Core-glycosylated Mucin-like Repeats from MUC1 Are an Apical Targeting Signal*DS

14 Pages·2011·4.93 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.286,NO.45,pp.39072–39081,November11,2011
©2011byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Core-glycosylated Mucin-like Repeats from MUC1 Are an
Apical Targeting Signal*□S
Receivedforpublication,August3,2011,andinrevisedform,September18,2011Published,JBCPapersinPress,September20,2011,DOI10.1074/jbc.M111.289504
CarolL.Kinlough‡,PaulA.Poland‡,SandraJ.Gendler§,PollyE.Mattila‡1,DiMo‡,OraA.Weisz‡,
andRebeccaP.Hughey‡2
Fromthe‡DepartmentofMedicine,Renal-ElectrolyteDivision,UniversityofPittsburghSchoolofMedicine,Pittsburgh,
Pennsylvania15261andthe§DepartmentofBiochemistryandMolecularBiologyandTumorBiologyProgram,MayoClinic
CollegeofMedicine,MayoClinicScottsdale,Scottsdale,Arizona85259
Background: MUC1 apical delivery in polarized MDCK cells employs a path used by proteins with glycan-dependent
targetingsignals.
Results:CoreO-glycansonmucin-likerepeatsofMUC1actasanapicaltargetingsignal.
Conclusion:MUC1apicaltargetingisO-glycan-dependent.
Significance:Wehaveidentifiedaspecificsequencewithapost-translationalmodificationthatcandirectapicaldeliveryof
MUC1orareporterprotein.
MUC1isefficientlydeliveredtotheapicalsurfaceofpolarized MUC1 is a transmembrane glycoprotein that functions in
Madin-Darbycaninekidney(MDCK)cellsbytransitthroughapi- signaltransductionthroughinteractionwithcytoplasmicpro-
calrecyclingendosomes,arouteassociatedwithdeliveryofapical teins and by modifying transcription factor activity after
proteinswithglycan-dependenttargetingsignals.However,arole nuclear targeting of its small subunit (for reviews, see Refs. 1
forglycansinMUC1sortinghasnotbeenestablished.Akeyfeature and2).BecauseMUC1isnormallyrestrictedtotheapicalsur-
ofMUC1isaheavilyO-glycosylatedmucin-likedomainwithavari- faceofpolarizedepithelialcellsbutisaberrantlylocalizedtoall
ablenumberofnearlyperfecttandemrepeatsandadjacentimper- cellsurfacesorintracellularmembranesintumorcells(3,4),we
fectrepeats.Metaboliclabeling,cellsurfacebiotinylation,immobi- are interested in identifying the features of this protein that
lizedlectins,andconfocalimmunofluorescencemicroscopywere regulateitsmembranetrafficking.Ourpreviousstudiesidenti-
usedtocharacterizethepolarizeddeliveryofMUC1mutantsand fiedsitesintheMUC1cytoplasmicdomainforadaptorprotein
chimerasinMDCKcellstoidentifytheapicaltargetingsignal.Both binding (AP-1, AP-2, and Grb2) and Cys palmitoylation that
theinterleukin-2receptor(cid:1)subunit(Tac)andachimerawherethe altogethermodulateitsrelativelyslowclathrin-mediatedendo-
TacectodomainreplacedthatofMUC1weredeliveredprimarily cytosisandfastrecyclingfromendosomes(5,6).Glycosylation
tothebasolateralsurface.AttachmentoftheMUC1mucin-like ofthemucin-likeectodomainalsoaffectscellsurfaceexpres-
domaintotheNterminusofTacenhancedapicalbutnotbasolat- sion because we found that modification with shorter O-gly-
eral delivery when compared with Tac. Conversely, deletions cansinglycosylation-defectiveCHOcellsslowsdeliverytothe
within the mucin-like domain in MUC1 reduced apical but not cellsurfacefromthebiosyntheticpathwayandincreasesMUC1
basolateral delivery when compared with MUC1. In pull-down internalization(7).
assayswithlectins,wefoundanotabledifferenceinthepresenceof Glycansontransmembraneproteinscanactasapicaltarget-
core1O-glycans,butnotpoly-N-acetyllactosamine,inapicallytar- ingsignalsaswellasenhancingsurfacedeliveryfromthebio-
getedMUC1andchimeraswhencomparedwithTac.Consistent syntheticpathway(forreviews,seeRefs.8and9).Inlinewith
withthesedata,wefoundnoeffectonMUC1targetingwhengalec- thesestudies,wefoundthatMUC1isefficientlytargetedtothe
tin-3,withpreferenceforpoly-N-acetyllactosamine,wasdepleted apical surface of polarized Madin-Darby canine kidney
frompolarizedMDCKcells.However,wedidblocktheapicaltar- (MDCK)3cellsviaapicalrecyclingendosomes(AREs),apath-
getingactivityofthemucin-likerepeatswhenweoverexpressed wayselectivelyusedfordeliveryofapicalproteinswithglycan-
CMP-Neu5Ac:GalNAc-R(cid:1)2,6-sialyltransferase-1 to block core dependenttargetingsignals(10).Galectin-3(Gal-3)isthought
O-glycansynthesis.Thecumulativedataindicatethatthecore-gly- toplayakeyroleinapicaltargetingofglycoproteinswithlipid
cosylatedmucin-likerepeatsofMUC1constituteanapicaltarget- raft-independent targeting signals by cross-linking cargo in
ingsignal. trans-Golginetwork-derivedvesicles,andwerecentlyreported
thatMUC1synthesizedinMDCKcellspreferentiallybindsto
*Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth canine Gal-3 (11–13). However, there is no direct proof that
GrantsDK54787(toR.P.H.),DK054407(toO.A.W.),CA64389(toS.J.G.)
MUC1apicaltargetingiseithergalectin-orglycan-dependent.
andP30DK079307(PittsburghCenterforKidneyResearchCoreC).This
workwasalsosupportedbytheGenzymeRenalInnovationsProgram(to
R.P.H.).
