Autoantibodies to transcription intermediary factor (TIF)1β associated with dermatomyositis

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Title:
Autoantibodies to transcription intermediary factor (TIF)1β associated with dermatomyositis
Series Title:
Arthritis Research & Therapy
Physical Description:
Book
Language:
English
Creator:
Satoh, Minoru
Chan, Jason Y. F.
Ross, Steven J.
Li, Yi
Yamasaki, Yoshioki
Yamada, Hidehiro
Vazquez-del Mercado, Monica
Petri, Marcelo H.
Jara, Luis J.
Saavedra, Miguel A.
Cruz-Reyes, Claudia
Sobel, Eric S.
Reeves, Westley H.
Ceribelli, Angela
Chan, Edward K. L.
Publisher:
BioMed Central
Publication Date:

Notes

Abstract:
Introduction: Myositis specific autoantibodies are associated with unique clinical subsets and are useful biomarkers in polymyositis/dermatomyositis (PM/DM). A 120 kD protein recognized by certain patients with DM was identified and clinical features of patients with this specificity were characterized. Methods: The 120 kD protein recognized by a prototype serum was purified and identified by mass spectrometry and immunological methods. Autoantibody to this 120 kD protein was screened in sera from 2,356 patients with various diagnoses from four countries, including 254 PM/DM, by immunoprecipitation of 35S-methionine labeled K562 cell extracts. Clinical information of patients with this specificity was collected. Results: The 120 kD protein, which exactly comigrated with PL-12, was identified as transcription intermediary factor TIF1β (TRIM28) by mass spectrometry and validated by immunoassays. By immunofluorescence, anti-TIF1β positivity showed a fine-speckled nuclear staining pattern. Four cases of anti-TIF1β were identified; all are women, one each in a Japanese, African American, Caucasian, and Mexican individual. Three had a diagnosis of DM and one case was classified as having an undifferentiated connective tissue disease with an elevated CPK but without significant muscle symptoms. This individual also had a history of colon cancer, cervical squamous metaplasia and fibroid tumors of the uterus. Myopathy was mild in all cases and resolved without treatment in one case. The anti- TIF1b specificity was not found in other conditions. Conclusions: Anti-TIF1β is a new DM autoantibody associated with a mild form of myopathy. Whether it has an association with malignancy, as in the case of anti-TIF1γ, or other unique features will need to be evaluated in future studies.

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University of Florida
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University of Florida
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doi - 10.1186/ar3802
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AA00012627:00001


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Abstract
Introduction
Myositis specific autoantibodies are associated with unique clinical subsets and are useful biomarkers in polymyositis/dermatomyositis (PM/DM). A 120 kD protein recognized by certain patients with DM was identified and clinical features of patients with this specificity were characterized.
Methods
The 120 kD protein recognized by a prototype serum was purified and identified by mass spectrometry and immunological methods. Autoantibody to this 120 kD protein was screened in sera from 2,356 patients with various diagnoses from four countries, including 254 PM/DM, by immunoprecipitation of 35S-methionine labeled K562 cell extracts. Clinical information of patients with this specificity was collected.
Results
The 120 kD protein, which exactly comigrated with PL-12, was identified as transcription intermediary factor TIF1β (TRIM28) by mass spectrometry and validated by immunoassays. By immunofluorescence, anti-TIF1β positivity showed a fine-speckled nuclear staining pattern. Four cases of anti-TIF1β were identified; all are women, one each in a Japanese, African American, Caucasian, and Mexican individual. Three had a diagnosis of DM and one case was classified as having an undifferentiated connective tissue disease with an elevated CPK but without significant muscle symptoms. This individual also had a history of colon cancer, cervical squamous metaplasia and fibroid tumors of the uterus. Myopathy was mild in all cases and resolved without treatment in one case. The anti-TIF1β specificity was not found in other conditions.
