resPrPDfragmentsarecommonlyreferredtoasPrP27308Aremarkablydifferen - PDF document

resPrPDfragmentsarecommonlyreferredtoasPrP27–30[8].AremarkablydifferentelectrophoreticprofileisobservedinGSS,wherethemostprominentandbyfarbestcharacterizedresPrPDspeciesisa6–8kDafragmentencompassinginternalresiduesfromwithinthe~70–150region[12,25,27,33,34].Highermolecularweight(hmw)bandsofvariableestimatedmolecularweightshavealsobeenreported,occasionallypromptingthehypothesisthattheyrepresentmultimers.However,themolecularna-tureofthePrPfragmentsgivingrisetothesebandsre-mainsenigmatic[12,17,23].Tobridgethisgap,herewehaveperformeddetailedanalysisofpurifiedresPrPDpreparationsfromGSScasesharboringtheA117V(GSSA117V)andF198S(GSSF198S)PrPmutations.OurdatademonstratethathighmolecularweightspeciesseeninelectrophoreticprofilesofGSSA117VandGSSF198SresPrPDrepresentcovalently-linkedmulti-mersofthesameinternalPrPfragmentsthatarepresent(asmonomers)inthe~7and~8kDabands.MaterialsandmethodsReagentsandantibodies
–mercaptoethanol,Dithiothreitol,LaemmliSampleBuf-fer,Non-fatdrymilk,Sodiumdodecylsulfate(SDS),TrisBufferedSaline(TBS),Tris/Glycine/SDSbuffer,Tris/Gly-cineBuffer,Tween20and15%CriterionTris-HClpoly-acrylamideprecastgelswerepurchasedfromBio-RadLaboratories(Hercules,CA,USA).Benzonase,Calciumchloride,CompleteUltraProteaseInhibitorCocktailTab-lets,Dulbecco’sPBS(D-PBS),KodakBiomaxMRandXARfilms,NaCl,NonidetP-40,N-Lauroylsarcosineso-diumsaltsolution(Sarkosyl),Phenylmethanesulfonylfluoride(PMSF),Polyvinylidenedifluoride(PVDF)mem-brane(Immobilon-P),ProteinaseK,SodiumdeoxycholateandTris-HClcamefromMilliporeSigma(Burlington,MA,USA)whereasEthylenediaminetetraaceticacid(EDTA)fromPromega(Madison,WI,USA).Glycerol-freePNGaseFwasfromNewEnglandBiolabsInc.(Ipswich,MA,USA);Acetonitrile,ColloidalBlueStainingKit,Etha-nol,FormicAcid,8MGuanidine-HClSolution,Methanol,PierceECL2WesternBlottingSubstrateandTrypsinfromThermoFisherScientificInc.(Waltham,MA,USA).Thefollowingantibodies(Abs)wereusedinthestudy:8B4(tohumanPrPresidues36–43)[22],SAF32(tohu-manoctarepeatregion)[14](CaymanChemical,AnnArbor,MI,USA),3F4(tohumanPrPresidues106–110)[19,42],F89(tohumanPrPresidues139–142)(ThermoFisherScientificInc.,Waltham,MA,USA),L42(tohu-manPrPresidues145–150)[18,38](R-Biopharm,AG,Darmstadt,Germany)andSAF70(tohumanPrPresidues156–162)[14].SecondaryAbwassheepanti-mouseIgG,HRP-linkedwholeantibody(GEHealthcareLifeSciences,Chicago,IL,USA).BraintissuesAllfrozenbraintissueswereobtainedfromtheNationalPrionDiseasePathologySurveillanceCenter(NPDPSC).FrontalcortexsamplesfromtwocaseseachofGSSA117V–129V(129MVand129VV)andF198S-129V(129MV)wereused.Allcaseswereusedforimmuno-blotting,butonlyonecaseofGSSA117VandGSSF198S,bothwith129MVgenotype,wereusedformassspec-trometry.AcaseofsCJDMV1wasusedascontrol.PreparationofbrainhomogenatesBrainhomogenates(BH)werepreparedaspreviouslydescribed[13].ProteinaseKdigestionSampleswereincubatedfor1hat37°CwithdifferentamountsofPK,asfollows:GSSA117V:purifiedPrPD(PK2U/ml);GSSF198S:BH(PK5U/ml),purifiedPrPD(PK10U/ml);sCJDMV1:purifiedPrPD(PK10U/ml).Thereactionwasstoppedbytheadditionof3mMPMSF.EpitopemappingTheoptimalPKconcentrationsandthevolumesofpuri-fiedGSSF198S,GSSA117VandsCJDMV1resPrPDloadedintothegelwereselectedtoobtainaclearvisibilityofallthebandswith3F4.RecombinanthumanPrPfull-length(23–231)andtruncated(90–231)specieswereusedasmolecularweightmarkers.Methanol/ethanolprecipitationMethanolprecipitationwasperformedaspreviouslyde-scribed[13].Forethanolprecipitationthesameprocedurewasfollowedwiththeexceptionof9volumesofpre-chilledethanolinsteadof5volumesusedtodilutethesample.PrPdeglycosylationSamplesweretreatedwithglycerol-freePNGaseFinac-cordancewithmanufacturer’sinstructions.ElectrophoresisandimmunoblotProteinsampleswererunon15%CriterionTris-HClpolyacrylamideprecastgelsandthensubjectedtoimmu-noblot(60Vfor2h)usingImmobilon-PPVDFmem-branes.After1hblockingin5%non-fatdrymilkinTBSwith0.1%Tween20(TBS-T),themembraneswerein-cubatedwithprimaryAbovernightat4°C;theyw

erethenwashedwithTBS-T,incubatedfor1hatRTwiththesecondaryHRP-conjugatedAbandthenwashedagainpriortodevelopingbyenhancedchemilumines-cencereactionusingECL2westernblottingsubstrate,aspermanufacturer’sinstructions.KodakMRandXARfilmswereusedtocapturethesignal.Craccoetal.ActaNeuropathologicaCommunications (2019) 7:85 Page2of9 ResPrPDpurificationResPrPDpurificationwasperformedasreportedinBol-tonetal.[7]andadaptedbyZouetal.[40].RotorSW55Ti(BeckmanCoulter,Brea,CA,USA)wasusedforultracentrifugation.In-geltrypsindigestionofpurifiedresPrPDIn-geltrypsindigestionwasperformedaccordingtoanestablishedprotocol[31].Briefly,theproteinbandsofinterest(visualizedbyColloidalCoomassiebluestaining)wereexcisedfromthegel,cutintosmallcubesanddestained.Thedestainedgelpieceswerethendehy-dratedandfollowedbyreduction/alkylationstep.Fordi-gestion,trypsinsolution(15ng/l)wasaddedandgelpieceswereincubatedovernightat37°Cwithshaking.ThesupernatantwasthentransferredintoanewEppen-dorftubeandtheremainingpeptidesingelpieceswereextracted,supernatantswerepooled,concentratedandstoredat80°CuntilanalyzedbyNanoLC-MS.NanoLC-MS/MSNanosprayLC-MS-MSanalysiswasperformedusinganLTQOrbitrapXLmassspectrometerequippedwithnanoelectrospraysource(ThermoScientific,SanJose,CA,USA).Trypsin-digestedsampleswereloadedontoaC-18trapcolumn(toremovesalts)andseparatedonaC-18columnconnectedtoanemitter.Separationwasper-formedusingaDionexUltiMate3000system(ThermoScientific,SanJose,CA,USA)andagradientofaceto-nitrileinwatercontaining0.1%formicacid.Theflowratewas300nl/min.ThemassspectrometerwasexternallycalibratedusingaPierceLTQESIpositiveioncalibrationsolution(ThermoScientific,catalognumber88322).Fullscanexperimentswereacquiredinthem/z300–1800rangeataresolutionof30,000(FWHMatm/z400).Thefollowingsourcesettingswereused:sprayvoltage=4.2kV;capillarytemperature=200°C.Data-dependentMSn(n=2)wereacquiredatITMSusingcollisioninduceddis-sociation(CID);thetop14intenseionsweresubjectedforfurtherfragmentation.Calculationofelementalformulaewasperformedonthemono-isotopicpeakofeachionclusterusingXcalibursoftwarev2.2withamasstoleranceof3to5ppm.MS/MSrawfilesweresearchedusingMASCOTDeamonengineagainstthedatabasecontain-ingsequenceofhumanprionprotein129M/Vwithmuta-tionA117VorF198S.Trypsin/PsearchparametersforMascotpeptideidentificationincludedonemissedtrypticcleavage,fixedcarbamidomethylation(+57Da,Cys),andvariableoxidation(+16Da,Met).Masstolerancesof2.0and1.0Dawereusedforparentandmonoisotopicfrag-mentions,respectively.TheresultingfilesgeneratedbyMASCOTwereusedforpeptideidentificationwiththeconstraintsthatonlyMASCOTionscoresgreaterthan10wereconsidered.Thepercentageof129Mand129VPrPinresPrPDsampleswascalculatedbythespectralcount-ingmethod[4].ResultsThecharacteristicsofresPrPDinGSSF198SandGSSA117VwereexaminedandcomparedwiththoseofsCJDMV1resPrPD,usedascontrol,bycombiningepitopemappingwithenzymaticdeglycosylationonimmunoblotsofpurifiedresPrPDpreparations(Fig.1).Overall,GSSF198SandGSSA117VshowedsimilarelectrophoreticprofilesthatwereeasilydistinguishablefromtheprofileofsCJDMV1(Fig.1).GSSF198SandGSSA117Vdisplayedlowmwbandsof~8and~7kDaaswellastwobroadbandscomprisedbetween17-2023–24kDaforGSSF198S,and16–18and22–23kDaforGSSA117V;additionalbandsandsmearsofhighermwwerealsopresentmoreprominentlyinGSSF198S(Fig.1).Bycontrast,resPrPDprofileinsCJDMV1includedthetypicalthreeglycoformbandsof~30,~27,and~21kDaofresPrPDtype1.Twoadditionaluniquecharacteristicsfur-therdistinguishedGSSA117VandGSSF198SfromsCJDMV1.VirtuallyallresPrPDbandsassociatedwiththesetwoGSSconditionswerereadilydetectedonlywithAbstoepitopeswithinthePrP~51–150region,butnotwithAbsrecogniz-ingmoreC-terminalepitopes(Fig.1,panelsb-f).Further-more,GSSA117VandGSSF198SresPrPD(aswellasresPrPDfromsCJDMV1)showednoimmunoreactivitywith8B4AbthatrecognizestheN-terminalepitope36–43(Fig.1,panela).ThisfindingexcludesthepresenceoffulllengthresPrPDinthesetwoGSSvariants,a

ndisatvariancewiththepres-enceoffull-lengthresPrPDreportedinGSSH187R[12].Inadditiontoepitopemapping,asecondmajordistinguishingfeaturewasthat,incontrasttosCJDMV1resPrPD,deglyco-sylationhadnosignificanteffectontheelectrophoreticmo-bilityofanyofthemajorresPrPDbandsobservedinGSSF198SandGSSA117V,stronglysuggestingthatresPrPDpopulatingthesebandsisnotglycosylated(Fig.1).Inasep-arateexperimentcarriedoutinGSSF198S,wealsoobservedthattheresPrPDprofiledidnotchangeafterpretreatmentofsampleswithstrongdenaturantssuchasguanidinehydrochlorideandurea(Additionalfile1:FigureS1).Col-lectively,thesedatasuggestthatatleasttwohmwbandsseeninimmunoblotsofGSSA117VandGSSF198SresPrPDrepresentcovalently-linkedmultimersofthe~7and~8kDafragments(Fig.1andAdditionalfile1:FigureS1).Todefinitelyassesswhetherthehighermolecularbandsrepresentedmultimers(likelycovalently-linked)ofthe~7and~8kDainternalfragments,asourepitopemappingandglycosylationstudystronglysuggested,weperformedtrypsinin-geldigestionofproteinfragmentsonindividualelectrophoreticbandsofpurifiedresPrPDfromGSSA117VandGSSF198S(~7,16–18and22–23kDainthecaseofGSSA117Vand~8,17–20and23–24kDainthecaseofGSSF198S)anddidaminoacidsequencingusingmassspectrometry(NanoLC-MS).Craccoetal.ActaNeuropathologicaCommunications (2019) 7:85 Page3of9 Thetrypticdigestofthe~7kDaresPrPDfragmentex-tractedfromGSSA117VandanalyzedbyNanoLC-MSre-vealedthepresenceofpeptidesexclusivelyfromthecentralregionofPrPbetweenresidues78and152,withnodetectablefragmentsfromotherpartsoftheprotein.Trypsinisknowntocleavepolypeptidechainsatthecarboxylsideoflysine(K)orarginine(R).AsshowninFig.2,sevenpotentialtrypsincleavagesitesareavailablewithinthecentralregionofPrPbetweenresidues70and153.NanoLC-MSanalysisoftheGSSA117V~7kDafrag-mentusingtheMascotDeamonsearchingengineidenti-fiedfourteentrypsin-generatedpeptideswithN-terminibeforethefirstpotentialtrypsincleavagesite,twoin-ternaltrypticfragments,andtwelvetrypsin-generatedpeptideswithN-terminusatthecleavagesitefollowingR136(Fig.2aandAdditionalfile2:TableS1).Altogether,thesedatademonstratedthatinGSSA117Vthe~7kDafragmentencompassedresidueswithinthe78–152re-gionandhadraggedN-andC-terminicorrespondingtoresidues78/82/85–88and141–152,respectively.ThisisgenerallyconsistentwithpreviousMALDI-deriveddatafromextractsofPrPamyloidplaquecores,eventhoughourNanoLC-MSanalysis(whichismoreaccurate)re-vealedthattheN-terminusofthisfragmentmayextendasfarasuptoresidue78versusresidue85previouslyreportedbasedontheMALDIanalysis[33].Asimilarsequencinganalysisofthe16–18kDaand22–23kDabandsrevealedthepresenceofessentiallyidenticaltrypticfragments,withtheexceptionthatthemostN-terminalfragmentstartedatresidue82,andthe Fig.1EpitopemappingcombinedwithdeglycosylationofpurifiedresPrPDassociatedwithGSSF198S,GSSA117VandsCJDMV1usedascontrol.PNGaseF-treatedoruntreated,PK-resistantPrPD(resPrPD)purified(seeMaterialsandMethods)fromGSSF198S,GSSA117VandsCJDMV1controlwereblottedandprobedwiththeAbstoPrPdenoted,withtheirepitopes,atthetopofeachpanel.Panela:Onlyfull-length(PK-untreated)recombinantPrP(23–231)wasdetectedbythisAbtotheproximalN-terminalregionconfirmingtheabsenceofresPrPDwithcompleteN-terminus.Panelsbtoe:BothGSSF198SandGSSA117VresPrPDconformerswereselectivelydetectedbythesameAbstoPrPN-andC-terminalregions;bothalsoshowednomajorvariationoftheresPrPDbandingpatternfollowingdeglycosylationindicatingthatmostoftheresPrPDisunglycosylatedinbothconditions.Althoughoverallsimilar,thetworesPrPDprofilesdifferedespeciallyinthehighermolecularweightregionsuggestingadistinctrepertoire,orrelativeproportions,ofpolymersinthetwoGSSvariants.TheGSSbandingpatternsclearlydifferedfromthethree-bandpatternofsCJDMV1,twoofwhichareglycosylated(SeeResultsfordetaileddescription).Panelcincludesthebandsofboth23–231and90–231recombinantPrPwhichhavebeenusedasmolecularweightmarkers.InbtheportionofthepanelwiththeGSSA117Vsamplesrequiredlongerexposure.Inpanelf,*ind

