The glycomic effect of N-acetylglucosaminyltransferase III overexpression in metastatic melanoma cells : GnT-III modifies highly branched N-glycans

GlycoconjugateJournal(2018)35:217–

231https://doi.org/10.1007/s10719-018- 9814-y

ORIGINALARTICLE

TheglycomiceffectofN-

acetylglucosaminyltransferaseIIIoverexpressioninmetastaticmelano macells.GnT-IIImodifieshighlybranchedN-glycans

PawełLink-Lenczowski^{1,2 &M}onikaBubka^2&CrinaI.A.Balog^3&CarolienA.M.Koeleman^3&TerryD.Butters^4&

ManfredW uhrer^3&AnnaLityńska²

Received:14December2017/Revised:15January2018/Accepted:30January2018/Publishedonline:3March2018

#TheAuthor(s)2018.Thisarticleisanopenaccesspublication

Abstract

N-acetylglucosaminyltransferaseIII(GnT-III)isknowntocatalyzeN-

glycanBbisection^andtherebymodulatetheformationofhighlybranchedcomplexstructureswithintheGolgiapparatus.Whileactive,iti nhibitstheactionofotherGlcNActransferasessuchasGnT-IVandGnT-V.Moreover,GnT-

IIIisconsideredasaninhibitorofthemetastaticpotentialofcancercellsbothinvitroandinvivo.However,theeffectsofGnT- IIImaybemorediverseanddependonthecellularcontext.WedescribethedetailedglycomicanalysisoftheeffectofGnT- IIIoverexpressioninWM266–4-GnT-IIImetastaticmelanomacells.WeusedMALDI-TOFandESI-ion-trap- MS/MStogetherwithHILIC-HPLCof2-AAlabeledN-glycanstostudytheN-glycomeofmembrane-

attachedandsecretedproteins.WefoundthattheoverexpressionofGnT-IIIinmelanomaleadstothemodificationofabroadrangeofN- glycantypesbytheintroductionoftheBbisecting^GlcNAcresiduewithhighlybranchedcomplexstructuresamongthem.Thepresence oftheseunusualcomplexN-glycansresultedinstrongerinteractionsofcellularglycoproteinswiththePHA-

L.BasedonthedatapresentedhereweconcludethatelevatedactivityofGnT-

IIIincancercellsdoesnotnecessarilyleadtoatotalabrogationoftheformationofhighlybranchedglycans.Inaddition,themodificationofp re-existingN-glycansbytheintroductionofBbisecting^GlcNAccanmodulatetheircapacitytointeractwithcarbohydrate-

bindingproteinssuchasplantlectins.OurresultssuggestfurtherstudiesonthebiologicalfunctionofBbisected^oligosaccharidesinca ncercellbiologyandtheirinteractionswithcarbohydrate-bindingproteins.

KeywordsMelanoma. Glycosylation. N-acetylglucosaminyltransferaseIII. Glycome. Cancer

Abbreviations

AA 2-aminobenzoicacid

Fuc,F fucose

Hex,H hexose

HILIC

hydrophilicinteractionliquidchromatographyHexNAc,N N-acetylhexosamine

mDCIR2 mousedendriticcellinhibitoryreceptor2 NeuAc,SA N-acetylneuraminicacid

ElectronicsupplementarymaterialTheonlineversionofthisarticle(http s://doi.org/10.1007/s10719-018-9814-

y)containssupplementarymaterial,whichisavailabletoauthorizedusers .

*PawełLink- Lenczowskip.link- lenczowski@uj.edu.pl

1 DepartmentofMedicalPhysiology,FacultyofHealthSciences,Ja giellonianUniversityMedicalCollege,Michałowskiego12,31- 126Kraków,Poland

2 DepartmentofGlycoconjugateBiochemistry,InstituteofZoologyand BiomedicalResearch,JagiellonianUniversity,Kraków,Poland 3 CenterforProteomicsandMetabolomics,LeidenUniversityMedicalC

enter,Leiden,TheNetherlands

4 OxfordGlycobiologyInstitute,Oxford,UK

NeuNAcLac lactonizedN-

acetylneuraminicacidPAG pregnancy- associatedg lycoprotein

Introductio n

Glycosylationisthemostfrequentandabundantpost- translationalmodificationofmembraneandsecretedproteins inEukaryota.Becauseoftheirstructuralcomplexity,glycans bearanenormousinformationalcapacity,stronglyenhancet hediversityofproteins,andthuscanregulatecellbiologyatm anylevels[1].TheN-

glycosylationofproteinstakesplacewithinthesecretorypat hway,whichinvolvesendoplasmicreticulum(ER)andGol giapparatus(GA).Theformationof

GlycoconjJ ( 2018)35:217–231 3 matureglycansattachedtotheconsensussequencewithinthepr

oteinbackboneisstronglydependentontheactionofsev- eralglycosylationenzymes(glycosidasesandglycosyltrans- ferases),theaccessibilityofactivatedmonosaccharidedonorsa ndtheefficiencyoftheirtransportationintothelumenofERandG A[2].Althoughthegeneralorchestrationoftheglyco-

sylationmachineryhasbeenwelldescribed[3],thedetailsofther egulationoftheprocessarestillunderinvestigation.Oneofthem ostinterestingaspectsisthemutualrelationshipbe-

tweenglycosyltransferaseswithintheGolgiapparatus.

N-AcetylglucosaminyltransferaseIII(GnT-III)isanen- zymeactingwithinthemedial-

GolgiandisresponsibleforthetransferofasingleN- acetylglucosamine(GlcNAc)inaβ1,4linkagetotheβ- mannoseonN-

glycans,thusformingtheBbisecting^GlcNAcstructure.The presenceoftheBbisecting^GlcNAc,whichcausesacharacteri sticconforma-

tionalchangeoftheglycan[4],hasbeenrecognizedasinhib- itorytowardsotherGlcNActransferasessuchasGnT- IVandGnT-V,preventingtheformationofhighly- branchedspecies,suchastheβ1-6-

GlcNAcbranchingcatalyzedbyGnT-V[5–

7].Moreover,recentdatashowthatoverexpressionofGnT- IIIcandramaticallysuppressα2-3-sialylationatapost- transcriptionallevel[8].

TheactionofGnT-

IIIalsohasimportantimplicationsincancerbiology,sincethet ransformationandprogressionofmanytypesofcancerareacco mpaniedbychangesinproteinglycosylation,someofwhichare consideredcancerbio-markers[9–11].Inthisregard,GnT- IIItraditionallyispicturedasasuppressorofmalignancyandther eismuchevidencethatoverexpressionofGnT-

IIIinhibitsthemetastaticpotentialofcancercellbothinvitroan dinvivo[6,12,13].Thisanti-cancereffectofGnT-

IIIactivityisoftenattributedtothefactthatthepresenceofBbisec ting^GlcNAconmembraneadhe-

sionproteinssuchascadherinsandintegrinsmodulatestheirfun ctionandthusinfluenceadhesionrelationsbetweencancercells themselvesaswellasbetweencellsandtheextracellularmatrixp roteins[14,15].PreviousstudiesclearlyshowthatGnT- IIIactivitypromoteshomotypicinteractionsofE-

cadherinsonmammarycarcinomaandmelanomacells[14,16]

.DownregulationofMgat3inthemousemodelofPyMT- inducedmammarycarcinomaacceleratesmigratoryproperties ofcancercellspromotingthemetastasestothelung[17].Fin ally,itisknownthatintroductionofBbisecting^GlcNActointeg rinsreducescellmigration[18].Themetastasis-

inhibitoryeffectofGnT-

III,alsointhecontextofadhesionproteinmodulationislinkedto itsabilitytoinhibittheforma-tionofhighlybranchedN-

glycans,especiallyβ1-6-

GlcNAcbranchingwithpolylactosamineepitopes,whichcanp romotecellproliferation,migrationandinvasion[6].However

,thepictureofGnT-

IIIasauniversalmetastasismodulatorremainscontroversial,asits elevatedactivityisobservedinsomema-

lignanciessuchashepatoma,ovariancancer,multiplemyelo- maandchronicmyeloidleukemia[19–22].These

GlycoconjJ ( 2018)3 5:217–231 218

observationssuggest,thattheGnT-

IIIeffectincancerismorecomplex,andmaydependonthecellu larcontext.Hence,itsimpactontherepertoireofN-

glycansonthecellsurfaceandonsecretedproteinsneedsfurthe rstudy.

