Structural of two isolectins from the marine red alga Solieria filiformis (Kützing) P.W. Gabrielson and their anticancer effect on MCF7 breast cancer cells

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ContentslistsavailableatScienceDirect

International

Journal

of

Biological

Macromolecules

jo u r n al h om ep age :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

Structural

characterization

of

two

isolectins

from

the

marine

red

alga

Solieria

filiformis

(Kützing)

P.W.

Gabrielson

and

their

anticancer

effect

on

MCF-7

breast

cancer

cells

Renata

Pinheiro

Chaves

a

_,

_Suzete

_Roberta

_da

_Silva

a

_,

_Luiz

_Gonzaga

_Nascimento

_Neto

a,b

_,

Romulo

Farias

Carneiro

a

_,

_André

_Luis

_Coelho

_da

_Silva

c

_,

_Alexandre

_Holanda

_Sampaio

a

_,

Bruno

Lopes

de

Sousa

d

_,

_Maria

_Guadalupe

_Cabral

e

_,

_Paula

_Alexandra

_Videira

f

_,

Edson

Holanda

Teixeira

b

_,

_Celso

_Shiniti

_Nagano

a,∗

a_Laboratório_de_{Biotecnologia}_Marinha_–_BioMar-Lab,_Departamento_de_Engenharia_de_Pesca,_Universidade_Federal_do_Ceará,_Campus_do_Pici_s/n,_bloco

871,60440-900Fortaleza,Ceará,Brazil

b_Laboratório_Integrado_de_{Biomoléculas}_–_LIBS,_Departamento_de_Patologia_e_Medicina_Legal,_Universidade_Federal_do_Ceará,_Monsenhor_Furtado,_s/n,

60430-160Fortaleza,Ceará,Brazil

c_Laboratório_de_{Biotecnologia}_Molecular_–_LabBMol,_Departamento_de_Bioquímica_e_Biologia_Molecular,_Universidade_Federal_do_Ceará,_Campus_do_Pici,

bloco907,60440-900,Fortaleza,Ceará,Brazil

d_Faculdade_de_Filosofia_Dom_Aureliano_Matos,_Universidade_Estadual_do_Ceará,_Av._Dom_Aureliano_Matos,_2060,_Limoeiro_do_Norte,_CE,_62930-000,_Brazil e_CEDOC,_NOVA_Medical_School,_Universidade_NOVA_de_Lisboa,_1150-082,_Lisbon,_Portugal

f_UCIBIO,_Departamento_de_Ciências_da_Vida,_Faculdade_de_Ciências_e_Tecnologia,_Universidade_NOVA_de_Lisboa,_2829-516,_Caparica,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received21July2017 Accepted28September2017 Availableonline29September2017

Keywords: Marinealga Lectin Anticancereffect

a

b

s

t

r

a

c

t

Asdescribedintheliterature,Solieriafiliformislectin(SfL)fromthemarineredalgaS.filiformiswasfound tohaveantinociceptiveandanti-inflammatoryeffects.Inthisstudy,wecharacterizedtwoSfLvariants, SfL-1andSfL-2,withmolecularmassof27,552Daand27,985Da,respectively.Theprimarystructures ofSfL-1andSfL-2consistoffourtandem-repeatproteindomainswith67aminoacidseach.SfL-1and -2showedhighsimilaritytoOAAH-familylectins.3DstructurepredictionrevealedthatSfL-1and-2are composedoftwo␤-barrel-likedomainsformedbyfiveantiparallel␤-strands,whichareconnectedbya shortpeptidelinker.Furthermore,themixtureofisoforms(SfLs)showedanticancereffectagainstMCF-7 cells.Specifically,SfLsinhibited50%ofviabilityinMCF-7cellsaftertreatmentat125␮g.mL−1_,_while theinhibitionofHumanDermalFibroblasts(HDF)was34%withthesametreatment.Finally,24hafter treatment,25%ofMCF-7cellswereinearlyapoptosisand35%inlateapoptosis.Evaluationofpro-and anti-apoptoticgeneexpressionofMCF-7cellsrevealedthatSfLsinducedcaspase-dependentapoptosis within24h.

1. Introduction

Lectins,whichareubiquitouscarbohydrate-bindingproteinsor glycoproteins,havereceivedspecialattentionincancerprognosis, diagnosis,andtreatmentbytheirbiologicalactivities.Lectinscanbe usedastoolstoidentifyaberrantglycansonthemembranesurface ofneoplasiccellsandasantitumormoleculestoinduceapoptosis andautophagythroughvariousmechanisms[1,2].

∗ Correspondingauthorat:BioMar-Lab,DepartamentodeEngenhariadePesca, UniversidadeFederaldoCeara,Av.HumbertoMontes/n,60440-900,Fortaleza, Ceara,Brazil.

E-mailaddress:naganocs@gmail.com(C.S.Nagano).

Marine algaeare good sources of newlectins. Algae lectins possess unique molecular structures and important biological activities.Inparticular,theirhighspecificityforcomplex carbohy-dratesandglycoconjugatesmakesthemusefulforbiochemicaland biologicalapplications[3,4].Biochemicalstudiesrevealthatmany lectinsisolatedfrommarineredalgaesharecommonproperties withprokaryotelectins[5].Theselectinscouldbegroupedintoa familyoflectinsknownasOscillatoriaagardhiiagglutinin homo-logue(OAAH)lectinfamily,amongwhichisalectinisolatedfrom thecyanobacteriumO.agardhii[6].

