Structural characterization of coagulant Moringa oleifera Lectin and its effect on hemostatic parameters

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ContentslistsavailableatSciVerseScienceDirect

International

Journal

of

Biological

Macromolecules

j ou rn a l h o m e pa g 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

Structural

characterization

of

coagulant

Moringa

oleifera

Lectin

and

its

effect

on

hemostatic

parameters

Luciana

de

Andrade

Luz

a,1

_,

_Mariana

_Cristina

_Cabral

_Silva

b,2

_,

_Rodrigo

_da

_Silva

_Ferreira

b,2

_,

Lucimeire

Aparecida

Santana

b,2

_,

_Rosemeire

_Aparecida

_Silva-Lucca

c,3

_,

_Reinhard

_Mentele

d

_,

Maria

Luiza

Vilela

Oliva

b,2

_,

_Patricia

_Maria

_Guedes

_Paiva

a,1

_,

Luana

Cassandra

Breitenbach

Barroso

Coelho

a,∗

a_Departamento_de_Bioquímica,_Universidade_Federal_de_Pernambuco,_Av._Prof._Moraes_Rego_s/n,_50670-901_Recife,_PE,_Brazil

b_Departamento_de_Bioquímica,_Universidade_Federal_de_São_Paulo,_Rua_Três_de_Maio,_100,_04044-020_São_Paulo,_SP,_Brazil

c_Centro_de_Engenharias_e_Ciências_Exatas,_Universidade_Estadual_do_Oeste_do_Paraná,_Rua_da_Faculdade,_Jardim_La_Salle,_85903-000_Toledo,_PR,_Brazil

d_Institute_of_Clinical_{Neuroimmunology,}_LMU,_Munich,_Germany_and_Department_for_Protein_Analytics,_Max_Planck_Institute_for_{Biochemistry,}

Martinsried,Germany

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20February2013

Receivedinrevisedform12March2013 Accepted14March2013

Available online 26 March 2013

Keywords:

CoagulantMoringaoleiferalectin Primarysequence

Circulardichroism

AnticoagulantlectinandHemostatic parameters

a

b

s

t

r

a

c

t

Lectinsarecarbohydraterecognitionproteins.cMoL,acoagulantMoringaoleiferaLectin,wasisolated fromseedsoftheplant.Structuralstudiesrevealedaheat-stableandpHresistantproteinwith101 aminoacids,11.67theoreticalpIand81%similaritywithaM.oleiferaflocculentprotein.Secondary structurecontentwasestimatedas46%␣-helix,12%␤-sheets,17%␤-turnsand25%unorderedstructures belongingtothe␣/␤tertiarystructureclass.cMoLsignificantlyprolongedthetimerequiredforblood coagulation,activatedpartialthromboplastin(aPTT)andprothrombintimes(PT),butwasnotsoeffective inprolongingaPTTinasialofetuinpresence.cMoLactedasananticoagulantproteinoninvitroblood coagulationparametersandatleastonaPTT,thelectininteractedthroughthecarbohydraterecognition domain.

1. Introduction

Lectinsareagroupofproteinswhichrecognizeandbind mono-andoligosaccharides[1,2].Theycanbindtocarbohydratemoieties onthesurfaceoferythrocytesandagglutinatethecells,without altering the carbohydrate structure [3]. Lectins exist in viruses andallformsoflife,howeverthebestknownareextractedfrom plants,especiallyseeds, organofstock,whichisa majorsource toobtainthesemolecules[4].Hundredsoflectinshavebeen puri-fiedandtheirsugarspecificitiesidentified,whichhasenabledtheir developmentintopowerfultoolsforthepurification,separation, andstructuralanalysisofglicoproteins[5],aswellasrecognition

∗Correspondingauthor.Tel.:+558121268540; fax:+558121268541/8121268576.

E-mailaddresses:lucianaluz16@yahoo.com.br(L.d.A.Luz),

roseslucca@hotmail.com(R.A.Silva-Lucca),mentele@biochem.mpg.de(R.Mentele), olivaml.bioq@epm.br(M.L.V.Oliva),lcbbcoelho@gmail.com,

luanacassandra@terra.com.br(L.C.B.B.Coelho). 1 _Tel.:₊₅₅_8121268540;_fax:₊₅₅_8121268576.