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains 3Theabbreviationsusedare:MDCK,Madin-Darbycaninekidney;ARE,apical
supplementalFig.S1. recyclingendosome;IP,immunoprecipitate;LEA,L.esculentumagglutinin
1SupportedbyindividualNRSAF32DK082109. (tomato lectin); PNA, peanut agglutinin; polylactosamine, poly-N-
2Towhomcorrespondenceshouldbeaddressed:Dept.ofMedicine,Renal- acetyllactosamine; R, imperfect repeat; ST6GalNAc-1, CMP-Neu5Ac:Gal-
ElectrolyteDivision,S933ScaifeHall,3550TerraceSt.Pittsburgh,PA15261. NAc-R(cid:1)2,6-sialyltransferase-1; TR, nearly perfect tandem repeat; WGA,
Tel.:[phone];Fax:[phone];E-mail:[email protected]. wheatgermagglutinin.
39072 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER45•NOVEMBER11,2011
This is an Open Access article under the CC BY license.
Glycan-dependentApicalTargetingofMUC1
AprominentfeatureoftheMUC1ectodomainisthevariable MN)),oranti-ST6GalNAc-1(monoclonal2C3fromUllaMan-
numberofmucin-likenearlyperfecttandemrepeats(TRs)of del (19) (University of Copenhagen, Denmark). MDCK cells
20aminoacidswithfiveorsixSer/Thrresiduesthatserveas expressing ST6GalNAc-1 (neo) (named MDCK(cid:1)ST6) were
sitesforO-linkedglycosylationwithGalNAc(14).TheTRsare stablytransfectedwithpcDNA(hygro)encodingTacand0TR-
flankedbyfiveproximalandtwodistalimperfectrepeats(Rs) Tac. Cells were grown in G418 (0.5 mg/ml) with or without
with 4–9 Ser/Thr residues each (see Fig. 1A for details). In hygromycin(0.4mg/ml)asindicatedandDulbecco’smodified
previousstudies,bothMUC1with30TRandamutantMUC1 Eagle’s medium (DMEM) and Ham’s F-12 (1:1) with 5% fetal
lackingitscytoplasmictailwerelocalizedusingconfocalimmu- bovineserumat37°Cinahumidifiedincubatorwith5%CO
2
nofluorescencemicroscopytotheapicalsurfaceofMDCKcells (Invitrogen). Clonal cell lines were isolated and screened by
growingoncoverslips,whereasthelymphocyte-specificmodel SDS-PAGEaftermetaboliclabelingandimmunoprecipitation.
proteinCD2waslocalizedtothebasolateralsurfaceofMDCK Surface Delivery in Polarized MDCK Cells—Polarized cul-
cells(15).AchimeraoftheCD2ectodomainandMUC1trans- turesofMDCKcellsweregrownon12-mmCostarpermeable
membraneandcytoplasmictailwasalsopresentatthebasolat- supports(CorningGlass)with0.4-(cid:2)mmembraneson12-well
eralsurface,whereasachimeraoftheMUC1ectodomainand platesfor4daysbeforeuse.Cellsurfacedeliverywasassayedas
CD2transmembraneandcytoplasmictailwasapical,suggest- describedpreviously(6,7,10).Briefly,cellswerestarvedfor30
ingthatanapicaltargetingsignalispresentintheMUC1ect- mininmediumlackingMet/Cys(Sigma),metabolicallylabeled
odomain(seeFig.1B)(15). for 30 min with [35S]Met/Cys (Easy Tag Express-[35S]Protein
Inthepresentstudy,weusedmetaboliclabeling,cellsurface LabelingMixture,PerkinElmerLifeSciences),andchasedupto
biotinylation,immobilizedlectins,andconfocalimmunofluo- 120mininnormalculturemedium.Allstepswerecarriedout
rescencemicroscopytodissectthefeaturesoftheMUC1ect- ina37°Cincubatorwith5%CO .Duplicatefiltersweremoved
2
odomain that target it to the apical cell surface in polarized toiceateachtimepoint.Afteralltimepointswerecollected,
MDCKcellsgrowingonpermeablesupports.Surprisingly,we thecellsonduplicatefiltersweretreatedwithsulfo-NHS-SS-
foundnoroleforGal-3oritsligand,poly-N-acetyllactosamine biotinoneithertheapicalorbasolateralsurfaceonice(6,7,10).
(polylactosamine), in apical targeting of MUC1. Rather, core Cell extracts were prepared, and recombinant proteins were
O-glycansonmucin-likerepeatsofMUC1constituteanapical recoveredbyimmunoprecipitationwitheitheranti-MUC1TR
targetingsignal. mouse monoclonal antibody B27.29 for MUC1, MUC1–2TR,
and 22TR-Tac; mouse monoclonal anti-FLAG epitope anti-
EXPERIMENTALPROCEDURES
body (Sigma) for MUC1–0R-FLAG; or mouse monoclonal
Recombinant cDNAs and Adenovirus—The cDNAs for anti-Tac (CD25) antibody (Ancell) for Tac-MUC1, 0TR-Tac,
MUC1 with 22 tandem repeats (MUC1–22TR), MUC1 with andTac.Armenianhamstermonoclonalanti-MUC1cytoplas-
two tandem repeats (MUC1–2TR), and Tac-MUC1 chimera mictailantibodyCT-2(20)wasincludedforimmunoprecipi-
werepreparedandsubclonedintopcDNA3(neo)(Invitrogen) tation of MUC1, MUC1–2TR, and Tac-MUC1. Immunopre-
as described previously (5, 6, 16). See Fig. 1 for models of all cipitates(IP)werewashedandelutedbyboilinginto60(cid:2)lof1%
constructsanddetailsonsequencesofnearlyperfectTRsand SDS.Analiquot(25%)wasretainedtocalculatethetotalIP,and
adjacent imperfect repeats (R). The cDNA for MUC1–0R- the remaining 75% was diluted 20-fold with a solution of
FLAGinpCI(neo)(Promega,Madison,WI)wasobtainedfrom HEPES-buffered0.5%TritonX-100andincubatedwithavidin-
TonyHollingsworth(UniversityofNebraskaMedicalCenter, conjugated beads to recover biotinylated proteins. Both the
Omaha, NE) (17). The cDNA for full-length wild-type Tac totalIPandbiotinylatedproteinsweresubjectedtoSDS-PAGE
(interleukin-2receptor(cid:1)-subunitfromMichaelS.Marks(Uni- on4–15%gradientgelsbeforedryingandanalysiswithaKodak
versityofPennsylvania,Philadelphia,PA))wassubclonedinto TR screen and a Bio-Rad Personal Imager and Quantity One
pcDNA3(neo)toproduceatype1transmembraneproteinof software.DataarepresentedasthepercentageofthetotalIP
251 amino acids after cleavage of the 21-residue signal delivered to the cell surface at each time point for multiple
sequence.ThesignalsequenceofTacwasreplacedwiththeN experiments(mean(cid:2)S.E.).
