KeywordstypeIVcollagenrecombinantproteinHEK293cellheterotrimerAlportsyndromeReceivedforpublicationApril172003andinrevisedformJuly152003AcceptedforpublicationJuly3020032003bytheInternationalS ID: 940871
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KidneyInternational,Vol.64(2003),pp.1986Ð1996CharacterizationofassemblyofrecombinanttypeIVcollagen4,and5chainsintransfectedcellstrainsAKEHIROOBAYASHIandMAKOTOCHIYAMADivisionofPediatrics,DepartmentofHomeostaticRegulationandDevelopment,NiigataUniversityGraduateSchoolofMedicalandDentalSciences,NiigataCity,JapanCharacterizationofassemblyofrecombinanttypeIVcollagen4,and5chainsintransfectedcellstrains.Background. Keywords:typeIVcollagen,recombinantprotein,HEK293cell,het-erotrimer,Alportsyndrome.ReceivedforpublicationApril17,2003andinrevisedformJuly15,2003AcceptedforpublicationJuly30,20032003bytheInternationalSocietyofNephrologybiopsiesfromAlportsyndromepatientsshowstypicalul-trastructuralchangessuchasregionalthinning,thicken-ing,andsplittingintheglomerularbasementmembrane(GBM).AlportsyndromeisgeneticallyheterogeneouswithX-linked,autosomal-recessive,andmuchlesscom-monautosomal-dominantformsofinheritance[1].MostcasesofAlportsyndromearecausedbymutationsinthetypeIVcollagen5gene()ontheXchromo- brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Elsevier - Publisher Connector KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrainsonecontaining4(IV),and5(IV)chains,andtheothercontaining5(IV)and6(IV)chains.InX-linkedAlportsyndrome,mostmutationsinthegeneleadtothedisappearanceofnotonly5chainbutalso4(IV),and6(IV)chainsfromthebasementmembranesinthekidney[8].Ontheotherhand,inautosomalAlportsyndrome,mutationineitherthegeneleadstothedisap-pearanceof4(IV),and5(IV)chainsfromtheGBM,butthe5(IV)chainisretainedinthebasementmembraneofBowmanscapsules[9].Thesendingssup-porttheabovespeculation.Inthisstudy,toconrmtheassemblyof4(IV),and5(IV)chains,weestablishedsevencelllinesthatstablyexpressrecombinantmouse4(IV)and/or5(IV)chains,andweanalyzedtheassemblyofthesechainsincellextractsandcultureme-diaofthecellsusinganimmunoprecipitationmethod.Wealsointroducedtwoindividualsubstitutionmuta-tions,G1182R(GlytoArg)andC1573R(CysArg),intothe5(IV)expressionvector.Thesetwomuta-tionscorrespondtothosepreviouslyreportedinX-linkedAlportsyndromepatients[8,10].InordertounderstandthemolecularmechanismsunderlyingthepathogenesisofAlportsyndrome,weestablishedcelllinesthatcoex-pressthemutant5(IV)chainwiththewild-type4(IV)chains,andweanalyzedtheassemblyofthese(IV)chains.ConstructionofplasmidsThemousecDNAencodingNC1domainsofthe4(IV),and5(IV)chainswereampliedbypolymerasechainreaction(PCR)fromaZAPIImousekidneycDNAlibraryusingthreepairsofprimersbasedonpublishedsequencedata[11].MousecDNAclonesforthe4(IV),and5(IV)chainswereisolatedfromthelibraryusingtheampliedcDNAsasprobes.The5regionsoftheclonesweresuccessivelyamplibyPCRforprobes,andscreeningsofthecDNAlibrarywererepeatedusingtheprobes.Full-lengthmousecDNAsforeachchainwereconstructedfromaseriesofsomeoverlappingclones.PCRamplicationoftheregionofeachcDNAwasperformedinordertoalterthestopcodontotheDNAsequenceencodinganepitop