2 _Tel.:₊₅₅₁₁_55764445;_fax:₊₅₅₁₁_55723006.

3 _Tel.:₊₅₅₄₅₃₂₂₀_3000;_fax:₊₅₅₄₅₃₃₂₄_4590.

moleculesinsidecells,oncellsurfaces,andinphysiologicalfluids [6].Theyhaveshowninhibitoryactivityagainstfungiandbacteria [7,8],insects[9],viruses[10]andcytotoxiceffectsagainsttumor cellslines[11].

Physical-chemistrycharacterizationoflectinsisimportantto explainthefunctionindifferentbiologicalprocesses[12]. Struc-tural biology of macromolecules searches products potentially usefulforsolvingbiochemicalproblemsandeventuallydevelops new therapeutical agents [13]. To study structures of macro-molecule,spectroscopictechniquessuchascirculardichroism(CD) isused,whereopticallyactivesubstanceswillabsorbdifferentlyleft andrightcircularlypolarizedlight,andthedifferenceinabsorption ofcomponentsismeasured.Themethodhasbeenextensivelyused tounravelsecondarystructureofproteinsgivinginformationon theeffectofaddedligands[14].Proteinswithhigh␣-helical con-tentshowsCDspectruminthefarUVregionastwonegativeCD bandsaround208–210nmand222–228nmaswellasonepositive bandnear190–195nm[15].

Moringa oleifera,known ashorseradishor drumstick tree,is widelyfoundthroughoutIndia,Asia,somepartsofAfricaand Amer-ica,belongingtotheMoringaceaefamily[16,17].Itsconstituents suchas leaf,flower,fruit and barkhavebeen anecdotallyused

http://dx.doi.org/10.1016/j.ijbiomac.2013.03.044

Open access under the Elsevier OA license.

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32 58 (2013) 31–36

asherbalmedicinesintreatmentsforinflammation,paralysisand hypertension[18].Theseedscontainedibleoilsandwater solu-blesubstances(coagulantproteins)thatcanbeusedindrinking waterclarification[19–22]orfortreatmentofselecteddyeing efflu-ents[23].Lectinswithcoagulant propertieswerepurified from M.oleifera seeds. Santos et al. [24] purified and partially char-acterizedcoagulantM.oleiferalectin,cMoL,thefirstlectinwith coagulantpropertiesforcontaminantsin water.cMoLis abasic protein,activeatpHrange4.0–9.0andcomplexsugarspecificity whichrecognizedmainlytheglycoproteinsazocaseinand asialofe-tuin.WSMoL, Water-SolubleM.oleiferaLectin,wasdetectedby Santosetal.[25]asanacidglycoprotein,withhigher hemagglu-tinationactivityatpH4.5,recognizingmainlyfructoseandporcine thyroglobulin.Ferreiraetal.[26]referredthatthislectinhas coag-ulantactivityandisanaturalcoagulantforcontaminantsinwater, reducingturbidityandbacterialproliferation.WSMoLandcMoL showedinsecticidalactivityagainstAedesaegypti[27]andAnagasta kuehniella[28],respectively.Katreetal.[29]reportedthepresence ofaM.oleiferalectin,MoL,ahomodimerwithmolecularmassof 14kDaandsubunits(7.1kDa)linkedbydisulfidebond(s).MoLis alsoaglycoproteinwithhighstabilityandagglutinateshumanas wellasrabbiterythrocytes.

cMoLisaproteinwithimportantbiologicalactivities[24,28], howeveritsstructuralcharacterizationwasnotcompletely elu-cidated. In this article, we report the primary structure, CD characterizationandforthefirsttimetheanticoagulant proper-tiesofacoagulantlectinfromM.oleiferaoninvitrohemostatic parametersofhumanbloodcoagulation.