icatesthe13kDacomponentoftheglycosylatedandanchorbearingPrP12/13C-terminusfragmentswithN-terminiatresidues162–167and154–156,respectively[24,41]Craccoetal.ActaNeuropathologicaCommunications (2019) 7:85 Page4of9 mostC-terminalfragmentendedatresidue150(Fig.2a).Remarkably,wecouldnotdetectanytrypticpeptidesfromtheregionC-terminaltoresidue150.Giventhatnumer-ouspeptidesfromthelatterregionarereadilydetectablebyMSuponproteolyticdigestionofresPrPDfromsCJDcasesandmouseprionstrains[1,30],onecandefinitelyconcludefromthesedatathatthe16–18and22–23kDaspeciesinGSSA117VresPrPDindeedrepresentoligomers(likelytrimersandtetramers,respectively)ofinternalfrag-mentsencompassingresidueswithinthe82–150region.Itisofnotethatfragmentsintheoligomersaresomewhatshortercomparedtothoseinthe~7kDaband.MS-basedsequencinganalysisofproteinpresentintheresPrPD~8kDabandfromGSSF198SdemonstratedthatthisbandcontainedruggedinternalresPrPDfrag-mentsfromthe70–152region,withN-terminibetweenresidues70and90andC-terminibetweenresidues141and152(Fig.2bandAdditionalfile2:TableS2).Again,MSanalysisdidnotrevealthepresenceofpeptidesfrompartsofPrPotherthanthecentralregionbetweenresi-dues70and152.Previoussequencingstudiesofthe~8kDafragment(orfragmentsofsimilarkDa)extractedfromPrPamyloidplaquesandanalyzedbyEdmandeg-radationchemistryaloneorcombinedwithautomatedsequencingidentifiedthemajorN-terminusatresiduesG58,G74andG81whiletheC-terminuswasreportedatresidue150[27,34,35].Verysimilarinternalfragmentswereidentifiedinhighermolecularbandsof17–20and23–24kDainGSSF198SresPrPD,withtheexceptionthatthelongestofthesefragmentshadN-terminiatresidue74and78inthe17–20and23–24kDabands,respectively(Fig.2b).Importantly,akintothefindingforGSSA117VresPrPD,nopeptidesfromtheregionC-terminaltoresidue152weredetectedbyMSintrypticdigestsofproteininthesetwohighermolecularweightbands.Thus,alsoinGSSF198SresPrPD,thelatterbandscontainedcovalently-linkedoligomersoftheinternalPrPfrag-mentswithinthe74–78/142–152region,which,asinGSSA117V,aresomewhatshorterthanthe~8kDamonomer.UsingNanoLC-MSwealsodeterminedtherelativerep-resentationinresPrPDofthe129Mand129Vpolymorphicformsoftheprionprotein.Consistentwithapreviousre-port,resPrPDfromGSSA117Vwasinvariably100%129V,withnodetectable129Mpolymorph[33].Bycontrast,andatvariancewithapreviousreport,inGSSF198SresPrPD,allthreebandsexaminedconsistentlyshowedthepresenceof Fig.2Massspectrometry(MS)-basedsequencingofPrPfragmentsinthematerialextractedfromindividualgelbandsoftwoGSSvariants.GSSA117VresPrPD(a)andGSSF198SresPrPD(b).Thematerialpresentinthesebandswassubjectedtotrypsindigestion,followedbyMSanalysisoftrypticfragments.ThefragmentsidentifiedbyMSforspeciesextractedfromindividualgelbandsareshownasblue,redandgreenlines.Aminoacidsequencewithinthe70-152regionisshownabovethelines.ResiduesmarkedinredrepresentpotentialcleavagesitesintherelevantregionofPrP;129M/VpolymorphicresiduesaremarkedinblueCraccoetal.ActaNeuropathologicaCommunications (2019) 7:85 Page5of9 bothpolymorphicvariants,withalargedominance(76–88%)ofthe129Vpolymorph(Fig.3)[35].DiscussionThepresentresultsindicatethatthemechanismofresPrPDaggregationinGSSA117VandGSSF198Sinvolvesformationofcovalently-linkedmultimersofthe~7-8kDainternalfragments.Furthermore,ithasbeenpreviouslyshownbyEdmandegradationchemistrythatthe7and14kDafrag-mentsinGSSH187RresPrPDsharetheN-terminus,suggest-ingthatformationofcovalently-linkedmultimersmayalsotakeplaceinthelatterGSSvariant[12].PrPbandssuggest-iveofdimerizationhavebeenreportedincellsystemsandbraintissues[13,29].However,theresistancetoPKdiges-tionofthesePrPspeciesand,moreimportantly,theirmo-lecularnaturehavenotbeendetermined.Inthiscontext,thepresentdataprovidethestrongestevidencetodateforthepresenceofcovalentlycross-linkedspeciesofresPrPDinpriondiseases,suggestingthatthesepreviouslyunrecognizedspeciesmayplayamajorroleinthepatho-genesisofhumanpriondiseases.Ofnote,covalentcro

ss-linkinghasbeenreportedfortoxicaggregatesof-synucleinandA
involvedinParkinson’sandAlzhei-mer’sdiseases,respectively[2,3,9,10,32].Thisnovelfindinghasimportantimplicationsforunder-standingphenotypicvariabilityinhumanpriondiseases.EventhoughresPrPDaggregatesassociatedwithbothCJDandGSSphenotypeshavebeenshowntobetransmissible,thedramaticdifferencebetweenthesespeciessuggestsfun-damentallydifferentstructuralmechanismsofprionpro-teinconformationalconversioninGSSascomparedwiththoseinCJD[6,28].Indeed,theabsenceoftheconstraintsimposedbyglycansandtheGPIanchor(thataremissingin~7–8kDafragmentsandtheircovalently-linkedmulti-mers)islikelytoallowfordifferenttypesofresPrPDassem-blies.Apotentialstructuralmodelforthe~7–8kDafragmentsandtheirmultimersisprovidedbysyntheticamyloidfibrilsgeneratedfromPrP23–144,aproteinmatch-ingthesequenceoftheC-terminallytruncatedPrPhar-boredinGSSY145Stop[11,17].Thesesyntheticamyloidfibrilsadoptaparallelin-register
-structure[36]thatisfundamentallydifferentfromthe4-rungsolenoidmodelproposedfortheCJD-associatedresPrPD[5,37].Remark-ably,diseasedmiceinoculatedwithPrP23–144fibrilsaccu-mulateGSS-likeresPrPDaggregates(consistingof~7kDafragment)thatlikelysharethestructuralmotifofamyloidfibrilsusedastheinoculum[11].ThenatureofthecovalentcrosslinksbetweenPrPfrag-mentsandtheresiduesinvolvedinthesecrosslinksareatpresentunknown.However,ourdatastronglysuggestthatatleasttwodifferenttypesoflinkages(betweendifferentresidues)arelikelyinvolvedincouplingthemonomersthatformtrimersandtetramers,sinceasinglelinkagetypewouldprecludemassspectrometricidentificationofalltrypticfragmentsfromthe~80–150region.AlthoughthebasicstructuralcharacteristicsandmodeofaggregationofresPrPDinGSSA117VandGSSF198Sap-peartobesimilar,atleasttwofeaturesdistinguishthesetwoconditions.First,substantiallymoreresPrPDpopu-latesthehmwregionsoftheimmunoblotinGSSF198SthaninGSSA117V,suggestingastrongerpropensityforcovalentpolymerizationinGSSF198S.Second,resPrPDassociatedwithGSSA117Vconsistssolelyoffragmentscontainingthe129VpolymorphicformofPrP,whereasbothpolymorphsarepresentinGSSF198S,eventhoughthe129Vform