terminusofMUC1,includingfiveproximalRandonedistalR, Binding to Lectins Conjugated to Beads—Polarized MDCK
withorwithout22TRs(550aminoacidsor108aminoacids, cellsexpressingMUC1–22TR,22TR-Tac,0TR-Tac,orTacand
respectively,plustheMUC1cleavablesignalsequence),topro- growing on permeable supports were metabolically labeled
duce the chimera 22TR-Tac or 0TR-Tac, respectively. The with [35S]Met/Cys for 30 min and chased for 90 min as
cDNA for CMP-Neu5Ac:GalNAc-R(cid:1)2,6-sialyltransferase-1 describedabove.Foranalysisoflectinbinding,immunoprecipi-
(ST6GalNAc-1)withaC-terminalvesicularstomatitisvirus-G tateswerepreparedasalreadydescribedandelutedintoSDS
epitope tag was obtained from Joyce Taylor-Papadimitriou (analiquotsavedastotalIPwhereindicated),dilutedwith1%
(Guy’s Hospital, London, UK) and subcloned into pcDNA3 TritonX-100inHEPES-bufferedsaline,andincubatedwith50
(18). (cid:2)lofa50%slurryofeithertomatolectin(Lycopersiconesculen-
StablyTransfectedCellLines—MDCKcellsweretransfected tum;LEA)orwheatgermagglutinin(Triticumvulgare;WGA)
with(i)pcDNA3(neo)encodingeitherMUC1–22TR,MUC1– conjugated to beads (4–5 mg of lectin protein/ml slurry, EY
2TR, Tac-MUC1, 22TR-Tac, 0TR-Tac, Tac or ST6GalNAc-1 Laboratories,Inc.(SanMateo,CA))overnightwithend-over-
or (ii) pCI(neo) encoding MUC1–0R-FLAG. Clonal cell lines end mixing. Immunoprecipitates were treated with recombi-
were screened with anti-MUC1 antibody B27.29 (Fujiribio, nant Clostridium perfringens neuraminidase (New England
Malvern, PA), anti-Tac (anti-CD25 from Ancell (Bayport, Biolabs,Ipswich,MA)orreactionbuffer(mock)beforeelution
NOVEMBER11,2011•VOLUME286•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 39073
Glycan-dependentApicalTargetingofMUC1
fromProteinGconjugatedtoSepharoseandsubsequentbind- Cellswerestarvedfor30min;pulse-labeledwith[35S]Met/
ingtoaslurryofpeanutlectin(Arachishypogaea;PNA)conju- Cysfor30min;andchasedfor30,60,90,and120minpriorto
gated to beads (EY Laboratories). Material bound to lectin- biotinylation of proteins on the apical or basolateral cell sur-
beadswasanalyzedbySDS-PAGEandaBio-Radimagerwith face.Asexpected,MUC1–22TRwasefficientlydeliveredtothe
QuantityOnesoftware.Thedataarepresentedasthepercent- apicalsurface(58%oftotalIPwasapicalat120min),whereas
ageofthetotalconstructboundtotheimmobilizedlectin(per- onlyasmallfractionreachedthebasolateralsurface(maximum
centagebound(cid:3)(amountofconstructrecoveredfromimmo- of9%at60min)(Fig.2),asweobservedpreviously(10).Tac-
bilizedlectin/totalconstructadded)(cid:4)100). MUC1wasdeliveredprimarilytothebasolateralsurface(max-
SurfaceStainingofPolarizedMDCKCells—MDCKcellssta- imumof26%basolateralat60min)withonly12%reachingthe
blytransfectedwithMUC1–22TR,22TR-Tac,0TR-Tac,orTac apicalsurfaceby120min.ThedataobtainedforTac-MUC1are
wereplatedon12-mmCostarpermeablesupports.Fourdays consistent with the loss of an apical targeting signal present
after plating, cells were gently rinsed on ice five times with withintheMUC1ectodomainandthelackofanyapicaltarget-
HEPES-buffered minimum essential medium and blocked on ingsignalwithinthetransmembraneorcytoplasmicdomainof
iceinHEPES-bufferedminimumessentialmediumcontaining MUC1.ApicaldeliveryofMUC1–2TR(48%apicalat120min)
1% BSA for 30 min. Cells were incubated with either mouse andMUC1–0R-FLAG(25%apicalat120min)wasnoticeably
anti-Tac(CD25,2.5(cid:2)g/ml)ormouseanti-MUC1TR(B27.29,5 reducedascomparedwithdeliveryofMUC1–22TR,butbaso-
(cid:2)g/ml)primaryantibodiesfor1honice,washedfivetimes,and lateral delivery was not affected. Any statistically significant
incubated with Alexa 488-conjugated goat anti-mouse anti- reductionsinapicaldeliveryandincreasesinbasolateraldeliv-
body(dilution1:500;Invitrogen)for30minonice.Cellswere erywhencomparedwithMUC1–22TRareindicatedinFig.2A
fixedoniceatroomtemperaturein100mMsodiumcacodylate with an asterisk. Interestingly, apical delivery of MUC1–2TR
buffer, pH 7.4, for 15 min and then permeabilized with 0.1% appears delayed when compared with MUC1–22TR because
apicaldeliverywassignificantlydifferentonlyatthe30and60
TritonX-100inphosphate-bufferedsalinecontainingglycine
min time points. Altogether, the data support the possibility
andNH Clfor5minatroomtemperature.Cellsweresubse-
4
that the mucin-like repeats (TR and/or R) are necessary for
quentlyincubatedwithratanti-ZO-1hybridomatissueculture
MUC1 delivery selectively to the apical surface of polarized
supernatantR40.76(giftofGerardApodaca(Pittsburgh,PA))
MDCKcells.
andAlexa647-conjugatedsecondaryantibody(Invitrogen)for
TheMucin-likeRepeatsofMUC1ContaintheApicalTarget-
30 min at room temperature. Images were acquired using a
Leica SP5-STED CW confocal microscope with a (cid:4)100 oil ingSignal—Wenextdesignedexperimentstodetermineifthe
mucin-likerepeatsofMUC1aresufficientforapicaltargeting
immersionobjective(numericalaperture1.40)equippedwith
ofamodelprotein.TheNterminusofMUC1,including22TRs
argon,greenhelium-neon,andredhelium-neonlasersinCore
andsixadjacentRswereaddedtotheNterminusofTac(22TR-
CofthePittsburghCenterforKidneyResearch.Imageswere
Tac)todetermineifthemucin-likedomaincouldreversethe
processed with Adobe Photoshop (Adobe Systems, San Jose,
polarizeddeliveryofthismodelproteininapulse-chaseassay.