OAA, which contains 132 residues with two tandem-repeat domains[7],issimilartoPFAfromthebacteriumPseudomonas fluo-rescens[8].Ontheotherhand,BOAfromthebacteriumBurkholderia oklahomensis EO147[9],MBHA fromthebacteriumMyxococcus

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Xanthus[10],ESA-2fromthemarinealgaEucheumaserra[11], EDA-2fromthemarinealgaEucheumadenticulatum[12],KSA-2from themarinealgaKappaphycusstriatum[13],andKAA-1/-2fromthe marinealgaKappaphycusalvarezii[14]allcontainfour tandem-repeatsequences246–269residuesinlength.

OAAH-family members share high identity of amino acid sequenceandpossessduplicateorquadruplicatedomainsintheir sequencesthatleadtosingular␤-barrel-liketopologyinvolvedin thetightbindingtooligomannosides[7,15].Interestingly,amino acidsequencesofOAAHlectinsarecompletelydistinctfromother highmannose-bindinglectins.

SolieriafiliformisisamarineredalgafoundalongtheBrazilian northeastcoast.Itslectin,SfL,at29kDawasisolatedbyBenevides andcollaborators[16].Abreuandcoauthors[17]showedthatSfL enhancestheproductionofIL-6athighlevels,butwithno cyto-toxicactivityonsplenocytes,thuspromotinganti-inflammatory effect.Inaddition,SfLseemstohaveimportantinvivo antinocicep-tiveactivityandcouldrepresentapotentialtherapeuticagentfor invitroandinvivoimmunomodulatoryeffects[18].However,other biologicalactivitieshavenotbeenexploited,includingthislectin’s antitumoractivity.

Breastcancer,theforemostcauseofdeathinwomen,isa het-erogeneousdiseasecharacterizedbya varietyofmolecularand geneticalterationsthatinducegrowthandsurvival[19,20]. Cur-rently,chemotherapyisthemostcommonlyusedmethodtotreat cancer.However,chemotherapeutictechniquesarelimitedbytheir highcellulartoxicity,aswellastheoccurrenceofsideeffects[21]. Therefore,selectivebiologicaltreatmentshaveemergedtotreat certaintypesofcancersortotargetspecificdeterminantsexpressed bymanydifferenttumors[22].

Inthiswork,weidentifiedandthenconductedstructuralstudies ontwoisolectinsofthemarineredalgaS.filiformis:SfL-1and SfL-2.Theirprimarystructuresweredetermined,theirtridimensional structureswerepredicted,andmoleculardockingwasperformed. ThecytotoxiceffectoftheisolectinsonMCF-7cellswasalso eval-uated.

2. Methods

2.1. Algaecollection

SpecimensfromthemarineredalgaSolieriafiliformis(Kützing) P.W.GabrielswerecollectedintheintertidalzoneofPachecoBeach, Ceará,Brazil.Algaeweretransportedtothelaboratoryiniceand keptat−20◦_C _until_use. _A_small _portion_of _the_specimens _was storedat−80◦CforRNAextraction.Collectionswereauthorized

andcertifiedbyresponsibleenvironmentalinstitutions(SISBIOID: 33913-8).

2.2. Purificationoflectin

ThelectinfromthemarineredalgaS.filiformis(SfL)waspurified followingtheprotocoldescribedbyBenevidesandcollaborators [16]withminormodifications.Frozenalgaeweregroundtoafine powderbymortarandpestleinthepresenceofliquidnitrogen. Thepowderwasstirred(1:3w/v)with20mMphosphatebuffer, pH7.0,containing150mMNaCl(PBS),for4hatroomtemperature. Insolublealgaematerialwasremovedbycentrifugationat5000×g for30minat4◦_C._The_supernatant_(crude_extract)_was_collected_and assayedforhemagglutinatingactivityand proteinconcentration [23].

Thecrude extract wassubmitted toprecipitation withsolid ammoniumsulfate(70%ofsaturation)for4handcentrifugedat 5000×gfor30minat4◦_C._The_resulting_precipitate_(F_0/70)_was recoveredinPBSanddialyzedagainstdeionizedwaterandthen

against20mMphosphatebuffer,pH7.0(PB).Aftercentrifugation, F0/70wasappliedtoaDEAE-SephacelColumn(1.0cm×3.0cm)

previouslyequilibratedwithPB.Thecolumnwaswashedwiththe samebufferataflowrateof1mLmin−1_until_that_column’s

efflu-entshowedabsorbanceat280nm<0.02(P1).Theadsorbedfraction

(P2)waseluted withPBcontaining1MNaCl.Two-mLfractions

werecollectedandtestedforhemagglutinatingactivityagainst3% trypsin-treatedrabbiterythrocytes.Fractionsshowing hemagglu-tinatingactivity(P1)werepooled,dialyzedagainstdistilledwater,

andfreeze-dried.

2.3. Molecularmassdetermination

The isotopicaveragemolecularmass of SfLwasdetermined byliquidchromatographycoupledtoElectrosprayionization-mass spectrometry(LC-ESI–MS)usingahybridSynaptHDMSmass spec-trometer (Waters Corporation, MA, USA). Two ␮g of SfL were appliedtoaC-18nanocolumn(75␮m×100mm)andelutedwith

gradientof10% to85% ofACNcontainingformicacid(FA)0.1% ataflowrateof0.6␮Lmin−1_._Eluates_were_directly_infused_into themassspectrometerusingananoAcquitysystemconnectedtoa nanoelectrospraysource.