etagand/oranadditionalrestrictionendonucle-asesiteforsubcloning.DNAsequenceanalyseswereperformedtoexcludethepossibilityofnucleotidemiss-incorporationinPCR.ThemycepitopeEQKLISEEDL,astopcodonandanIsitewereintroducedintocDNAusingapairofPCRprimers,S3/R3(5-TCATCTTCACCCGACACAGT-3TAGATTACAGATCCTCTTCTGAGATGAGTTTTTGTTCATGTCTTTTCTTCATGCACA-3).Themod-cDNA,includingtheentireopenreadingframe,wasclonedintotheexpressionvectorpcDNA3.1/Hygro(Invitrogen,Carlsbad,CA,USA)containingahygromycinresistancegene.ThecDNA,includingtheentireopenreadingframe,wasclonedintotheexpressionvectorpcDNA6/V5-HisA(Invitrogen),whichcontainsacarboxyl-terminaltagencodingtheV5epitopeGKPIPNPLLGLDSTandablasticidinresistancegene,aftermodicationofastopcodontotheIrestrictionsiteusingapairofPCRprimers,S4/R4(5-CTGGGGCACCTGGCGAGAAG--TCTGAGCTGTGCTTCATGCAAAC-3).TheFLAGepitopeDYKDDDDK,astopcodonandIrestrictionsitewereintroducedintothecDNAusingapairofPCRprimers,S5/R5-CATTCATTAGTCGATGTGCA-3-CTCGAGCTACTTGTCATCGTCGTCCTTGTAATCTGTCCTCTTCATGCATACTT-3).TheresultantcDNA,includingtheentireopenreadingframe,wasclonedintotheexpressionvectorpCAGGScontainingacytomegalovirusenhancer,achicken-actinpromoter,andaneocassette[12].CellcultureandtransfectionsThehumanembryonickidneycellline293(HEK293)wasculturedinDulbeccosmodiedEaglesmedium(DMEM)containing10%fetalcalfserum(FCS).HEK293cells(5cells)weretransfectedwithgofthe,orcDNAexpressionplasmidbyusingLipofectaminePLUSreagent(GibcoBRL,Gaithersburg,MD,USA).Trans-fectantswereselectedinmediumcontaining200g/mLofHygromycinB(GibcoBRL)forg/mLofblasticidinS(Funakoshi,Tokyo,Japan)forand400g/mLofG418(GibcoBRL)forResistantcloneswerescreenedforproteinexpressionfromcellextractsbyWesternblotanalysisusinganti-myc(Invitrogen),anti-V5(Invitrogen)andanti-FLAG(SigmaChemicalCo.,St.Louis,MO,USA)monoclonalantibodies.Thesecloneswerenamed4,and5cellsaccordingtotheirtransfectedcDNAs.3cellsweretrans-fectedwiththecDNAexpressionplasmid,andestablishedpermanenttransfectantswere34and35cells,respectively.4cellsandcellsweretransfectedwiththecDNAexpres-sionplasmid,andstablytransfectedcloneswerenamed45and345cells,respectively.ImmunopuriÞcationof5(IV)proteinandassociatedproteinsTheclonalcelllinesweregrowntoconuenceon100mmPetridishesandextractedin1mLofNETN[50mmol/LTris-HCl,pH7.8,150mmol/LNaCl,1mmol/Lethyelenediaminetetraaceticacid(EDTA), KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrains1%NonidetP-40,1mmol/Ldithiothreitol(DTT),0.5mmol/Lphenylmethylsulfonyluoride(PMSF),g/mLpepstatinA,and1g/mLleupeptin].Conentcellswerealsoincubatedforanadditional24hoursin3mLofserum-freemedium.Themediumwascol-lectedandequilibratedto1mmol/LEDTA,0.5mmol/LPMSF,1g/mLpepstatinA,and1g/mLleupeptin.Onemilliliterofthecellextractorculturemediumwasthenincubatedwith30Lofanti-FLAGM2afgel(SigmaChemicalCo.)overnightat4C.Thegelwasvetimeswith10Vofice-coldNETN.Theboundproteinswereelutedfromtheafnitygelbycompet-ingFLAG-peptide(SigmaChemicalCo.
)ataconcentra-tionof200g/mLinTris-bufferedsalineandsubjectedto6%sodiumdodecylsulfate-polyacrylamidegelelec-trophoresis(SDS-PAGE)undernonreducingconditions.TheboundproteinswerealsoextractedbyboilinginSDSsamplebuffercontaining6%2andthenseparatedby7.5%SDS-PAGE.Proteinswerethenanalyzedbyimmunoblottingwithanti-myc,anti-V5,andanti-FLAGmonoclonalantibodiesandthenwithanalkalinephosphatase-conjugatedhorseantimouseIgGantibody.Colordevelopmentwasperformedusingnitrobluetetrazolium(NBT)and5-bromo-4-chloro-3-indolylphosphate(BCIP).Co-cultureof4,and5cells3cells(14cells(1),and5cells(1)wereco-culturedona100mmPetridishfor48hoursuntilconuence.Attheendoftheculture,cellswereextractedinNETN.Conuentcellswerealsoincubatedforanadditional24hoursinserum-freemedium,andthemediumwascollected.InvitromutagenesisInvitromutagenesisreactionswerecarriedoutcDNAinthepCAGGSvectorbyPCRbasedonamethoddescribedbyImaietal[13]withcations.Briey,oligonucleotideprimersweredesignedininvertedtail-to-taildirectionstoamplifythecloningvectortogetherwiththeinsert.Apointmutationforanaminoacidsubstitutionwasintroducedintothemiddleofoneprimer.Adeletionwasgeneratedbycationwithprimersthathaveacorrespondinggapbetweentheir5ends.Theprimersusedwere5TGTTGCCCATGGATTCCT-3-ACTGCAGTCACATTTTGATCforCOL(deletionof42-1462aminoacidresidues),5-GATTACAAGGACGACGATGA--GGTGGTTCCAGGGGGTCCTG-3(deletionof1463-1691aminoacidresidues),5-AGGACAAAAGCGAGAACCAGG-3-GCTGGCCCAGGAATGCCATC-3forG1182R,and5-CTGCAACATTAACAACGTTT-3-CAGCTGGCGCCTCACGCACTG-3forC1573R.Afterphosphorylationoftheendofeachprimer,parentalcDNAinsertedinthepCAGGSvectorwasampliedusingKOD-PlusDNApolymerase(Toyobo,Osaka,Japan)withtheseprimersfteenconsecutivecycles.AfterdigestionoftheparentalDNAwithI,theampliedDNAwasself-ligatedusingaligation-highkit(Toyobo)andtrans-fectedintoDH5-competentcells.Theplasmidswereprepared,andDNAsequencingconrmedobjectivemutationsandexcludedthepossibilityofadditionalmutationshavingbeenintroducedelsewhereinthecDNA.Mutant-typecDNAexpressionplasmidswereconstructedbyreplacementofapartofthewild-typecDNAwiththemutant-typecDNAusingappropriaterestrictionendonucleases.RESULTSRecombinantproductionof4(IV),andThecDNAs,includingtheentireopenreadingframesforthemouse4(IV),and5(IV)chains,wereclonedintotheeukaryoticexpressionvectorspcDNA3.1/Hygro,pcDNA6/V5-HisA,andpCAGGS,respectively.TheseexpressionplasmidsweretransfectedintoHEK293cells,andstabletransfectantswereestab-lished.Sinceeachrecombinantproteinwasexpressedasafusionproteincontainingashortepitopetag,mycforthe3(IV)chain,V5forthe4(IV)chain,andFLAGforthe5(IV)chain,onthecarboxyl-terminalregion,thepresenceoftherecombinantproteinwascon-rmedusinganti-epitopetagantibodies.Immunoblotanalyseswithanti-myc,anti-V5,andanti-FLAGmon-oclonalantibodiesrevealedtheexpressionsofthere-4(IV),and5(IV)chainsbothinthecellextractsandinserum-freeculturemediathathadbeenconcentratedtenfoldbyMicroconYM-100(Millipore,Bedford,MA,USA)(datanotshown).The
seresultsindicatethatthetransfectedHEK293cellspro-ducedtherecombinant4(IV),or5(IV)chain,whichwassecretedintotheculturemedium.Thesta-blytransfectedHEK293cellswerenamed4,andcells,respectively.Then,3cellsweretransfectedwiththeexpressionplasmidforrecombinant4(IV),andancelllinethatexpressedboth3(IV)and4(IV)chainswasestablished.Inthesamemanner,35cellsthatex-pressedboth3(IV)and5(IV)chains,45cellsthatexpressedboth4(IV)and5(IV)chains,and345cellsthatexpressed4(IV),and5(IV)chainswereestablishedandusedforfurtherexperiments.Figure1showstheexpressionsofrecombinant5(IV)chainsinthecellextractsorculturemediaof45,and345cellsunderreducingconditions.Expectedrecombinantproteinswerefoundbothineachcellextractandineachculturemedium.Inthe345cells,4(IV),and5(IV)chainswereexpressedto-gether.Thesizeofeveryrecombinantproteinwithalmost KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrains 199kD128199199kD1281283453(IV)4(IV)5(IV)293 A 199199kD199kD1281281283452934(IV)3(IV)5(IV) B Fig.1.Synthesisandsecretionofrecombinant4(IV),and5(IV)chainsintransfectedhumanembryonickidney293(HEK293)cells.Cellextracts()andtenfoldconcentratedculturemedia()ofHEK293cellstransfectedwithmouse4(IV),and5(IV)cD-NAsweresubjectedto7.5%sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)underreducingconditionsfollowedbyimmunoblotanalysesusingmonoclonalantibodiesagainstepitopetags,mycfor3(IV),V5for4(IV),andFLAGfor5(IV).Thenu-meralsin35,and45meanthekindof(IV)cDNAstransfectedintoHEK293cells.Thenumeral293meansuntransfectedHEK293cellsasacontrol.185kDwascompatibletotheproteinsizededucedfromaminoacidsequences.Theexpressionof4(IV)chainwasweakcomparedtothoseof3(IV)and4(IV)chainsbutwassufcienttoanalyzetheassemblyof4(IV),and5(IV)chains.Moreover,theamountof4(IV)chainintheculturemediumofeither345orcellswaslessthanthatintheculturemediumof34cells.Otherclonesofthesecellsalsoshowedsimilarexpres-sionpatterns(datanotshown).Thereasonforthisisnotclear.Thecoexpressed5(IV)chainmighthavehadaneffectonthesecretionofthe4(IV)chain.Immunoblotanalysesofthewhole-cellextractsandconcentratedculturemediaundernonreducingconditionsshowedhighbackground,andbandswithhighmolecularweightwereindistinct(datanotshown).Coimmunoprecipitationof4(IV)withInordertoanalyzecomplexformationof4(IV),and5(IV)chains,weexaminedtheinteractionamongtheseproteinsbycoimmunoprecipitationanaly-sesusingananti-FLAGantibody.Whole-cellextractsorculturemediaofHEK293cellstransfectedwithchainswereimmunoprecipitatedwiththeanti-FLAGantibody.TheimmunoprecipitantswereanalyzedbySDS-PAGEandsubsequentimmunoblottingwithanti-myc,anti-V5,andanti-FLAGantibodies.AsshowninFigure2,twobandsof5(IV)chainwithamolecularmassofeitherapproximately185kDor580kDappeared345cells,whereasonlyasinglebandwithamolec-ularmassofalmost185kDwasseenin5cellsundernonreducingconditions.The3(IV)and4(IV)chainswerecoimmunoprecipitatedwiththe5(IV)cha
inanddetectedasbandsmigratingaround580kD,theexpectedsizeofaheterotrimer,in345cells.Thesendingsindi-catethatthe4(IV),and5(IV)chainsformaheterotrimerandthatthe5(IV)chaindoesnotformahomotrimer.In345cells,theintensityofthe580kDbandofaheterotrimerwasmuchfainterthanthatofthe185kDbandofan5(IV)chainmonomerinimmunoblotanalyses.WhenthepolyacrylamidegelwasstainedwithCoomassiebrilliantblue,themonomerbandwasob-servedclearly,buttheheterotrimerbandwashardlydetectable(datanotshown).Thesendingsindicatethatthemajorityof5(IV)chainsexistasmonomersinboththecellextractandculturemediumof345cells.Un-derreducingconditions,eithercoimmunoprecipitated3(IV)or4(IV)chainwiththe5(IV)chainappearedasasinglebandwithamolecularmassof185kD(Fig.3).Sincethebandsunderreducingconditionswereclearerthanthosewithamolecularmassof580kDundernonre-ducingconditions,immunoprecipitationanalysesofvar-iouscellstrainsweredoneunderreducingconditions.AsshowninFigure3,neitherthe3(IV)chainnorthe4(IV)chainwascoimmunoprecipitatedwiththechainbytheanti-FLAGantibodyinthecellextractsandculturemediaof35and45cells,incontrasttotheingsforcellextractandculturemediumof345cells.Theseresultsindicatethatthe5(IV)chaindoesnotformacomplexwitheitherthe3(IV)or4(IV)chainalone.Todeterminewhetherthe4(IV),andmonomersinthemixtureofwhole-cellextractsorculturemediaof4,and5cellsformaternarycomplex,the KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrains 123456789 123456789 Fig.2.Formationofaheterotrimerof4(IV),and5(IV)chainsincells.Cellextracts()andcultureme-dia()of345(lanes1,4,and7),(lanes2,5,and8)andcontrolhumanem-bryonickidney293(HEK293)(lanes3,6,and9)cellswereincubatedwithanti-FLAGM2afnitygel.Afterwashingthegel,theboundproteinselutedbycompetingFLAG-peptidewereanalyzedby6%sodiumdode-cylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)undernonreducingconditionsfollowedbyimmunoblotanalyseswithanti-FLAG,anti-myc,andanti-V5monoclonalan-tibodies.Thepositionsofthemonomer(M)andheterotrimer(T)areindicated.whole-cellextractandculturemediumofco-cultured4,and5cellswereimmunoprecipitatedwiththeanti-FLAGantibody.AsshowninFigure4,the3(IV)and4(IV)chainswerenotcoimmunoprecipitatedwiththe5(IV)chainbytheanti-FLAGantibodyeitherinthecellextractorculturemediumofco-cultured4,and5cells.Theseresultssuggestthatacomplexof4(IV),and5(IV)chainsmaybeformedin345cellsfollowedbysecretionintotheculturemedium.Effectofdeletionin5(IV)onternarycomplexformationof4(IV),andEachchainof4(IV),and5(IV)consistsofalongcollagenousdomainofapproximately1400residueswithGly-X-YrepeatsandanNC1domainofabout230residuesatthecarboxylterminus.Todeter-minewhether5(IV),inwhicheachdomainisdeleted,formsaternarycomplexwith3(IV)and4(IV)chainsinthisassaysystem,weestablishedcelllinesNC1cellsexpressmutantproteinlackingtheNC1domainwith3(IV)andchains,andCOLcellsexpressmutant5(IV)lack-ingthecollagenousdomainwith3(IV)andchains.Asshownbytheresultsofimmunoblotanaly-sispresentedinFigure5A,thetrunc