CA), and single images were processed utilizing the available
AsshowninFig.3,Tacwasdeliveredprimarilytothebasolat-
rangeofgraylevelswithoutsaturation.
eralsurfaceinpolarizedMDCKcells(22%ofthetotalIPwas
KnockdownofGalectin-3(Gal-3)withsiRNAs—MDCKcells
basolateral,and9%wasapicalat120min),whereasthechimera
with stable expression of MUC1–22TR were treated with
22TR-Tac was delivered primarily to the apical surface (42%
siRNA duplexes directed to either firefly luciferase (negative
was apical at 120 min). Interestingly, a chimera of the six Rs
control)orGal-3beforeplatingcellsonpermeablesupportsas
attachedtotheNterminusofTac(0TR-Tac)wasalsodelivered
describedpreviously(21).ThepolarizeddeliveryofMUC1and
primarilytotheapicalsurface(29%apicalat120min).There
levels of Gal-3 (R&D Systems, Inc., Minneapolis, MN) and
wasastatisticallysignificantdifferenceinapicaldeliveryofboth
(cid:3)-actin(Sigma)wereassessedafter4daysbyimmunoblotting.
22TR-Tac and 0TR-Tac when compared with Tac at all time
points(p(cid:5)0.05)butminimaldifferencesinbasolateraldelivery
RESULTS
of either 22TR-Tac or 0TR-Tac when compared with Tac.
The Signal for Apical Targeting Is within the MUC1 Although Tac was stable on the basolateral surface, levels of
Ectodomain—AprominentfeatureoftheMUC1ectodomainis both 22TR-Tac and 0TR-Tac declined significantly after a
thevariablenumberofmucin-likeTRsof20aminoacidswith 60–90-minchase.Therewasnostatisticallysignificantdiffer-
five Ser/Thr residues that serve as sites for glycosylation ence between 22TR-Tac and 0TR-Tac delivery to either the
throughO-linkedGalNAc.Toassesstheroleofthemucin-like apicalorbasolateralsurface.
repeatsinMUC1apicaltargeting,weusedmetaboliclabelingof The polarized distribution of surface 35S-labeled MUC1–
MDCKcellsexpressingeitherwild-typeMUC1–22TR(with22 22TR,Tac,andchimerasobservedat120minofchase(Figs.2
TRs and seven adjacent Rs), MUC1–2TR (with two TRs and and3)alsoreflectsthesteadystateexpressionoftheseproteins
sevenRs),MUC1–0R-FLAG(withaFLAGepitopetagreplac- whentheirsurfaceexpressionisanalyzedbyconfocalimmuno-
ingatractcontainingalloftheTRs,fiveRs,andtwoN-glyco- fluorescence microscopy (Fig. 4). Although MUC1–22TR,
sylationsites),andTac-MUC1withtheectodomainofthenor- 22TR-Tac,and0TR-Tacexhibitprimarilyapicalstaining,Tac
mally basolateral Tac (interleukin-2 receptor (cid:1) subunit) exhibits primarily lateral staining. At steady state, 0TR-Tac
replacing the entire MUC1 ectodomain (see models and appearstohaveaslightlylesspolarizeddistributioncompared
sequencesinFig.1,AandB). with22TR-Tac.Thismayreflectreducedstabilityof0TR-Tac
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Glycan-dependentApicalTargetingofMUC1
FIGURE1.ModelsofMUC1mutantsandchimeras.MUC1isatype1transmembraneproteinwithacleavedsignalsequence,anectodomainincludingRsand
avariablenumberofTRs,aSEAmodulewithautocatalyticcleavage65residuesfromthemembrane,a23-residuetransmembranedomain,anda72-residue
cytoplasmictail.A,aminoacidsequencesoftheTRsandadjacentRsareshowninsingle-lettercode.TherearefivevariantsofTR,asindicated,whereresidues
Asp-ThrarereplacedbyGlu-SerorwhereProisreplacedbyGln,Ala,orThr(allhavefiveSer/Thrresiduesexceptthelast,whichhassix).Conservedresiduesare
highlightedingray.TherearefiveproximalRsbetweenthecleavedsignalsequenceandtheTRsandtwodistalRsbetweentheTRsandaSer/Thr/Pro-richlinker
totheSEAmodule.ThenumberofSer/ThrresiduesineachRvariesasindicatedbetweenfourandnine.N-Linkedglycosylationconsensussites(NX(S/T))are
underlined.B,schematicsofconstructsusedinthisstudy,includingMUC1(with22TRor30TR),MUC1–2TRwith2TR,MUC1-CD2chimera,Tac-MUC1chimera,
andMUC1–0R-FLAG(withFLAGreplacingatractwithallTRs,fourRs,andtwoN-glycans).MUC1RwithorwithoutTRwereappendedtotheNterminusofTac
togenerate0TR-Tacand22TR-Tac,respectively.
attheapicalsurface,assuggestedbytheslightlyreducedlevels MDCK cells stably expressing MUC1–22TR were treated
of0TR-Tacapicalexpressionobservedat120minascompared withsiRNAduplexesdirectedtoeitherfireflyluciferase(con-
withMUC1–22TR(Fig.2)and22TR-Tac(Fig.3).Altogether, trol)orGal-3andplatedonpermeablesupportsasdescribed
weconcludedthatanapicaltargetingsignalispresentwithin previously(21).After4daysinculture,wemeasuredMUC1–
themucin-likerepeatsfromMUC1. 22TRdeliverytotheapicalandbasolateralsurfacebymetabolic
MUC1ApicalTargetingIsNotDependentonGalectin-3—A labeling and surface biotinylation and found no difference in
keyfeatureofthemucin-likedomainofMUC1isthepresence MUC1–22TRpolarizeddeliveryincellstreatedwithcontrolor
of O-linked glycans on Ser and Thr residues (see Fig. 5A). Gal-3siRNAs,despiteefficientknockdownofGal-3(Fig.5B).