Melanomaisahighlyinvasivetumor,whichcandevelopwi thintheskin,uveaandgastricmucosa[23].Manystudiessugge sttheimportantroleofβ1-6-GlcNAcbranchedN-

glycansinthepromotionofmetastaticpotentialofmelanomac ells,mainlythroughthemodulationofintegrin-

dependentadhesionandmigration[24–

27].Recently,wehavedevelopedaninvitromelanomamodeli nwhichweinducedtheoverex-pressionofGnT-

IIIinmetastaticmelanomacelllineWM266–

4[28].Inthepresentwork,wehaveinvestigatedtheN- glycosylationprofileofmembraneandsecretedproteinsofth esecellsindetail,providingevidencethatGnT-IIIupregu- lationdoesnotinhibittheformationofhighlybranchedN- glycansbutefficientlymodifiestheseglycansbytheintroduc- tionofaBbisecting^GlcNAc.

Materialsandmethods

s

NonidetP40waspurchasedfromRoche(Warszawa,Poland).

Acrylamide,APS,bisacrylamide,0.5MTrispH6.8buffer, 1.5M T rispH8.8buffer,LaemmliSampleBuffer,2- mercaptoethanol,TEMED,Tris-glycinebuffer,Tris- glycine-SDSbufferwereprocuredfromBio-

Rad(Warszawa,Poland).PageRulerPrestainedProteinLadd erwasobtainedfromFermentas(ThermoFisherScientific, Warszawa,Poland).DPBSandFBSwerepurchasedfromLif eTechnologies(Warszawa,Poland).2,5-

dihydroxybenzoicacid(2,5-

DHB)wasfromBrukerDaltonics(Bremen,Germany).Aceto nitrileHPLCgradeforfarUV,2-aminobenzamide(2- AB),anthranilicacid(2-

AA),sodiumcyanoborohydride,DMSOandtrifluoroacetica cid(TFA)werepurchasedfromSigma-

Aldrich(Poznań,Poland).Allothersalts,alcoholsandacidsw ereanalyticalgradechemicalsfromSigma-

Aldrich.WaterusedwasofMilli-Qgrade.

Cellsandculturecondition s

Asanexperimentalmodel,weusedpreviouslydescribedWM -266-4-GnT-

IIIhumanmetastaticmelanomacells,stablyoverexpressingth eMGAT3genetogetherwithmockcontrolcellsWM-266-4- pIRESneo[28].ThecellsweregrowninRPMI-

1640culturemediumsupplementedwith25mMHEPESand

L-alanine-L-glutamine(RPMI-1640Glutamax-

I;Gibco,LifeTechnologies),inthepresenceofstandardanti- bioticcocktail(100μg/mlstreptomycin,100U/mlpenicillin;Si gma-

Aldrich)and100μg/mlG418sulfate(Geneticin;Gibco,LifeT echnologies)asa selectionagent.Culture

mediumwassupplementedwith10%fetalbovineserum(Gibc o,LifeTechnologies)andcellsweregrownwith5%CO2a t3 7°C.CellsweresystematicallytestedbyPCRforthepresenceof Mycoplasmasp.

Isolationofmembraneandsecretedproteins

Cellsweregrownuntil~70%confluency,washed5timeswithDP BSandthenculturedinserum-

freemediumfor24h.Theculturemediacontainingsecretedprotei nswerecollected,fil-

teredthrougha0.4μmporesizesyringefilterandfrozenat

−70°C.Thecellswerewashed3timeswithcoldDPBS,scrapedcare fullywitharubberpolicemaninabout2mlofcoldDPBSandcentrif ugedat500×gfor10minat4°C.Themembraneproteinswereisola tedusingQProteomeCellCompartmentKit(Qiagen,Hilden,Ger many)accordingtomanufacturer’sproto-

colandtheobtainedfractionswerefrozenat−70°C.

Beforeelectrophoresisanddeglycosylation,thesecreteda ndmembraneproteinswereconcentratedbyprecipitation.Bri efly,thecollectedconditionedmediacontainingsecretedprote inswerelyophilized,resuspendedinaminimalvolumeof50m MTris-HCl,pH8.0containing0.5%Triton-X-

100anddialyzed3timesovernightagainstwater.Forproteinpre -cipitation,onepartofsecretedproteinssuspensionormem- braneproteinfractionwasmixedwith4partsofmethanol,follo wedby1partofchloroformand3partsofwater,withmixingate achstep.Aftercentrifugation(15,000×g,2min),theupperaque ouslayerwasremoved,leavingtheproteinsintactattheinterph ase.Then,4volumesofmethanolwereadded,andtheprecipit atedproteinswerespundown(15,000×g,2min)anddriedatro omtemperature(RT)underthefumehood,afterremovalofthesu pernatant.

PolyacrylamidegelelectrophoresisinpresenceofSDS(S DS-PAGE)

PrecipitatedproteinpelletswereresuspendedinLaemmlisamp lebuffer,andafterheatdenaturation(100°C,5min)theprot einswereseparatedon8%polyacrylamidegels,ac-

cordingtoLaemmli[29]inthepresenceof0.1%SDSandbeta- mercaptoethanol.Molecularmassesofproteinswerede- terminedusingaprestainedproteinladder(Fermentas).Afterel ectrophoresis,separatedproteinsweretransferredontoanIm mobilon-

PPVDFmembrane(MerckMillipore,Warszawa,Poland) overnightat4°Cwithaconstantcurrentof100mA(MiniTrans- BlotElectrophoreticTransferCell;Bio-

Rad)accordingtoTowbinetal.[30].

Lectindetectionofblottedglycoproteins

Theproteinbandscontainingstudiedoligosaccharideepitopeswe redetectedaccordingtoHaselbecketal.

[31]withfurthermodificationsmadebyOchwatetal.

[32].PVDFmembranes

withseparatedglycoproteinswereblockedin0.5%blockingrea gent(DIGGlycanDifferentiationKit;RocheDiagnostics,Man nheim,Germany)inTBSfor2–

3hatRT.BlotswerewashedtwiceinTBS- 0.1%Tween20andonceinlectinincu-

bationbuffer.Membraneswereincubated(1h,RT)withfol- lowingbiotinylatedlectins(VectorLabs,Burlingame,CA):

Phaseolusvulgariserythroagglutinin(PHA- E),Phaseolusvulgarisleucoagglutinin(PHA-

L),Sambucusnigraagglutinin(SNA),Maackiaamurensis agglutinin(MAA),Galanthusnivalisagglutinin(GNA)and Aleuriaaurantiaagglutinin(AAA)

(seeTable1).Alllectinswerediluted(1:4000)in50mMTr is-

HCl,pH7.5containing150mMNaCl,1mMMgCl2,1mMMn Cl2,1mMCaCl2.Afterincubationwithlectins,membranesw erewashed(3×15min)inTBS-

0.1%Tween20andincubated(1h,RT)withExtrAvidin- AP(Sigma-Aldrich)dilutedinTBS-

0.1%Tween20(1:4000).Afteranotherwashingseries(3×5 mininTBS-0.1%Tween20;3×5mininTBS),lectin-

reactivebandswerevisualizedonmembranesaftertransformati onofNBTandBCIP4-

toluidinesaltsubstrates(RocheDiagnostics)intocoloredproduc ts.