The instrument was calibrated with [Glu1]-fibrinopeptide-B collision-induceddissociation(CID)fragments.Massspectrawere acquiredbyscanningatm/zrangingfrom800to3000at1scan s−1_._The_mass_spectrometer_was_operated_in_positive_mode,_using asourcetemperatureof363Kandcapillaryvoltageat3.5kV.Data collectionandprocessingwerecontrolledbyMassLynx4.1 soft-ware (WatersCorporation,MA, USA).The deconvolution of ESI massspectrawasperformedusingtheMaxEnt1algorithminthe MassLynxsoftware.

2.4. Primarystructuredetermination

2.4.1. N-terminalaminoacidsequencing

The N-terminal aminoacidsequence of SfLwasdetermined by Edmandegradation ina Shimadzu model PPSQ-31Aprotein andpeptidesequencer(ShimadzuCorp.,Japan).PTH-aminoacids fromtheN-terminussequencewereseparatedona2.0×250mm

WakosilODS column(WakoPureChemicalCorp.,Osaka,Japan) connectedtoamodelLC-20ATpump.Theabsorbancewasdetected at269nmwithaUV–visSPD-20Adetector.

The sequence similarity of the N-terminus was evaluated online (http://www.ncbi.nlm.nih.gov/BLAST/), and homologous sequenceswereidentifiedwiththebasiclocalalignmentsearch toolprogram(BLAST)usingPROTEINBLASTfromtheNational Cen-terforBiotechnologyInformation(NCBI).

2.4.2. Tandemmassspectrometry(MS/MS)ofpeptides

SDSPAGEwasperformedasdescribedbyLaemmli[24].After staining, SfL spots were excised as described by Shevchenko and collaborators [25]. Discolored spots were subjected to digestion withtrypsin (Promega, Madison, WI, USA). Digestion was performed in ammonium bicarbonate 25mM at 1:50w/w (enzyme/substrate).Thedigestionwasmaintainedat37◦_C_for₁₆_h and then was stopped with2␮L of FA2%. The peptides were extracted,separatedbyreversephasechromatographyinaC-18 column,asdescribed above,and directlyinjectedintothemass spectrometer.

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evaluatedonlinebyPROTEINBLASTfromNCBItoidentify homol-ogoussequences.

2.4.3. RapidamplificationofcDNAends(RACE)

TotalcellularRNAfromS.filiformiswasisolatedwithAxyPrep MultisourceTotalRNAMiniprepkit(AxygenBiosciences,CA,USA), accordingtothemanufacturer’sprotocol.First-strandcDNAwas synthesized from total RNA using MMLV RT (Moloney Murine LeukaemiaVirusReverseTranscriptase;Sigma-Aldrich,MO,USA) andadaptorQt(5′-CCAGTGAGCAGAGTGACGAGGACTCGAGCT

CAAGCT(T)16-3′(Sigma-Genosys,TX,USA),accordingtothe

man-ufacturer’sinstructions.

cDNAwasPCR-amplifiedusingone-tenthoftheRTreactionas atemplate.Adegenerateprimerwasdesigned(primer-SfL3′_;₅′ -GTICAGAATCARTGGGGIGG-3′₎_based_on_the_amino_acid_sequences obtainedbyMS/MS:VQNQWGG.PCRforamplificationof3′_RACE (rapidamplificationcDNA3′ _ends)_was_carried_out_by_using_Taq DNApolymerase(ThermoScientific,MA,USA).Thereaction con-sistedofatotalvolumeof25␮L,containing2␮LofcDNA,0.4␮M ofeachprimer,0.4mMofdNTPmix(ThermoScientific,MA,USA), and1Uofenzymein1XPCRBufferwith3mMofMgCl2.PCRwas

performedwithprimer-SfL3′_and_Q_i₍₅′ -GAGGACTCGAGCTCAAGC-3′_)._{Amplification}_protocol_included_an_initial_denaturation_step_at 95◦_C_for₅_min,_followed_by₃₅_cycles_of_denaturation_at₉₅◦_C_for 30s,annealingat50◦_C_for₃₀_s,_extension_at₇₂◦_C_for₁_min,_and thefinalextensionstepat72◦_C_for₅_min._Then,_the_reaction_was quenchedto4◦_C_and_analyzed_by_agarose_gel_{electrophoresis.}

ThePCRproductwaspurifiedbyillustraTM_GFX_PCR_DNA_and

GelBand Purification kit (GE Healthcare, IL,USA). Purified PCR productwascloned intopGEM-T-easy vector, transformedinto Escherichiacoli strainDH5␣(Novagen,Germany), and screened with blue-white selection in LB agar, containing 100␮gmL−1

ampicillin (Thermo Scientific, MA, USA), 0.5mM IPTG (Iso-propyl␤- D D-1-thiogalactopyranoside; Thermo Scientific, MA, USA), and 80␮gmL−1 _X-Gal _{(5-bromo-4-chloro-3-indolyl-}_␤_-d

-galactopyranoside;ThermoScientific,MA,USA).Thecloningwas performedinabiosafetylaboratorycertifiedinaccordancewith governmentalrequirements(CQB:R007-2016).Therecombinant plasmidswereextractedbyillustraTM_plasmidPrep_Mini_Spin_kit

(GEHealthcare,IL,USA)and confirmedbydigestionwithEcoRI (Promega,WI,USA).

