B-N-Acetilhexosaminidase involvment in a-conglutin mobilization in Lupinus albus

(1)

ContentslistsavailableatSciVerseScienceDirect

Journal

of

Plant

Physiology

jou rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p l p h

Biochemistry

␤-N-Acetylhexosaminidase

involvement

in

␣-conglutin

mobilization

in

Lupinus

albus

Cláudia

N. Santos

a,b

_,

_Marta

_Alves

a

_,

_António

_Oliveira

a

_,

_Ricardo

_B.

_Ferreira

a,c,∗ a_Instituto_de_Tecnologia_Química_e_Biológica,_Universidade_Nova_de_Lisboa,_Apartado_127,_2781-901_Oeiras,_Portugal

b_Instituto_de_Biologia_Experimental_e_{Tecnológica,}_Apartado_12,_2781-901_Oeiras,_Portugal c_Instituto_Superior_de_Agronomia,_Universidade_Técnica_de_Lisboa,_1349-017_Lisboa,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8November2012

Receivedinrevisedform26February2013 Accepted7March2013

Available online 18 April 2013 Keywords:

␤-N-Acetylhexosaminidase ␣-Conglutin

Lupinusalbus

a

b

s

t

r

a

c

t

Glycosylationisanimportantpost-translationalmodiﬁcationinvolvedinthemodulationofawidevariety ofcellularprocesses.Becauseglycosydasesarecentral,theaimofthisstudywastoinvestigatetheglycosyl activitypresentinthecotyledonsoftheseedsofanimportantcroplegume,Lupinusalbus,aswellas potentialnaturalsubstratesofthedetectedenzymes.

Theglycosylactivitydetectedinthecotyledonsbeginningatseedimbibitionandcontinuinguntil9days after,wasduetoa␤-N-acetylhexosaminidase(␤-NAHase),whichwasmolecularlyandbiochemically characterizedafterpurification.Twoisoenzymeswithmolecularmassesof64and61kDaweredetected, eachhavingfiveisoenzymeswithpIs5.3–5.6.The64and61kDaisoenzymeshadthesameproteincore showingdifferentdegreesofglycosylation.TheN-terminalsequenceoftheenzymeproteincorewas determined[VDSEDLI(EN)AFKIYVEDDNEHLQGSVD]andtoourknowledge,isthefirstreportedprotein sequencefromaplant␤-NAHase.

L.albus␤-NAHase hadKm valuesof2.59mMand2.94mMandVvaluesof18.40␮Mmin−1 and

2.73␮Mmin−1,forpNP–GlcNAcandpNP–GalNAc,anoptimumpHof5.0and4.0andtemperatureof 50◦Cand60◦CweredetectedtowardpNP–GlcNAcandpNP–GalNAc.InthepresenceofAgNO3,CoCl2,

CuSO4,FeCl3,CdCl2andZnCl2theenzymaticactivitydecreasedmorethan50%,andwheninthepresence

ofsugars,anactivityreductionofnomorethan25%wasobserved.

Aphysiologicalrolefor␤-NAHaseinL.albusstorageproteinmobilizationwasinvestigated.␤-NAHase hasalreadybeenimplicatedinseveralbiologicalprocesses,namelyinglycoproteinprocessingduring seedgerminationandseedlinggrowth.However,thenaturalsubstratesusedbythisenzymearenotyet completelyclariﬁed.

Bygatheringinvivoandinvitrodatafor␤-NAHaseactivitytogetherwithglobulindegradation,we suggestthatL.albus␤-NAHaseisinvolvedinthemobilizationofstorageproteindegradation,with ␣-conglutinbeingapotentialnaturalsubstrateforthisenzyme.

Introduction

Glycosylation,ineukaryotes,isanessentialprocessinvolvedina

numberofcellularprocesses,includingcorrectproteinfoldingand

Abbreviations: DMS, dimethylsuberimidate; DW, dry weight; FW, fresh weight; ␤-ME, ␤-mercaptoethanol; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine;␤-NAHase,␤-N-acetylhexosaminidase;pNP,p-nitrophenyl; pNP–GalNAc, p-nitrophenyl–␤-d-N-acetylgalactosamine; pNP–GlcNAc, p-nitrophenyl–␤-d-N-acetylglucosamine; pNP–(GlcNAc)2, p-nitrophenyl–␤-d-N,N -diacetylchitobiose; pNP–(GlcNAc)3, p-nitrophenyl–␤-d-N,N,N -triacetyl-chitotriose;pNP–Glcp,p-nitrophenyl–␣-d-glucopyranosyde.

∗ Correspondingauthorat:InstitutodeTecnologiaQuímicaeBiológica, Universi-dadeNovadeLisboa,Apartado127,2781-901Oeiras,Portugal.

Tel.:+351214469651;fax:+351214433644. E-mailaddress:rbferreira@itqb.unl.pt(R.B.Ferreira).

proteinprotectionagainstproteolyticdegradation(Bernard,2008;

Aebietal.,2009).Inparticular,N-acetylglucosamine(GlcNAc),an

abundanthexose,isknowntoplaymultiplerolesincellseitheras

amonomeroraspartofmacromolecules,especiallyasaresidueof

oligo-andpolysaccharidesandconjugatedwithlipidsorproteins

(Bernard,2008).

A typical glycoside hydrolase involved in the cleavage of

terminal GlcNAc, and alsoGalNAc, fromthe non-reducing end

ofoligosaccharides is␤-N-acetylhexosaminidase (␤-NAHase; EC

3.2.1.52). This enzyme has been shown to be universally

dis-tributedamongmosttypes oflivingorganisms and,in contrast

to the wellstudied mammalian, insect and fungal ␤-NAHases,

onlylimitedinformationisavailableonthecorrespondingplant

enzymes(Slamovaetal.,2010).

Threeputative␤-NAHasesencodedbytheArabidopsisthaliana

genomewereclonedandanalyzedwithrespecttotheirenzymatic

(2)

properties(Liebmingeretal.,2011;Strasseretal.,2007).Sincethese

enzymeswerefoundtobelocatedindifferentsubcellular

compart-ments,theywerehypothesizedtobeinvolvedindifferentcellular

processes(Strasseretal.,2007).

Inhigherplants,␤-NAHasesareinvolved intheformationof

paucimannosidicN-glycans,whicharegeneratedinlatestagesof

theN-glycosylationpathway,bytheremovalofGlcNActerminal

residuesofglycoproteins.PaucimannosidicN-glycansconstitute

themajorityofglycanspresentonvacuolarglycoproteins,suchas

beanphaseolin(Sturmetal.,1987)andoccurinsmalleramountson

extracellularplantglycoproteins(Dirnbergeretal.,2001;Takahashi

etal.,1986)andcellwallboundproteins(Kotakeetal.,2001).The

presenceoflargeamountsoftruncatedpaucimannosidicN-glycans

inplantglycoproteinsindicatesthattheremovalofterminal

Glc-NAc residues from N-glycans by ␤-NAHase, which presumably

takes place in a post-Golgi compartment like vacuoles, plasma

membrane,cellwallorapoplast(Strasser,2009),haveanimportant

roleinplantmetabolism.

Inadditiontotheposttranslationaltrimmingof

oligosaccha-ridechainsofstorageproteinsandlectinsdepositedintheprotein

bodiesduringseeddevelopment(VitaleandChrispeels,1984),

␤-NAHasehasbeenimplicatedinother biologicalprocesses,such

asglycoproteinprocessingandturnoverduringseedgermination

(HarrisandChrispeels,1975;Poultonetal.,1985).Togetherwith

otherglycosydases,␤-NAHasewasproposedtobeinvolvedin

N-glycanmetabolismduringfruitripening(JagadeeshandPrabha,

2002;Jagadeeshetal.,2004;YongandGross,1994),softeningof

climactericand non-climacteric fruits(Meli etal., 2010; Ghosh

etal.,2011)andprocessingofN-glycanspresentinsecretory

glyco-proteinsofvacuolesandplasmamembrane(Strasseretal.,2007).

Withendochitinase,␤-NAHasewassuggestedtoactintheplant

defensesystemagainstfungalpathogens,bydegradingchitin,or

byinhibitingthesporegerminationandmycelialgrowth(Hodge

etal.,1995;Boletal.,1990;Broekaertetal.,1988;Schlumbaum etal.,1986).

However,theexactphysiologicalrolesof␤-NAHaseinplant

N-glycanmetabolism,aswellasthenaturalsubstratesusedbythe

enzyme,arenotcompletelyclariﬁed.

Inordertocontributetothisarea,the␤-NAHasepuriﬁedfromL.

albusseedswasmolecularlyandbiochemicallycharacterized.