Conclusions
Anti-TIF1β is a new DM autoantibody associated with a mild form of myopathy. Whether it has an association with malignancy, as in the case of anti-TIF1γ, or other unique features will need to be evaluated in future studies.
http:purl.orgdcelements1.1creator
Satoh, Minoru
Chan, Jason YF
Ross, Steven J
Li, Yi
Yamasaki, Yoshioki
Yamada, Hidehiro
Vazquez-del Mercado, Monica
Petri, Marcelo
Jara, Luis J
Saavedra, Miguel A
Cruz-Reyes, Claudia
Sobel, Eric S
Reeves, Westley H
Ceribelli, Angela
Chan, Edward KL
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Satoh et al.; licensee BioMed Central Ltd.
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Arthritis Research & Therapy. 2012 Apr 18;14(2):R79
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RESEARCHARTICLE OpenAccessAutoantibodiestotranscriptionintermediary factor(TIF)1b associatedwithdermatomyositisMinoruSatoh1,2*,JasonYFChan1,StevenJRoss1,3,YiLi1,YoshiokiYamasaki4,HidehiroYamada4, MonicaVazquez-delMercado5,6,MarceloHPetri5,LuisJJara7,8,MiguelASaavedra9,ClaudiaCruz-Reyes9, EricSSobel1,2,WestleyHReeves1,2,AngelaCeribelli3andEdwardKLChan3AbstractIntroduction: Myositisspecificautoantibodiesareassociatedwithuniqueclinicalsubsetsandareusefulbiomarkers inpolymyositis/dermatomyositis(PM/DM).A120kDproteinrecognizedbycertainpatientswithDMwasidentified andclinicalfeaturesofpatientswiththisspecificitywerecharacterized. Methods: The120kDproteinrecognizedbyaprototypeserumwaspurifiedandidentifiedbymassspectrometry andimmunologicalmethods.Autoantibodytothis120kDproteinwasscreenedinserafrom2,356patientswith variousdiagnosesfromfourcountries,including254PM/DM,byimmunoprecipitationof35S-methioninelabeled K562cellextracts.Clinicalinformationofpatientswiththisspecificitywascollected. Results: The120kDprotein,whichexactlycomigratedwithPL-12,wasidentifiedastranscriptionintermediary factorTIF1 b (TRIM28)bymassspectrometryandvalidatedbyimmunoassays.Byimmunofluorescence,anti-TIF1 b positivityshowedafine-specklednuclearstainingpattern.Fourcasesofanti-TIF1 b wereidentified;allarewomen, oneeachinaJapanese,AfricanAmerican,Caucasian,andMexicanindividual.ThreehadadiagnosisofDMand onecasewasclassifiedashavinganundifferentiatedconnectivetissuediseasewithanelevatedCPKbutwithout significantmusclesymptoms.Thisindividualalsohadahistoryofcoloncancer,cervicalsquamousmetaplasiaand fibroidtumorsoftheuterus.Myopathywasmildinallcasesandresolvedwithouttreatmentinonecase.TheantiTIF1 b specificitywasnotfoundinotherconditions. Conclusions: Anti-TIF1 b isanewDMautoantibodyassociatedwithamildformofmyopathy.Whetherithasan associationwithmalignancy,asinthecaseofanti-TIF1 g ,orotheruniquefeatureswillneedtobeevaluatedin futurestudies.IntroductionAutoantibodiestocellularc onstituentsareclinically importantbiomarkersassociatedwithparticulardiagnoses, specificclinicalfeaturesorsu bsetsofdisease,helpingto establishadiagnosis,and/orpredictingorganinvolvement