Finally, constructions were sequenced in an automatic sequencer(MegaBACE;GEHealthcare,IL,USA),usingtheprimers T7promoter(5′_-_TAA_TAC_GAC_TCA_CTA_TAG_GG-3′₎_and_SP6₍₅′_-_ATT TAGGTGACACTATAG-3′_)._Sequencing_was_performed_with_at_least tenclonesfromPCRamplification.Thereadswereanalyzedbythe Phred-Phrap-Consedprogram[26–28].Thecontigsformedwere translatedwiththeExpasytranslationtool(http://web.expasy.org/ translate/).Aminoacidsequencealignmentswereperformedusing MultAlin[29]andESPript2.2[30].ThesequencesimilarityofSfL wasevaluatedonlinebyNCBI’sPROTEINBLAST.

2.5. StructuralanalysisofSfLisoforms

2.5.1. Proteinmodeling

StructuralpredictionofisoformsIandIIfromS.filiformislectin (SfL-1andSfL-2)wasperformedintheMODELLERprogram,suite v.9.16,usingthestructureofB.oklahomensisagglutininasthe tem-plate(BOA,PDBID4GK9)[9,31].Forstructuralmodeling ofSfL isoforms,MODELLERdefaultparameterswereused,andsequence alignmentcorrectionsweremanuallyedited.Initially,20 theoreti-calmodelsforeachisoformweregenerated,whichwereranked basedontheirDOPE(discreteoptimizedproteinenergy)scores [32].

The best modelswere then selected and analyzed for their stereochemicalproperties(Ramachandranplots, stericoverlaps,

Fig.1. Differentbranchedoligosaccharides.Oligosaccharidesselectedonthebasis ofpreviouslyreporteddataforhomologouslectins.Thecirclesrepresentmannosyl residues.

C␤deviationparameters,rotamers,andbondanglequality)with theMolProbityServer[33].Afterwards,thefinalstructureswere manuallyinspectedusingPyMolMolecularGraphicsSystem, Ver-sion1.8(Schrödinger,LLC).

2.5.2. Moleculardockingcalculations

BindingofSfLisoformstodifferentbranchedoligosaccharides wasanalyzedbymoleculardocking calculations.Toaccomplish this, seven branched oligosaccharides were selected based on previouslyreporteddataonalgallectins,whichsharethesame mannopentosecore,varyingbyextramannoseresiduesat differ-entbranches(Fig.1)[7,12].Thecarbohydratestructureswerebuilt throughthewebserverSWEETII[34,35].

CalculationswereperformedwithAutoDockVina,version1.1.2, whichappliesaniteratedlocalsearchglobaloptimizerforthe opti-mizationprocedure,wherethesuccessionofeachstepconsistsof amutationandlocaloptimization,withtheacceptancedecisions made according to the Metropolis criterion. It uses the effi-cientquasi-NewtonmethodBroyden–Fletcher–Goldfarb–Shanno (BFGS)forlocaloptimization[36].TheAutodockgraphicalinterface AutoDockTools,version1.5.6,wasusedtokeeppolarhydrogens and add partial charges to the proteins and ligands using the KollmanUnitedcharges[37].Theproteinand carbohydrate lig-andsweretreatedasrigidandflexiblemolecules,respectively.The searchspaceforthedockingcalculations,selectedbasedonthe lengthofmucinfragments,wasdefinedbya20Å×20Å×20Åcube

centredontheconservedcarbohydratebindingsites. Exhaustive-nesswassetto15,andallotherparameterswereusedasdefault. Foreachdocking,thetentop-rankedgenerationsbasedonthe pre-dictedbindingaffinity(inkilocalories permole)wereanalyzed. Thesolutionswerefirstchosenbasedonthecoordinationofthe mannopentosecoreco-crystallizedwithOAAandBOA.Themost suitableresultswerefurtherrankedbasedonthetheoretical bind-ingenergy,whichisgivenasanegativescoreinkcal/mol.

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theroot-mean-squaredeviation(RMSD)ofheavyatompositions betweenthedockedconformationandthecrystalstructurewas usedtoassesstheaccuracyofdockingpredictions.Basedon previ-ouslyreportedanalysis,wedefinedasolutionthathadamaximum RMSDvalueof2.0Åasanacceptabledockedresult[38,39].RMSD analysiswasperformedwiththeVisualMolecularDynamics(VMD) software[40].

2.6. Anticancereffect

2.6.1. Celllineandcultureconditions

Humanbreastadenocarcinomacellline(MCF-7)andprimary Human Dermal Fibroblasts (HDF) were both purchased from American Type Culture Collection (ATCC, USA). The cells were maintainedinT-25flaskscontainingDulbecco’smodifiedEagle’s medium (DMEM; Gibco®, TX, USA) supplemented with 10% of heat-inactivatedFetalBovineSerum(FBS;Gibco®

,TX,USA),1%ofl

-glutamine,100UmL−1_penicillin_and₁₀₀_␮_gmL−1_streptomycin_at

37◦_C_in_a_humidified_atmosphere_containing_5%_CO₂_._Medium_was routinelychangedevery3rdday,andcellsat90%confluencewere subculturedbytrypsinization(0.025%trypsin/0.1% EDTA).Inall experiments,cellswereusedbetweenthe3rdand14thpassage.

2.6.2. Cellviabilityassay

CellviabilitywasdeterminedusingCellTiter96®

AqueousMTS ReagentPowder(Promega,WI,USA),followingthemanufacturer´ıs instructions.Briefly,cells(2×104/200␮L/well)wereseededinto a96-well flatbottom microtiterplateinDMEMcontaining10% FBS and incubated overnight.Afterwards, the supernatant was removedandreplacedbyDMEMsupplementedwith2%FBS con-tainingdifferentconcentrationsofSfL.MTSassaywasperformed during24hforMCF-7cellsandHDF.

Fourindependentexperiments wereperformed intriplicate. Theopticaldensitywasreadat490nmonamicroplatereader.The viabilitywascalculatedas:

Cellviability (%)= _{averageOD490nm (control)}averageOD490nm (H3) ∗100

2.6.3. AnnexinVassay

CellapoptosisandnecrosiswereassessedwiththeAPCAnnexin V ApoptosisDetectionKitwith7-AADdouble staining method. Briefly,MCF-7cells(2.25×105/well)wereseededin6-wellplates

containingDMEMsupplementedwith10%(v/v)FBS andgrown for 24h. Then, cells wereharvested and incubated withSfL at 125␮gmL−1 _in_DMEM _containing_2%_FBS_for ₂₄_h. _Control_cells wereincubatedwithDMEMsupplementedwith2%FBSonly.After treatmentwithSfLintheaforementionedperiods,thecellswere detachedbytrypsinizationandthencentrifuged(206g/5min)and washedtwicewithphosphatebufferedsaline(PBS;pH7.4).The cells were resuspended in binding buffer (PBS,pH 7.4, 25mM CaCl2_),_and₅_␮_L_of_annexin_V-APC_(ImunoTools,_Germany)_and₅_␮_L

of7AAD(InvitrogenTM_,_CA,_USA)_were_added_to_each _well._Cells

wereincubatedinthedarkfor20minatroomtemperatureand thenthepro-apoptoticpotentialofSfLwasdeterminedbyflow cytometry(FCM).

2.6.4. mRNAextractionandqRT-PCR

Thequantificationofrelativegeneexpressionwasperformed byquantitativereversetranscriptase-realtimepolymerasechain reaction(qRT-PCR)aspreviouslydescribed[41].Briefly,after24h of treatmentwithSfL, MCF-7(106 _cells) _were _trypsinized,_and

totalmRNAwasextractedusingGenEluteMammaliantotalRNA Purificationkit(SigmaAldrich,MO, USA),andDNAse treatment wasperformedtoeliminateDNA(Nzytech,Lisboa,POR).Then,1␮g oftotal mRNAwasreversetranscribedusing theHighCapacity

cDNA Reverse Transcription kit (Applied Biosystems, CA,USA). qRT-PCRwasperformedina 7500FastSystem(Applied Biosys-tems, CA, USA), using TaqMan Fast Universal PCR Master Mix. PrimersandTaqManprobeswereacquiredfromApplied Biosys-tems and used following the manufacturer´ıs instructions. The analyzedgeneswereBAX(Hs00180269m1),BCL-2(Hs00608023), CASP 3(Hs00234387m1), CASP 8(Hs01018151m1), CASP 9(Hs00609647m1), and TP53(Hs01034249m1). Each reac-tionwasperformedinduplicate.Allgenes,includingendogenous controls (␤-actin and GAPDH) and treated or untreated cells, werealwaysanalyzedin thesame runtoexcludebetween-run variations.

Therelative expression for eachgene wascalculated by the 2−Ct_method_as_previously_reported_[42]_._The_{amplification}

effi-ciencyforeachprimer/probewasabove95%.

2.6.5. Statisticalanalysis

Allresultswereconfirmedbyatleastthreeindependent exper-iments.Statisticsarepresentedasthemean±SEM.Experimental

datawereanalyzedbyStudent´ıst-testandone-wayANOVA fol-lowedbyTukey´ıspost-hoc.P<0.01andP<0.05wereadoptedas thelevelofsignificance.TheIC₅₀valueswerecalculatedbyusing thesoftwareGraphPadPrism®

5tofitavariable sigmoidal-dose-responsecurve.

3. Results

3.1. PurificationSfL

Thelectinwaspurifiedbyacombinationof(NH4)2SO4

precipita-tionandion-exchangechromatographyonDEAE-SephacelColumn, asdescribedbyBenevidesandcollaborators[16].Apureproteinof relativemolecularmassof28kDawasobservedinSDSPAGE12.5% (datanotshown).

3.2. N-terminalaminoacidsequence

ThefirstfifteenaminoacidsofSfLweredeterminedbyEdman degradation.Heterogeneitieswereobservedinpositions4and5, indicatingtheexistenceofisoforms.ThrandAsnwereobserved in position 4, whereas Ala and Val were observed in position 5.Theresidue inposition13couldnotbeidentified.Therefore, theN-terminal ofSfL wasGRY(T/N)(A/V)QNQWGGSXAP.Search forsimilarityintheNCBIPROTEINBLASTshowedhighsimilarity betweenSfLandESA-2(P84331.1).

3.3. MolecularmassandaminoacidsequencingbyMS/MS

LC–MSshowedadistinctionseriesrelatedtoSfL(Fig.2).The majorionof27,553Daagreesverywellwiththeapparent molecu-larmassof28kDaobservedinSDS-PAGE.Othermolecularmasses wereobservedindeconvolutedspectra,indicatingthepresenceof isoformsinSfLpreparations.Smallvariationsaroundthese molec-ular masses were observed, suggesting the presence of adduct formations.

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Fig.2. MolecularmassdeterminationofSfL.ESI–MSdeconvolutedmassspectraofLC–MSofSfLs.Insert.Zoomofregionofmassbetween27,900and28,100onthe deconvolutedmassspectra.

Fig.3.AminoacidsequencesofSfL-1(A)andSfL-2(B).Primarystructureswere determinedbycDNAcloningsequence,N-terminaldeterminationandTandemmass spectrometryoftrypticpeptides(T).Molecularmassofpeptides,asdeterminedby massspectrometry,isinparentheses.