Fur-thermore,thisenzymeactivitywasscreenedduringL.albusseed

germinationandseedlinggrowthinthedifferentplantletorgans,

andthepuriﬁedenzymeincubatedwithoneofthemainstorage

proteinsfromL.albusseeds,␣-conglutin,toevaluatewhetherthis

enzymecanusethisstorageproteinassubstrate.

Materialsandmethods

Chemicals

N-Acetylgalactosamine (GalNAc), N-acetylglucosamine

(GlcNAc), N-acetylglucosamine–asparagine (GlcNAc–Asn),

␤-N-acetylhexosaminidase (␤-NAHase), 4-chloro-1-naphtol,

Concanavalin A (ConA), 3-(cyclohexylamino)-1-propanesulfonic

acid (CAPS), dichitobiose, p-dimethylaminobenzaldehyde,

dimethylsuberimidate(DMS), fucose(Fuc), galactosamine (Gal),

glucosamine(Glc),lactose(Lac),␣-d-mannopyranoside(␣-Man),

4-methylumbelliferyl-N-acetyl-␤-d-glucosamine, p-nitrophenyl

(pNP), p-nitrophenyl–␤-d-N-acetylgalactosamine (pNP–GalNAc),

p-nitrophenyl–␤-d-N-acetylglucosamine (pNP–GlcNAc),

p-nitrophenyl–␤-d-N,N_{-diacetylchitobiose} _{(pNP–(GlcNAc)}₂_),

p-nitrophenyl–␤-d-N,N_,N_{-triacetylchitotriose} _{(pNP–(GlcNAc)}₃_),

triﬂuoromethanesulfonic acid (TFMSA) were purchased from

Sigma(St.Louis,MO).ConcanavalinASepharose,MonoSHR5/50,

Superose12HR10/300,MonoQHR5/50,Superose6HR10/300,

PD-10 and ampholytes (pI 3.5–10.0) were obtained from GE

HealthcareBio-SciencesAB(Uppsala,Sweden).Allotherchemicals

wereofreagentgradeorpuregrade.

Plantmaterial

Dryseedsofwhitelupin(LupinusalbusL.cv.Mizak)were

sur-facesterilizedwith1%(w/v)HgCl2and0.02%(w/v)Tween-20for

15minandextensivelywashedwithsterilizedbi-distilledwater.

Germinationwasinitiatedbyseedimmersioninrunningtapwater

for2days,andseedlinggrowthcontinuedinpotsﬁlledwitha

mix-tureofsandandpeat(1:1)for7moredays.Inthegerminatedseeds,

thecoatswereremovedandtheintactcotyledonsdissectedfrom

theembryosandaxesandthenstoredat−80◦_C_until_required.

Enzymepuriﬁcation

Cotyledonswereground[13mL(gFW)−1]withanaqueous

solu-tion(10mMCaCl2 and10mMMgCl2)at pH8.0and stirredfor

4h.Afterpassingthroughtwolayersofcheesecloth,theextract

wascentrifugedat30,000×gfor1h.Theresultingpelletwasused

fortotalglobulinextractionandthesupernatantfortotalalbumin

extraction.

Forglobulinextraction,thepelletwassuspendedbystirringit

inasolutioncontaining10%(w/v)NaCl,10mMEDTAand10mM

EGTA[13mL(gFW)−1]for12h.Thesuspensionwascentrifugedfor

1hat30,000×gandtheresultingglobulinsolutionconcentrated

byammoniumsulphate(561gL−1)precipitation.Theprecipitated

globulins werecentrifuged at 30,000×g for 20minand

resus-pendedin20mM Tris–HClatpH7.5 andstored at−20◦_C_until

furtheruse.

Thesupernatant,correspondingtothealbuminfraction, was

subjectedto30–70%(w/v)ammoniumsulphateprecipitation.The

pelletobtainedaftertheprecipitationwasresuspended [0.5mL

(gFW)−1]inabuffersolutionatpH5.0(50mMNaCH3CO2,1mM

CaCl2,1mMMgCl2,0.5MNaCland10mMGlcNAc).Allofthesteps

describedwerecarriedoutat4◦C(Santosetal.,2012;Francoetal.,

1997).

Thesupernatant collectedfromthealbuminfraction, aftera

centrifugationof13,800×gfor15min,wassubjectedtoafﬁnity

chromatographyonaConA-Sepharosecolumn(elutedwith0.5M

␣-Maninabuffersolutionof50mMNaCH3CO2atpH5.0)atroom

temperature.Fractionscontaining␤-NAHaseactivitywerepooled

andappliedtoaMonoScolumninaFPLCsystem(GEHealthcare,

Uppsala,Sweden)andelutedwithanincreasinggradientofNaCl

(upto1MNaClusing thesamebuffersolution).Fractions

con-taininghigher␤-NAHaseactivitywerepooledanddesaltedbygel

ﬁltrationinaPD-10columnelutedwith50mMTris–HClatpH7.5.

Theresultingfractionsweresubjectedtoanionicchromatography

withaMonoQcolumnelutedwithincreasinggradientofNaCl(up

to1MNaClin50mMTris–HClatpH7.5).Unlessstatedotherwise,

allstepswerecarriedoutat10◦C.Throughoutthepuriﬁcation

pro-cedure,theenzymeactivitywasmonitoredbythereleaseofpNP

frompNP–GlcNAcasdescribedbelow.Fractionshaving␤-NAHase

activitywerestoredat4◦Cuntilfurtheruse.

Proteindetermination

TheproteinconcentrationwasdeterminedaccordingtoLowry’s

method (Lowry et al., 1951) using bovine serum albumin as

standard. Electrophoresis

SDS–PAGEcarriedoutbythemethodofLaemmli(1970)was

(3)

Tstacking gels.Sampleswereredissolved inSDS–PAGE loading

buffercontaining80mMTris–HClpH6.8,2%(w/v)SDS,0.1M

␤-mercaptoethanol(␤-ME),15%(v/v)glycerol,0.01%(w/v)m-cresol

purple.Thegelsweresilverstained(Blumetal.,1987)orstained

withCALRED(Hongetal.,1993)andscannedusingtheImageQuant

v3.3densitometer(MolecularDynamics,USA).

Determinationofnativemolecularmass

A Superose 12 HR 10/300 column (Amersham Pharmacia

BiotechAB,Uppsala,Sweden)wasequilibratedwith0.1MTris–HCl

atpH7.5usingaﬂowrateof0.5mLmin−1tocalculatethenative

molecularmassofpuriﬁed␤-NAHase.Acalibrationcurvewas

per-formedwithproteinstandards.

Enzymaticassayforˇ-NAHaseactivity

Enzymereactionswereperformedusingadequatediluted

sam-ples (1–30␮L) with 70␮L of a 2mM pNP–GlcNAc in a buffer

solutionof50mMNaCH3CO2,pH5.0at25◦Cfor30minandthe

reactionwasterminatedbyadding200␮Lof1MNa2CO3(Chang

etal.,1998).Thereactionwasmonitoredcolorimetricallyby

mea-suringtheabsorbanceat405nmandoneunitofenzymeactivity

(U)wasdeﬁnedastheamountofenzymethatliberated1␮molof

pNPperhourat25◦C.

Substratespeciﬁcity

The enzymatic activity of ␤-NAHase for hydrolysis of other

syntheticsubstrates,wasdeterminedaspreviouslydescribedfor

pNP–GlcNAc.InadditiontoexaminingpNP–GalNAc,theactivity

towardpNP–(GlcNAc)2andpNP–(GlcNAc)3wasalsoassessedusing

2mMofsubstrate.

Enzymekineticstudies

TheMichaelisconstant(Km)andvelocity(V)forpNP–GlcNAc

andpNP–GalNAchydrolysisweredeterminedatsubstrate

concen-trationsrangingfrom0.25to15mM.TheKmandVof␤-NAHasefor

bothsubstrateswerecalculatedusingtheMichaelis–Menten

equa-tionwithHyperv1.1w(CopyrightJSEasterby,Liverpool,UK)and

conﬁrmedbyEZ-FitEnzymeKineticsv5.03(PerrellaScientiﬁcInc.,

Amherst,USA)software.

DeterminationofoptimumpHandstability

ForoptimumpHandstabilitystudies,puriﬁedfractionsof

␤-NAHasewereused.TheoptimumpHwasassayedfrom2to12,

usingBrittonuniversalbufferduringactivitymeasurement(Britton

andRobinson,1931).ToassesspHstabilitytheenzymewas

pre-incubatedinuniversalbufferatdifferentpHfrompH2.6to12.4,

at 25◦C for 30min and the remaining enzymatic activity was

determinedatpH5.0.Theenzymatic assayswereperformedas

previouslydescribed,usingbothsyntheticsubstratespNP–GlcNAc

andpNP–GalNAc.