andprognosis[1,2].Inparticular,inpolymyositis/dermatomyositis(PM/DM)andscleroderma(systemicsclerosis, SSc)patientscanbeclassifiedintoseveralsubsetsassociatedwithcharacteristicclinicalfeaturesbasedonspecific autoantibodies,sincecoexistenceofotherdisease-specific autoantibodiesisuncommon[3].Eachmyositisspecific antibody(MSA)isassociatedwithauniqueclinicalsubset. Forexample,theanti-synthetasesyndromewasnamedfor thepresenceofanti-Jo-1andotherautoantibodiestoaminoacyltRNAsynthetasesfoundinasubsetofpatients withPM/DMwhoseclinicalpresentationwasdominated byinterstitiallungdisease(ILD),Raynaud sphenomenon, arthritis,fever,andmechanic shands[3,4].Althoughnew autoantibodyspecificitieshavebeenreported,approximately40%to50%ofpatientswithPM/DMarestillwithoutaknownMSAcomparedwithonlyapproximately 15%inSScwithoutassociationtoknownSScantibodies [2].Thus,identifyingnewMSAmayhelpinmonitoring PM/DMpatientsandseveralnewclinicallysignificant autoantibodiesassociatedwithDMincludinganti-p155/ 140[5-11],anti-CADM(clinicallyamyopathicDM)140/ *Correspondence:minoru.satoh@medicine.ufl.edu1DivisionofRheumatologyandClinicalImmunology,Departmentof Medicine,UniversityofFlorida,P.O.Box100221,1600SWArcherRd, Gainesville,FL32610-0221,USA FulllistofauthorinformationisavailableattheendofthearticleSatoh etal ArthritisResearch&Therapy 2012, 14 :R79 http://arthritis-research.com/content/14/2/R79 2012Satohetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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MDA5(melanomadifferentiationassociatedantigen5) [10,12-14],anti-SAE(smallubiquitin-likemoleculeactivatingenzyme)andanti-MJ/NXP-2havebeenreported recently[15,16].Amongthese,anti-p155/140hasbeen studiedextensivelyinaveryshortperiodoftimeduetoits strongassociationwithmalignancy[5-9,11]whichwas confirmedbyarecentmeta-analysis[17].However,this associationdoesnotappear toapplytochildren[7]or youngadults[11].p155wasi dentifiedastranscription intermediaryfactor1 g ,(TIF1 g ,alsoknownastripartite motif(TRIM)33)[18].ArecentstudyinJapanesepatients hasidentifiedthep140asTIF1 a andanotherrelated moleculeTIF1 b hasalsobeenidentifiedasatargetof autoantibodiesinDM[11].Inthepresentstudy,wehave independentlyidentifiedtheapproximately120kDautoantigenicproteinasTIF1 b bymassspectrometry.Thepresenceofanti-TIF1 b andclinicalfeaturesofAmerican, Mexican,andJapanesepatientswiththisspecificitywere characterized.MaterialsandmethodsPatientsAtotalof2,356sera,including1,966subjectsenrolledin theUniversityofFloridaCenterforAutoimmune Diseases(UFCAD)registryfrom2000to2010,werestudied.DiagnosesoftheUFCADpatientsinclude434systemiclupuserythematosu s(SLE),86PM/DM(51PM including12PM-SScoverlap,35DM),121SSc,and122 rheumatoidarthritis(RA). Additionally,serafrom 36PM/DM(13PM,20DM,3amyopathicDM)fromSt. MariannaUniversityHospital(Kawasaki,Japan),74PM/ DM(18PM,56DM)serafromGuadalajaraandMexico City(Mexico),58PM/DM(25PM,27DM,6overlap: 4PM-SSc,1DM-SLE,1PM-RA),57SSc,and113SLE, and52primaryanti-phospholipidsyndrome(PAPS)from SpedaliCivilidiBrescia(Brescia,Italy)werealso screened.DiagnosisofPM/DMisbyphysician sassessmentbasedonBohan