Fig.3Relativeabundanceof129Mand129VPrPvariantsinresPrPDassociatedwithGSSA117VandGSSF198S.a:GSSA117V;b:GSSF198S.TherelativeabundanceofresPrPDwithMorVatresidue129reflectstherepresentationofthePrPmutation,whichiscoupledwiththe129VinbothGSSvariants.Approximately10–25%ofresPrPDcouldbeidentifiedasnon-mutated(wildtype)inGSSF198SwhileonlymutatedresPrPDcouldbedetectedinGSSA117V.TherelativepopulationsweredeterminedbymassspectrometryusingthespectralcountingmethodCraccoetal.ActaNeuropathologicaCommunications (2019) 7:85 Page6of9 predominates.SincepathogenicmutationsinbothGSSvariantsareonthebackgroundof129VPrP,thisimpliesthatresPrPDinGSSF198ScontainsinternalPrPfragmentsderivedbothfromboththemutantaswellaswild-typeproteins.Bycontrast,theinternalPrPfragmentspopulat-ingGSSA117VresPrPDrepresentexclusivelythemutantprotein.ThissuggeststhattheA117Vmutation(whichiswithinthePK-resistantfragments)mayimpedethetem-platedconversionofwildtypePrP.NosuchimpedimentwouldbeexpectedinGSSF198Swherethepathogenicmu-tationisoutsidethePK-resistantregion.Recently,anoveleight-residueinsertioninthehydro-phobicregionofPrPhasbeenassociatedwithaGSSphenotype[23].Brainsoftransgenicmiceharboringthecorrespondingmouseinsertionvariationrevealedthepresenceofa8kDaresPrPDfragmentsimilartothoseobservedinotherGSSvariantsaswellasa16kDathermolysin-resistantfragmentmappingtoresidues~23–155.Basedonthisfindingandotherdata,itwassug-gestedthatthe8kDainternalfragmentderivedfromtheN-terminaltruncationofthe16kDafragmentandthatthismechanismwassharedbyotherGSSvariants.Whilethishypothesismayapplytothemousemodeland,pos-sibly,tothisspecificGSSinsertionvariant,ourdemon-strationthathmwbandsharboroligomersofthe~7and~8kDafragmentsinGSSA117VandGSSF198S(andpossiblyGSSH187R)makestheproposedmechanismnotapplicablet

otheseGSSvariants.ConclusionWedemonstratedforthefirsttimethattheformationofmultimersofasmallinternalfragmentistheprimarymechanismofprionaggregationinGSSA117VandGSSF198S,twoclassicalvariantsofGerstmann-Sträussler-Scheinkerdisease(GSS).Prionaggregationbycovalently-linkedmulti-merformationfromasmallglycan-andGPI-freefragmentislikelytoallowfornovelprionassembles.Thisfindingopensnewhorizonsandlikelywillstimulatenewresearch.Covalently-linkedmultimersformationhasbeenreportedinotherneurodegenerativediseasessuchasParkinson’sandAlzheimer’sdiseases.AdditionalfilesAdditionalfile1:FigureS1.FurtherbiochemicalcharacterizationofresPrPDassociatedwithGSSF198S.Lane1:PNGaseF-deglycosylatedresPrPDfrombrainhomogenateimmunoblottedwith3F4followingstandardconditions.Lane2:withadditionalboiling,freezing-thawingandsonicationpre-deglycosylation;lane3:41hPNGaseFtreatment;lanes4–7:incubationwithstrongdenaturants,8Murea(afterethanolormetha-nolprecipitation,lanes4,5)and8Mguanidinehydrochlorideat80°C(afterethanolormethanolprecipitation,lanes6,7)pre-deglycosylation.TreatmentshadnodetectableeffectontheresPrPDelectrophoreticpro-file.