Determinationofoptimumtemperatureandstability

For theoptimum temperature and stability studies, puriﬁed

fractionsof␤-NAHasewereused.Theoptimumtemperaturewas

assayedatpH5.0 from0 to100◦C. For theevaluation of

ther-malstability,theenzymesolutionswerepre-incubatedatdifferent

temperatures,from0to100◦C,for30minandtheremaining

enzy-maticactivitydeterminedat25◦Caspreviouslydescribed,using

bothsyntheticsubstratespNP–GlcNAcandpNP–GalNAc.

Effectsofpotentialeffectors

Theeffectsofsalts(AgNO3,CaCl2,CdCl2,CoCl2,CuSO4,FeCl3,

KI, MgCl2, MnSO4,Na2MoO4, NiCl2 and ZnCl2), chelating agent

(EDTAand EGTA)and carbohydrates(Fuc, Gal,Glc,Lac,␣-Man,

GalNAc and GlcNAc) wereevaluatedby measuringtheenzyme

activityafteranincubationof30mininabuffersolutionof50mM

Tris–HClatpH7.5containing0.1mM,1.0mMor10mMofeach

compound.Theenzymatic assayswereperformedaspreviously

describedusingpNP–GlcNAcassubstrate.Theenzymeactivitywas

measuredandexpressedastherelative activitypercentage

cal-culatedfromtheratioofactivityof␤-NAHasetreatedtothatof

␤-NAHaseuntreated.

ˇ-NAHasechemicaldeglycosylation

The puriﬁed ␤-NAHase was treated with TFMSA(Tams and

Welinder, 1995). Brieﬂy, anhydrous TFMSA was added to the

lyophilizedproteinsampleand incubatedat−10◦_C_for₅_min_in

aN2-richatmosphere.Thereactionwasneutralizedwithpyridine.

Thedeglycosylated␤-NAHasewasprecipitatedin4volumesofcold

acetonefor2hat−20◦_C._The_enzyme_was_recovered_after

centrifu-gation(13,800×gat4◦Cfor10min)andthepelletresuspendedin

SDS–PAGEloadingbuffer.

A glycoprotein detection assay was performed after

chemi-caldeglycosylation.Forthatpurpose,proteinswereseparatedby

SDS–PAGE,electrotransferredontoPVDFmembrane(Amersham

PharmaciaBiotechAB,Uppsala,Sweden)for1hat70Vand4◦C

anddetectedasdescribedbyFayeandChrispeels(1985).Brieﬂy,the

membranewasblockedinTTBS,asolutionofTBS(20mMTris–HCl,

0.5MNaClatpH7.5)containing0.1%(w/v)Tween-20,for1h.The

membranewasincubatedatroomtemperaturewithConA(30␮g

mL−1)inTTBS+Sbuffer(1mMMgC12,1mMCaCl2andTTBS)for

1hfollowedby4washing steps,10mineach,inTTBS+S.

Incu-bationwith50␮gmL−1horseradishperoxidaseinTTBS+Sfor1h

wasfollowedby4washingstepsinTTBS+Sand1inTBS+S.The

membranewasstainedbyincubationwith0.03%(v/v)H2O2and

0.5mgmL−14-chloro-1-naphtolforthecolordevelopingreaction

andphotographed.

Subunitcross-linkingreaction

Tostudythesubunitstructure,across-linkingmethod

accord-ing toDavies and Stark (1970)was used.The protein fraction,

previouslydesaltedinto100mMTris–HClatpH8.5, wasadded

toDMShydrochloridesolution(2:1)dissolvedinthesamebuffer.

Thereactionmixture,incubatedatroomtemperaturefor2h,was

precipitatedin4volumesofcoldacetonefor2hat−20◦_C._After

centrifugation(13,800×gat4◦Cfor10min),thepelletwas

resus-pendedintheSDS–PAGE loadingbuffersolution.Sampleswere

analyzedbySDS–PAGEorsubjectedtogelﬁltration

chromatog-raphyonaSepharose6columnwith100mMTris–HClatpH7.5

usingaﬂowrateof0.5mLmin−1.

DeterminationoftheN-terminalaminoacidsequence

The puriﬁed ␤-NAHase fractionobtainedfrom theSuperose

12 columnwasadsorbedonaProsorbcartridgepriortothe

N-terminal amino acid sequencing, which was performed on an

Applied Biosystems 477A sequencer (Applied Biosystems,

Fos-ter City,USA). Sequence data were compared withtheprotein

databasesusing theBLAST tool fromUniProtdatabase(October

(4)

Phylogeneticanalysis

A Multiple sequence alignment, using the ClustalW tool

fromGenomeNet(http://www.genome.jp/tools/clustalw)was

per-formedforalltheentriesfoundintheUniProtdatabase(October

2012,http://www.uniprot.org,Jainetal.,2009)forViridiplantae

N-acetylhexosaminidase(SupplementalTable1)andtheN-terminal

aminoacidsequenceobtainedfortheL.albus␤-NAHase(UniProt

databaseaccessionnumber:B3EWR5).

Supplementarymaterial related tothis article found, in the

onlineversion,athttp://dx.doi.org/10.1016/j.jplph.2013.03.009.

ˇ-NAHasetwo-dimensionalelectrophoresis

L.albus puriﬁed␤-NAHasefractionswereevaluatedby

two-dimensionalelectrophoresis(2-DE).Forisoelectricfocusing(IEF)

electrophoresis,theIPGphorsystem(AmershamBiosciences,

Upp-sala,Sweden)wasusedwithanon-linearpHgradientgelof3–10

(IPGstrips,GE,Uppsala,Sweden)loadedwith35␮gofprotein

resol-ubilizedin7Murea,2Mthiourea,0.4%(v/v)tritonX-100,4%(w/v)

CHAPS,60mMDTTand1%(v/v)IPGbuffer3–10NL(GE,Uppsala,

Sweden).TheIEFwascarriedoutat30Vfor12h,followedby200V

for1h,500Vfor1.5h,1000Vfor1.5h,and8000Vfor3h,at20◦C.

PriortoSDS–PAGE,theIPGstripswereequilibratedfor2×15minin

abuffersolutioncontaining50mMTris–HClpH8.8,6Murea,30%

(v/v)glyceroland2%(w/v)SDS.SixtymillimolarsDTTwereadded

totheﬁrstequilibrationstepand135mMiodoacetamidetothe

secondone.TheSDS–PAGEwasperformedonslabgels(Laemmli,

1970).Thegelsweresilverstained(Blumetal.,1987)andscanned

using theImageQuant v3.3 densitometer (Molecular Dynamics,

USA).Afterimaging,thegelswereanalyzedbyImageMaster2D

Platinumsoftwarev5.0(AmershamBiosciences,Uppsala,Sweden).

Puriﬁcationof˛-conglutin

L.albusseedglobulinswerepuriﬁedfrom2daysgerminating

cotyledonsas described above.Samplescontaining theisolated

globulinsweredesaltedat4◦ConaPD-10column(GE,Uppsala,

Sweden)previouslyequilibratedin0.05MTris–HClbufferatpH

7.5.AsampleofdesaltedglobulinswasloadedintoaMonoQHR5/5

column(GE,Uppsala,Sweden),previouslyequilibratedinthesame

buffer.Thebound␣-conglutinwaselutedwithacontinuous,

lin-eargradientbetween0.4and0.45MNaCl(Meloetal.,1994;Franco

etal.,1997).

Turbiditymeasurements

Theturbiditymeasurementsofthe␣-conglutinself-aggregation

weremadeaccordingtoanadaptedprocedurebasedontheone

describedbyOkuboetal.(1976).Brieﬂy,thepuriﬁed_{␣-conglutin}

fractionwas resolubilized in 0.05M Tris–HCl buffer, pH 6.5 in

a 1mL silica cuvette containing 0.5mg of proteinmL−1, 5mM

MgCl2 plus5mMCaCl2 and30ngofpuriﬁed␤-NAHase.

Turbid-itymeasurementsweremadespectrophotometricallyat590nm

inaBeckmanDU-70spectrophotometer(Beckman,Inc.,Huenden,

Germany)afterincubationperiodsof0,15and30minat25◦C.

N-Acetylglucosaminerelease

Fivemicrograms␣-conglutinwereincubatedwith30ngof

␤-NAHasepuriﬁedfractionfor1hat25◦C.TheGlcNAcreleasedwas

determinedbythemodiﬁedElson–Morganmethod(Reissigetal.,

1955).ThespeciﬁcityofthemethodwastestedforGlcNAc,GalNAc,

(Glc)2NAcandGlcNAc-Asn.

Statisticalanalysis

Datawerecomparedbyone-wayanalysisofvariance(ANOVA)

followedbytheFisherLSDmethodwhensigniﬁcantdifferences

werefound.Independentbiologicaltriplicateswereanalyzed.