ated5(IV)pro-teinwithasizeof160kDwasdetectedwiththe4(IV)chainsinNC1cellextract.Neither3(IV)chainnorthe4(IV)chainwascoimmuno-precipitatedwiththetruncated5(IV)proteinusingtheanti-FLAGantibodyinNC1cellextracts(Fig.5B).Figure5Cshowstheexpressionofthemutantproteinthathasadeletionofthecollagenousdomain3(IV)and4(IV)chainsinCOLcells.Thesizeofthetruncatedproteinwasabout28kD,consis-tentwiththesizeexpectedfromaminoacidsequence.Thismutant5(IV)proteinalsodidnotcoimmunopre-cipitatethe3(IV)and4(IV)chainsusingtheanti-FLAGantibodyinCOLcellextracts(Fig.5D).TheseresultsindicatethatboththecollagenousdomainandtheNC1domaininthe5(IV)chainareindis-pensablefortheformationofacomplexwith4(IV)chainsinthisimmunoprecipitationassay KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrains 45293 199kD199kD199kD2935(IV)3(IV)4(IV)B Fig.3.Coimmunoprecipitationof4(IV)with5(IV)inhumanembry-onickidney293(HEK293)cellstransfected(IV)cDNAs.Cellextracts()andcul-turemedia()ofHEK293cellstransfected(IV)cDNAswereincubatedwithanti-FLAGM2afnitygel.Afterwashingthegel,theboundproteinswereanalyzedby7.5%sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)underreduc-ingconditionsandimmunoblottingwithanti-FLAG,anti-myc,andanti-V5monoclonalan-tibodies.Effectofaminoacidsubstitutionof5(IV)onternarycomplexformationof4(IV),andSite-directedmutagenesiswasusedtogenerateaglycine-to-argininesubstitutionattheaminoacidresidue1182(G1182R)andacysteine-to-argininesubstitutionattheaminoacidresidue1573(C1573R)inthemouse5(IV)chain.Weestablishedcelllines345G1182Rand345G1182RcellsexpressamutantchaincontainingtheG1182Rsubstitutionwith4(IV)chains,and345C1573Rcellsexpressamu-5(IV)chainthatcontainstheC1573Rsubstitution3(IV)and4(IV)chains.Intheextractof345G1182Rcells,the3(IV)and4(IV)chainswerecoimmunoprecipitatedwiththe5(IV)chainusingtheanti-FLAGanti-body.Theamountsof3(IV)and4(IV)chainspre-cipitatedwiththemutant5(IV)chainwerealmostthesameastheamountsofthosechainsprecipitatedwiththewild-type5(IV)chain(Fig.6A).Inculturemedium,3(IV)and4(IV)chainswerecoimmunopre-cipitatedwiththeG1182R-type5(IV)chain,buttheamountsofthecoimmunoprecipitated3(IV)and4(IV)chainsin345G1182Rcellsweremarkedlyde-creasedcomparedtotheamountsofthosechainsincells(Fig.6B).TheseresultssuggestthattheG1182R-5(IV)chainisabletoformacomplexwith4(IV)chainsandthattheamountofsecretionand/ordegreeofstabilityoftheformedcomplexin345G1182Rcellsmightbedecreased.Intheextractof345C1573Rcells,theC1573R-type5(IV)chainwasimmunoprecipitatedbytheanti-FLAGantibody,butthecoimmunoprecipitaitonofeitherthe3(IV)chainor4(IV)chainwashardlyseen(Fig.6A).Moreover,theC1573R-type5(IV)chain,aswellasthe3(IV)and4(IV)chains,wasnotimmunoprecipitatedbytheanti-FLAGantibodyinculturemediumof345C1573Rcells(Fig.6B).ThesendingsindicatethattheC1573Rsub-stitutioninthe5(IV)chainimpairstheabilityofthe5(IV)chaintoformacomplexwith3(IV)andchainsandtobesecretedfromcellseveninmono
mericCollagenisthemostabundantproteininthemam-malianbody.Nineteendifferenttypesofcollagen, KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrains 199kD199kD1993453455(IV)3(IV)4(IV)A 1991991993453455(IV)3(IV)4(IV)B