scriteria(PM/DM).OtherdiagnoseswereestablishedbytheAmericanCollegeof Rheumatology(ACR)(SLE,SSc,RA)orEuropeancriteria (Sjgren ssyndrome).Clinicalinformationwasfrom databaseandmedicalrecords.Theprotocolwas approvedbytheInstitutionalReviewBoard(IRB).This studymeetsandisincompliancewithallethicalstandardsinmedicine,andinformedconsentwasobtained fromallpatientsaccordingtotheDeclarationofHelsinki.Materialsandmethods ImmunoprecipitationAutoantibodiesinserawerescreenedbyimmunoprecipitationusing35S-methioninelabeledK562cellextracts [19].Specificityofautoantibodieswasdeterminedusing previouslydescribedreferencesera.AnalysisofRNAcomponentsofautoantigenswasbyurea-PAGEandsilver staining(SilverStainPlus,Bio-Rad,Hercules,CA,USA) [20].IdentificationofproteinsbyLC-MS/MSThenewunidentifiedautoantigenof120kDproteinwas purifiedusingserumfromtheprototypepatient.ImmunoglobulinG(IgG)from20 lofserumwascrosslinkedto proteinASepharosebeadsus ingdimethylpimerimidate. Proteinwasthenaffinity-purifiedfromacellextractof4 108K562cellsandfractionatedbySDS-PAGE.The 120kDproteinbandwascutfollowingsilverstainingof thegel,trypsin-digestedandanalyzedbyliquidchromatography-tandemmassspectrometry(LC-MS/MS)analysis onahybridquadrupole-TOFmassspectrometer(QSTAR elite,AppliedBiosystems)atUFICBRProteinCore.TandemmassspectrawereextractedbyABIAnalystversion 2.0.AllMS/MSsampleswereanalyzedusingMascot (MatrixScience,London,UK;version2.2.2).Scaffold(versionScaffold-02-03-01,ProteomeSoftwareInc.)wasused tovalidateMS/MSbasedpeptide(>95.0%probability) andproteinidentification s(>99.0%probabilityandat leasttwoidentifieduniquepeptides).AffinitypurificationoftheTIF1 b andwesternblotIdentityoftheapproximately120kDproteinasTIF1 b was verifiedbyimmunoprecipitationfollowedbywesternblot (IP-WB)usinganextractfrom5106K562cellsand2 l ofhumanserum.Purifiedproteinswerefractionatedin8% acrylamideSDS-PAGE,transferredtonitrocellulosefilter andprobedwithmouseanti-TIF1 b monoclonalantibodies (mAb)(EMDMillipore,Billerica,MA,USA),followedby horseradishperoxidase-conjugated(HRP)goatanti-mouse Iglight-chainantibodies(JacksonImmunoResearch Laboratories,Inc.WestGrove,PA,USA)anddeveloped withSuperSignalWestFe mtoChemiluminescentSubstrate(ThermoScientific,Rockford,IL,USA). Inanotherexperiment,TIF1b wasaffinitypurified fromanextractof2108K562cellsusing10 gofantiTIF1 b mAb,andpurifiedproteinswerefractionatedby SDS-PAGEandtransferredtonitrocellulosefilter.Strips (2mmwidth)ofnitrocellulosefilterwereprobedwith mousemAbandhumanautoimmunesera.StripsincubatedwithmousemAbwerethenincubatedwithHRP goatanti-mouseIglight-chainantibodiesanddeveloped, whilesamplesprobedwithhumanserawereincubated withHRP-donkeyIgGF(ab) 2anti-humanIgG( g -chain specific)antibodies(JacksonImmunoResearchLaboratories,Inc.)anddeveloped.ImmunofluorescentantinuclearantibodiesImmunofluorescentantinucle ar/cytoplasmicantibodies (HEp-2ANAslides;INOVADiagnostics,SanDiego,CA, USA)weretestedusinga1:80-dilutedhumanserumor 2 g/mlmousemAbtoTIF1 b .Secondaryantibodieswere DyLight488donkeyIgGF(ab) 2anti-humanor-mouse IgG(1:200dilution, g-chain-specific,JacksonImmunoResearchLaboratories).Satoh etal ArthritisResearch&Therapy 2012, 14 :R79 http://arthritis-research.com/content/14/2/R79 Page2of8