2. Materialsandmethods

2.1. IsolationofcoagulantM.oleiferalectin(cMoL)

M.oleiferaseedswerecollectedonthecampusofUniversidade Federalde Pernambuco, in Recifecity, Northeast of Brazil.The extract,fractionandlectinwerepreparedaccordingtoSantosetal. [24],withmodifications.Seedsweregroundtoflour,whichwas extractedwith0.15MNaClatroomtemperature(25◦_C)_for₆_h, andasalineextractwasobtained.Proteinswereprecipitatedwith ammoniumsulphate(60%)atroomtemperaturefor4h;the frac-tionobtainedaftercentrifugation(12,000×gfor20minat4◦_C)_was dialyzedagainstwaterand0.15MNaCl.Thefractionwasapplied (10mgofprotein)onaguargelcolumn(10cm×1.0cm)previously equilibrated(20mL/hflowrate)with0.1MNaCl.cMoLwaseluted withasalinegradientof0.15,0.3,0.5and1MNaCl.UVabsorbance wasusedtomonitorsamples.cMoLactivefractionselutedwith 0.3MNaClwerepooled,analyzedbyHPLCandusedinthe exper-iments.cMoLproteinconcentrationwasevaluatedaccordingto Lowryetal.[30]usingbovineserumalbuminasstandardatarange of0–500␮g/mLandabsorbanceat720nm.

2.2. Reversed-phaseHPLC

cMoLwassubjectedtoreverse-phasecolumnC4onHPLC sys-tem(Shimadzu)forpurityanalysis.Thecolumnwasequilibrated withsolventA[0.1%(v/v)trifluoroaceticacid(TFA)inH2O] and elutedusingsolventB(90%acetonitrilein0.1%TFA)inalinear gra-dient,whereB=5%whent=0min,B=5%att=5min,B=100%at t=60min,B=0%whent=65min.Theelutionprofilewasmonitored at215and280nm.

2.3. Hemagglutinationactivity(HA)

Glutaraldehyde-treatedrabbiterythrocytes wereobtainedas describedbyBing,Weyand,andStavinsky[31].Thelectin(50␮L) was serially two-fold diluted in microtiter V-plates containing

0.15M NaCl beforeaddition of 50␮L 2.5% (v/v) suspension of treated rabbit erythrocytes. The results were read after about 45minwhenthecontrol,containingonlyerythrocytesfully pre-cipitated,appearedasadotatthebottomofthewell.HA(inverse ofthetiter)wasdefinedasthehighestsampledilutionshowingfull hemagglutination[4,32].

2.4. Primarysequencedetermination

Edman degradation [33] was performed with an automatic gas-phase sequencer (492cLC; Applied Biosystems) using condi-tionsrecommended by the manufacturer. Samples(0.8mg/mL) forsequencingwerereducedin200␮Lof0.25MTris–HClbuffer, pH8.5 containing 6Mguanidine–HCl,1mMEDTA and 5mg of DTT,andalkylatedwithiodacetamide[34].Then,theproteinwas separatedby reversed phasecromatography HPLC.The similar-ityofsequenceswassearchedusingtheBLASTproteinsequence database[35]andthesequenceswerealignedwiththeMULTALIN program[36].TheoreticalpIwascalculatedbyExPASyProtParam toolthroughtheprimarysequenceoftheprotein.

2.5. Spectroscopicmeasurements

CD data were recorded on a Jasco J-810 spectropolarime-ter. Samples were placed in a 0.5mm path length circu-lar quartz cuvette. Lectin concentration was 0.2mg/mL in phosphate–borate–acetate(PBA)bufferformeasurementsinthe farUVregion(250–190nm),asanaverageof8scans.Datawere expressedintermsofmeanresidueellipticity[].Thesecondary structureestimativewascalculatedbydeconvolutingtheCD spec-trum using the CDPro software package, which contains three CDanalysisprograms,CONTINLL,SELCON3andCDSSTR[37].The threeprogramswereusedwithareferenceproteinset consist-ingof56proteins,thusincreasingthereliabilityofdeconvolution. Resultswereexpressedasameanofthethreeprograms.CDPro packagealsocontainstheClusterprogramthatwasusedto deter-mine the tertiary structure class of cMoL [38]. To study the pH effect on cMoL, the protein (0.2mg/mL) was incubated in phosphate–borate–acetatebuffer(PBA),10mM,for10min,atpH valuesof2.0,4.0,6.0,7.0,8.0,10and12.Thetemperatureeffecton cMoLsecondarystructurewasalsoanalyzed.Proteinsampleswere heatedat40,60,80and100◦_C_for₃₀_min_and_at₁₀₀◦_C_for₁_h._CD measurementswererecordedasdescribedabove.