Isolationandlabelingofglycoproteinderived N-linkedoligosaccharides

Precipitatedproteinswereresuspendedin20μlofreducingbu ffer(0.5%SDS,1%2-

mercaptoethanol),denaturedat100°Cfor10minandaftercoo lingtoRT,SDSwasneutral-izedbyadding4μlof10%NP- 40.Afterthat,4μlof0.5Msodiumphosphatebuffer,pH7.5was addedfollowedby2μlofPNGaseF(500,000U/ml,NewEngla ndBiolabs,Ipswich,MA)andwaterupto40μl.Theglycoprot einsamplesweredeglycosylatedat37°Covernight.Therele asedN-

glycansweredesaltedbysolidphaseextraction(SPE)onnon- porousgraphitizedcarbonSPEcolumns(Grace,Alltech,Colu mbia,MD)accordingtoPackeretal.

[33]andelutedglycansweredried-

downonSpeedVac.Next,samplesweremadeupto30μl withwaterandthereleasedoligosaccharideswerela- beledwithanthranilicacid(2-

AA)accordingtoAnumulaandDhume[34]modifiedbyNev illeetal.[35].FluorescentlylabeledN-

glycanswerethenpurifiedonSpe-edAmide-

2SPEcolumns(PelicanScientific,Tattenhall,U.K.).Thecol- umnswerewashedwith1mLofACNfollowedby1mLofwater andequilibratedwith2mLofACN.Thesamplesweredilutedw ith1mLof97%

(v/v)ACNinwaterandloadedoncolumns.Afterwashingtwot imeswith95%(v/v)ACNinwaterthelabeledN-

glycanswereelutedwith1.5mLofwaterandkeptfrozenat−20°

C.

SeparationofchargedandneutralN- glycanspriortheHPLCanalysis

Negativelychargedoligosaccharideswereseparatedfromneu tralspeciesbySPEusinganionexchangeresin(QAE-

GlycoconjJ ( 2018)35:217–231 7

Table1Listofplantlectinsused

inlectinblotassay Lectin

source Bindingspecificity

SNA-I Sambucusnigra MAA-I

Maackiaamurensis PHA-L

Phaseolusvulgaris PHA-E

Phaseolusvulgaris GNA

Galanthusnivalis

AAA

Aleuriaaurantia

NeuNAcα2-6Gal- NeuNAcα2- 6GalNAc-

NeuNAcα2-3Galβ1-4GlcNAc

Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-2)Manα- Galβ1-4GlcNAcβ1-2Manα1-4(GlcNAcβ1-4)Manα-

Manα1-2Manα- Manα1-6Manα- Manα1-3Manα- Fucα1-2Galβ- Fucα1-6GlcNAcβ- Fucα1-3GlcNAcβ-

Sephadex,Sigma-Aldrich,Poole,U.K.).1mLplasticmini- columnswereloadedwith200μloftheresinandwashedwith2m Lofwater.Aliquotsof2-AAlabeledN-

glycanswereloadedoncolumnsandafterwashingwithwaterth eneutralglycanswereelutedwith0.5Maceticacidandthechar gedfractionwith0.5Mammoniumacetate.Theneutralswer epurifiedbylyophilizationandthechargedspeciesweredesal tedusingPGCSPE(HyperSep®HyperCarb®;ThermoFisher Scientific,Paisley,UK)accordingtoAlonzietal.anddriedunde rvacuum[36].

HPLCanalysisoffluorescentlylabeledN-glycans Purified2-AAoligosaccharideswereseparatedbyNP- HPLCusinga4.6×250mmTSKgel-

Amide80column(5μmbeadsize)

(Anachem,Luton,Beds,U.K.)accordingtoNevilleetal.

[37].ThechromatographysystemconsistedofWatersAllia nce2695separationsmoduleandanin-

lineWaters474fluorescencedetectorsetatExλ360nmandEmλ 425nm.Allchromatographywasperformedat30°C.SolventA wasace-tonitrile.SolventBwasMilli-

Qwater.SolventCwascom-

posedof800mMammoniumhydroxide,titratedtopH3.85with aceticacid,inMilli-

Qwater.Sampleswereloadedin70%ACNandseparatedusin ggradientconditionsasfollows:time=0min(t=0),71.6%A,25 .9%B,2.5%C(0.8mL/

min);t=6,71.6%A,25.9%B,2.5%C(0.8mL/min);t=45, 46.2%A,51.3%B,2.5%C(0.8mL/min);t=46,35%A, 62.5%B,2.5%C(0.8mL/min);t=48,35%A,62.5%B, 2.5%C(0.8mL/min);t=49,71.6%A,25.9%B,2.5%C (0.8mL/min);t=51,71.6%A,25.9%B,2.5%C(1.2mL/

min);t=64,71.6%A,25.9%B,2.5%C(1.2mL/min);t=65,

71.6%A,25.9%B,2.5%C(0.8mL/min).Allchromatogra- phywascontrolled,anddatawerecollectedandprocessedusing WatersEmpowersoftware,andtheglucoseunitvalues

220 GlycoconjJ ( 2018)3 5:217–231

weredeterminedfollowingcomparisonwitha2AA- labeledglucoseoligomerladderexternalstandard(GlykoPro zyme,Hayward,CA).

MALDI-TOF(/TOF)- MS

Dried2-

AAlabeledglycanswerereconstitutedinaminimalvolumeof wateranddesaltedusinga C18ZipTip™(Millipore)follow ingthemanufacturer’sinstructions.Glycanswereelutedwi th1.5μlof2,5-

dihydroxybenzoicacid(10mg/mlin50/50,ACN/watercontai ning0.1%TFA)direct-

lyontoaMALDItargetplateanddriedatRT.

MALDI-TOF-

MSwasperformedonUltrafleXtreme™massspectrometer controlledbyFlexControl3.1software(BrukerDaltonics, Bremen,Germany).Theinstrumentwasexternallycalibrate dusingtheBrukerpeptidescalibrationkit.Thespectrawerea cquiredbothinthenegativeionreflectronmodeandlinearm odeoverthem/zrangefrom700to5000foratotalof5000sho ts.

LC-ESI-iontrap- MS/MS

Nano-liquidchromatography- tandemmassspectrometry(nanoLC-

MS/MS)wasperformedwiththeuseofanUltimate3000 LCsystem(Dionex,Amsterdam ,TheNetherlands).Ali quotsofthe2-AAlabeledN-

glycans(1μl)wereappliedtoaC18PepMap™0.3×5mmtrappi ngcolumn(Dionex)andwashedwith100%solventA(0.1

%formicacidinwaterand0.4%ACN)for10minataflowrateof 25μl/min.Next,AA-

labeledglycanswereseparatedonareversephaseanalyticalco lumn(C18PepMap100Å,3μm,75μm×150mm;Dionex)atafl owrateof300nl/minwiththeUVdetection.Themobilephaseg radientwasasfollows:0–

25%eluentB(95%ACN,5%water)in15minand25–

70%eluentBinthenext10min,followedbyanisocraticelutio nwith70%eluentBfor5min.TheLCsystemwascoupledvi aanonlinenanospraysourcetoanEsquireHCTUltraESI- iontrap-MS(BrukerDaltonics).Forelectrospray(1100–

1250V),stainlesssteelcapillarieswithaninnerdiam-

eterof30μm(Proxeon,Odense,Denmark)wereused.Thedryg astemperaturewassetto165°C,andthenitrogenstreamwassett o7 l/min.Theglycanspectrawereacquiredinpositive- ionmodeandthemassspectrometerwascarefullytunedtomin imizeglycandecayintheiontransferregion,thankstothefoll owingsettings:skimmer,40V;capillaryexit,106V;octopole1 DC,6V.TheAA-

labeledglycanswereanalyzedusingthedata- dependentMS/MSmodeovera 300–

1500m/zrange.FiveofthemostabundantionsinanMSspectr umwereselectedforMS/MSanalysisbycollision-

induceddissociation(CID).TheLC-

MSsystemwastunedtominimizetheeffectofin-

sourcedecayofsialylatedstructuresasdescribedpreviously[38 ].

Statisticalmethods

Unlessotherwiseindicated,theStudent’st-

testwasperformedtodeterminestatisticalsignificancebetwee ntheaverageof3replicates.