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ELISAAntigen-captureELISAforanti-TIF1 b wasperformed using2 g/mlmousemAbtoTIF1 b (Millipore),following aprotocolthatwasusedforotherautoantibodysystems [21].AntibodiestoTIF1 a (p140ofp155/140),TIF1 b ,and TIF1 g (p155)werealsotestedusingfull-lengthrecombinantproteinsfromAbnova(TIF1 a and b ,Taipei,Taiwan) andOriGeneTechnologies(TIF1 g ,Rockville,MD,USA), respectively.Wellsofmicrotiterplateswerecoatedusing 0.5 g/mlofproteinandELISAwasperformedfollowing standardprotocolasdescribed[22].ResultsFirst,the120kDproteinwasaffinitypurifiedusingthe humanprototypeserumandtheidentityoftheprotein wasdeterminedbyLC-MS/MS.Sixteenunique(totalof 18)peptidesthatcontainedsequencesidenticaltoTIF1 b wereisolatedasfollows:aa128-136DIVENYFMR( n =2), aa254-261KLLASLVK,aa283-290QVSDVQKR,aa297304MAILQIMK,aa310-319GRVLVNDAQK,aa312-319 VLVNDAQK,aa312-327VLVNDAQKVTEGQQER, aa331-337QHWTMTK,aa408-427IVAERPGTNSTGPAPMAPPR,aa473-483SGEGEVSGLMR( n =2),aa493507LDLDLTADSQPPVFK,aa508-524VFPGSTTEDYNLIVIER,aa751-767LSPPY SSPQEFAQDVGR,aa775-790 LTEDKADVQSIIGLQR,aa780-790ADVQSIIGLQR,and aa796-804MNEAFGDTK.Thesepeptidescovered19.5% (163/835aminoacids)ofthesequenceofTIF1 b .Sincethe reportedmolecularweightofTIF1 b isapproximately120 kD,itwasconsideredagoodcandidateanditsidentity wasvalidatedusingmAbtoTIF1 b .ImmunoprecipitationThe120kDproteinsimmunoprecipitatedbyfoursera (Figure1A,lanes1-4),bothinsizeandappearance, appearedidenticaltotheTIF1 b proteinimmunoprecipitatedbythemAb,consistentwiththeidentityofthe 120kDproteinsasTIF1 b .TIF1 b showedamigrationpatternsimilartotheknownmyositisautoantigenPL-12but hadabroaderbandincontrasttothesharpbandofPL-12 (Figure1A).Twosera(lanes1-2)hadstrongreactivity, whiletheothertwo(lanes3-4)wereweak.When20%of thesampleofcasetwowasrun(lane2),itshowedan appearancethatwasverysimilartotheweakonesinlanes 3-4,suggestingthattheyarethesameproteins.Although TIF1 b /TRIM28andTIF1 g /TRIM33areinthesamefamily ofrelatedproteinsandtheirinteractionshavebeen reported[23],anti-TIF1 b mAbdidnotimmunoprecipitate TIF1 g /p155(Figure1A,laneTIF1 b mAb).Also,antiTIF1 g (p155/140)positiveserumdidnotimmunoprecipitateTIF1 b (Figure1A,lanep155/140),consistentwitha lackofcrossreactivityandinteractionsbetweenTIF1 b and TIF1 g undertheconditionsusedforimmunoprecipitation. Noneofthesefourhumanserawithanti-TIF1 b antibodies clearlyimmunoprecipitatedTIF1 g / a (p155/140)(Figure 1A).However,theserumfromcasethreewaspositivefor anti-TIF1 a byELISAusingrecombinantprotein(Table1). CasefourwasnegativeforantibodiestoecombinantTIF1 g and a byELISA.Also,noneofthe23anti-p155/140positiveserainourcohortimmunoprecipitatedTIF1 b (data notshown).Casefourhadcoexistinganti-Mi-2antibodies (Figure1A,laneMi-2),casestwoandfourhadanti-Su/ Ago2,andcasetwoalsohadanti-RoandU1RNP.Sequentialserumsamplesavailableforcasetwoweretestedby