Results

Glycosidaseactivity

TheglycosidaseactivitypresentinL.albuswasinvestigatedin

thecotyledons ofgerminatingseeds.For thatpurpose,thetotal

proteinextractofthecotyledonswasassayedwiththree

differ-entsubstrates:pNP–GlcNAc,pNP–␣-ManandpNP–Glcp.Because

pNP–GlcNAcwastheonlysubstrateactivelyhydrolyzed,the

subse-quentstudiesonglycosidaseactivitywerefocusedonthedetection

of␤-NAHase.

ˇ-NAHasepuriﬁcation

ThetotalproteinextractofL.albusseedcotyledonswasfurther

separatedintoglobulinandalbuminfractions.Because␤-NAHase

activitywasmainlydetectedinalbumins,thisproteinfractionwas

usedforfurtherenzymepuriﬁcation.Afterammoniumsulphate

precipitation,theproteinfractionobtainedwassubjectedto

afﬁn-itychromatographywithConAsepharose(Fig.1a)andtheactivity

ofthecollectedfractionmonitoredwiththepNP–GlcNAc.The

frac-tionswiththehigherenzymaticactivityweregatheredandapplied

inaMonoSHR5/50columnforcationexchangechromatography

forfurtherpuriﬁcation(Fig.1b).Again,thefractionsshowingthe

highestglycosidaseactivitywerecollectedandappliedinaMono

QHR5/50columnforanionexchangechromatographytoobtaina

sharppeakofenzymeactivity(Fig.1c).

Thisenzymeactivitywasstablethroughoutallthepuriﬁcation

stepsandwasfoundtobestableafterlyophilizationorifkeptin

solutioneitherat4◦Corat−20◦_C_for_several_days._The_resulting

puriﬁcationwas14.6-fold,witha speciﬁcactivityof153Umg−1

proteinandarecoveryyieldof1.3%(Table1).

ASDS–PAGEforthedifferentfractionscollected(albumin

frac-tion,fractionprecipitatedwith30–70%of ammoniumsulphate,

ConA-Sepharosecolumn,MonoScolumnandMonoQcolumn)was

performed(Fig.2).

MolecularcharacterizationofL.albusˇ-NAHase

PuriﬁcationbygelﬁltrationchromatographywithSuperose12

(Fig. 3a)showeda peak withmolecularmass ofapproximately

153kDaandtheSDS–PAGEofthisfraction(Fig.3b)showedtwo

polypeptideswithapproximatemolecularmassesof64and61kDa.

ThesameSDS–PAGEproﬁlefortheenzymewasfoundunder

reduc-ing (with ␤-ME) and non-reducing (without ␤-ME) conditions

(datanotshown).EnzymefractionstreatedwithDMS,acovalent

cross-linkingagenttoprotein–proteininteractions,showedno

dif-ferencestotheuntreatedfractionswhenanalyzedbygelﬁltration

chromatography(Superose6)orSDS–PAGE(datanotshown).

A2-DEapproachwascarriedoutwiththepuriﬁedfractionof

theenzymeforfurthercharacterization.TenisoenzymeswithpI’s

between5.3and5.6weredetected(Fig.4),correspondingﬁveof

them(a1–5)tothe 64kDapolypeptides and ﬁveother(b1–5)to

61kDapolypeptides.

Thedeglycosylationof␤-NAHasewasperformedwithTFMSA

and theabsenceof glycidicresidueswas conﬁrmedin a

mem-brane by the ConA/Peroxidase method (Fig. 5a, lane 2). Both

electrophoretic patterns, from native _␤-NAHase (Fig. 5b, lane

1) and deglycosylated enzyme (Fig. 5b, lanes 2 and 3) were

(5)

Table1

Puriﬁcationof␤-NAHasefromthecotyledonsofLupinusalbusseeds.

Step Totalactivity(U) Speciﬁcactivity(Umg−1₎ _{Puriﬁcation}_(-fold) _Recovery_(%)

Totalalbumins 60,800 10.5 1.0 100

Ammoniumsulphateprecipitation(30–70%) 18,200 6.0 0.57 30

ConA-Sepharose 1380 36 3.4 2.3

MonoSHR5/50 790 52 5.0 1.3

MonoQHR5/50 810 153 14.6 1.3

Fig.1. Chromatographicseparationof␤-NAHasefromthecotyledonsofLupinus albusseeds.Chromatographicprofileoftheammoniumsulphatefractionpurified by(a)aConA–Sepharosecolumn.Thefractionsselectedfromtheprevious chro-matographyashavingthehighestenzymeactivitywerefurtherpurifiedby(b) cationexchangechromatography(MonoSHR5/50)and(c)anionexchange chro-matography(MonoQHR5/50).Theblacklinecorrespondstotheabsorptionprofile at280nm,thegreylinetotheNaClgradient,andthedashedlinetothe␤-NAHase activitydetectedforpNP–GlcNAc.Thefractionsselectedforthesubsequentanalysis aremarked( ).

The N-terminal amino acidsequence for L. albus ␤-NAHase

obtained was: VDSEDLI(EN)AFKIYVEDDNEHLQGSVD (UniProt

databaseaccession number:B3EWR5).No signiﬁcanthomology

foroursequence withthoseannotatedinUniProtdatabasewas

foundwhenasimilarproteinsequencesearch(BLAST)wascarried

out. Thus, a multiplesequence alignment with the94 insilico

translationproteinentriesannotatedinUniProtdataset

(Supple-mentalTable1)wasperformedwithClustalWtool(Supplemental

Fig.1),revealingthatL.albus␤-NAHaseisembeddedinacluster

comprising Ricinus communis(B9T146), Zea mays (B6TP85) and

Vitisvinifera(F6GYZ3andF6H7W3).

Supplementarymaterial related tothis article found, in the

onlineversion,athttp://dx.doi.org/10.1016/j.jplph.2013.03.009.

Fig.2.ProteinelectrophoreticproﬁlebySDS–PAGEofthedifferentproteinpuriﬁed fractionsofLupinusalbusseedcotyledons.Thegellanesrepresent(1,7) molecu-larweightmarkers,(2)thetotalalbuminfraction,(3)thefractionprecipitatedwith 30–70%ammoniumsulphateandthefractionselutedfrom(4)ConA–Sepharose col-umn,(5)cationexchangechromatography(MonoSHR5/50)and(6)anionexchange chromatography(MonoQHR5/50).Thegelwasloadedwith20␮gofproteinper laneandwasstainedwithCALRED.

BiochemicalcharacterizationoftheL.albusˇ-NAHase

Several biochemical parameters for L. albus ␤-NAHase were

characterized:apparentMichaelisconstant(Km),velocity(V),

opti-mumpH,pHstability,optimumtemperature,temperaturestability

andeffectorsinﬂuenceonenzymaticactivity.

The enzyme was inactive toward pNP–(GlcNAc)2 and

pNP–(GlcNAc)3, evenwhen usingdifferentconcentrations (data

notshown)butwasabletoreleasepNPfrombothpNP–GlcNAc

andpNP–GalNAc(Table2).TheKmandVvaluesforpNP–GlcNAc

andpNP–GalNAcaresummarizedin Table2.L.albus␤-NAHase

showedanoptimumpHof5.0with50%oftheactivityatpH3.0

and6.5towardpNP–GlcNAcandanoptimumpHof4.0with50%

of theactivityat pH2.5 and 5.5 toward pNP–GalNAc(Fig.6a).

␤-NAHase kept90% or more of themaximum enzyme activity

frompH6.5to8.0toward pNP–GlcNAcandfrompH6.0to7.5

towardpNP–GalNAc(Fig.6b).

L. albus _␤-NAHase optimum temperature proﬁles toward

pNP–GlcNAcandpNP–GalNAcwerequitesimilar,withtheenzyme

optimum temperature of 50◦C toward pNP–GlcNAc and 60◦C

toward pNP–GalNAc, with the enzyme activity higher than

50% between 35◦C and 70◦C. Temperature stability toward

Table2

Kinetic parameters of the Lupinus albus ␤-NAHase toward 2mM p-nitrophenyl–␤-d-N-acetylglucosamine (pNP–GlcNAc) and p-nitrophenyl–␤-d-N-acetylgalactosamine(pNP–GalNAc).