Result s

Initially,tochecktheeffectoftransfectiononglycosylationstat usinstudiedcellsweanalysedtheMALDI-TOF-MSspec- traofparentandtransfectedcells(SupplementaryFig.2).Wedid notobserveanysignificanteffectofthetransfectionitself.Infurt herstudiesweusedmock-transfectedcelllineasaneg-

ativecontrol.

CharacterizationofN-

glycanepitopesonmembraneandsecretedproteinsu singplantlectins

AsaninitialcharacterizationoftheimpactofupregulationofGn T-IIIontheN-

glycanrepertoireinmelanomacells,weevaluatedthebin dingofselectedplantlectinstomembraneandsecretedglycopr oteinsblottedontoPVDFmembrane.Wechoseanarrayoflectin srecognizingmostcharacteristicgly-

canepitopes(seeTable1).Asthebuffersusedforextractionofsu bcellularproteinfractionswerenotcompatiblewithanyofthepr oteinassay,toavoidtheeffectofinequalityofproteinloadthelect inbindingstrengthwascalculatedasarelativeopticaldensityo fallreactiveproteinbandsineachlanewiththeCoomassieBrilli antBlue(CBB)stainedproteinlanesasaloadingcontrol.Weobs

ervedsimilarlectinbindingpatternsinthecaseofmembraneandse cretedproteins,suggestingasim-

ilarglycosylationstatusofglycoproteinsinbothproteingroups.

OverexpressionofGnT-IIIdidnotappeartoinfluence

thebindingstrengthofSNAandM AAtomembraneandse cretedproteins,suggestingnodifferencesintheamountofα2–

6andα2–

3sialylatedglycans,respectively(Figs.1and2).Asimilarobse rvationwasdoneinthecaseofGNAstain-

ing,recognizinghigh-mannoseN-

glycans(Figs.1and2)andAAA,whichspecificallybindstof ucose(Figs.1and2).PHA-

Estaining(detectingBbisecting^GlcNAc)wassignifi- cantlyincreasedinthecaseofbothproteingroupsisolatedfro mWM266–4-GnT-

IIIcellsincomparisontocontrolcells(Figs.1and2).Surprising ly,thestainingpatternwithPHA-

L(Figs.1and2),whichspecificallyrecognizesβ1–

6branchingofcomplexN-

glycanswassimilar,suggestinganelevationofPHA-L- reactiveglycoproteinsorglycansuponoverexpres- sionofGnT-III.

HILIC-HPLCanalysisofN-

glycansderivedfrommembraneandsecr etedproteins

TostudytheglycosylationchangesuponGnT-IIIoverexpres- sioninmoredetails,weperformedHILIC-HPLCof2- AAlabeledN-

oligosaccharidesderivedfrommembraneandse- cretedproteins.Incaseofmembraneprotein- derivedneutralN-

glycansthemostabundant,bothincaseoftransfectedcellsandi nthecontrol,were5peaksa,b,d,e,fandg(Fig.3a)withGUvalues of6.13,7.00,7.87,8.72,9.40and10.05,respec-

tively.Similarly,incaseofsecretedproteinglycans,themostin tensivefluorescencerepresentedpeaksa,b,d,e,fwithnopeakg present,butwithadditionalpeakcwithGUof7.43(Fig.3b).O verexpressionofGnT-

IIIcausedtheappearanceofadditionalpeaks(Fig.3),whichinc aseofmembraneproteinshaveGUvalueso f 6.78,7.22and7.

52( peaksi ,j andk,respectively;Fig.3a)andincaseofsecreted proteinderivedglycanshaveGUvaluesof6.41,6.76,7.22,7.53 ,8.22(peaksh,i,j,k,andl,respectively,Fig.3b).Theseaddition alpeaksrepresentedapproximately9%ofallneutralspeciesfr ommembraneproteins,whereasincaseofsecretedproteinsap -proximately47%ofallneutraloligosaccharides.

Asimilaranalysiswasmadewhenitcomestonegativelycha rgedN-glycansfrombothproteingroups(Fig.4).GnT- IIIupregulationresultedintheappearanceofadditionalpeaks1 3and14withGUvaluesof8.39and9.07,respectively.There lativeabundanceoftheseadditionalpeaksrepresentsappro x.32%ofallchargedglycansincaseofmembranepro-

teins,andapprox.12%ofchargedspeciesfromsecretedglyco proteins.

Toelucidatethestructuresofglycansrepresentedbyselect- edpeaks,HILIC-

HPLCanalysisofglycanpoolsorcollectedindividualpeakstr

eatedwithspecificexoglycosidaseswasperformed(forenzy mesusedinthestudyseeTable2).Afterdigestionofneutralspec ieswithAspergillussaitoiα1–

2mannosidase(ASM)thepeaksb,d,eandfcollapsedtoasing lepeakof6.13GUcorrespondingtoMAN5,relyingontheribon ucleaseBglycanlibrarydigestionasanexternal

222 GlycoconjJ ( 2018)3 5:217–231

Fig.1L ectindetectionofglycoproteinsblottedonPVDFmembrane.Mem braneproteins(a)andsecretedproteins(b)isolatedfromcontrolcells(1:W M266–4-pIRESneo)andGnT-IIIoverepressingcells(2:WM266–4- GnT-III)wereresolvedbySDS-

PAGEandblottedglycoproteinswereprobedwithspecificlectins(seeT able1).CBB–

CoomasieBrilantBlue-

stainedblot.Asthebuffersusedforextractionofsubcellularproteinfract ionswerenotcompatiblewithanyoftheproteinassay,thesamplesw erepreparedbyvolumeandtheopticaldensityofpositiveproteinbands wasthannormalizedtoCBBstaining(Fig.2)

standard(Fig.5aandb).Thus,theidentityofthepeaksb,d,eandfa shighmannosestructures(MAN6,MAN7,MAN8andMAN9,re spectively)wasconfirmed.Thesinglepeakspecificonlyforthe membraneproteinglycanfraction(peakg),wasalsosusceptibl etoASMtreatmentandcollapsedtoasinglepeakof8.61GU(Fig .5a).Thissuggestedthepresenceoftheterminalglucoseonthe MAN9glycan(GlcMAN9)inthiscase.Toprovethis,thepeakw asmanuallycollectedandtreat-

edadditionallywithratliverα1–

3glucosidaseII(RLGII),andafterdigestiontheGUshiftfrom 10.05to9.40(corre-

spondingtoMAN9)wasobserved(Fig.5c).

Thepeaks,thatwerecharacteristiconlyfortransfectedcells(W M266–4-GnT-

III)werealsopartiallycharacterizedwiththeuseofbovinetestesβ1- 3-4galactosidase(BTG),Arthrobacterureafaciensα2-3- 6sialidase(ABS)andASM.Asthemain3peaksofneutralspecies(p eaksi,jandk,Fig.3)didnotshiftaftermannosidasetreatment(Fig.5 a),theywereprobablycomplexN-

glycans.OnthebasisoftheeffectofBTGdigestionandGUvaluesofi solatedpeaksiandk,theywereidentifiedasdiantennaryfucosylat edstructureswithBbisecting^GlcNAcandone(Hex4NexNA c5Fuc1-2-

AA)ortwoterminalgalactoses(Hex5HexNAc5Fuc1- AA),respectively(Fig.5d).Intermsof

Fig.2Relativeo pticald ensityo f l ectin-

positiveg lycoproteinb andsonPVDFmembrane.Membraneproteins(a) andsecretedproteins(b)isolatedofcontrolcells(WM266–4-

pIRESneo)andGnT-IIIoverepressingcells(WM266–4-GnT- III)wereresolvedbySDS-

PAGEandblottedglycoproteinswereprobedwithspecificlectins.Optical

GlycoconjJ ( 2018)35:217–231 3

densityofeachlanewasmeasuredandpresentedinrelationtoopticalden sityofloadingcontrol(CBBstainedblots).Threeindependentexperime ntswereperformed.Dataarepresentedasmeans+/

−standarddeviation(SD).Asteriskindicatesstatisticallysignificantdiffe rence(*p<0.05,**p<0.01;Studentt-test)

Fig.3 H ILIC-HPLCs e p a rationofn e u tralN -

glycans.M e mbraneproteins(a)andsecretedproteins(b)ofcontrolcells(

WM266–4-pIRESneo)andGnT-IIIoverepressingcells(WM266–4- GnT-III)weredeglycosylatedand2-

AAlabelledneutralglycanswereseparatedon

TSKgelAmide-

80column.Themajorpeaksareindicatedwithletters.Theblacklettersrepr esentthepeakspresentincaseofbothcelllines,redlettersindicatepeakschar acteristiconlyforcellsthatoverexpressGnT-

III.GU-glucoseunits

chargedN-

glycansoftransfectedcellsthetwocharacteristicpeaks(peak13and 14)bothcollapsedafterABStreatmentand,onthebasisoftheirGUv aluestheywereannotatedasdiantennaryfucosylatedstructures withBbisecting^GlcNAcandone(Hex5NexNAc5Fuc1Sia1-2- AA)ortwo(Hex5NexNAc5Fuc1Sia-2-

AA)terminalsialicacidresidues(Fig.5e).