3.4. PrimarystructuredeterminationofSfL-1andSfL-2

ThesequencingofthePCR3′_RACE_product_revealed_a_partial aminoacidsequencefrom5to268,indicatingtwoisolectins,SfL-1 andSfL-2.TheprimarystructureofSfL-1andSfL-2wasdetermined byoverlap of sequencesobtainedby cDNA cloning,N-terminal determinationbyEdmandegradationandpeptidessequencedby MS/MS(Fig.3).

Thecalculated molecularmasses of thededucedaminoacid sequencesofSfL-1andSfL-2were27,552Daand27,985Da, respec-tively,whichwereconsistentwithions27,553Daand27,988Da, asdeterminedbyESI–MS.

The primary structure of SfL-1 and SfL-2 has four tandem-repeatdomains,consistingof67aminoacidseach.InSfL-1,the domainsshareatleast53.7%ofidentityeachother,whereasin SfL-2,identityamongdomainswasatleast44.7%(Fig.4).SfLisoforms sharedsequencesimilaritywithseverallectins(Fig.5),including OAA(P84330),PFL(WP016985694),BOA(WP010112459),MBHA

Table1

Theoreticalbindingenergy(kcal/mol)basedondockingcalculations.

Oligosaccharide SfL-1 Site SfL-2 Site

1 −10.7 I −9.8 I

1 −9.1 II −10.1 II

1 −9.9 III −9.9 III

1 −8.7 IV −10.5 IV

2 −6.5 I −4 IV

3 −8.3 I −6 IV

4 −6.9 I −5.8 IV

5 −9.5 I −7.8 IV

6 −7.4 I −7.3 IV

7 −10.5 I −9.7 IV

(WP 011556980),ESA-2(P84331),EDA-2(BAR91516)andKSA-2 (BAR91206).

3.5. Structuralmodeling

ThestructureofB.oklahomensisagglutinin(BOA,PDBID:4GK9) presentedthehighestrankingstructuralsimilaritytoSfL-1,withan identityof63%,accordingtoPSI-BLASTsearchfortemplateproteins onPDB(63%identity,74%positivity,and1%gaps).Therefore,the three-dimensionalstructureofSfLisoformsbuiltthrough compar-ativemodelingusingBOAstructureastemplatewasdeterminedto besuitableforstructuralandbindinganalysis(Fig.6).Theamino acidsequenceofBOAcrystalstructurecoversthefulllengthofSfL isoforms,exceptforthefirstfiveaminoacidsintheNterminus.

TheRamachandranplotforSfL-1showedthat100%ofresidues wereplacedinallowedregionswith98.11%onthefavoredzone. ForSfL-2,99.7%ofresidueswereplacedinallowedregionswith 98.11%onthefavoredzone,aswell.Asareference,thetemplate crystalstructureofBOAhad100%and98.0%residuesinthefavored andallowedzones,respectively.ThesedataindicatethatbothSfL modelledstructures,SfL-1andSfL-2,shouldbestableandreliable foranalyzingSfL-oligosaccharidebindingmodesthrough molecu-lardocking.

3.6. Docking

Dockingsystem(AutodockVina)suitabilityforcomplexligands, suchasoligosaccharides,wasanalyzedbasedonaredocking calcu-lationinvolvingthecrystalstructureofthecomplexbetweenBOA and␣3-␣6-mannopentaose[9].ThecalculatedRMSDbetweenthe bestdockingresultandthecrystalstructurewas1.3,whichattests tothereliabilityoftheadoptedsystemforthecurrentpurpose, despitetheconsiderableamountofrotatablebondspresentinthe usedoligosaccharides.

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Fig.4.AlignmentoftherepeateddomainsofSfL-1(A)andSfL-2(B).Blackandwhiteboxesrepresentidenticalandnon-identicalaminoacids,respectively.

Fig.5. AlignmentofSfL-1andSfL-2andotherOAAH-familymembers.TheaminoacidsequencesofSfL-1and-2werealignedwithEucheumaserraagglutinin(ESA-2), Kappaphycusstriatusagglutinin(KSA-2),Eucheumadenticulatumagglutinin(EDA-2),Myxococcusxanthusagglutinin(MBHA),BurkholderiaoklahomensisEO147agglutinin (BOA),Oscillatoriaagardhiiagglutinin(OAA)andPseudomonasfluorescensPf0-1lectin(PFL).ResiduesunderlinedareaminoacidsofthebindingsiteaccordingtoWhitley etal.[9].Blackandwhiteboxesrepresentidenticalandnon-identicalaminoacids,respectively.

Fig.6. TheoverallstructureofSfL-1(A)andSfL-2(B).I,II,IIIandIVrepresentthecarbohydrate-bindingsites.

theothersixligands.For SfL-1,site Ipresentedthebestresult, whilesiteIVwasselectedforSfL-2.Despitethepresenceof con-servedresidues,subtledifferences inthecompositionof amino acidsdirectlyinvolvedinligandcoordinationamongthesitesin eachisoformandbetweenthem,beyondavariableloop structura-tion,mayinfluencethebindingspecificityofeachsite(Fig.7).