Fig.4.Immunoprecipitationinco-cultured4,and5cells.Cellextracts()andcul-turemedia()of345cellsandco-cultured4,and5cells(5)wereimmuno-precipitatedwithananti-FLAGmonoclonalantibodyandanalyzedby7.5%sodiumdode-cylsulfate-polyacrylamidegelelectrophore-sis(SDS-PAGE)underreducingconditionsfollowedbyimmunoblotanalyseswithanti-FLAG,anti-myc,andanti-V5monoclonalantibodies.consistingofmorethan30kindsofcollagenchains,havesofarbeenidentied.Threecollagenchainsareassem-bledandformacollagenmoleculewithatripleheli-calstructure.Whetherthemoleculeisahomotrimerorheterotrimerdependsonthetypeofcollagen.Collagenmoleculesdifferinstructure,expression,tissuedistri-bution,andfunction.Mutationshavebeenreportedinmorethanhalfoftheknowncollagengenesandhavebeenshowntoresultinhumangeneticdisorderssuchasosteogenesisimperfecta,Ehlers-Danlossyndrome,anddystrophicepidermolysisbullosa[14].TypeIVcolla-gens,whichcontainsixdistinctchains,5(IV),and6(IV),arefoundinbase-mentmembraneswithatissue-specicdistributionpat-tern.Two1(IV)chainsandone2(IV)chainformaheterotrimer,[2(IV)andexistinalmostallbasementmembranes[15,16].Inimmunohistochemicalanalysesofkidneysections,4(IV),andchainsshowasimilarstainingpatternintheGBM.InAl-portsyndromepatients,mutationsineitherthe4(IV),or5(IV)chainusuallyleadtoanabsenceofallthreechainsintheGBM.ThesefactssuggestthatthethreechainsformaheterotrimerintheGBMandthatmostmutationsofthechainsinAlportsyndrome KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrains 199kD128199199345NC12935(IV)3(IV)4(IV)A 199199199345NC12935(IV)3(IV)4(IV) B Fig.5.Effectofdeletionin5(IV)onternarycomplexformation4(IV),and34cellsweretransfectedwith5(IV)thatlackseithertheNC1domainorcollagenousdomain,andstabletransformantswereestablished(NC1andcells).ImmunoblotanalysesofextractsfromNC1()andCOLcells()wereperformed.ExtractsfromNC1(COLcells()wereimmunoprecipitatedwithananti-FLAGmonoclonalantibodyandthensubjectedto7.5%sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)underreduc-ingconditionsfollowedbyimmunoblotanalyseswithanti-FLAG,anti-myc,andanti-V5monoclonalantibodies.Arrowheadsin5(IV)chains.Twostronglyreactedbandsseeninalllanesin(D)areheavyandlightchainsofanti-FLAGantibodyelutedfromafnitygel.causeadefectinassemblyofthethreechains[69].Re-cently,Gunwaretal[17]analyzedtruncatedtriple-helicalmoleculespuriedfromtheGBMandidentiedanoveldecross-linkednetworkof4(IV),and 42kD1991993452935(IV)3(IV)4(IV) 32C 423234529385 1991991993455(IV)3(IV)4(IV)D Fig.5.(Continued). KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrains G1182R
G1182R 5(IV)3(IV)4(IV)B Fig.6.Effectofaminoacidsubstitutionof5(IV)onternarycom-plexformationof4(IV),and34cellsweretrans-fectedwitheitherG1182RorC1573Rmutant-type5(IV),andstabletransformantswereestablished(345G1182Rand345C1573Rcells).Cellextracts()andculturemedia()of345G1182R,and345C1573Rcellswereimmunoprecipitatedwithananti-FLAGmon-oclonalantibodyandthensubjectedto7.5%sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)underreducingcon-ditionsfollowedbyimmunoblotanalyseswithanti-FLAG,anti-myc,andanti-V5monoclonalantibodies.5(IV)chains.TheyalsoshowedthattheNC1domainmaycontainarecognitionsequencefortheassemblyof4(IV),and5(IV)chainsbyusingnativeandrecombinantNC1domainsof1(IV)to6(IV)chains[18].Ontheotherhand,Heikkilaetal[19]showedthatarecombinanthuman5(IV)chain,whichwasexpressedbyusinganadenovirusvectorinhumanHT1080cellsthatnormallyexpress1(IV)to5(IV)chains,assembledinaheterotrimerwithendogenous3(IV)andchains.Inthisstudy,weestablishedHEK293transfectantsexpressingrecombinantmouse3(IV)and/or5(IV)chains.HEK293cellsareknowntosynthesizefew,ifany,extracellularmatrixproteinsandtoproducerecombinantproteinsinlargequantityusinggeneticengineeringtechniques[2022].Inthepresentstudy,weusedmouse,nothuman,cDNAs.TissuedistributionsoftypeIVchainsinmicearethesameasthoseinhu-mans[11,23].ElectronmicroscopicanalysisoftheGBMknockoutmiceshowedtypicalchangesfoundinpatientswithAlportsyndrome,andnostaining4(IV),and5(IV)chainswasdetectedintheGBM.Ithasalsobeenshownthatoutmicedevelopproteinuriaat1to2monthsofageanddieofrenalfailureby3to4monthsofage[24,25].Thesendingsindicatethattheremightbenodifferencesintheessentialrolesof4(IV),and5(IV)chainsintheGBMinmiceandhumans.Eachchainof4(IV),and5(IV)waspresentbothinthecellextractsandculturemediaof4,and5cells.Theabilityof4(IV),and5(IV)chainstobesecretedisinagreementwithndingspreviouslyre-portedbyLeinonenetal[26].TheyestablishedHEK293cellsthatstablyexpressthe3(IV)chainandshowedthattherecombinant3(IV)chainwassecretedasasin-glepolypeptidechain.Ithasalsobeenreportedthatthe1(IV)chainwassecretedasarandomcoilstructureinCHOcellsoverexpressingthe1(IV)chain[27].Inthepresentexaminationofimmunoprecipitationusingcellextractsandculturemediabyananti-FLAGmonoclonalantibody,the3(IV)and4(IV)chainswerecoimmunoprecipitatedwiththe5(IV)chainincells,butneitherthe3(IV)chainnorthe4(IV)chainwascoimmunoprecipitatedwiththe5(IV)chainin45cells.Thesendingssuggestthatcoexpressionof4(IV),and5(IV)chainsisnecessaryforformationofacomplex,whichisaheterotrimer.Acom-plexwasnotformedincellextractorculturemediumof4,and5cells,indicatingthat4(IV),and5(IV)chainsmightbeassembledintoahet-erotrimerin345cellsandthensecretedintotheculturemedium.Thisspeculationiscompatiblewiththefactthatcollagenchainsassociateandarefoldedwithsomechap-eroneproteinssuchasheatshockprotein47(HSP47)inthecytoplasmandthenthecorrectlyformedheterotrimerissecreted[14].HSP47specicallyinteractswithvarioustypes
ofcollagenandactsasacollagen-specicmolecularchaperonethatfacilitatesnormalcollagenbiosynthesisbiosynthesisSincetheassociationamongcollagenchainsoccursinacarboxyl-terminaltoamino-terminaldirection,theNC1domain,whichislocatedinthecarboxyl-terminalend,isthoughttoplayacrucialroleincomplexformation.Infact,ininvitroassemblystudiesusingpuriednativeand1(IV)to5(IV)NC1monomersofabout230aminoacids,twokindsofcomplexwereformed.Onecontained1(IV)and2(IV)NC1monomers,andtheothercontained4(IV),and5(IV)NC1monomers[18].Inourexperiment,5(IV)lackinganNC1domaindidnotformacomplexwith3(IV)and4(IV)chains.Thisndingsupportsthespeculationthat KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrainstheNC1domainisimportantfortheassemblyof4(IV),and5(IV)chains.Ontheotherhand,lackingacollagenousdomainwasnotcoimmunoprecip-itatedwith3(IV)and4(IV)chains,indicatingthatifNC1domainsof4(IV),and5(IV)chainsareassociated,thestabilityoftheassemblyisnotsufforcoimmunoprecipitationinourpresentexperimentalconditions.IntypeIVcollagen,singletriple-helicalmoleculesas-sociatedatthecarboxyltermini(NC1-to-NC1)formingdimmersandattheaminoterminiformingtetramers[5].Inthe7Sdomainattheaminoterminus,therearenumeroushydroxylysine-linkeddisaccharideunitsandanasparagine-linkedoligosaccharideunit.Thesecarbo-hydrateunitsplayanimportantroleintheformationandstabilizationofthetetramer[29,30].Itisnotclearwhethertherecombinant(IV)chainsinthisstudywereglycosylatedornot.Alportsyndromeiscausedbymutationsinthe,andgenes[2,3].Themuta-tionscancausestructuralandpathophysiologicchangesincollagenchains,resultinginGBMabnormalities.Thechangesmayaffecttheassemblyandfoldingofthree(IV)chainsintoatriple-helicalmoleculeaswellasthestabilityofthehelixstructure.AlmostallofthepointmutationspreviouslyreportedinAlportsyndromeweregeneratedbysubstitutionofeitherglycineresiduesinthecollagenousdomainorconservedaminoacidsintheNC1domain[1].WeintroducedaG1182RorC1573Rsubsti-tutionintothemouse5(IV)chain.Theglycineataminoacid1182inthemouse5(IV)chain,correspondingtoglycineataminoacid1182inthehuman5(IV)chain,existsinthecollagenousdomainandcorrespondstoGlyofaGly-X-Yrepeat.Thecysteineataminoacid1573inthemouse5(IV)chainisinthemiddleoftheNC1do-mainandcorrespondstocysteineataminoacid1567inthehuman5(IV)chain[23,31].BoththeG1182Rsubsti-tutionandthecysteine-to-argininesubstitutionataminoacid1567(C1567R)inthehuman5(IV)chainhavebeenedinX-linkedAlportsyndromepatients.AmalepatientwiththeG1182Rsubstitutionexhibitedearlyoc-currenceofhematuriaandocularabnormalitiesbutnodeafnessandonlymildrenalimpairmentattheageof16years[8].Ontheotherhand,amalepatientwiththeC1567Rsubstitutionhadaseverephenotype.Heshowedearlyoccurrenceofhematuria,hearingloss,andocularle-sions,andend-stagerenalfailureoccurredattheageof16years[10].Inthepresentstudy,theG1182R-type5(IV)chainhadtheabilitytoformacomplexwith3(IV)and4(IV)chainstoalmostthesamedegreeasthatofthewild-type5(IV)chaininthecellex-tr
acts.However,theamountofcomplexescontainingthe5(IV)chainwasmuchlessthanthatofcom-plexescontainingthewild-type5(IV)chaininthecul-turemedium.Consideringglycineistheonlyaminoacidsmallenoughtotintothecenterofthetriplehelix,the5(IV)chainmightleadtoreducedsta-bilityofthecomplexcomparedtothewild-typechain.TheC1573R-type5(IV)chainhardlyassociated3(IV)and4(IV)chains.Moreover,themutantchainwasnotsecretedfromcellseveninmonomericform.ItislikelythattheC1573Rmutationaltersthepro-teinconformationandthattheabilitytoformacomplexandtobesecretedisimpaired.Thesendingssuggestthatadefectintrimerformationby4(IV),and5(IV)chainsisoneofthemolecularmechanismsunder-lyingthepathogenesisofAlportsyndrome.Ontheotherhand,itisinterestingthatthepatientwithaC1567R-5(IV)chain,whichmightpossessnoabilitytoformtrimerswith3(IV)and4(IV)chains,exhibitedamoreseverephenotypeofAlportsyndromethandidthepatientwithaG1182R-type5(IV)chain,whichmightpossessresidualabilitytoformtrimerswith3(IV)and4(IV)chains.Resultsoffurtherstudiesusingvariousre-combinantmutantsof4(IV),and5(IV)chainsmightprovideinformationonthecorrelationsbetweenmoleculardefectsandclinicalphenotypesofAlportsyndrome.ACKNOWLEDGMENTSWethankDr.IchiroSatokataforkindlyprovidingamousekid-neycDNAlibrary,Dr.MasanariNakayamaforkindlyprovidingPCRprimers,andDr.Jun-ichiMiyazakiforkindlyprovidingpCAGGSexpressionvector.WealsothankDr.ToshioKakiharaforhishelpfuladvice.ReprintrequeststoTakehiroKobayashi,M.D.,DivisionofPedi-atrics,DepartmentofHomeostaticRegulationandDevelopment,NiigataUniversityGraduateSchoolofMedicalandDentalSciences,1-757Asahimachi-dori,NiigataCity,951-8510,Japan.E-mail:takekoba@med.niigata-u.ac.jp1.TK,MP:Alportsyndromeandbasementmem-branecollagen,inTheMetabolicandMolecularBasesofInherited(vol.4),editedbySCR,BAL,SWS,VD,NewYork,McGraw-Hill,2001,pp54532.BDF,HSL,Zetal:Identicationofmuta-tionsintheCOL4A5collagengeneinAlportsyndrome.1226,19903.MT,LHH,Metal:Identicationofmutationsinthe3(IV)and4(IV)collagengenesinautosomalrecessiveAlportsyndrome.NatGenet82,19944.MM,ZK,YTL,RST:Colocaliza-tionofthegenesforthe3(IV)and4(IV)chainsoftypeIVcol-lagentochromosome2bandsq35-q37.813,19925.HBG,RST,TK:TypeIVcollagen:struc-ture,geneorganization,androleinhumandisease.MolecularbasisofGoodpastureandAlportsyndromesanddiffuseleiomyomatosis.JBiolChem26036,19936.ZJ,RST:ThechainsoftypeIVcollagen,inPathologyandGeneticsofAlportSyndrome,editedbyTK,Basel,Karger,1996,pp807.NY,KM,Ietal:Differentialexpres-sionoftwobasementmembranecollagengenes,COL4A6andCOL4A5,demonstratedbyimmunouorescencestainingusing KobayashiandUchiyama:RecombinanttypeIVcollagenintransfectedcellstrainscmonoclonalantibodies.JCellBiol8.NI,KS,Netal:RelationshipbetweenCOL4A5genemutationanddistributionoftypeIVcollageninmaleX-linkedAlportsyndrome.KidneyInt311,19969.GMC,KB,Betal:AutosomalrecessiveAlportsyndrome:ImmunohistochemicalstudyoftypeIVcollagenchaindistribution.KidneyInt1147,199510.KB,BC,Fetal:Spectrumofmuta-tion
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