IdentificationofN-

glycansfrommembraneandsecretedprotein sbymassspectrometry

2-AAlabeledN-

glycansfrombothcelllinesandbothproteingroupswerefurther purifiedandanalyzedbyMALDI-TOF-

MS(Figs.6and7).Inaddition,toelucidatestructuralfeaturesoft heN-glycansall4sampleswerestudiedbyLC-ESI-iontrap- MS/MS.ThefragmentationspectraofselectedN-

glycansaregatheredinSupplementaryFig.1.Theglycansidenti

fica-tionwasbasedonfragmentationofselectedglycansandcom- monknowledgeofglycobiology.TheConsortiumforFuncti onalGlycomics(CFG)notationwasusedforthe

schematicrepresentationofN-

glycanswithinmostcasesnospecificationofthelinkagepositi onincaseofisomericstruc-tures,unlessindicatedotherwise.

ThestructuralannotationofglycansissummarizedinSup plementaryTable1.Ingeneral,90differentN-

glycanstructureswereidentified,with29ofthempresentonlyin caseofWM266–4-GnT-

IIIcells.Thus,theappearanceofthesestructuresmaybeatt ributedtotheoverexpressionofGnT-

IIIinmelanomacells.ThemajorpeakswithintheN-

glycansofmembraneproteinsisolatedfromcontrolcellswere identifiedashighmannosestructuresMAN6,MAN7,MAN8an dMAN9withm/z1516.45,m/z1678.55,m/z1840.60andm/z 2002.64,respectively.Thisisconsistentwiththedatafro mHILIC-

HPLCanalysis,wherethesestructureswerealsoidentifieda smajorpeakswithinthechromatogram(Fig.5).Anotherhig hmannoseglycanwasalsoidentifiedatm/z1354.39asMAN

5,togetherwiththemonoglucosylatedMAN9glycanatm/z21 64.72,inlinewiththeresultsoftheHPLCanalysis(Fig.5).T hesumofrelativeabundancesofallhigh-

Fig.4 H I L I C-HPLCs e p arationo f n e g a tivelyc h a r g e d N - g l ycans.Membraneproteins(a)andsecretedproteins(b)ofcontrolcells (WM266–4-pIRESneo)andGnT-IIIoverepressingcells(WM266–4- GnT-III)weredeglycosylatedand2-AAlabelledchargedglycanswere

separatedonTSKgelAmide-

80column.Themajorpeaksareindicatedwithnumbers.Theblacknumbers representthepeakspresentincaseofbothcellines,rednumbersindicatepea kscharacteristiconlyforcellsthatoverexpressGnT-III.GU-glucoseunits

mannoseN-glycanpeakswithintheMALDI-

MSspectrumofmembraneproteinderivedglycanswas58%.

Additionally,unusualsmallpaucimannosidicstructureswith orwithoutcorefucosewereidentified(eg.Hex4HexNAc2- AAandHex3HexNAc2dHex1-2-

AAatm/z1192.35andm/z1176.36,respectively).Othermajor peaksthatwereobservedincaseofthemembraneproteinfractio nofcontrolcellswereatm/z

2051.69,m/z2197.76,m/z2488.92,m/z2562.88,m/z 2854.07,m/z2927.98,m/z3145.19andm/z3219.15.Thesepe aksweremainlycore-fucosylatedcomplexN-

glycanswithterminalsialicacidresidues(1–

3)anddifferentnumberofN-

acetyllactosaminyl(LacNAc)unitsorantennae(2–4) (SupplementaryTable1),andthesumoftheirrelativeabun- danceswithinthespectrumwas23%.Amongtheseglycans,the peakatm/z3145.19wasannotatedastriantennarystruc- t ureco ntaini n g3term i n alsi ali caci dresi d ues

Table2Listof

exoglycosidasesusedtos tudythestructuresofN-

Exoglycosidasesource Abbreviation (Hex6HexNAc5dHex1NeuNAc3-

AA).Anotherpeakwith3or4LacNAcunitscanrepresenteith ertri-ortetra-antennary

glycansbyHILIC-HPLC α1-2mannosidase Aspergilluss aitoi β3-

4galactosidasebo vinetestes α,1-3glucosidaseII ratliver

α2- 3- 6sial idase Arthr obacteru r eafaciens

ASM BTGRTG ABS

UND

ABS ^GU

13

14

UND ABS

G

glycansordiantenn arystructureswithp oly-LacNAcexten- sionsoftheantenna e.Takingintoaccou

nttheMS/MSdata weobserveinthesec asesclearintensives ignalsatm/z366.0[

M+ H]⁺

(He x1H

ex N A

c1)andm/z657.2[M+ H]⁺

(Hex1HexNAc1NeuNAc1)indicatingthepresenceofindivid- ualantennae(seespectra41–

48inSupplementaryFig.1).Moreover,inthecaseofallthelarg estructures,thefragmen-

tationspectradidnotcontainoxoniumionswhichwould

a

UND

GU

6 7 8 9 10

b

UND

GU

6 7 8 9 10

GU6,13 GU7,00

c

7 8 9 101 1 12 UND

GU6,13

GU7,84GU8,72 GU9,40

GU6,13

ASM

GU8,6 1

ASM

ASM

GU8,61

GU9,40

RLGII

d e

UND

BTG ^GU6.13

GU6.76

GU7.53

UND

BTG ^GU6.13

Fig.5HILIC-HPLCcharacterisationofselectedneutralandchargedN- glycanpeaksfromWM266–4-GnT-

IIIcells.Proteinsweredeglycosylatedand2-AAlabelledN- glycanswereseparatedonTSKgelAmide-

80columnbeforeandafterdigestionwithexoglycosidases(seeTable 2).PanelarepresentstheeffectofneutralN-glycanpooltreatedwithα1–

2mannosidase(ASM).Panelbshows

theeffectofdigestionofribonucleaseBhighmannoseglycanstandardwith ASM.PanelCrepresentstheeffectofASMandα1–

3glucosidaseII(RLGII)treatmentonisolatedpeakg.Paneldrepresentsthee ffectofβ1–

3,4galactosidase(BTG)treatmentonisolatedpeaksiandk.Panelerepre sentstheeffectofα2-3-

6sialidase(ABS)treatmentonisolatedpeaks13and14.UND–

undigested,GU-glucoseunits

suggestthepresenceofpoly-

LacNAcextensionsoftheb r anches( e .g.co m p os i t i on sH e x2H e x N Ac2orHex2HexNAc2NeuNAc1).Basingontheseobser vation,wecansafelyconclude,thatinthesecasesweobservedtri -

andtetraantennarycomplexstructuresratherthandiantennary N-

glycanswithLacNAcrepeats.Intermsofsecretedproteinsfracti onofcontrolWM266–4-

pIRESneocells,themajorpeakswithintheN- glycanprofiledidnotrepresenthigh-

mannosespecies.Sevenmajorpeakswerepresent,withthepredo minantsignalsatm/z2198.01(approx.31%ofallgly-

cans)andm/z2489.19(approx.15%ofallglycans),whichwerei

dentifiedascorefucosylateddiantennarycomplexstructures

withone(Hex5HexNac4dHex1NeuNAc1-AA)and two(Hex5HexNAc4dHex1NeuNAc2-2-

AA)terminalsialicacids,respectively.Theotherstructureswer eidentifiedmainlyascomplexglycanswithcorefucose,termin alsialicacidres-

iduesandadifferentnumberofLacNAcunits,similarlyasinthec aseofmembraneproteinfraction.Alongwiththesemainpeaks,a lsosomeweakersignalsweredetected,bothincaseofmembrane andsecretedprotein-

derivedglycans,mainlyasfucosylatedcomplexstructures withadifferentnumberofterminalsialicacids(1–

3)andLacNacunits(4–6)

(Figs.6and7andSupplementaryTable1).Thesepeakswerepre sentatm/z3510.57,m/z3584.16andm/z3658.20.Finally,weal soobservedanunusualcore-fucosylatedMAN5N-glycanatm/z 1500.54withinthemembraneprotein-derivedfraction.