Galectins (Gal) are small proteins that bind glycoconjugates Consistent with this finding, we also did not find evidence of
containing(cid:3)-galactose,suchaspolylactosamine,andGal-3is differential levels of polylactosamine on our model proteins
reportedtoplayakeyroleinapicaltargetingofglycoproteinsby that exhibit primarily apical (MUC1–22TR, 22TR-Tac, and
cross-linkingcargointrans-Golginetwork-derivedvesicles(11, 0TR-Tac) or basolateral (Tac) delivery (Fig. 5C). Polarized
12). Using a pull-down assay with the canine galectins MDCKcellsexpressingeitherMUC1–22TR,22TR-Tac,0TR-
expressedinMDCKcells(GST-taggedGal-1,-3,-4,-7,-8,and Tac,orTacweremetabolicallylabeledwith[35S]Met/Cysfor30
-9)werecentlyfoundthatMUC1–22TRexpressedinMDCK minandchasedfor90minbeforeimmunoprecipitationofthe
cellsbindspreferentiallytoGal-3(13).Therefore,weaskedif proteins.Immunoprecipitateswereeluted,andequalaliquots
MUC1 apical targeting was Gal-3-dependent by following wereincubatedwitheitherWGA(controlwithspecificityfor
MUC1–22TR surface delivery in polarized MDCK cells after GlcNAc or sialic acid) or LEA conjugated to beads (Fig. 5C).
Gal-3depletionbytreatmentofcellswithsiRNAs. Although MUC1–22TR (68%), 22TR-Tac (63%), 0TR-Tac
NOVEMBER11,2011•VOLUME286•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 39075
Glycan-dependentApicalTargetingofMUC1
FIGURE2.Mucin-likerepeatsarenecessaryforMUC1apicaldelivery.PolarizedMDCKcellsstablyexpressingMUC1–22TR,MUC1–2TR,MUC1–0R-FLAG,or
Tac-MUC1werestarvedfor30min,pulse-labeledfor30minwith[35S]Met/Cys,andchasedupto120minbeforesurfacebiotinylationattheapicalor
basolateralsurface.MUC1andmutantswereimmunoprecipitatedfromcellextracts,and25%wassavedastotal(Total),whereas75%wasincubatedwith
avidin-conjugatedbeadstorecoverthebiotinylatedprotein(Surface).TotalandsurfacesampleswereanalyzedafterSDS-PAGEwithaBio-Radimager.A,the
percentageofthetotalMUC1ormutantfoundonthecellsurfaceispresentedformultipleexperimentsasmeanandS.E.(errorbars)(n(cid:3)3–6),andstatistically
significantdifferencesbetweenthemutantsandMUC1–22TRarenotedbyanasterisk(*,p(cid:5)0.05).RepresentativeSDS-gelprofilesareshownforMUC1–22TR
(B),MUC1–2TR(C),MUC1–0R-FLAG(D),andTac-MUC1(E).MobilityofBio-Radmolecularmarkersisindicatedontherightofeachpanel.
(55%),andTac(61%)allboundsimilarlytoWGA-beads(con- supplementalFig.S1)(22–24).Thesesamethreepredominant
trol),wefoundcomparablebindingofMUC1–22TR(25%)and sitesarepredictedsitesofglycosylationwhentheaminoacid
22TR-Tac(18%)toLEA-beadsbutnegligiblebindingofeither sequences of the TR variants are analyzed with specificity
0TR-Tac (4%) or Tac (3%) (percentage bound (cid:3) (amount of parametersdevelopedforthepolypeptide-GalNActransferases
construct recovered from immobilized lectin/total construct T1,T2,T3,T5,T10,T12,andcore1Galtransferase(26,27).A
added)(cid:4)100).WeconcludedthatthepresenceofO-glycans similar analysis of the sequences of the proximal and distal
with terminal polylactosamine on the mucin-like repeats of imperfectR(andlinker)indicatesthatnumerousSerandThr
MUC1doesnotcontributetoapicaltargetingofMUC1. areprobablyglycosylatedsuchthat1–6SerorThrresiduesare
MUC1ImperfectRepeatsAreO-Glycosylated—Wenextcon- modifiedineachR.Theresultsofthisanalysisareconsistent
sideredthepossibilitythatshorterO-linkedglycanscouldrep- withthepresenceofO-glycansonsixRappendedtoTacinboth
resent a key component of the apical targeting signal from 22TR-Tacand0TR-Tac.
MUC1. Although O-linked glycosylation of the TR has been ToconfirmthatO-glycansarepresentonthesixRof0TR-
well characterized on both full-length MUC1 and synthetic Tac, we first compared binding of MUC1–22TR, 22TR-Tac,
peptides based on MUC1 tandem repeats (22–25), glycosyla- 0TR-Tac,orTactoimmobilizedPNAwithspecificityforthe
tionoftheadjacentRhasnotbeenaddressed.WithintheTR, non-sialylated O-linked core 1 glycan (Gal(cid:3)1,3GalNAc-Ser/
theThrwithinthesequenceVTSAandboththeSerandThr Thr)(seeFig.5A).PolarizedMDCKcellsexpressingeachcon-
within the sequence GSTA are modified, whereas the structweremetabolicallylabeledwith[35S]Met/Cysfor30min
remaining two sites are used less frequently (see Fig. 1A and andchasedfor90minbeforeimmunoprecipitationofthepro-
39076 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER45•NOVEMBER11,2011
Glycan-dependentApicalTargetingofMUC1
FIGURE3.Mucin-likerepeatsfromMUC1aresufficientforapicaltargetingofamodelprotein.PolarizeddeliveryofTac,22TR-Tac,or0TR-Tacstably
expressedinMDCKcellswasanalyzedasdescribedinthelegendtoFig.2.ThepercentageoftotalTac(A),22TR-Tac(B),or0TR-Tac(C)ispresentedforthree
experimentsasmeanandS.E.(errorbars)RepresentativeSDS-gelprofiles(D–F)areshownwithmature(M)andprecursor(P)formsnotedontheleftand
mobilityofBio-Radmolecularweightmarkersindicatedontherightofeachpanel.Theincreaseinapicaldeliveryof22TR-Tacand0TR-Tacwasstatistically
significantlydifferentfromapicaldeliveryofTacatalltimepoints,whereasbasolaterallevelsweresignificantlyreducedwhencomparedwithTaconlyatlater
timepoints(*,p(cid:5)0.01).Therewerenostatisticallysignificantdifferencesbetweenapicalandbasolateraldeliveryof22TR-Tacand0TR-Tac.