3.7. EffectofSfLonMCF-7cellviabilityandcellapoptosis

ToassesstheeffectofSfLoncellviability,weincubatedHDFand MCF-7cellswithincreasingdosesofthelectinat24h.Asshownin Fig.8,SfLdecreasedtheviabilityofMCF-7cells.Significantlosses of43.40%,58.27%and61.20%inviabilitywereobservedatdoses of62.5,250and500␮g.mL−1_,_respectively_(*_P_<_0.05)._However,_in

HDFcells,treatmentwithSfLdidnotcausereductionofmorethan

50%ofthecellpopulation.TheviabilityofHDFcellstreatedwith SfLat125and250␮gmL−₁_was_decreased_by_only_34%_and_40.2%, respectively.At500␮gmL−1_,_a_slight_increase_in_the_population offibroblastswasobserved,suggestingthatthelectincan stimu-latecytoproliferationinlargerdoses.Afterwards,theconcentration that promoted50% inhibition of tumorcell viability(IC50)was

established,revealingthattreatmentwith125␮gmL−1 _resulted

in50%inhibitionofMCF-7viability.

InordertoevaluatetheapoptoticpotentialofSfL,MCF-7cells weredoublestainedwithannexinV-APC/7AAD.Twenty-fourhours aftertreatmentwithSfLat125␮gmL−1_,_results_showed_that_33.87%

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Fig.7. Analysisofligand7bindingtositeIonSFL-1andSFL-2.AandC–SurfacerepresentationofSfL-1andSfL-2,respectively,highlightingthebindingsitegrooveand ligandstructuration.BandD–Residuesinvolvedinthecoordinationofligand7onSfL-1andSfL-2(siteI),respectively,arerepresentedassticks,whilehydrogenbondsare presentedasdashedlines.Ligandsarerepresentedassticks.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionof thisarticle.)

Fig.8. EffectofSfLonproliferationandcellviability.Humandermalfibroblasts andMCF-7cellsweretreatedwithserialconcentrationsofSfLduring24h.The cellviability(%)wasmeasuredbyMTSassay.(n=3,Mean±SEM).(*P<0.05and** P<0.01).

3.8. EffectofSfLonpro-apoptoticgeneexpression

TheeffectofSfLontheexpressionofpro-andanti-apoptotic genesofMCF-7cellswasanalyzed24haftertreatment.The rel-ativeexpressionofCASP3,CASP8,CASP9,BCL-2,BAX,andTP53 wasevaluatedbyreal-timePCR.MCF-7cells24haftertreatment withSfLwerecomparedwithuntreatedcells.AsshowninFig.10, after24h,theanti-apoptoticgeneBcl-2wasdownexpressed. How-ever,pro-apoptoticgenes,suchasBax,underwentover-expression. Interestingly,theexpressionofCASP3,-8and-9,whichareinvolved inintrinsicandextrinsicapoptosispathways,wereoverexpressed at24h(P=0.0139).These resultsindicatethat Solieriafiliformis

lectininducedcaspase-dependentapoptosiswherecaspase-8and -9arestronglyactivated.

4. Discussion

Inthecurrentstudy,wehaveisolatedanddeterminedthe pri-marystructureoftwoisolectinsfromS.filiformis,SfL-1andSfL-2. Weperformedastructuralpredictionforbothsequencesand ana-lyzedtheiranticancereffectonMCF-7breastcancercells.

Thedeterminedsequencesofbothisoforms,SfL-1andSfL-2, presentfourtandem-repeatdomainswithapproximately67amino acids,thuspresentinghighsequenceidentitytootherlectinsfrom theOAAH-family.SfL-1showed83%,83%,82%,62%and62%of iden-titywithESA-2,KSA-2,EDA-2,MBHAandBOA,respectively.SfL-2 showed80%,79%,78%,60%and60%ofidentitywithESA-2,KSA-2, EDA-2,MBHAandBOA,respectively.SfL-1andSfL-2showed82% ofidentitywitheachother.

Moreover,structuralmodelingandthebindinganalysis demon-stratethatSfL-1andSfL-2exhibitstructuralfeaturesandbinding specificitysimilartothelectinsoftheOAAH-family,indicatingthat SfL-1and-2couldbegroupedintothislectinfamily.

SimilartoBOA,SfL-1andSfL-2presentedstructurescomposed oftwo␤-barrel-likedomainsformedbyfiveantiparallel␤-strands, whichareconnectedbyashortpeptidelinker[9].Thelectin’sability tospecificallybindto3␣,6␣mannopentoseoligosaccharideswas assessedbymoleculardocking calculations,andfavorable bind-ingenergiesweredetectedforbothlectinisoforms,SfL-1(siteI) andSfL-2(siteIV),suggestingthesamespecificitytohigh-mannose oligosaccharidesasthatpresentedbylectinsoftheOAAH-family. Theaminoacidcompositioninvolved inthecarbohydrate bind-ingofSfL-1(siteI)wasW9G10G11N17D18S94R95E123G124P125

I126.InSfL-2(siteIV),itwasW211G212G213N219P220D161 R162

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Fig.9.SfLcausedapoptosisinductioninMCF-7cells.MCF-7cellswereincubated with125␮g/mlSfLfor24h.AnnexinV-APC/7-AADdoublestainingwasperformed andanalyzedbyflowcytometry.(A)Dotblotsshowstagesofapoptosis/necrosis. R1(necroticcells),R2(lateapoptosis),R3(viablecells)andR4(apoptotic).(B) SfLinducesapoptosisinMCF-7cellswhencomparedtountreatedcells.(n=4, Mean±SEM).(*P<0.05).