1354,371354,39 1395.251395.28 1516.211516.27 1541.241582.241541.26 1582.30 1678.221678.29 1760.261760.32 1801.27 1840.251840.31 1906.291906.34 1947.29 2002.272003.30 2035.26 2068.302052.38 2110.30 2165.26 2198.302164.33 2198.38 2243.372255.32 2344.302360.31 2401.362417.362417.41 2475.36 2490.41 2563.412563.50 2604.42 2693.382709.38 2766.44 2782.42 2840.47 2855.58 2929.462929.39 2969.44 3058.31 3132.47 3147.76 3220.86 3424.74 3497.36

a

2x2x 2x

2x 3x

3x

4x

b

3x 3x

2x 4x

2x 2x

2x

1500 1750 2000 2250 2500 2750 3000 3250 3500

m/z

Fig.6NegativeionmodeMALDI-TOF-MSspectraofAA-labeledN- glycansreleasedfrommembraneproteinsofWM266–4-pIRESneo (a)andfromWM266–4-GnT-

III(b)melanomacells.ProposedcompositionoftheN-

glycanstructuresdeducedfromtheMALDI-TOF-MSandLC-ESI-ion- trap-

MS/MSarelistedinsupplementaryTable1.Onlymajorstructuresaredepi cted.Onlythem/zvalueswhichareconsideredasantransfectioneffectare annotatedinb.Insomecases

structuralisomersarepossible.Fucoselinkageisspecifiedonlyincaseswher eLC-ESI-ion-trap-

MS/MSgivesevidence.G lycans chemeswerepreparedu singGlycoWorkbe nch.T hes pectraandm /zv alueso btainedintheMALDI-

TOFlinearmodearepresentedhere.Redtriangle,fucose;yellowcircle,gala ctose;greencircle,mannose;bluesquare,N-

acetylglucosamine;purplediamond,sialicacid

TheoverexpressionofGnT-

IIIinthesemelanomacellsresultedintheappearanceof29N -

glycans,whichwerenotobservedincaseofcontrolcells.Mos tofthemwereBbisected^structuresandwerepresentbothwith inmembraneprotein-derivedN-

glycansaswellassecretedprotein-

derivedfraction.ThepresenceoftheBbisecting^GlcNAcresid uewasinmajorityofcasesconfirmedinfragmentationspectraa ndthefragmentionsatm/z911.4[M+H]⁺⁽H1N3-AA),m/z 1056.4[M+H]⁺⁽H1N3Fuc1-AA)andm/z1276.6[M+

H]⁺⁽H2N4-

AA)wereindicativeinthiscase(SupplementaryFig.1,Supple mentaryTable2).Themajorsignal,whichis

stronglyupregulatedinthetransfectantswasobservedatm/z 2400.7andidentifiedasdiantennaryfucosylatedcomplexstru cturewithsingleterminalsialicacidresidueandBbisecting

^GlcNAc(Hex5HexNAc5dHex1NeuNAc1-2-AA) (SupplementaryTable1,Figs.6and7).Therelativeabun- danceofthisoligosaccharideisapproximately15%ofmembra ne-derivedN-glycansandabout23%forthesecreted

protein-

derivedfraction.OthermajorpeakswereidentifiedasBbisected

^d iantennaryg lycanswitho r w ithoutc oref ucoseandw i thd if ferentl engtho f t hea n tennae.I nterestingly,t headditiono f t h e B bisecting^G l c N Aca s a r e s u lto fG n T-

IIIoverexpressiona lsoo ccurredi n c aseo f h ybridg lycans,a nd 4differentspecieswereidentifiedatm/z1922.5,m/z2068.6,m/

z2213.6a ndm /z2359.7( Hex6HexNAc4witho r w ithoutsialica cid,andwitho rwithoutcorefucose).Finally,thead-

ditionofBbisecting^N-acetylglucosamineinWM266–4-GnT- IIIc ellsi nvolveda lsoN -

glycansc ontainingm oret hantwoL acNAcr epeats.T hem ajor p eaksw ithint hisg roupo f o l i g osa c c ha r i d e sw er ed e t ec t eda tm / z2 4 7 4 . 7( H e x6H e x N Ac6d H ex1-

A A ),m / z2 7 6 5. 8(Hex6H exNAc6dHex1Neu NAc1- A A),m/z3056.9

(Hex6H exNAc6dHex1NeuNAc2- A A),m / z3130.9 (Hex7HexNAc7dHex1NeuNAc1-

AA)andm/z3422.0(Hex7HexNAc7dHex1NeuNAc2- AA).ThesecanbeidentifiedasBbisected^tri-

ortetraantennarycomplexstructuresfrom

1354,311354,59 1395.01 1516.241515.96 1582.221597.91 1639.261678.22 1677.92 1744.20 1759.95 1785.27 1831.971840.14 1906.251905.95 1947.271946.91 2001.89 2035.262069.242051.93 2110.25 2178.90 2198.232197.94 2239.30 2238.972271.91 2313.25 2343.99 2383.27 2401.322416.89 2475.192470.93 2489.96 2547.252562.99 2604.27 2674.36 2693.37 2766.38 2840.332855.02 2928.02 2969.45 3058.72 3132.173146.24 3219.73 3423.81

a

2x 2x

2x 3x

2x

2x 3x

b

3x

2x 3x

2x 2x

2x 2x

1500 1750 2000 2250 2500 2750 3000 3250 3500

m/z

Fig.7N egativeionmodeMALDI-TOF-MSspectraofAA-labeledN- glycansreleasedf romp roteinss ecretedb y W M266–4-

pIRESneo( a)andWM266–4-GnT-

III(b)melanomacells.ProposedcompositionoftheN-

glycanstructuresdeducedfromtheMALDI-TOF-MSandLC-ESI-ion- trap-

MS/MSarelistedinsupplementaryTable1.Onlymajorstructuresaredep icted.Onlythem/zvalueswhichareconsideredasantransfectioneffectare annotatedinb.Insomecasesstructuralisomers

arepossible.FucoselinkageisspecifiedonlyincaseswhereLC-ESI-ion-trap- MS/MSgivesevidence.GlycanschemeswerepreparedusingGlycoWorkbe nch.Thes pectraa ndm /zvalueso btainedint heMALDI-

TOFlinearmodearepresentedhere.Redtriangle,fucose;yellowcircle,galact ose;g reenc i rcle,m a n nose;b l u e sq uare,N -

acetylglucosamine;purplediamond,sialicacid

thes imilarr easonas in caseo fc ontrolc ellsa nda llo ft hemwerec haracteristicf orb othm embranea nds ecretedp rotein-

derivedglycans.Interestingly,inbothcaseswealsoidentifiedtrisi alylatedstructures:t riantennaryBbisected^glycanat m/z 3348.2(Hex6Hex NAc6d Hex 1NeuNAc3)andtri-

ortetraantennaryB bisected^structureatm/z3713.2(

Hex7HexNAc7dHex1NeuNAc3-2- AA)amongsecretedprotein-

derivedglycans.SomeotheroftheidentifiedN-

glycanswerepresentonlyincaseofoneoftheproteinfrac- tions.Forexample,aninterestingBbisected^glycanatm/z 2645.8(Hex4HexNAc7dHex3-

AA),werefoundonlyincaseofthesecretedproteinfraction(Su pplementaryTable1).Bycontrast,twohighlybranchedcomple xstructureswithpoly-

LacNAcextensionsandBbisecting^GlcNAcwerefoundatm/z 3787.1(Hex8HexNAc8dHex1NeuNAc2-AA)andm/z 3861.2(Hex9HexNAc9dHex1NeuNAc1-AA)onlyincaseof

themembraneprotein-derivedN- glycans(SupplementaryTable1).

