducts(lane1).B.50RACEproductsamplifiedusing
3
3distinctgene-specificprimers(lanes1,2and3).LaneM(AandB):molecularmarkers.
doi:10.1371/journal.pone.0119871.g001
0 0
TheCDSandthe5 and3 UTRs
WithineachcDNAsequence,oneopenreadingframe(ORF)wasfoundinframes1(Mo-
CBP –2)and3(Mo-CBP –1,Mo-CBP –3andMo-CBP –4).TheORFsizevariedfrom483nu-
3 3 3 3
cleotides(nt)(inMo-CBP –3)to492nt(inMo-CBP –1,Mo-CBP –2andMo-CBP –4),as
3 3 3 3
summarizedinTable1.TheaveragesequenceidentityamongtheCDSswas~88%,ranging
from83.9%(Mo-CBP –1andMo-CBP –3)to99.3%(Mo-CBP –1andMo-CBP –4).Inthese
3 3 3 3
ORFs,thecontextsequencearoundtheATGstartcodon(AUGinthemRNA)wasinagree-
mentwiththeconsensussequenceAAAA/CAAUGGCofthetranslationalinitiationsite(TIS),
whichwasderivedfromtheanalysisof3643plantgenes[39].Therefore,thefollowingse-
quenceswerefoundforthesegmentspanningthenucleotidepositionsfrom−5(immediately
upstream)to+5(immediatelydownstreamoftheATGstartcodon):TTACTatgGC(Mo-
CBP –1andMo-CBP –4),TTACCatgGC(Mo-CBP –2),andTTACAatgGC(Mo-CBP –3)(nu-
3 3 3 3
cleotidesthatmatchthosefoundintheconsensussequenceoftheTISareunderlined).TheA
andGnucleotidesatpositions−3and+4aroundtheATGstartcodon,asobservedinthe4
Mo-CBP cDNAsequences,havebeensuggestedtobeparticularlyimportantforgreatertrans-
3
lationalefficiency[40],[41].
Table1.GeneralfeaturesofthecDNAsequencesencodingMo-CBP .
3
cDNAregions
cDNA GenBankaccessionnumber Length(nt) 50UTR(nt) CDS(nt)/preproproteinsize(aa) 30UTR(nt) Poly(A)site
Mo-CBP –1 KF616830 695 1–56 57–548(492)/163 549–695(147)1 n.d.2
3
Mo-CBP –2 KF616832 797 1–56 57–548(492)/163 549–751(203) 752
3
Mo-CBP –3 KF616833 819 1–62 63–545(483)/160 546–747(202) 748
3
Mo-CBP –4 KF616831 827 1–56 57–543(487)/163 544–747(204) 748
3
1Partial30UTRsequence
2Notdetermined
doi:10.1371/journal.pone.0119871.t001
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CloningofcDNAsEncodingaChitin-BindingProteinofMoringaoleifera
Fig2.ThecDNAsequenceanddeducedaminoacidsequenceofpreproMo-CBP –3.ThededucedaminoacidsequenceofthepreproMo-CBP –3is
3 3
shownbelowthecDNAsequence.Numbersforthefirstnucleotideandthelastaminoacidresidueineachrowareshownontheleftandright,respectively.
TheN-terminalsignalpeptide,aspredictedbytheSignalP4.1program[27],isshadedingray.TheN-terminalsequenceofthelargechainofMo-CBP ,as
3
determinedbyEdmandegradation,isunderlinedwithadashedline.Thestopcodonisindicatedbyanasterisk.Thepoly(A)signalnearesttothepoly(A)tail
isboxed,andtwootherupstreampoly(A)signalsareunderlined.
doi:10.1371/journal.pone.0119871.g002
The50UTRsintheMo-CBP cDNAswere51(Mo-CBP –2),56(Mo-CBP –1and
3 3 3
Mo-CBP –4)and62(Mo-CBP –3)ntlong.ThesequenceidentityamongtheseUTRswas80%
3 3
onaverage,varyingfrom100%(50UTRsofMo-CBP –1andMo-CBP –4)to66.6%(50UTRs
3 3
ofMo-CBP –1vsMo-CBP –3andofMo-CBP –3vsMo-CBP –4).Thelengthsofthe50UTRs
3 3 3 3
oftheMo-CBP cDNAsthusfallwithinthesizerangethatwasreportedforthisregioninasur-
3
0
veyof1,615Viridiplantaegenes,whichwereshowntohave5 UTRsthatare116ntlongonav-
0
erage[42].Theentire5 UTRandthefirst32ntoftheCDSoftheMo-CBP sequenceswere
3
predictedtofoldintoasecondarystructurecharacterizedby2or3hairpinsradiatingfroma
centralloop(Fig.3A).TheΔGoftheminimumfreeenergy(MFE)secondarystructuresranged
from−14.0kcal/molto−6.0kcal/mol,suggestingthatevenundertheassumptionthatthese
structurescouldoccurinvivo,theywouldnotbesufficientlystableenoughtoinhibittransla-
tion[43].