immunoprecipitation(Figure1B).ThelevelsofantiU1RNPantibodiesthatwereweaklypositiveattheinitial visitincreasedfollowedbythedevelopmentofadditional autoantibodiestoanunidentifiedproteinofapproximately 160kD(whitearrowhead). SincemanyautoantigensinPM/DMareRNA-protein complexes,RNAcomponentsinimmunoprecipitateswere alsoanalyzedbysilverstaining.NocommonRNAcomponentwasdetected,suggestingTIF1b isnotcomplexed withspecificRNA(datanotshown).Confirmationofthe120kDproteinasTIF1 bTheidentityofthe120kDproteinimmunoprecipitated byhumanseraasTIF1 b wasconfirmedbyIP-WB (Figure2A).Theproteinsimmunoprecipitatedby humanautoimmuneserawererecognizedbymAbto TIF1 b ,confirmingtheidentityoftheprotein. Antigen-captureELISAusingmouseantiTIF1 b mAb wasalsoperformedusingseriallydilutedanti-TIF1 b positivesera(Figure2B).Allfouranti-TIF1 b positivesera werepositiveinELISAintitersupto1:12,500to1: 312,500.Controlseraincludinganti-TIF1 g oranti-Mi-2 positiveseraandNHS,wereallnegative.WesternblotusingaffinitypurifiedTIF1 bStripsofnitrocellulosefilterwithTIF1 b affinity-purifiedby mAbwereprobedwithmousemAbanti-TIF1 b and humanautoimmunesera.Mo usemAbstronglyreacted withthepurifiedTIF1 b protein;however,humanautoimmuneserawerenegativewhenthewesternblotwasdevelopedusingSuperSignalWestPico.Onlytheserumfrom caseonewasveryweaklypositivewhendevelopedusing moresensitiveSuperSignalWestFemto(Figure2C).Immunofluorescencestainingofanti-TIF1 b positiveseraHEp-2ANAslideswerestainedwithmouseanti-TIF1 b mAborhumansera(Figure2D).MousemAbshoweda finenuclearspeckledpatternsparingthenucleoliand withoutchromosomalstaining(panela).RelativelymonospecificserumfromaJapanesepatient(caseone)showed apatternverysimilartothatofmAb(panelb).Othersera (casestwotofour)alsoshowedafinespecklednuclear stainingpattern(panelsc-e);however,oneserum(case three)alsohadlargenuclearspeckles(paneld).AserumSatoh etal ArthritisResearch&Therapy 2012, 14 :R79 http://arthritis-research.com/content/14/2/R79 Page3of8

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Figure1 Immunoprecipitationusing35S-methioninelabeledK562cellextract A .8%SDS-PAGE.35S-methioninelabeledK562cellextract wasimmunoprecipitatedasfollows:Anti-TIF1 b mAb;lanes1to4,anti-TIF1 b positivehumansera;20%2,20%loadingofprototypeserumin lane2;PL-12,p155/140,NHS,normalhumanserum;Mi-2,referenceserumforeachspecificity.PositionsofTIF1 b,Su/Ago2,Ro60,U1snRNPA (U1-A),andmolecularweightmarkersareshownontheleft.WhitearrowheadsindicatePL-12(lanePL-12)andp155(TIF1 g )andp140(TIF1 a ) (lanep155/140). B .12.5%SDS-PAGE.Sequentialserafromcasetwoweretestedbyimmunoprecipitation.PositionsofcomponentsofUsnRNPs, TIF1 b,andRo-60andmolecularweightmarkersareshown.NHS,normalhumanserum;Sm,U1,anti-Smandanti-U1RNPreferenceserum, respectively;TIF,transcriptionintermediaryfactor. Table1Clinicalfeaturesofpatientswithanti-TIF1 b autoantibodies.1234 DiagnosisDMUCTDDMDM Symmetricalmuscleweakness P N P Y Musclebiopsy Y ND ND NA Elevatedmuscleenzyme Y Y Y Y EMG