Substrate Km(mM) V(␮Mmin−1)

pNP–GalNAc 2.94±1.16 2.73±0.42

(6)

Fig.3.Chromatographicpurificationof␤-NAHaseresultingfromanionexchange chromatography(MonoQHR5/50)andSDS–PAGEelectrophoreticprofileofthe purifiedfraction.Chromatographicprofileofthefractionpurifiedby(a)Superose 12usingasmolecularstandards(a)ferritin(440kDa),(b)catalase(232kDa),(c) aldolase(158kDa),(d)alcoholdehydrogenase(150kDa),(e)malatedehydrogenase (73kDa),(f)trypsin(23.8kDa)and(g)cytochromec(12.4kDa),and(b)theresulting SDS–PAGEprofile(lane2)loadedwith5␮gofprotein;molecularweightmarkers areinlanes1and3andthegelwassilverstained.

Fig.4. Two-dimensionalelectrophoreticproﬁleforthe64and61kDaisoenzymes ofL.albus␤-NAHase.Thegelcorrespondsto35␮gofproteinloadedintheIEFand theSDS–PAGEwassilverstained.

pNP–GlcNAcandpNP–GalNAcassays revealedthat␤-NAHaseis

stableunder30◦C.

TheactivityproﬁleofL.albus␤-NAHaseinthepresenceof

var-iousconcentrations(0.1,1.0and10mM)ofpotentialeffectorsand

chelatingagentswasstudiedbymeasuringtheenzymeactivity

towardpNP–GlcNAc (Table 3).No effects wereobserved inthe

enzymeactivityduetothepresenceof0.1mMofCoCl2,MnSO4

andNa2MoO4andhigherconcentrationsofNa2MoO4(10mM).The

othersaltstestedreducedtheenzymeactivity,whichweremore

dramaticwithincreasedsaltconcentrations(Table3).Ofthose,the

onlysaltthatinducedadecreaseofmorethan50%oftheenzyme

activitywasAgNO3at0.1mM.Withincreasingconcentrationsof

thosecations(10mM),areductionofmorethan50%oftheenzyme

Fig.5. PuriﬁedL.albus␤-NAHaseelectrophoreticproﬁleafterchemical deglycosyla-tion.Detectionof(a)theglycidicresiduesinmembrane,usingtheConA/peroxidase method,innative␤-NAHase(lane1),deglycosilated␤-NAHase(lane2)and oval-bumin(lane3);(b)SDS–PAGEofnative␤-NAHase(lane1)anddeglycosilated ␤-NAHase,1and5minafterthechemicalreaction,respectively(lanes2and3). Fortheglycidicdetectioninthemembrane5␮gofproteinperlanewereusedand forthegel2.5␮gofproteinperlane;thegelwassilverstained.

Rela

ti

ve

acti

v

it

y (

%)

120

100

80

60

40

20

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13

pH

Rela

ti

ve

acti

v

it

y (

%)

120

100

80

60

40

20

0

1

2

3

4

5

6

7

8

9

10 11 12

13 pH

a

b

Fig.6. OptimumpHandpHstabilityofLupinusalbus␤-NAHaseusingpNP–GlcNAc and pNP–GalNAc as substrates: (a) optimal pH and (b) pH stability for p-nitrophenyl–␤-d-N-acetylglucosamine(pNP–GlcNAc,)and p-nitrophenyl–␤-d-N-acetylgalactosamine(pNP–GalNAc, ).Verticalbarsrepresent±SD.

activitywasalsoobservedforCoCl2,CuSO4andFeCl3withahuge

reductioninenzymeactivityobservedforCdCl2andZnCl2.By

con-trast,MgCl2 wastheonly saltthat produceda slightactivation

of_␤-NAHasewhenpresentinlowconcentrations(0.1mM).

How-ever,increasingconcentrationsofthiscompound(10mM)slightly

(7)

Table3

EffectontheactivityofLupinusalbus␤-NAHasetoward p-nitrophenyl–␤-d-N-acetylglucosamine(pNP–GlcNAc)inthepresenceofdifferentsaltsandsugars. Signiﬁcantdifferentvaluesa_relatively_to_the_control_are_marked_as_***_for_P_<_0.001,

**forP<0.01and*forP<0.05. Relativeactivity(%) 0.1mM 1.0mM 10mM Cations AgNO3 48.8±2.5*** 46.31±0.80*** 38.77±0.79*** CaCl2 90.7±2.0** 86.3±4.4*** 77.58±0.54*** CdCl2 82.8±2.3*** 71.3±2.7*** 10.20±0.32*** CoCl2 98.9±4.4 81.8±4.7*** 42.6±1.4*** CuSO4 82.8±1.9*** 75.6±2.8*** 38.4±2.0*** FeCl3 93.8±3.3 77.5±4.5*** 17.5±1.3*** KI 76.33±0.32*** 82.0±2.4*** 82.7±1.5*** MgCl2 104.9±1.4* 99.9±1.8 86.97±0.47*** MnSO4 96.7±2.7 87.6±1.9*** 82.3±2.4*** Na2MoO4 98.4±2.4 101.1±1.2 100.1±2.3 NiCl2 84.10±0.32*** 75.3±2.4*** 62.6±1.6*** ZnCl2 79.1±5.3*** 52.9±1.8*** 3.22±0.12*** Sugars Fuc 85.4±6.3** 83.96±0.93*** 91.6±2.3* Gal 97.2±1.7 98.6±2.7 93.4±2.3 Glc 84.1±2.4** 87.2±4.5* 95.0±8.3 Lac 81.2±3.5*** 82.1±2.9*** 77.8±2.1*** ␣-Man 81.5±5.9*** 82.6±4.7*** 87.6±2.4** GalNAc 101.4±3.1 96.9±3.4 81.78±0.78*** GlcNAc 103.8±4.0 106.78±0.74 99.2±4.1

a_Data_were_compared_by_one-way_analysis_of_variance_followed_by_Fisher_LSD

methodwhensigniﬁcantdifferenceswerefound.

Theinﬂuenceofseveralcarbohydrates(Fuc,Gal,Glc, Lac,

␣-Man, GalNAcand GlcNAc) wasalsoevaluatedin theactivityof

theenzyme.Thepresenceofdifferentsugarconcentrations

pro-ducednoeffect,asobservedforGlcNAc,GalNAcandGal,orsmall

decreasesinenzymeactivity,withactivityreductionsnomorethan

25%.

Potentialphysiologicalsubstratesforˇ-NAHase

Inanattempttofurtherelucidatethephysiologicalrole ofL.

albus␤-NAHase,theprogression ofits enzymeactivityin early

plant development was examined. No enzymatic activity was

detectedinL.albusleaves.Inthehypocotyls, ␤-NAHaseactivity

ranged from 0.3 to 1.2Umg−1 DW 5–9 days after seed

imbi-bition and in the roots from 0.2 to 0.4Umg−1 DW 3–9 days

afterseed imbibition. In thecotyledons, theenzymatic activity

was foundto ranges from 0.5Umg−1 DW in the dry seedsto

3.5Umg−1DW9daysafterseedimbibition.Bygatheringthe

pre-viouslyknownSDS–PAGE proﬁleof globulindegradation (0–11

daysafterseedimbibiton,Ferreiraetal.,1995)withtheobserved

␤-NAHase activityproﬁlein the cotyledons, anincrease in the

enzymeactivitywasdetected5daysafterseedimbibition(Fig.7a).

Thisincreasewascoincidentwiththeperiodinwhichthe

degra-dation of these reserve proteinsis more accentuated (Fig.7b).

Thisobservation leadsus to investigatethe possibility of

stor-age proteinsbeing ␤-NAHase substrates.For that purpose, the

well-describedaggregation–disaggregationbehaviorofglobulins

(Ferreiraet al.,1999,2003a),proposedtoparticipateinstorage

proteinmobilization(Santosetal.,2012),wasusedtoevaluate

␤-NAHaseinterference.Inthepresenceofincreasingconcentrations

ofCaCl2andMgCl2globulinsareabletoself-aggregate.However,

thisbehaviorcanbereversedwithevenhigherCaCl2 andMgCl2

concentrations.Thisaggregation–disaggregationbehavior,which

isdescribedtobeelectrostaticinnature,isdependentuponthe

storageproteinexternalenvironment,meaningthatalterationsin

thisproteinpropertycanresultinalteredglobulinself-aggregation.

␣-Conglutin,oneofthemajor,glycosylatedL.albusseed

glob-ulins (Ferreira et al., 2003b) was tested for this behavior. By

Fig.7. Evaluationof␤-NAHaseactivityandglobulinprofileduringLupinusalbus development.Profileof(a)␤-NAHaseactivitydetectedinthecotyledonsofL.albus duringseedgerminationandseedlinggrowth;thedaysthataremarkedcorrespond tothetimeperiodwherethe(b)globulinelectrophoreticprofilewasevaluated whicharefromday5untilday8afterseedimbibitionloadedwith15␮gofprotein perlane,molecularweightmarkersareinlane5;thegelwassilverstained.

promoting␣-conglutin self-aggregationprior totheadditionof

␤-NAHase,noalterationintheglobulinaggregateswasobserved

(Fig.8).However,analterationin␣-conglutinself-aggregationwas

observedwhentheglobulinwasincubatedwith␤-NAHasepriorto

thepromotionofaggregation(Fig.8).