0
The3 UTRsequencesoftheMo-CBP cDNAswerelongerandmoredivergentthanthese-
3
0
quencesofthe5 UTRs.Theirlengths(excludingthesegmentsofthepoly(A)tail)were203
(Mo-CBP –4),204(Mo-CBP –2)and202nt(Mo-CBP –3).Thepartialsequenceobtainedfor
3 3 3
the30UTRofMo-CBP –1was147ntlong.ThemeansequenceidentityamongtheMo-CBP
3 3
30UTRswasapproximately67.7%,rangingfrom60.2%(30UTRofMo-CBP –2vsMo-CBP –
3 3
4)to99.2%(30UTRofMo-CBP –1vsMo-CBP –4).ThelengthsoftheMo-CBP 30UTRsare
3 3 3
0
comparabletotheaveragelength(~240nt)ofthe3 UTRsof1,826Viridiplantaegenes[42].
Inthe30UTRsofMo-CBP –3andMo-CBP –4,atypicaleukaryoticpolyadenylationsignal
3 3
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CloningofcDNAsEncodingaChitin-BindingProteinofMoringaoleifera
Fig3.Predictedsecondarystructuresofthe50UTRand30UTRsequencesoftheMo-CBP mRNAs.A.ThepredictedMFEsecondarystructuresofthe
3
entire50UTRandthefirst32ntoftheCDSoftheMo-CBP –2,Mo-CBP –3andMo-CBP –4mRNAsareshown.B.ThepredictedMFEsecondarystructures
3 3 3
oftheentire30UTRsoftheMo-CBP –2,Mo-CBP –3andMo-CBP –4mRNAsareshown.Heatcolorgradationfrombluetoredrepresentsthebase-pairing
3 3 3
probabilityfrom0to1.
doi:10.1371/journal.pone.0119871.g003
sequence(AATAAA)wasfound16and25ntupstreamofthefirstAofthepoly(A)tail,respec-
tively(Fig.2).Inthe30UTRofMo-CBP –2,aclosevariant(GATAAA)ofthecanonicalpolya-
3
denylationsignalsequencewasobserved20ntupstreamofthepoly(A)tail.Furthermore,in
the30UTRsofMo-CBP –2andMo-CBP –3,thefirstAofthepoly(A)tailisprecededbyC,
3 3
whereasinthe30UTRofMo-CBP –4,thepoly(A)tailisprecededbyT.Thisagreeswiththe
3
0
findingthatinplantgenes,cleavageofthepre-mRNAduring3-endprocessingusuallyoccurs
0 0
3 toanadenosineresidueatthedinucleotideYA(Y=CorT)[44].Thefull-length3 UTRsof
theMo-CBP transcriptswerepredictedtobeabletofoldintostablesecondarystructures
3
(Fig.3B).ThecalculatedΔGoftheMFEsecondarystructuresrangedfrom−49.0kcal/molto
PLOSONE|DOI:10.1371/journal.pone.0119871 March19,2015 9/24
CloningofcDNAsEncodingaChitin-BindingProteinofMoringaoleifera
−55.0kcal/mol.The50and30UTRsofmanymRNAscharacteristicallycontaincis-regulatory
elementsthatplaycrucialrolesinpost-transcriptionalregulation.Manyofthesecis-regulatory
elementsarestructured,andtheyinfluencedistinctaspectsofthemRNAlifecyclesuchassta-
bility,transport,localization,andtranslationactivationandrepression[45–47].Thesecondary
structurespredictedtooccurintheUTRsofMo-CBP mRNAscouldplaysimilarroles.