Finally,wealsoobservedpeakssatellitetosome sialylatedcomplexN-

glycansandbasingonthemassshiftof18Daweidentifiedthe masstructurescontainingl ac t oniz edN -

a c et y l n eur am i n i ca c i dr e s i d ues(NeuNAcLac) (Figs.6and7andSupplementaryTable1).Astheinternal esterificationconcernsα2-3-

l i nk e ds i a l i ca c i d ,t h eg l y c a nsa tm /z2 1 9 7 . 6(Hex5Hex NAc4dHex1NeuNAc1- 2-

AA),m/z2400.7(Hex5Hex NAc5dHex 1NeuNAc1- 2 - A A),m/z2488.8

(Hex5H e x NAc4d Hex1NeuNAc-2-AA),m/z2691.8 (Hex5Hex NAc5dHex 1NeuNAc2- 2 -

A A),m/z2853.9(Hex6HexNAc5dHex1NeuNAc-2- AA)andm/z3056.9(Hex6HexNAc6dHex1NeuNAc2-2- AA)wereidentifiedascontainingatleastoneterminalα2-3- linkedNeuNAc.

Discussion

N-AcetylglucosaminyltransferaseIII(GnT- III)catalyzesthesynthesisofBbisected^N-

glycanstructuresbythetransferofasingleGlcNAcresiduetothe β-

mannoseofthecore.Thisreactionisoneofthecriticalstepsofthe glycosylationpath-

way,andtheresultingBbisecting^GlcNAchasuniquefea- turesamongallotherN-

glycansmodification.Firstofall,incontrasttootherGlcNAcbra nches,itisnotfurtherelongatedbyothertransferases[6].Second ly,intermsofthetopographyofglycosylationmachinerywithin Golgiapparatus,theactionofGnT-

IIIisveryoftenconsideredasaBstop^signalbecausethepresen ceofitsproductpreventstheactionofotherGlcNActransferas essuchasGnT-II,IVandVbutalsocoreα1-

6fucosyltransferaseandαmannosidaseII[39].Thisgivestheen zymethepotencytobeakindofaBswitcher^,whichthroughthe changesofitsexpressionlevelandtheactivity,couldregulatet heformationofcomplexN-

glycanstructuresofthecellularglycoproteins.Thus,thebiologi calroleofGnT-

IIIisunderintensiveinvestigation,partiallyintermsofitsimp actoncancercellbehavior.

Previously,wedescribedthemelanomacellularmodel,inwhi chWM266–

4metastaticmelanomacellswerestablytransfectedwithMAG T3gene[28].AlthoughtheupregulationofGnT-

IIIisfunctionalbecauseithasresultedinthemodifica-

tionofMelanomaCellAdhesionMolecule(MCAM)glycansbyi ntroducingBbisecting^GlcNAcresidue,ithasnotsignifi- cantlyinfluencedthetransendothelialinvasivenessofthecellsinv itro.TheWM266–

4melanomacelllinewaschosenasatransfectionhostpartiallybec auseitwasdescribedasexpress-

ingahighlevelofhighlybranchedoligosaccharides(tri- andtetraantennaryN-glycans)

[40].Theaimofthepresentstudywastocharacterizeindetailthein fluenceofGnT-IIIoverex-pressionontheN-

glycanrepertoireofmembraneandsecretedproteinsinthesemeta staticmelanomacells.WeusedWM266–4-

pIRESneocellsasatransfectioncontrolandWM266–4-GnT- IIIcelllineasamodelofGnT-IIIoverexpression[28].

ThestrongerreactionofglycoproteinswithPHA- EuponGnT-

IIIoverexpressionisalogicalconsequenceoftheen- zymeupregulationandisusuallytreatedasapositivecontrolofs uccessfultransfectionofcells[7,8,41,42].Surprisingly,wedid observealsostrongerstainingwithPHA-

L,whichwouldsuggestthatGnT-IIIup- regulationledalsototheele-vationofβ1–

6branchingofcomplexN-

glycanstructures.Thisremainsincontrasttothefact,thataction

ofGnT-IIIisconsideredasinhibitorytowardsGnT- Venzyme,whichwasobservedbyweakenedPHA-

LstaininginmanycasesofMGAT3transfectants[7,13,43].Toou rknowledge,thisre-

porttogetherwithourpreviousstudies[28]aretheonlyde- scriptionsofelevatedPHA-

LstainingofglycoproteinsuponGnT-

IIIoverexpression.Theoneexplanationforthisphenom- enonisthepossibleinfluenceofGnT-IIIontheGnT-

Vactivityoritsexpressionlevel.However,previouslyweshowe dno

impactofMGAT3transfectioneitherontheexpressiono fGnT-Vnoritsactivityinvitro[28].Theotherpossibleexpla- nationisthattheintroductionofBbisecting^GlcNAcresidues causesmoreglobalglycomiceffects,whichinturnschangesth ebindinginteractionsofsomepreviouslyexistingN- glycansstructureswithPHA-

L.Toinvestigatethiseffect,weperformeddetailedglycomica nalysisofN-

glycansisolatedfromcellularglycoproteinsofstudiedcells.

TheelevatedpresenceofBbisected^biantennaryglycans, whichweobservedincaseofGnT-

IIIoverexpressingWM266–4-GnT-

IIIcells,hasbeenoftendescribedasthere-sultofGnT- IIIoverexpression,buttherearenotmanydetaileddescription sofglycomicchangesinthesecases.Iharaetal.showedtheap pearanceofdiantennarystructurewithBbisecting^GlcN AcresidueonserumglycoproteinsinGnT-

IIItransgenicmouse[41].Koyotaetal.presentedtheupreg- ulationofBbisected^diantenneryoligosaccharidesinswine endothelialcellsoverexpressingGnT-IIIwiththesimulta- neousdownregulationoftri-

andtetraantennarysugars[44].Intermsofcancerbiology,th ebiologicalfunctionofsuchmodificationsofcellularglyco mehasbeendescribedmainlyasmodulatorofcellularmigrati onandinvasiveness.Inthelightofthesedata,GnT- IIIindeedcanact asametastasisinhibitor,butinthemajor ityofpublishedcases,thiseffectislinkedtothesimultaneous,al mosttotaldown-regulationofN-

glycanbranchingasaresultofcompetitionbetweenGnT- IIIandGnT-Vforanacceptorsubstrate.