2.6. Determinationofactivatedpartialthromboplastintime– aPTTandprothrombintime–PT

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58 (2013) 31–36 33

3independentprotocols(±s.e.m).Allexperimentswereapproved bytheEthicsCommitteeoftheUniversidadeFederaldeSãoPaulo, numberCEP1793/11,accordingtoBrazilianfederallaw.

2.7. Statisticalanalysis

Differencesbetweenmeansvalueswereanalyzedusing one-way ANOVA followed by Tukey’s multi-comparison test in the coagulationassays.Apvalue<0.05wasconsideredsignificant.

3. Resultsanddiscussion

3.1. StructuralanalysisofcMoL

cMoLisacoagulantM.oleiferalectin,purifiedbySantosetal. [24] after saline extraction and guar gel column chromatogra-phy.Thecoagulantpropertyofthislectinisrelatedtoreduction of contaminants in water; cMoL promoted turbidity reduction ofapproximately92%(inrelationtonegativecontrol)similarto thepositivecontrolaluminumsulphateonwaterwithhighand lowturbidityofkaolinclay[24].Byhemagglutinationassays,the proteinis resistanttochangein pH,thermostable, agglutinates erythrocytesfromrabbitand humanblood types[24]and have insecticidalactivity[28].Byanewandsimpleprotocol,Santosetal. [24]purifiedandpartlycharacterizedalectindifferentfromthose alreadyreported,WSMoL [25]and MoL[29].The knowledgeof thestructureandphysicochemicalpropertiesofcMoLisneeded tounderstandthemechanismsofactioninvolvedinthe biologi-calactivitiesdevelopedbythelectinandthusintegratethestudy ofstructure-functionoftheprotein.Reversephase chromatogra-phywasmadetoassessthepurityofthefractionsobtainedinguar gelcolumn.Fig.1showsthecMoLprofileobtainedbyHPLC.The peakrepresentscMoLfractionsfromguargelcolummelutedwith 0.3MNaCl.Thedetectionofasinglepeakshowshomogeneityinthe purification,sothissteppurifiedefficientlythelectin,biologically active.

Highlypurefractionsofproteinsareimportantforthe perfor-manceofaminoacidsequenceandcirculardichroismanalysis.In thisstudy,thecompletesequenceofcMoLwasobtainedand com-paredwithotherproteinsequencesoftheNCBI-BLASTdatabank; thesearchindicatedsimilaritywiththesequenceofM.O2.1,a floc-culentactiveproteinfromM.oleifera[19].Thesesequenceswere alignedusingMULTALINprogram(Fig.2).Thesequencerevealed thatcMoLisaproteinwith101aminoacids,twochains,andhas 81%ofsimilaritywithM.O2.1,whichpresents6.5kDaandisoelectric pointabovepH10[19].ItisimportanttonotethatcMoLhaveeight cysteineresidues,whichmaybeinvolvedinthedisulfidebonds.