The ability of ␤-NAHase to excise the exogenous sugar

residues fromindividualized ␣-conglutin wasevaluated by the

Morgan–Elsonmethod,whichresultedina releaseofGlcNAcof

3.8±1.8␮gGlcNAch−1.ThismethodologywastestedforGlcNAc,

GalNAc,(Glc)2NAcandGlcNAc-Asn,andwasspeciﬁcforGlcNAc

andGalNAc.

Discussion

A_␤-NAHaseactivitywasdetectedinbothdryseedsand

germi-natingseedsofL.albuscotyledonsasthemainglycosidaseactivity.

TheSDS–PAGEofthesuccessivepuriﬁedfractionsallowedusto

visualizethesimpliﬁcationoftheproteinpatternsthatculminated

in two polypeptides by the end of this procedure (Fig. 2).The

puriﬁedenzymewasthenmolecularlyandbiochemically

charac-terized.

MolecularcharacterizationofL.albusˇ-NAHase

Themolecularmassofthenativeenzymewasestimatedbygel

(8)

Fig. 8.Monitoring of ␣-conglutin self-aggregation, when in the presence of ␤-NAHase,byturbiditymeasurements.Themeasurementsweremadefor com-binationsof5␮g␣-conglutin,cations(5mMofCaCl2plus5mMMgCl2)and30ng ␤-NAHase.Verticalbarsrepresent±SDandletterssigniﬁcantdifferentvaluesfor P<0.001.

Moreover,twopolypeptideswithapproximatemolecularmasses

of64and61kDaweredetectedbySDS–PAGEforthesamefraction

(Fig. 3b).These observationssuggest theexistence of two

sub-unitsin thestructureof thenativeenzyme.However,thesame

SDS–PAGE proﬁle was obtained for the enzyme treated under

reducingandnon-reducingconditionsaswellaswithand

with-outDMS.Therefore,wemayconcludethatweareinthepresence

oftwomonomericisoenzymes.

There are reports for molecular masses of plant ␤-NAHase

thatresultedindifferentdeterminationswhenusingdiverse

tech-niques.Ithasbeenreportedthat_␤-NAHasepuriﬁedfromLupinus

luteusseedshasamolecularmassof69kDawhendeterminedby

SDS–PAGE,62.5kDa byBio-Gel P-60 ﬁltrationand anapparent

molecularweightof15.3kDabyaSephadexG-75ﬁltration(Pocsi

etal.,1990).Thispatternmayarisefromthedescribed

glycopro-teinnatureoftheenzyme.Regardless,there isabroadrangeof

molecularmassesfor␤-NAHase,rangingfrom40kDainL.luteus

(McFarlaneetal.,1984)to236kDainMalusdomestica(Yongand Gross,1994).

Similarly, there is a wide range of subunit compositions

describedfor␤-NAHase,asforinstance,monomersforL.luteus

seeds(McFarlaneet al., 1984), heterodimers for Pisum sativum

seeds(HarleyandBeevers,1987),heterotrimersforBrassica

Oler-acealeaves(Changetal.,1998),homopentamersorhomohexamers

forTrigonellafoenum-graecumseeds(Bouqueletand Spik,1978)

andhomooctamersforM.domestica(YongandGross,1994).There

arealsoseveralreportsfor␤-NAHaseisoenzymes,namelytwofor

Vignaradiataseeds(Prakash,1984),threeforC.annumandL.luteus

seeds(McFarlaneetal.,1984;JagadeeshandPrabha,2002),ﬁvefor

Triticumaestivumleaves(BarberandRide,1989)andsevenforO.

sativaseeds(Jinetal.,2002).

FurthercharacterizationoftheisoenzymesofL.albus␤-NAHase

wasperformed by2-DE (Fig.4).Theﬁve isoenzymes withpI’s

between5.3and5.6correspondingtothe64kDa(a1–5)andﬁve

otherforthe61kDa(b1–5)polypeptidesmayarise froma

num-berofdifferentposttranslationalmodiﬁcations.ThepI’sdescribed

forotherplant␤-NAHasesareintheacidicrangeas5.13–5.36for

Triticumaestivum(Barberand Ride,1989), 5.5forCaricapapaya

(Chenetal.,2011)and6forZeamays(Oikawaetal.,2003).

␤-NAHaseasaglycosylatedproteincanbeardifferentglycosyl

residuesattachedtotheproteincore.Sincetheelectrophoretic

pro-teinpatternobtainedforthedeglycosylated␤-NAHaseissimilar

forboth64and61kDaisoenzymes(Fig.5),the3kDadifference

detectedbetweenthesetwoisoformssuggestsdistinctdegreesof

glycosylationofthesameproteincore.Withrespecttotheprotein,

theN-terminalsequenceforL.albus␤-NAHasewasobtained

(VDS-EDLI(EN)AFKIYVEDDNEHLQGSVD),andcomparedwiththeother

plant␤-NAHases.ThisistheﬁrstN-terminalsequencereportedfor

aplant␤-NAHase(UniProtdatabaseaccessionnumber:B3EWR5).

Fromthe94insilicoproteinsequencesannotatedintheUniProt

databaseforviridiplantaeN-acetylhexosaminidase(Supplemental

Table1),noneresultedfromadirectproteinsequence.Allofthem

werepredictedproteins,ofwhich22werefoundattranscriptional

level.ThismayjustifytheabsenceofhomologyfortheN-terminal

sequenceofL.albus␤-NAHasewiththeproteinsannotatedinthis

database.ThemultiplesequencealignmentrevealedthatL.albus

␤-NAHase(SupplementalFig.1),isembeddedina cluster

com-prisingenzymesofimportantcropplants,R.communis(B9T146),Z.

mays(B6TP85)andV.vinifera(F6GYZ3andF6H7W3).TheL.albus

N-terminalsequencealignsafewaminoacidsafterthebeginning

ofseveralinsilicotranslatedproteinsequences,suggesting

post-translationprocessinguntilthematureprotein.Thisobservationis

furtherreinforcedbythefactthattheN-terminalaminoacidofthis

enzymeisnotmethionine.

BiochemicalcharacterizationoftheL.albusˇ-NAHase

L.albus␤-NAHaseactivitytowardpNP–GlcNAcandpNP–GalNAc

andtheinabilitytoremovedimmerand trimmerresiduesfrom

pNP–(GalNAc)2 and pNP–(GalNAc)3 is consistent with an

exo-glycosidase activity for this enzyme. Many other crop plant

␤-NAHases,likethoseofC.annuum(Ghoshetal.,2011),O.sativa

(Jinetal.,2002),R.communis(SuzanneandHarry,1985)andT.

foenum-graecum(Bouqueletand Spik,1978), arereportedtobe

abletohydrolyzedbothpNP–GlcNAcandpNP–GalNAc.Forthe

␤-NAHaseofT.foenum-graecumalowactivitytowardpNP–(GlcNAc)2

wasreportedwhencomparedtopNP–GlcNAc(BouqueletandSpik,

1978),whereasforO.sativa,nohydrolysisofchitinwasobserved

(Jinetal.,2002).

TheKmvalueobtainedforL.albus␤-NAHasewithpNP–GlcNAc

isintherangereportedforthissubstrate(JagadeeshandPrabha,

2002;BouqueletandSpik,1978)whereasforpNP–GalNAcisabove (BouqueletandSpik,1978;HarleyandBeevers,1987).Thefactthat

KmvaluesweresimilarforbothsubstrateswhiletheVvaluefor

pNP–GlcNAcwasoversixtimeslargerthanthatforpNP–GalNAc

reﬂectsahighercatalyticefﬁciencytowardpNP–GlcNAc.

The optimum pH and pH stability, as well as the optimum

temperatureand temperaturestabilityproﬁlesobservedforthis

enzymeareanalogoustothosedescribedintheliteratureforother

cropplant␤-NAHases(JagadeeshandPrabha,2002;Yi-Chingetal.,

2004;BouqueletandSpik,1978;Gers-Barlagetal.,1988;Jinetal., 2002;Changetal.,1998).

Thestability of ␤-NAHaseto awide range of pHs and

tem-peraturescanbeattributedtotheenzymeglycosylationcontent,

sinceN-glycansaredescribedtohaveanimportantcontributionfor

enzymefolding(Shental-BechorandLevy,2008)andthusstability.