Fig.10.SfLupregulatesmRNAexpressionofpro-apoptoticgenes.MCF-7cellswere treatedwithSfLataconcentrationof125␮g/mlfor24h.ThemRNAwasextracted andconvertedintocDNA.AnalysisofthemRNAlevelrelativetountreatedcellswas determinedbyreal-timePCR,usingthe2−Ct_method._(**P_<_0.01).

residuesW152G153G154,R236andE264G265P266areinvolvedinthe

structurationofcarbohydratebindinginsiteIVinBOA.Theseamino acidsarehighlyconservedbetweendomainsandprimary struc-turesoflectinsfromtheOAAH-family.TheyarepresentinSfL-1 andSfL-2,participatingdirectlyincarbohydratebinding,as sug-gestedbymoleculardockingcalculations,pointingoutthesame specificityforSfLandtheOAAH-family.

Thedifferenceintheoreticalbindingenergyamongthesites betweentheisoformsoccursasaresultofminorchangesinthe

compositionandstructureofeachsite.SitesIandIIIofSfL-1and SfL-2havethesameaminoacidcomposition;however,inSfL-2, siteIisnarrower.SitesIIandIVaresimilarinstructureand vol-ume,buttheypresentadifferentaminoacidcomposition,suchas S26andA86insiteIIofSfL-1andG26andP86insiteIIofSfL-2.On

theotherhand,siteIVpresentsD161forSfL-1andS161forSfL-2.

Forthesimulationsinvolvingtheremainingoligosaccharides, the terminal GlcNAc residues were removed in order to avoid previously detected steric hindrances. Differently, the addition ofterminalmannoseresiduesonMan-7,Man-8andMan-9was accompanied by a decreased binding energy. These resultsare corroboratedby previousdataobtained forhomologous lectins [7,9,11,43,44].

LectinsfromtheOAAH-familyareknowntopresentantiviral andanticanceractivities,asreportedintheliterature[7–9,43,45]. Regarding theanticancer effect,SfL presented significant activ-ity, inhibiting 50% of MCF-7 cell viability with a treatment of 125␮gmL−1_. _Recently, _increasing _interest _has _been _shown in marine lectins becauseof their pro-apoptotic,cytotoxic and antiproliferation effects over differentcell lines [46–49]. These lectinspresenthighspecificityforcomplexcarbohydrates. Con-sequently, their interaction with malignant, as compared to non-malignantcells,isbetterinviewofthelargenumberof glyco-proteinreceptorsontumorcellmembranes[50,51].Thus,theeffect ofSfLonnon-malignantcellsindicatesagreaterselectivityofSfL forMCF-7cells.Thisisaveryimportantpoint,sincemany anti-cancerdrugsgenerallydonotdifferentiatebetweennormaland malignantcells[52].Thepresentdatacorroboratewithpreviously reportedresultsforsimilarlectins,whichshowactivitiesagainst othercancercelllines[8,45].

Sugaharaandcollaborators[45]verifiedthatESAinducedcell death against Colo201 (Human Colon Cancer) and HeLa cells (humancervixcancer)atconcentrationsabove1.2␮gmL−1_.

Nev-ertheless,MCF-7cellsshowedrelativelyhightolerancetoESA.The deathofColo201cellswasinvestigatedbyDNAladderdetection andcaspase-3activity,indicatingthatESAisabletoinduce apo-ptosisincancercellsafter3days.Thepro-apoptoticpotentialof SfLoverMCF-7cellswasanalyzedafter24h.SfLinducedthedeath of60.23%ofthecellpopulation,betweenearlyandlate apopto-sis,althoughmostcellswerein lateapoptosis.Manyterrestrial lectinshavetheabilitytoprovokecelldeath[53].However,littleis knownabouthowmarinelectinsinducenecrosisorapoptosis.To addressthisquestion,quantitativePCRassayswereperformedto verifyifthemechanismunderlyingcelldeathinducedbySfLtakes placebyintrinsicorextrinsicpathways.TheeffectofSfLafter24h ontheexpressionofpro-andanti-apoptoticgenesinMCF-7cells showedacaspase-dependentmechanismrelyingonCASP-8and -9,whichareproteinsrelatedtobothapoptosispathways.These datacorroboratethereportthatlectinswhichspecificallybindto N-glycancarbohydratemoietiesoncancercellsmayactaspotential therapeuticagentsviaapoptosisinduction[51].

Satoetal.[8]verifiedthatPFLpresentedasignificanteffecton decreasingthecellviabilityofMKN28cells(HumanGastricCancer) post-treatmentof0.5␮M(approximately6.94␮gmL−1₎_or_higher by72h,whichwasaccompaniedbythelossofcelladhesion,inturn triggeringasignalingpathwaythatinducedanoikis-likecelldeath. Furthermore,treatmentwithlow doses(0.1–0.3uM)stimulated theproliferationofMKN28cells.

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carbo-hydrateligands[8,45].Therefore,thatfactthatSfLisalsoableto specificallybindtohigh-mannoseoligosaccharidespresentinthe MCF-7cellsstronglysuggeststhatitcanpotentiallyexertantitumor activity.However,togainabetterunderstandingofthatpotential, weneedtoundertakeindependentstudiesofeachisoform.

Acknowledgments

ThisworkwassupportedbytheBrazilianagenciesCNPq (Con-selho Nacional de Desenvolvimento Científico e Tecnológico), FUNCAP(Fundac¸ãoCearensedeApoioaoDesenvolvimento Cientí-ficoeTecnológico)andFINEP(FinanciadoradeEstudoseProjetos). TheauthorsaregratefultoProfessorDavidMartinforhelpingwith textediting.A.H.S., C.S.N.,and E.H.T.aresenior investigators of CNPq.

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