Fig.1.cMoLprofilebyreversephasechromatographyusingVYDACC4columnin aHPLCsystem.Fractionselutedwith0.3MNaClonanaffinityguargelcolumn wereassessedforhomogeneityevaluation.Absorbancewasperformedat280nm. Theproteinfractionwaselutedusingalineargradient:solventB(90%acetonitrile in0.1%TFA),whereB=5%whent=0min,B=5%att=5min,B=100%att=60min, B=0%whent=65min.

cMoLisabasicproteinwithatheoreticalpIof11.67indicatinga strongpositivechargeonthesurfaceandconfirmingitscationic nature.Highcontentsofglutamine(26.7%),alanine(6.9%),proline (6.9%)and17positivelychargedaminoacids(16argininesand2 histidines)arealsopresentinthelectinstructure.Severalprotein familieswithflocculent/coagulantactivityarepresentinM.oleifera seeds[19,39].cMoLmustdevelopitscoagulantactivityinwater duetointeractionsamongcharges,asproposedtoMO2.1[19,20] andnotthroughbindingtocarbohydraterecognitionsite.Further studiesonthegenomeandproteomeofM.oleiferaseedscould con-tributetounravelthefunctionofproductsfromauniquegene(s) intheplant[24].

ThenativecMoLmolecularweightobtainedbySantosetal.[24] throughSephacrylS-300sizeexclusionchromatographyrevealed a single peak correspondingto an apparentmolecularmass of 30,000Da; however, the molecular weight estimated to cMoL throughtheprimarysequencerevealedthattheproteinshowed 11,928Da.TheseresultssuggestedthatcMoLisatrimerconsisting ofthreesubunitsof11,928Da.cMoLisnotaglycosylatedprotein, whichwasconfirmedthroughSchiff’sreagentstainingbySantos etal.[24].

AnalysesofCDspectrumshowsatypicalshapeandfeaturesofa mainly␣-helicalsecondarystructure,i.e.,twonegativebandsat222 and209nm,andapositivebandat192nm(Fig.3).Thesecondary structurecontentofcMoLwasestimatedat46%␣-helix,12%␤ -sheets,17%␤-turnsand25%unorderedstructureswithrootmean

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34 58 (2013) 31–36

Fig.3. CDspectrumofcMoLin10mMPBA,pH7.0.Measurementswererecorded asanaverageof8scansforproteinsolutionsof0.2mg/mL,at25◦_C._CD_spectrum

deconvolutionindicated46%␣-helix,12%␤-sheet,17%␤-turn,25%unordered struc-ture.

squaredeviation(RMS)lowerthan2%forallstructures.The Clus-teranalysisshowedthatcMoLbelongstothe␣/␤tertiarystructure class,corroboratedbyresultsofsecondary structureestimative. For␣/␤proteins,the222nmbandgenerallyhasahigherintensity than208–210nmband,whereasforthe␣+␤proteinsthereverse isobserved[38].Thecontentof␣-helicesestimatedforcMoLwas similartothatdeterminedforMO,thecoagulantproteinpurified byNdabigengesereandNarasiah[40].KwaambwaandMaikokera [41]reportedthatthisproteinpresentssecondarystructural com-ponentsof58%␣-helix,10%␤-sheetand33%unorderedstructures, withRMSlowerthan4%forallstructures.ButdifferentfromMoL, analpha–betalectin,isolatedbyKatreetal.[29]whichcontaining 28%␣-helix,23%␤-sheet,20%turnand28%ofunordered struc-ture.ThesecondarystructurecontentofcMoLandMoLrevealed thattheselectinshavedifferentcontentofsecondarystructures butconsistofamixtureofalphaandbetastructures.

Generally,thefunctionalstabilityisaccompaniedbystructural stability.Fig.4ashowsCDspectraofcMoLinpHvaluesof2.0,7.0 and12.Thesecondarystructureoftheproteindidnotchangein acidicandalkalineconditionsasshownintheCDspectra, corrobo-ratingwiththedataonthestabilityofbiologicalactivityinfunction ofpH.ThecontentofeachsecondarystructureofcMoLat differ-entpHrangeis showninTable1.Nosignificantchangedueto pHwasobservedintheestimatedsecondarystructuresfromCD spectra.SamplespectrafromotherpHvalueswereomittedsince

Table1

cMoLsecondarystructureestimatedfromCDspectraatdifferentpHvalues.

pH ␣-Helix(%) ␤-Sheet(%) ␤-Turn(%) Unordered(%)

2.0 43 13 18 26

7.0 46 12 17 25

12.0 39 15 19 27

QuantitativepredictionswereperformedusingCDProsoftware.

Table2

cMoLsecondarystructureestimatedfromCDspectraatdifferenttemperatures.

Temperatures ␣-Helix(%) ␤-Sheet(%) ␤-Turn(%) Unordered(%)

40◦_C ₅₁ ₉ ₁₆ ₂₅

60◦_C ₅₁ ₈ ₁₆ ₂₅

80◦_C ₄₂ ₁₀ ₂₁ ₂₇

100◦_C₍₃₀_min) ₁₈ ₂₅ ₂₃ ₃₃ 100◦_C₍₁_h) ₇ ₂₆ ₂₇ ₄₀

QuantitativepredictionswereperformedusingCDProsoftware.

smallchangesweredetected.cMoLandMoLhaveasacommon fea-tureactivityinacidicandalkalineconditions[24,29].Otherlectin withsimilarpHstabilityisachitin-bindinglectinfromrhizomes ofSetcreaseapurpurea(SPL).SPLisahomotetramericproteinwith hemagglutinatingactivitystableinpHrangeof2.0–9.0[42].

Treating cMoL at 100◦_C _and _subsequent _evaluation _by _HA revealedthelectinasathermostableprotein[24],howeverby accu-rateCDanalysis,theproteinconformationismaintainedonlyuntil 80◦_C_for₃₀_min₍_Fig.₄_b)._When_cMoL_was_subjected_to_heating_at 80◦_C,_change_in_␣_-helix_content_was_verified,_followed_by_a_gentle increaseof␤-structureswhileunorderedstructuresdidnotalter. TheestimatedunorderedstructurecontentincreasedwhencMoL washeatedat100◦_C_for₃₀_min_and₁_h₍_Table₂_)._High_temperature isapowerfuldenaturingagentleadingtoproteinunfoldingthrough breakingofhydrogenbondsthatmaintainproteinstructure[43]. ThermostabilityisacommonfeatureofproteinsfromM.oleifera seeds.MOCP,acoagulantproteinisolatedbyGhebremichaeletal. [21]remainedactiveafter5hheattreatmentat95◦_C._Katre_et_al.

[29]alsoreportedthatMoLisaheat-stableprotein,whichretains theactivityevenafterincubatingat85◦_C_up_to₃₀_min_at_pH_7.2.

3.2. cMoLeffectonhemostaticparameters

TheinfluenceofcMoLonblood coagulationwasdetermined byactivatedpartialthromboplastintime(aPTT)andprothrombin time(PT)(Fig.5).Thepositivecontrolusedintheassayswasplasma ofhealthyvolunteerdonorswithnormalvaluesforclottingtimes. Avarietyofnewanticoagulantsarebeingdeveloped andtested

Fig.4. (a)CDspectraofcMoL(0.2mg/mL),in10mMPBAbuffer,atpH2.0(),7.0(䊉)and12()and(b)cMoLspectraafterheatingat40(),60(䊉),80()and100◦_C_for

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58 (2013) 31–36 35

Fig.5. InvitroeffectofcMoLinhemostaticparameters.Activatedpartial thrombo-plastintime(aPTT,a)andprothrombintime(PT,b)weredetermined.Thestatistical significancewasevaluatedusingone-wayANOVA,followedbyTukey’stest. A p-value<0.05wasconsideredtoindicatesignificance.p-Value<0.05(*)and p-Value<0.001(**).R:ratioofsamplecoagulationtimewithcontrolcoagulationtime. Datarepresentmeans±s.e.m.,n=3.

toinhibitthevariousstepsin thecoagulationcascade[44].The determinationofaPTTisparticularlyusefulinmonitoringtheeffect ofheparinanddeterminingdeficienciesoffactorsVIII,IX,XIandXII. TPreflectstheactivityoffactorII(prothrombin),V,VIIandX,whose deficiencyisaccompaniedbyaprolongationoftimerequiredfor clotformation[45].