Generally, inhibitory effects of AgNO3, CaCl2, CdCl2, CoCl2,

CuSO4,FeCl3,KI,MgCl2,MnSO4,Na2MoO4,NiCl2 andZnCl2 onL.

albus␤-NAHaseareinagreementwiththose reportedforother

plant␤-NAHases,suggestingsimilarreactionmechanisms

proba-blyduetocommonactivesitesoftheenzymes(LiandLi,1970;

Chenetal.,2011;Oikawaetal.,2003;Orlacchioetal.,1985;Yi,

1981).Inhibitoryeffects ofAgNO3,CdCl2 and CuSO4 are

indica-tivethatsulfhydrylgroupscanbeimportantfortheactivityand/or

conformationalstabilityofL.albus␤-NAHaseactivesite,similarto

whatwaspreviouslyreportedforP.sativum_␤-NAHase,wherethe

sulfhydrylgroupsweredescribedtoparticipateintheenzymatic

(9)

Thereareseveralreportsofenzymaticactivationinthe

pres-enceof MgCl2 as for C. annuum (Jagadeesh and Prabha, 2002)

andP.sativum(HarleyandBeevers,1987).Despitethefactthat

MgCl2 canactasanenzymaticactivator,theactivityofL.albus

␤-NAHaseisnotdependentonthiscation,sincethepresenceof

chelatingagents(EDTAandEGTA)doesnotaffectenzyme

activ-ity.

Severaleffectsaredescribedforsugars’inﬂuenceinplants

␤-NAHasesactivity that goesfrom inhibition to activation.These

generallyonlyslightlyaffecttheenzymeactivity(Gers-Barlagetal.,

1988;JagadeeshandPrabha,2002),similartoobservationsforL.

albus␤-NAHaseinthepresenceofFuc,Gal,Glc,Lac,␣-Man,GalNAc

andGlcNAc.

Potentialphysiologicalsubstratesforˇ-NAHase

The prevailing and higher enzymatic activity found in the

cotyledons during seed germination and seedling growth

sug-geststhatthemainphysiologicalactionofthisenzymeoccursin

theseorgans at thesestages of development.These are

impor-tantreservoir organs thatcontainessential storageproteinsfor

seed germination and seedling growth. A relatively large

frac-tionofthesereservesinL.albusiscomposedofglobulins,which

aredescribed asglycosilated proteins(Durantietal., 1981).An

increasein␤-NAHaseactivitycoincidentwithglobulin

degrada-tion(Fig.7)suggeststhat theseeventscouldbephysiologically

relatedduringseedgerminationandseedlinggrowth.Inorderto

testthishypothesisstudieswereconductedtoevaluatethe

possi-bilityof␤-NAHaseusingglobulinsasnaturalsubstrates.Ourinvitro

studyrevealedthat_␤-NAHasewasabletodisturbtheaggregation

of␣-conglutin,aprocessassociatedtostorageprotein

mobiliza-tion(Fig.8)(Santosetal.,2012).Moreover,␤-NAHasewasable

toliberateGlcNAcfrom␣-conglutinwhennotintheaggregated

form.

Together, these observations indicate the activity of this

enzyme as hexosaminidase toward this potential natural

sub-strate.

Conclusions

Duringseedgermination,storageproteinshavetobemobilized

tonourishtheembryo.Thepartialproteolysisofstorageproteins

byproteinaseAwasreportedasanimportantregulatorypointfor

storageproteinmobilization.Nevertheless,severalgapsremainto

beelucidatedinthisprocess(Müntzetal.,2001).The(i)

detec-tionof␤-NAHaseactivityinthedryseeds,togetherwiththe(ii)

highestactivitydetectedintheplantletcotyledonsandwiththe

(iii)increasingenzymeactivityintheseorgans beingcoincident

withglobulindegradation,in additiontothe(iv)invitroability

of␤-NAHasetoexciseexogenoussugarmoietiesfrom

individual-ized␣-conglutin,stronglyindicatethat␤-NAHaseisinvolvedin

theremovalofGlcNAcofaL.albusstorageproteinduringseedling

growth.

The presence of ␤-NAHase in dry seeds, together with the

invitroinabilityofthisenzymetoassess␣-conglutinaggregates

maysuggestthat␤-NAHaseand storageproteinaggregatescan

betiledwithoutanyeffectiveglobulin–enzymeinteraction.Upon

seedgermination,globulindisaggregationmayleadtotherelease

ofindividualizedconglutins(Santosetal.,2012),whichinturncan

beusedasapotentialsubstrateof␤-NAHasefortheremovalof

Glc-NAcresidues.Thisistheﬁrstreportthathasputativelyidentiﬁed

aphysiologicalsubstratefor␤-NAHase.However,furtherstudies

shouldbeperformedtogaindeeperinsightinto_␤-NAHase

involve-mentinthemobilizationanddegradationoflegumeseedstorage

proteins,forinstanceraisingpolyclonalantibodiesfortheenzyme

andconglutinsforsubcellularco-localization.Moreover,transcript

expressionduringtheseprocessescouldbefollowedbyRT-PCRby

usingdegeneratedoligonucleotidesdesignedbasedontheprotein

sequenceobtained.

Acknowledgements

ThisworkwassupportedﬁnanciallybytheFundacãopara a

Ciência ea Tecnologiathroughgrant PEst-OE/EQB/LA0004/2011

andalsobyﬁnancialsupportofC.N.S.(SFRH/BPD/84618/2012)as

wellasM.A.(SFRH/BPD/76646/2011).

References

AebiM,BernasconiR,ClercS,MolinariM.N-glycanstructures:recognitionand processingintheER.TrendsBiochemSci2009;35:74–82.

BarberMS,RideJP.Puriﬁcationandpropertiesofawheatleaf N-acetyl-beta-d-hexosaminidase.PlantSci1989;60:163–72.

BernardM.TheroleofGlcNAcinformationandfunctionofextracellularmatrices. CompBiochemPhysiolBBiochemMolBiol2008;149:215–26.

BlumH,BeierH,GrossHJ.Improvedsilverstainingofplantproteins,RNAandDNA inpolyacrylamidegels.Electrophoresis1987;8:93–9.

BolJF,LinthorstHJM,CornelissenBJC.Plantpathogenesis-relatedproteinsinduced byvirus-infection.AnnRevPhytopathol1990;28:113–38.

Bouquelet S,Spik G. Properties of four molecular forms of N-acetyl-beta-d-hexosaminidase isolated from germinating seeds of fenugreek (Trigonella foenumgraecum).EurJBiochem1978;84:551–9.

BrittonH,RobinsonR.Universalbuffersolutionsandthedissociationconstantof veronal.JChemSoc1931;CXCVIII:1456–62.

BroekaertWF,VanparijsJ,AllenAK,PeumansWJ.Comparisonofsomemolecular, enzymaticandantifungalpropertiesofchitinasesfromthorn-apple,tobaccoand wheat.PhysiolMolPlantPathol1988;33:319–31.

Chang CT, Young FP, Chang MH, Sung HY. Puriﬁcation and properties of beta-N-acetylhexosaminidasefromcabbage.BiochemMolBiolInt1998;45: 371–80.

Chen L-C, Chung Y-C,Chang Y-M, Chang C-T. Characterisation of a beta-N-acetylhexosaminidasefromacommercialpapayalatexpreparation.FoodChem 2011;124:1404–10.

DaviesGE,StarkGR.Useofdimethylsuberimidate,across-linking reagent,in studyingthe subunitstructure ofoligomericproteins. Proc Natl AcadSci 1970;66:651–6.

DirnbergerD,SteinkellnerH,AbdennebiL,RemyJ-J,vandeWielD.Secretionof biologicallyactiveglycoformsofbovinefolliclestimulatinghormoneinplants. EurJBiochem2001;268:4570–9.

DurantiM,RestaniP,PoniatowskaM,CerlettiP.TheseedglobulinsofLupinusalbus. Phytochemistry1981;20:2071–5.

FayeL,ChrispeelsMJ.CharacterizationofN-linkedoligosaccharidesbyafﬁnoblotting withconcanavalinA-peroxidaseandtreatmentoftheblotswithglycosidases. AnalBiochem1985;149:218–24.

FerreiraRB,MeloTS,TeixeiraAN.Catabolismoftheseedstorageproteinsfrom Lupinusalbus:fateofglobulinsduringgerminationandseedlinggrowth.AustJ PlantPhysiol1995;22:373–81.

FerreiraRB,FrancoE,TeixeiraAR.Calcium-andmagnesium-dependentaggregation oflegumeseedstorageproteins.JAgricFoodChem1999;47:3009–15. FerreiraRB,FreitasRL,TeixeiraAR.Self-aggregationoflegumeseedstorageproteins

insidetheproteinstoragevacuolesiselectrostaticinnature,ratherthan lectin-mediated.FEBSLett2003a;534:106–10.