teins. Immunoprecipitates were pretreated with (or without)
C.perfringensneuraminidasefor1hpriortoelutionandincu-
bation with PNA-conjugated beads. We found appreciably
greaterbindingofMUC1–22TR(15and84%),22TR-Tac(35
and75%),and0TR-Tac(25and60%)toPNA-beads(beforeand
after neuraminidase treatment, respectively) than Tac (2 and
19%) (Fig. 6). The levels of protein binding to PNA (MUC1–
22TR,22TR-Tac(cid:6)0TR-Tac Tac)suggestthatboththeTR
andtheRappendedtoTacaremodifiedwithcore1O-glycans.
Weconcludethatcore1glycansareclearlypresentontheper-
fectandimperfectrepeatsof22TR-Tacand0TR-Tacandthat
O-glycans could contribute to the apical targeting of these
constructs.
FIGURE4.ImmunofluorescencemicroscopyofMUC1–22TRandchimeras
CoreO-GlycansonMucin-likeRepeatsAreaKeyFeatureof
inpolarizedMDCKcells.PolarizedMDCKcellsstablyexpressingMUC1–
22TR,Tac,orchimeras,asindicated,wereblockedwith1%BSAoniceand theApicalTargetingSignal—BecauseMUC1isdeliveredtothe
thenincubatedwithanti-MUC1TR(B27.29)oranti-Tacantibodies(Ab)onice apicalsurfacebytransitthroughtheARE,apathusedbyother
todetectcellsurfaceexpression(green).Afterincubationwithsecondaryanti-
bodiesandextensivewashing,cellswerefixed,permeabilized,andprocessed apical proteins with glycan-dependent signals (10), and the
forindirectimmunofluorescencetodetectZ0–1(red).Singlemergedpanelsof mucin-likerepeatson0TR-Tacthatdirectitsapicaldeliveryare
representative apical, lateral, and xz sections of each stable cell line are
O-glycosylated(Fig.6),wedesignedexperimentstodetermine
shown.Notethatanti-Tacantibodyisapparentlymoresensitiveatdetecting
lateralstainingof22TR-TaccomparedwithB27.29.Scalebar,10(cid:2)m. if O-glycans are a key feature of the apical targeting signal
NOVEMBER11,2011•VOLUME286•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 39077
Glycan-dependentApicalTargetingofMUC1
FIGURE5.PolarizeddeliveryofMUC1inMDCKcellsisnotGal-3-dependent.A,pathwaysandenzymesinvolvedinsynthesisofO-linkedglycans.Notethat
PNArecognizesnon-sialyatedcore1,whereasLEArecognizespoly-N-acetyllactosamineoncore2(seeboxedstructures).ST6GalNAc-1(ST6)competeswith
T-synthaseandcore3synthase(C3GnT)forthesubstrateGalNAc(cid:1)-Ser/Thr.B,MDCKcellsstablyexpressingMUC1weretreatedwithsiRNAduplexesdirected
tofireflyluciferase(control)orGal-3andplatedonpermeablesupports.After4daysinculture,thepolarizeddeliveryofMUC1wasassessedbymetabolic
labelingfor30minwith[35S]Met/Cys.Aftervaryingchasetimes,apicalandbasolateralsurfacesweretreatedwithsulfo-NHS-SS-biotin.BiotinylatedMUC1was
recoveredwithavidin-conjugatedbeadsfromtheMUC1immunoprecipitatesandanalyzedwithaBio-RadimagerafterSDS-PAGE.MUC1deliverytotheapical
orbasolateralsurfaceispresentedasthefractionofthetotalimmunoprecipitatefoundonthecellsurface(onerepresentativeexperimentisshown,n(cid:3)2).
ImmunoblotsforGal-3and(cid:3)-actinincellextractsaftertreatmentwithcontrol(Con),Gal-3(G-3),orno(NA)siRNAsindicateefficientdepletionofGal-3inthis
experiment.C,polarizedMDCKcellsexpressingMUC1–22TR,22TR-Tac,0TR-Tac,orTacweremetabolicallylabeledfor30minwith[35S]Met/Cysandchasedfor
90minbeforedetergentextractionandimmunoprecipitation.Immunoprecipitateswereresuspended,andequalaliquotswereincubatedovernightwith
beadsconjugatedtoWGA(W)orLEA(L)orreservedastotal(T)beforeanalysisafterSDS-PAGEwithaBio-RadImager.Notethattheorderoftotal,WGA,andLEA
variesbetweengelprofiles.Thenumbersbelowthegelprofilesindicatethepercentageoftotalboundtoeachlectin(percentagebound(cid:3)(amountof
constructrecoveredfromimmobilizedlectin/totalconstructadded)(cid:4)100).Resultsfromonerepresentativeexperimentareshown(n(cid:3)2).