cMoLsignificantlyprolongedaPTT(tomorethan300s)andPT (Fig.5b)coagulationtime,attheassayedconcentrations(3.0,15,30, 37.5,45and60␮g/mL)butthemostaffectedparameterwasaPTT (Fig.5a).ProlongationofaPTTsuggestsinhibitionoftheintrinsic pathwayand/orcommonbloodcoagulation,whileprolongationof PTsuggestsinhibitionoftheextrinsicpathway[46].Theintrinsic andextrinsicpathwaysconvergeintheformationoffactorXa[47]. Otherlectinsalsosignificantly prolongsbothcoagulationtimes. Cratyliamollisseedlectin(Cramoll1,4),amannose/glucose bind-inglectin,promotesalmosttwo-foldincreaseofcoagulationtimes [48].Bauhiniaforficatalectin,BfL,increasedonlyaPTTcoagulation timebutthiseffectwasnotrelatedtohumanplasmakallikreinor humanfactorXainhibition[49].ThuscMoLshowedan anticoagu-lantactivity,sinceindeterminingtheR,theratiobetweensample coagulationtimeandcontrolcoagulationtime[50]washigherthan 1.0.

Significant correlation between recognition and binding of lectinstocarbohydratesorglycoconjugatesoncellsurfaceorfree inbiologicalfluidsareinvolvedinmanylectineffects,suchas activ-ityagainstinsects,viruses,bacteriaandcytotoxicity[27,42,51,52]. Then, to investigate cMoL interaction with coagulationfactors, blood coagulation assays were performed in the presence of asialofetuin. This glycoprotein blocks the site of carbohydrate recognitionofthelectin.aPTTassaysshowedareduction inthe

Fig.6.BloodcoagulationassaysinthepresenceofcMoLinhibitedbyasialofetuin (0.5mg/mL).Lectinwaspreviouslyincubated(15min)withasialofetuin.(a) Acti-vatedpartialthromboplastintime(aPTT);(b)prothrombintime(PT).Thestatistical significancewasevaluatedusingone-wayANOVA,followedbyTukey’stest.A p-value<0.05wasconsideredtoindicatesignificance.p<0.05(*)andp<0.001(**) representcomparativeanalysisbetweencMoLinhibitedbyasialofetuinand con-trols.StatisticalsignificanceofcMoLinthepresenceandabsenceofglycoprotein wasalsoshowed.R:ratioofsamplecoagulationtimewithcontrolcoagulationtime. Datarepresentmeans±s.e.m.,n=3.

timerequiredforbloodcoagulationinthepresenceoftheinhibited lectinbyglycoproteininthesameassayedconcentrations(Fig.6a), whilePTwasnotaffected(Fig.6b).TheseresultssuggestthatcMoL interactswiththecarbohydrateportionofserineproteasesofthe coagulationcascadefromintrinsicpathway.NodecreaseinPTin thepresenceofinhibitedlectinsuggeststhatthelectinshouldact onthefactorsoftheextrinsicpathwayand/orunderthefactorXa byadifferentdomainfromthecarbohydraterecognition.

4. Conclusions

InthisarticlestructuralcharacteristicsofcMoLweredescribed; also,forthefirsttime,theanticoagulantactivitywasdetectedon humanbloodcoagulationprocessbyacoagulantM.oleiferaseed lectin.Structuralanalysisrevealed cMoLcommonfeatures with anothercoagulantproteinfromM.oleifera.cMoLinteractionsin bloodcoagulationmustoccuratleastpartially,bythecarbohydrate recognitiondomain.cMoListheonlycurrentlydescribedprotein fromM.oleiferaseedsthatexhibitsanticoagulantactivity.

Authorcontributions

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36 58 (2013) 31–36

Acknowledgements

TheauthorsexpresstheirgratitudetotheConselhoNacional deDesenvolvimentoCientíficoeTecnológico(CNPq)forresearch grantsandfellowship(PMGP,MLVOandLCBBC).Also,theFundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE),theCoordenaçãodeAperfeiçoamentodePessoaldeNível Superior(CAPES)andtheFundacãodeAmparoàPesquisadoEstado deSãoPaulo(FAPESP)areacknowledgedforfinancialsupport.

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