3
Therefore,thesesequenceanalysesdemonstratedthattheMo-CBP cDNAshavethegener-
3
alstructuralfeaturesusuallyfoundinplantgenes.
TheproteinsencodedbytheMo-CBP cDNAs
3
ThepolypeptidesencodedbytheMoCBP cDNAswerepredictedtocontain160(Mo-CBP –
3 3
3)and163aaresidues(Mo-CBP –1,Mo-CBP –2andMo-CBP –4).Thepercentagesequence
3 3 3
identityamongthe4putativeprimarytranslationproductswas~81%onaverage,ranging
from76.4%(betweentheproductsofMo-CBP –1andMo-CBP –3)to98%(betweentheprod-
3 3
uctsofMo-CBP –1andMo-CBP –4),indicatingthattheencodedpolypeptidesarecloselyre-
3 3
latedtoeachother.WhenthesesequenceswereanalyzedwithSignalPsoftware,thesegment
comprisingthefirst20aaresiduesofeachproteinwaspredictedtobeasignalpeptide(SP).
TheSPsofthe4encodedproteinshaveuniqueaasequences,andtheaveragesequenceidentity
betweenthemisapproximately77.5%.EachMo-CBP SPhastheclassicaltripartitestructure
3
comprisingapositivelychargedN-terminalregion(3aaresidueslong,containingaLysresi-
due)followedbyacentralhydrophobiccore(13aaresidueslong,withahighproportionof
Leuresidues)andaneutralbutpolarC-terminalregion(4aaresidueslong)containingthepu-
tativesignalpeptidase(SPase)cleavagesite.Positions−1and−3relativetotheSPasecleavage
siteareoccupiedbyAlaandAla(in3sequences)orAlaandThr(inonesequence),respective-
ly;thisfindingagreeswiththespecificityofSPasecleavagesite[48].Indeed,seedstoragepro-
teinsaretypicallysynthesizedwithanN-terminalSPthatiscleavedastheproteinsare
translocatedintothelumenoftheendoplasmicreticulumwheretheyaresubjectedtopost-
translationalprocessingandthentransportedtoproteinstoragevacuoles[49].
TofurtherclarifytheidentityoftheproteinsencodedbytheMo-CBP cDNAs,thededuced
3
aasequencesoftheirputativeprecursors(proMo-CBP ,excludingtheirN-terminalSPs)were
3
submittedtoBLASTsearchesagainstproteindatabases.ThesearchesagainsttheConserved
DomainDatabaserevealedthateachproMo-CBP sequencepossessesasingleAAI_LTSSdo-
3
main(CDDsuperfamilyaccessionnumber:cl07890),whichischaracteristicoftheprolamin
superfamily.Thissuperfamilyisuniquetohigherplantsandincludesi)cereal-typeα-amylase
inhibitors(AAI),trypsininhibitors,andbifunctionaltrypsin/α-amylaseinhibitors;ii)lipid
transferproteins(LTPs),suchasthenon-specifictype1LTP(nsLTP1)andtype2LTP
(nsLTP2);iii)seedstorage(SS)proteins,suchas2Salbumins,γ-gliadin,andprolamin;andiv)
otherrelatedproteins[50],[51].Morespecifically,theproMo-CBP sequenceswereclustered
3
inthesubfamilyAAI_SS(conserveddomainmodelaccessionnumber:cd00261),whichin-
cludestheα-amylaseinhibitorsandseedstorageproteinssuchas2Salbumins.Searchesagainst
thenon-redundantproteinsequencedatabaseoftheNCBIusingBLASTprevealedthatthe
proMo-CBP sequencesweremorecloselyrelated(45–47%sequenceidentity;E-value=
3
4×10-25–1×10-19;95–97%querycoverage)totheaasequenceofaprecursorofthesweetpro-
teinMabinlinII,a2SalbuminfromtheseedsofCapparismasaikai[52].Alltheotherproteins
thatshowedsignificantalignmentwiththeproMo-CBP sequenceswere2Sseedstoragepro-
3
teinsfromdifferentspecies.Tofurthersupportthesefindings,aphylogenetictreewasgenerat-
edusingprimarystructuresfromrepresentativensLTP1,nsLTP2,AAIs,and2Salbumins.As
showninFig.4,foursupportedcladeswererecovered,correspondingtothensLTP1,nsLTP2,
PLOSONE|DOI:10.1371/journal.pone.0119871 March19,2015 10/24