within the mucin-like repeats. Because there are no specific (Fig. 5, sialyl Tn), thereby preventing synthesis of the core
inhibitors of O-linked glycosylation, we stably overexpressed O-glycans (e.g. core 1, core 2, core 3, or core 4). MDCK cells
the enzyme ST6GalNAc-1 in MDCK cells to compete with expressing ST6GalNAc-1 (MDCK(cid:1)ST6) and either 0TR-Tac
enzymessuchascore1Galtransferaseandcore2andcore3 or Tac were pulse-labeled with [35S]Met/Cys for 30 min and
GlcNAc transferases for the substrate GalNAc-Ser/Thr (see chased for 30, 60, 90, and 120 min prior to biotinylation of
pathways in Fig. 5A) (18, 19, 28). Overexpression of proteins on the apical or basolateral cell surface (Fig. 7). We
ST6GalNAc-1 produces primarily SA(cid:1)2,6GalNAc-Ser/Thr observed that Tac was primarily delivered to the basolateral
39078 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER45•NOVEMBER11,2011
Glycan-dependentApicalTargetingofMUC1
FIGURE6.Core1O-glycansarepresentontheimperfectrepeatsof0TR-Tac.PolarizedMDCKcellsexpressingMUC1–22TR,22TR-Tac,0TR-Tac,orTacwere
metabolicallylabeledfor30minwith[35S]Met/Cysandchasedfor90minbeforedetergentextractionandimmunoprecipitation.Immunoprecipitateswere
treated((cid:1))ormock-treated((cid:7))withneuraminidase(N’ase)priortoresuspensionandincubationovernightwithbeadsconjugatedtoPNA(P)ornobeadsas
total(T;notedifferenttotalsforwithandwithoutneuraminidase)andanalysisafterSDS-PAGEwithaBio-Radimager.Thenumbersbelowthegelprofiles
indicatethepercentageofthetotalboundtoPNA(percentagebound(cid:3)(amountofconstructrecoveredfromimmobilizedlectin/totalconstructadded)(cid:4)
100).Resultsfromonerepresentativeexperimentareshown(n(cid:3)2).
FIGURE7.TerminalprocessingofO-linkedglycansisakeycomponentoftheMUC1apicaltargetingsignal.PolarizedMDCKcellsstablytransfectedwith
ST6-GalNAc-1aswellasTacor0TR-Tacwerestarvedfor30min,pulse-labeledfor30minwith[35S]Met/Cys,andchasedupto120minbeforebiotinylationat
theapicalorbasolateralsurface.Tac(AandB)and0TR-Tac(CandD)wereimmunoprecipitatedfromcellextractswithanti-Tacantibodies,and25%wassaved
astotal(Total),whereas75%wasincubatedwithavidin-conjugatedbeadstorecoverthebiotinylatedprotein(Surface).Totalandsurfacesampleswere
analyzedafterSDS-PAGEwithaBio-Radimager.AandC,thepercentageofthetotalMUC1ormutantfoundonthecellsurfaceispresentedasmeanandS.E.
(errorbars)(n(cid:3)3).BandD,representativeSDS-gelprofilesareshownwithmature(M)andprecursor(P)formsnotedontheleftandthemobilityofBio-Rad
molecularweightmarkersontherightofeachpanel.BothapicalandbasolateralTacdeliveryhadasmallbutsignificantincreaseinMDCK(cid:1)ST6cellswhen
comparedwithMDCKcells,whereasapical0TR-Tacdeliverywassignificantlydecreased,andbasolateral0TR-Tacdeliverywasincreased(p(cid:5)0.05;compare
withFig.3).E,polarizedMDCKcells,MDCK(cid:1)ST6cells,orMDCK(cid:1)ST6cellsexpressingTacor0TR-Tacwerestarvedfor30min,pulse-labeledfor30minwith
[35S]Met/Cys,andchasedfor120min.AliquotsfromtheapicalandbasolateralmediumwereanalyzedafterSDS-PAGEwithaBio-Radimager.Thethree
radiolabeledbandsbetween30and50kDarepresentingcleavedgp80werequantified.Thenumbersbelowthegelarethepercentageofthetotalgp80
(apical(cid:1)basolateral)foundintheapicalmedium.Arepresentativegelprofileispresented(n(cid:3)2).
surfaceofMDCK(cid:1)ST6cells(36%basolateraland20%apicalat endogenous protein gp80. The apical targeting of gp80 is
120 min of chase), similar to its delivery in wild-type MDCK dependent on the presence of N-linked glycans (29). Both
cells (Fig. 3A). However, 0TR-Tac delivery to the surface of MDCK(cid:1)ST6cells(non-transfectedorstablytransfectedwith
MDCK(cid:1)ST6 cells was non-polarized (19% apical and 16% either 0TR-Tac or Tac) and control MDCK cells were pulse-
basolateralat120min),whichwasverydifferentfromitspri- labeledwith[35S]Met/Cysfor30minandchasedfor120min
marilyapicaldeliveryinwild-typeMDCKcells(Fig.3C). beforerecoveryoftheculturemedium.Ineverycase,[35S]gp80
ToensurethatMDCK(cid:1)ST6cellsexpressing0TR-Tacwere wassecretedprimarilyintotheapicalmedium(68–78%),indi-
still capable of apical targeting, we assessed secretion of the cating that overexpression of ST6GalNAc-1 does not disrupt
NOVEMBER11,2011•VOLUME286•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 39079
Glycan-dependentApicalTargetingofMUC1
thepolarityofMDCKcells(Fig.7E).Weconcludedthatcore thepolypeptide-GalNActransferasespredictednumeroussites
O-linked glycosylated mucin-like repeats of MUC1 can func- for O-linked glycosylation, we subsequently found that Tac
tionasanapicaltargetingsignal. bindingtoimmobilizedpeanutlectinwithspecificityforcore1
O-glycanswasnotablyenhancedbytheadditionoftheimperfect
DISCUSSION
repeatstotheNterminusofTac(0TR-Tac).However,attachment
Inthisstudy,weobservedthat(i)deletionofthemucin-like of the imperfect repeats to Tac did not enhance its binding to
repeatsfromMUC1reducedapicaldeliverywithoutaffecting immobilized tomato lectin with specificity for polylactosamine
basolateraldelivery,(ii)addingthemucin-likerepeatsofMUC1 thatrepresentsterminalprocessingofcore2glycans,indicating
toTacenhanceditsapicaldeliverywithoutaffectingbasolateral that core glycosylation rather than polylactosamine was more
delivery,and(iii)overexpressionofST6GalNAc-1blockedthe likelyakeyfeatureoftheapicaltargetingsignal.