(TIF17802kb)Additionalfile2:TableS1.Trypticpeptidesidentifiedfor~7KDabandinGSSA117VresPrPD.Residueatposition129ismarkedinblue.TableS2.Trypticpeptidesidentifiedfor~8KDabandinGSSF198SresPrPD.(PDF122kb)AbbreviationsAb:Antibody;BH:Brainhomogenates;CJD:Creutzfeldt-Jakobdisease;GPI:Glycosylphosphatidylinositol;GSS:Gerstmann-Sträussler-Scheinkerdisease;hmw:Highermolecularweight;K:Lysine;kDa:Kilodaltons;M:Methionine;MS:Massspectrometry;mw:Molecularweight;NanoLC-MS/MS:Nanoliquidchromatographymassspectrometry;NPDPSC:NationalPrionDiseasePathologySurveillanceCenter;PK:ProteinaseK;PMSF:Phenylmethanesulfonylfluoride;PrPD:Disease-relatedprionprotein;PVDF:Polyvinylidenedifluoride;R:Arginine;resPrPD:Disease-relatedproteinaseK-resistantprionprotein;SDS:Sodiumdodecylsulfate;TBS:Trisbufferedsaline;V:ValineAcknowledgementsTheauthorsthanktheCJDFoundation,thepatients’familiesandtheNationalPrionDiseasePathologySurveillanceCenter,inparticularBrianS.Appleby,MD,NPDPSCDirector,aswellasMses.JanisBlevins,KatieGlisicandMr.AaronFoutz.FundingThisstudywassupportedbytheUSNationalInstitutesofHealthGrantsR01NS083687,P01AI106705(toWKSandPG),P01AI077774(toPG),R01NS103848(toWKS)andTheCharlesS.BrittonFund(toPG).AvailabilityofdataandmaterialsThedatasetsusedand/oranalysedduringthecurrentstudyareavailablefromthecorrespondingauthoronreasonablerequest.Authors’contributionsConceptionanddesignoftheproject:LC,WKS,PG.Acquisitionofdata:LC,XX,SKN,JL,IC.Analysisandinterpretationofthedata:LC,XX,SKN,SN,WKS,PG.Writingofthemanuscript:LC,XX,SKN,WKS,PG.Revisionofthemanuscript:LC,XX,SKN,JL,IC,BG,SN,WKS,PG.Allauthorsreadandapprovedthefinalmanuscript.EthicsapprovalandconsenttoparticipateAllprocedureswereperformedunderprotocolsapprovedbytheInstitutionalReviewBoardatCaseWesternReserveUniversity.WritteninformedconsentforresearchwasobtainedfromallpatientsorlegalguardiansaccordingtotheDeclarationofHelsinki.Allpatients’dataandsampleswerecodedandhandledinaccordancewithNIHguidelinestoprotectpatients’identities.ConsentforpublicationNotapplicable.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Publisher’sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.Authordetails1DepartmentofPathology,CaseWesternReserveUniversity,Cleveland,OH,USA.2DepartmentofPhysiologyandBiophysics,CaseWesternReserveUniversity,Cleveland,OH,USA.3NationalPrionDiseasePathologySurveillanceCenter,CaseWesternReserveUniversity,Cleveland,OH,USA.4DepartmentofPathologyandLaboratoryMedicine,IndianaUniversitySchoolofMedicine,Indianapolis,IN,USA.Received:22April2019Accepted:9May2019 References1.Aguilar-CalvoP,XiaoX,BettC,EranaH,SoldauK,CastillaJ,NilssonKP,SurewiczWK,SigurdsonCJ(2017)Post-translationalmodificationsinPrPCraccoetal.ActaNeuropathologicaCommun

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