Interestingly,ourdatashowthatbesidesthediantennaryg lycans,intowhichtheBbisecting^GlcNAcisintroducedu ponGnT-IIIoverexpression,othertypesofglycansaremod- ifiedinthismanneraswell.IncaseofbranchedBbisected^str uctures,onetriantennaryandoneatleasttriantennarysialylat edcomplexglycanswereidentifiedatm/z3348.2(Hex6H e x

NAc6d H ex1N e u N Ac3)andm / z371 3.2(Hex7HexNAc7dHex

1NeuNAc3-2-

AA),respectively.Addingtothat,abroadgroupofBbisecte d^N-

glycanscontainingLacNAcunitswereannotated,startingfr omsimplecorefucosylatedandnon-

sialylatedstructureatm/z2474.8(Hex6HexNAc6dHex1-2- AA)uptomonosialylatedoligosac-

charidecontainingsixLacNAcunitsatm/z3861.2(Hex9

NeuNAc9dHex1NeuNAc1-2-AA).Basingonmassanal- ysisandfragmentationdataitwasnotpossibleheretomakeacle ardistinctionbetweenhighlybranchedstructuresanddiante nnaryglycanswithpoly-

LacNAcextensions.However,astheLC-ESI-iontrap- MSdataindicatesahighlevelofsialylationandthefragmentat ionspectradonotgiveanevidenceforthepresenceofLacNAc repeatsinstudiedsamples,itismorelikelythatthemultianten naryN-

glycansprevail.Addingtothat,Kinoshitaetal.havedescribed

aglycomeofWM266–

4melanomacellswhichweusedasatransfectionhost,ascontain ingahighamountoftri-andtetra-antennarycomplexN- glycans[45].Basingonmassanalysis,ourresultsconcerningth eglycosylationofcontrolcells

(WM266–4-

pIRESneo)areinlinewiththesedata.Incaseofthetransfectants(

WM266–4-GnT-

IIIcells),wepresumethatthesehighlybranchedN- glycanswerealsomodifiedbyGnT-

III.ItisevidentlyvisiblewhencomparingtheMALDI-

TOFspectraofcontrolandtransfectedcells,whilethereisasignif -

icantshiftofmainpeaks(besideshighmannoseglycans)ofthec onstantvalueofm/z203,whichcorrespondstoasingleGlcNAc residue(Figs.6and7).

ThepresenceofBbisecting^GlcNAcinthetri-andtetra- antennaryglycansisveryunusual,becauseoftheabilityofthis modificationtoblocktheactivityofotherGlcNActrans- ferases,whichwasmentionedabove[46].AfewyearsagoKlis chetal.describedthehighcontentoftri-andtetra-

antennary,corefucosylatedN-

glycanscontainingBbisected^GlcNAconpregnancy- associatedglycoproteins(PAGs)inru-

minants[47].Theoccurrenceofsuchstructurescouldbetheresu ltofthespecificspatialdistributionofglycosyltransfer- aseswithintheGolgicompartments.Theintroductionofsin- gleGlcNAcresiduetotheβ-

mannoseofthecoreofhighlybranchedstructureswouldbeena bledbythelocalizationofGnT-IVand-

VearlierwithinthesecretorypathwaythanGnT- III. Interestingly,itwasproved,thatcaveolin- 1byformingcomplexeswithGnT-

IIIcanactasaregulatorofitsdistribu-

tionwithintheGolgi[48].Theexpressionofcaveolin- 1inmelanomacellshasbeenstudiedbyBełkotetal.,andtheir workshowedthatincaseofWM266–

4melanomacellsitsexpressionisrelativelylowandthedistri butionwithinthecellsdonotshowlocalizationoftheproteinin ERandGolgiapparatus[49].Itispossible,thatinWM266–4- GnT-IIImel-anomacellsstudiedhere,thelevelofcaveolin- 1isthustoolowtolocalizeGnT-

IIIintheearlycompartmentsofthese-

cretorypathway.Fromtheotherhand,weobservethepres- enceofhybrid,Bbisected^glycans,whichformationprobablyo ccurswithintheearlyGolgiandcouldbetheeffectofαmann osidaseIIinhibitionbyGnT-IIIproduct[50].

Incaseofmelanomamodeldescribedhere,GnT-

IIIactionseemstoinvolvealmostallhybridandcomplexN- glycansregardlessoftheirmolecularmass,leadingtoabroadglyc omiceffect.But,themechanismsandbiologicalconsequences ofsuchmodificationremainschallengingtostudyindetail,par- tiallybecausethemutualinterplayofglycosyltransferaseswith- insecretorypathwayisstillapuzzle.Firstofall,anambiguouspictu reoftheroleofBbisected^N-

glycansincancerallowstoraisetheassumption,thatthecellular context(forexampleoveralllevelsofdifferentbranchingglyc osyltransferases)maycontributetodeterminingstructureand functionofBbisected^glycans[51].Thereisalsogrowingnumbe

rofev-

idencethatglycosyltransferasescanformhomomersandhetero mers,andthebalanceoftheformationofthesespeciescaninfluence theactivityoftheenzymesitself[52].Theele-

vatedreactivityofcellularglycoproteinswithPHA-

LthatweobservehereisforsuretheconsequenceofelevatedGnT- IIIactioninthecells.Itisknown,thattheintroductionof

Bbisecting^GlcNAcresiduechangesthespatialshapeofN- glycansandthuscanmodulateitsbiologicalproperties[4].Asin glesugarsubstitutionoftheN-

glycancoreisespeciallystudiedinthiscontext,givingevide ncethatBbisecting^GlcNAcandcorefucosecanbetreatedas molecularswitchesofconformationalbehaviourofglycanst ructures[53].Recently,ithasbeenshown,thatBbisecting^Glc NAccanre-

stricttheconformersofbranchedstructuresbytheformationoft he‘back-fold’conformation[54].Asaconsequence,thegly- canbindingtospecificlectincanbechanged.Itispossiblethatthe strongerbindingofglycoproteinsfromWM266–4-GnT- IIIcellstothePHA-

Listheresultofaconformationalchangemadebytheintroducti onofBbisecting^GlcNAcintohighlybranchedstructures,whic hleadstothepromotionofPHA-

Lbinding.Itcannotbeexcluded,thatsimilarmodifyingeffectasa resultofGnT-IIIoverexpressionwouldbeobservedinmela- nomacellsinvivo,ifothercarbohydrate-

proteininteractionsweretakenintoaccount.Incaseofbranchedg lycansthestudiesconcerningtheirinteractionswithmammalian lectinsarehow-

everlimited[55].But,thepotentialofBbisecting^GlcNActobe amodificatoroftheseinteractionsseemstobeworthtoconsid er,asweknow,forexample,thatmousedendriticcellinhibitoryr eceptor2(mDCIR2)recognizesnotonlytheα1–

3branchbutalsoaBbisecting^GlcNAcresidueofdiantennaryc omplexglycans[56].

Toconclude,ourstudiesprovidethedetailedglycomicdata oftheeffectofGnT-

IIIoverexpressioninmelanomacells.Weshowedthattheenzy mecanmodifyhighly-branchedN-gly-

cansbytheintroductionoftheBbisecting^GlcNAcresidue.Itp robablystrengthensthebindingcapacityofsomeofthesestru cturestoPHA-

L,whichisthephenomenonthathasnotbeendescribedsofar.

OurresultssuggesttheneedforfurtherstudiesofPHA- LbindingspecificitytowardsdifferenttypesofcomplexN- glycans.Moreover,inouropinion,theglycomiceffectseen herecouldleadtothechangesofotherprotein-

carbohydrateinteractions,alsoinvivo.Thus,itwouldbeimpor tanttostudythebiologicalimpactofintroducingoftheBbisecti ng^GlcNAcresidueintocomplexN-

glycansontheirbiologicalfunction,forexample,specificityt owardglycan-

bindingproteins,especiallyinthecontextofcancercellbiolo gy.

AcknowledgementsWewishtothankDomAlonzifromOxfordGly cobiologyInstituteforhelpandadviceforHPLCanalysis.

FundingThisworkwassupportedbythePolishNationalScienceCentre(

NCN),grantnumber3046/B/P01/2009/37.

Compliancew ithe thicalstandards

Conflictof interestTheauthorsdeclaret hattheyh aveno conflictofinterest.

EthicalapprovalT hisarticledoesnotcontainanystudieswithhumanpartici pantsoranimalsperformedbyanyoftheauthors.

GlycoconjJ ( 2018)35:217–231 17

OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCo m mo nsAt tribu t ion4. 0In t ernat iona lLicen se(ht tp://creativecommo ns.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedyougiveappropriatecredittotheori ginalauthor(s)andthesource,providealinktotheCreativeCommonslicens e,andindicateifchangesweremade.

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