apicaltargetingactivityofthemucin-likerepeats.Thecumula- Thelackofpolylactosamineontheapical0TR-Tacwasalso
tive data indicate that core-glycosylated mucin-like repeats consistent with our finding that Gal-3 plays no role in apical
withinMUC1representanapicaltargetingsignal. targeting of MUC1–22TR. Several facts were consistent with
N-Linked Glycans Are Not Required for MUC1 Apical the possibility that Gal-3 might play a role in MUC1 apical
Targeting—Pemberton et al. (15) previously concluded that targeting: (i) Gal-3 has been implicated in apical targeting in
apicaltargetinginformationwasfoundwithintheMUC1ect- MDCK cells of the glycoprotein phlorizin lactate hydrolase
odomain by characterizing chimeras of MUC1 and CD2 in with glycan-dependent targeting (31); (ii) Gal-3 is the most
MDCKcells.Concurrently,theresultsofourstudieswithchi- abundantgalectininMDCKcellsandexhibitsapreferencefor
merasofMUC1andTacalsoindicatedthatanapicaltargeting polylactosamine on both N-linked and O-linked glycans with
signalwaspresentwithintheMUC1ectodomain.Wealsosus- eitherterminalbloodgroupA,sialicacid((cid:1)2,6),orfucose(13,
pectedthattheapicaltargetingsignalwouldbeglycan-depen- 32);and(iii)MUC1–22TRsynthesizedinMDCKcellsexhibits
dentbecauseadominantfeatureofMUC1isitsheavilyglyco- polylactosamineandbindspreferentiallytoGal-3inpull-down
sylated mucin-like domain. Also, we had already found that assayswithalloftheendogenouscaninegalectins(13).How-
MUC1transitstheapicalrecyclingendosome,apathusedby ever,weconsistentlyobservedthatefficientdepletionofGal-3
otherapicallydestinedproteinswithglycan-dependenttarget- inMDCKcellsusingtreatmentwithsiRNAshadnoeffecton
ing signals, such as the sialomucin endolyn (10). Although MUC1apicaldelivery.
endolynexhibitsbothN-linkedandO-linkedglycans,Potteret CoreO-GlycansAreaKeyFeatureoftheMUC1ApicalTar-
al.(30)foundthatitsapicaltargetingwasfullydependenton getingSignal—BecausethepolylactosaminefoundonMUC1–
terminalprocessingoftwospecificN-glycans.However,inpre- 22TRand22TR-TacwasseeminglyirrelevanttoMUC1apical
liminary experiments, we found that treatment of cells with targeting,wefocusedouranalysisonthepresenceofcoregly-
kifunensinetoblockterminalprocessingofN-glycanshadno cans by overexpression of ST6GalNAc-1 to terminate O-gly-
effect on MUC1 apical delivery.4 Our subsequent analysis of cans at SA(cid:1)2,6GalNAc-Ser/Thr. The absence of core glycans
MUC1mutantslackingallfiveconsensussitesforN-glycosyla- on0TR-TacinMDCK(cid:1)ST6cellsdidblockitsapicaldelivery,
tionwasimpairedbyitsconsistentlylowlevelofexpression,but whereas Tac polarized delivery in MDCK(cid:1)ST6 cells was
immunoblotanalysisofpolarizedMDCKcellsrevealedthatthe unchanged,indicatingthatcoreglycans(e.g.core1,2,3,or4)on
mutant was still expressed primarily on the apical surface.5 theimperfect(andmaybenearlyperfect)repeatsrepresented
Overall,thesedataledustofocusontheroleofO-linkedgly- theapicaltargetingsignalforMUC1.Wedidtestthisconclu-
cosylationinMUC1apicaltargeting. sionbyanalyzingMUC1–22TRsynthesisincellsoverexpress-
MUC1 Apical Targeting Is Not Galectin-3-dependent—The ingST6GalNAc-1,butwedidnotobserveareductioninapical
primaryfeatureoftheMUC1mucin-likedomainisthepres- delivery.4BecausewefoundthatMUC1–22TRbindingtoLEA-
ence of Ser/Thr/Pro-rich nearly perfect tandem repeats that conjugatedbeadswasreducedonly70%inMDCK(cid:1)ST6cellsas
exhibit at least three sites for O-glycosylation in each repeat. compared with MUC1–22TR expressed in wild-type MDCK
Because we found that mutations that removed most (2TR- cells,wesuspectthatthelevelofST6GalNAc-1expressionwas
MUC1)orallofthetandemrepeats(MUC1–0R-FLAG)corre- notsufficienttoblockterminalprocessingofenoughO-glycan
spondingly reduced apical delivery, the data were consistent sitesinMUC1–22TR(66sitesin22TRsplus18sitesinseven
withthepresenceofsynergisticapicaltargetingsignalswithin Rs). However, this level of ST6GalNAc-1 overexpression was
thetandemrepeats.However,thedeletionwithintheMUC1– adequatetoblockterminalprocessingoftherelevantO-glycan
0R-FLAG mutant also removed imperfect repeats, which fits sitesin0TR-Tac(16sitesinsixRs)(seesupplementalFig.S1for
withoursubsequentdiscoverythattargetinginformationwas predictionofSerandThrmodifiedbyO-glycosylation).
presentwiththeselattertracts.Weweresurprisedtofindthat MechanismofGlycan-dependentApicalTrafficking—O-Gly-
apicaltargetinginformationwaspresentwithintheimperfect cans on apical glycoproteins have been implicated previously
repeats of MUC1 because it was not known whether these (33, 34) as a key feature of the apical targeting signal of the
sequences were actually modified with O-linked glycans. neurotrophin receptor p75 and sucrase-isomaltase. More
Althoughanalysisoftheaminoacidsequenceoftheseimper- recently, Lee et al. (35) reported that terminal processing of
fectrepeatswithspecificityparametersdevelopedforseveralof O-glycansbyspecificallycore2GlcNActransferase-2isessen-
tialforsurfaceexpressionoftheapicalsucrase-isomaltaseand
4C.L.KinloughandR.P.Hughey,unpublisheddata. dipeptidylpeptidase IV in intestinal epithelial cells (35). They
5S.J.Gendler,C.L.Kinough,andR.P.Hughey,unpublisheddata. foundthatcellsurfacelevelsofsucrase-isomaltaseanddipeptidyl-
39080 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER45•NOVEMBER11,2011
Glycan-dependentApicalTargetingofMUC1
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1R–16R
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L.,Dar,I.,Jeanneau,C.,Defrees,S.,Krustrup,D.,Vogel,L.K.,Kure,E.H.,
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