FerreiraRB,FreitasRL,TeixeiraAR.ThestructureofLupinusseedstorageproteins. Recentdevelopments.CurrTopPlantBiol2003b;4:139–50.

FrancoE,FerreiraRB,TeixeiraAR.Utilizationofanimprovedmethodologytoisolate Lupinusalbusconglutinsinthestudyoftheirsedimentationcoefﬁcients.JAgric FoodChem1997;45:3908–13.

Gers-BarlagH,BartzI,RudigerH.beta-N-Acetylhexosaminidasefromsoybean. Phy-tochemistry1988;27:3739–41.

Ghosh S, Meli VS, Kumar A, Thakur A, Chakraborty N, Chakraborty S, etal. TheN-glycanprocessingenzymesalpha-mannosidaseand beta-d-N-acetylhexosaminidase are involvedinripening-associated softeninginthe non-climactericfruitsofcapsicum.JExpBot2011;62:571–82.

HarleySM,BeeversL.Isozymesofbeta-N-acetylhexosaminidasefrompeaseeds (PisumsativumL.).PlantPhysiol1987;85:1118–22.

HarrisN,ChrispeelsMJ.Histochemicalandbiochemicalobservationsonstorage proteinmetabolismandproteinbodyautolysisincotyledonsofgerminating mungbeans.PlantPhysiol1975;56:292–9.

HodgeA,AlexanderIJ,GoodayGW.ChitinolyticactivitiesofEucalyptuspilularisand Pinussylvestrisrootsystemschallengedwithmycorrhizalandpathogenicfungi. NewPhytol1995;131:255–61.

HongHY,ChoiJK,YooGS.Detectionofproteinsonpolyacrylamidegelsusing cal-concarboxylicacid.AnalBiochem1993;214:96–9.

Jagadeesh BH, Prabha TN. beta-Hexosaminidase, an enzyme from ripening bell capsicum (Capsicum annuum var. variata). Phytochemistry 2002;61: 295–300.

(10)

JagadeeshBH,PrabhaTN,SrinivasanK.Activitiesofbeta-hexosaminidaseand alpha-mannosidaseduring developmentandripeningofbellcapsicum(Capsicum annuumvar.variata).PlantSci2004;167:1263–71.

JainE,BairochA,DuvaudS,PhanI,RedaschiN,SuzekBE,etal.Infrastructureforthe lifesciences:designandimplementationoftheUniProtwebsite.BMCBioinform 2009;10:136.

JinYL,JoYY,KimKY,ShimJH,KimYW,ParkRD.Puriﬁcationand characteri-zationofbeta-N-acetylhexosaminidasefromriceseeds.JBiochemMolBiol 2002;35:313–9.

KotakeT,TonariA,OhtaM,MatsuuraF,SakuraiN.Smallcomplex-typeN-linked glycansareattachedtocell-wallboundexo-beta-glucanasesofbothmungbean andbarleyseedlings.PhysiolPlant2001;112:308–14.

LaemmliUK.Cleavageofstructuralproteinsduringtheassemblyoftheheadof bacteriophageT4.Nature1970;227:680–5.

LiSC,LiYT.Studiesontheglycosidasesofjackbeanmeal,3.Crystallizationand propertiesofbeta-N-acetylhexosaminidase.JBiolChem1970;245:5153–60. Liebminger E, Veit C, Pabst M, Batoux M, Zipfel C, Altmann F, et al.

beta-N-AcetylhexosaminidasesHEXO1andHEXO3areresponsibleforthe for-mationofpaucimannosidicN-glycans in Arabidopsisthaliana.JBiolChem 2011;286:10793–802.

LowryOH,RosebroughNJ,FarrAL,RandallRJ.ProteinmeasurementwiththeFolin phenolreagent.JBiolChem1951;193:265–75.

McFarlane EF, McFarlane DR, Kennedy IR. Isolation of beta-N-acetyl-d-hexosaminidasesfromlupinseed.Phytochemistry1984;23:2431–3. MeliVS,GhoshS,PrabhaTN,ChakrabortyN,ChakrabortyS,DattaA.Enhancement

offruitshelflifebysuppressingN-glycanprocessingenzymes.ProcNatlAcad Sci2010;107:2413–8.

MeloTS,FerreiraRB,TeixeiraAN.TheseedstorageproteinsfromLupinusalbus. Phytochemistry1994;37:641–8.

MüntzK,BelozerskyMA,DunaevskyYE,SchlerethA,TiedemannJ.Stored pro-teinasesandtheinitiationofstorageproteinmobilizationinseeds during germinationandseedlinggrowth.JExpBot2001;52:1741–52.

OikawaA,ItohE,IshiharaA,IwamuraH.Puriﬁcationandcharacterizationof beta-N-acetylhexosaminidasefrommaizeseedlings.JPlantPhysiol2003;160:991–9. OkuboK,MyersDV,IacobucciGA.Bindingofphyticacidtoglycinin.CerealChem

1976;53:513–24.

Orlacchio A, Maffei C, Emiliani C, Reinosa JA. On the active site of beta-hexosaminidasefromlatexofFicusglabrata.Phytochemistry1985;24:659–62. PocsiI,KissL,NanasiP.StudiesontheN-acetyl-beta-d-hexosaminidaseBfrom

ger-minatingLupinusluteusL.seeds.I.Puriﬁcationandcharacterization.Biochim BiophysActa1990;1039:110–8.

PoultonJE,ThomasMA,OttwellKK,McCormickSJ.Partialpuriﬁcationand charac-terizationofabeta-N-acetylhexosaminidasefromblackcherry(Prunusserotina EHRH.)seeds.PlantSci1985;42:107–14.

PrakashMD.Occurrenceofglycoproteinglycosidasesinmatureseedsofmungbean (Vignaradiata).Phytochemistry1984;23:257–60.

ReissigJL,StormingerJL,LeloirLF.Amodiﬁedcolorimetricmethodfortheestimation ofN-acetylaminosugars.JBiolChem1955;217:959–66.

SantosCN,AlvesM,BentoI,FerreiraRB.Missingpiecesinproteindepositionand mobilizationinsidelegumeseedstoragevacuoles:calciumandmagnesiumions. SeedSciRes2012;22:249–58.

SchlumbaumA,MauchF,VogeliU,BollerT.Plantchitinasesarepotentinhibitorsof fungalgrowth.Nature1986;324:365–7.

Shental-BechorD,LevyY.Effectofglycosylationonproteinfolding:acloselookat thermodynamicstabilization.ProcNatlAcadSci2008;105:8256–61. SlamovaK,BojarovaP,PetraskovaL,KrenV.beta-N-Acetylhexosaminidase:what’s

inaname...?BiotechnolAdv2010;28:682–93.

StrasserR.LocalizationofplantN-glycanprocessingenzymesalongthesecretory pathway.PlantBiosyst2009;143:636–42.

Strasser R, BondiliJS, Schoberer J, SvobodaB, Liebminger E, Glossl J, et al. Enzymatic properties and subcellular localization of Arabidopsis beta-N-acetylhexosaminidases.PlantPhysiol2007;145:5–16.

SturmA,VanKuikJA,VliegenthartJF,ChrispeelsMJ.Structure,position,and biosyn-thesisofthehighmannoseandthecomplexoligosaccharidesidechainsofthe beanstorageproteinphaseolin.JBiolChem1987;262:13392–403.

SuzanneHM,HarryB.Characterizationandpartialpuriﬁcationofthreeglycosidases fromcastorbeanendosperm.Phytochemistry1985;24:1459–64.

TakahashiN,HottaT,IshiharaH,MoriM,TejimaS,BlignyR,etal.Xylose-containing commonstructuralunitinN-linkedoligosaccharidesoflaccasefromsycamore cells.Biochemistry1986;25:388–95.

TamsJW,WelinderKG.Mildchemicaldeglycosylationofhorseradishperoxidase yieldsafullyactive,homogeneousenzyme.AnalBiochem1995;228:48–55. VitaleA,ChrispeelsMJ.TransientN-acetylglucosamineinthebiosynthesisof

phy-tohemagglutinin:attachmentintheGolgiapparatusandremovalinprotein bodies.JCellBiol1984;99:133–40.

YiCK.Increaseinbeta-N-acetylglucosaminidaseactivityduringgerminationof cot-tonseeds.PlantPhysiol1981;67:68–73.

Yi-ChingC,Wei-LiangL,Hui-ChingH,Yung-AnL,Chiang-SanC.Puriﬁcationand characterizationofisoformsofbeta-N-acetylhexosaminidasefrommungbean seedlings.BotBullAcadSin2004;45:275–83.

YongCS,GrossKC.N-Acetyl-beta-d-glucosaminidasefromgoldendeliciousapples. Phytochemistry1994;36:1–6.