myopathicpatternmyopathicpatternmyopathicpattern NA Dermatologicfeatures Y(G) N P(S) Y(G,H,S) Malignancy N Y N N Interstitiallungdisease N N N N Dysphagia Y N N N Raynaud sphenomenon N N N N Arthritis N Y N N CPK(U/L)initial /afterTx(lowest) 654 167 341 239 314 2414 InitialTx none PSL10mg PSL40mg PSL50mg HCQ200mg NSAIDs HCQ200mg MTX20mg/w HCQ150mg ResponsetoTx NA good good good Otherautoantibodies Ro,Su,U1RNP(Sm) Mi-2,Su ELISA(RP)TIF1 a --+TIF1 b ++-TIF1 g ---antigen-captureELISA TIF1 a ---TIF1 b + + weak+ weak+DM,dermatomyositis;ELISA,enzyme-linkedimmunosorbentassay;EMG,electromyogram;G,Gottron;H,heliotrope;HCQ,hydroxychloroquine;MTX, methotrexate;N,no;NA,notavailable;ND,notdone;NSAIDs,non-steroidanti-inflammatorydrugs;P,possible;PSL,prednisolone;RP,recombinantprotein;S, shawlsign;TIF,transcriptionintermediaryfactor;Tx,treatment;UCTD,undifferentiatedconnectivetissuedisease;Y,yes;Satoh etal ArthritisResearch&Therapy 2012, 14 :R79 http://arthritis-research.com/content/14/2/R79 Page4of8

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withcoexistinganti-Mi-2(casefour)alsohadfine specklednuclearstaining(panele).Clinicalmanifestationsofpatientswithanti-TIF1 b antibodiesClinicalfeaturesofanti-TIF1 b antibodypositivecasesare summarizedinTable1.Allfourwerewomen,threewitha diagnosisofDMandoneclassifiedasundifferentiated connectivetissuedisease(UCTD)withelevatedcreatine phosphokinase(CPK)butwithoutsignificantmuscle symptoms.Thus,anti-TIF1 b wasfoundin3/130DM(1/ 35Americans,1/23Japanese,1/56Mexicans)but0/92 PMandnoneinSLE,SSc,orotherconditions,suggesting thatthisspecificitymaybecloselyassociatedwithDM, similartoantibodiestop155/140(TIF1 g / a ).TheJapanese DMpatient(caseone)hadelevatedCPK(654IU/L),mild muscleweakness,myalgia,apositivemusclebiopsyand electromyogram(EMG);however,hermyopathyresolved withouttreatment.TheUC TDcase(casetwo)alsohad elevatedmuscleenzymes,butmusclesymptomswerenot clear.Moreover,shehadleukopenia,lymphopeniaand autoimmunehemolyticanemia.Thiscasealsohadahistoryofcoloncancer(atage41),cervicalsquamousmetaplasiaandfibroidtumorsoftheuterus.TwoAmerican patients(casestwoandthree)hadmildlyelevatedlevelsof CPK(approximately300IU/L)thatwerecontrolledwith lowtomoderatedosesofsteroids.TheMexicancasealso hadhighCPKbutrespondedwelltoinitialtreatment.DiscussionInthepresentstudy,a120kDproteinrecognizedbysera fromfourpatientswithDMorUCTDwasidentifiedas TIF1 b basedonmassspectrometricanalysisand immunologicalconfirmation,anditiscloselyrelatedtoa knowncancer-associatedDMautoantigenp155/140 (TIF1 g / a ).SincethemobilityofTIF1 b inSDS-PAGEis identicaltothatofPL-12(F igure1),approximately120 kDproteinbandsseeninIPwillneedtobecautiously interpreted.However,TIF1 b andPL-12havedistinctive immunofluorescencepatterns,whichshouldhelptodifferentiatethespecificities.Anti-TIF1 b producesafine nuclearspeckledpattern(Figure2D)whileanti-PL-12 givesacytoplasmicpattern[24]. TIF1 a ,TIF1 b ,andTIF1 g belongtotheTIFfamilyof transcriptioncofactorsandar epartofatripartitemotif superfamily(TRIM24,28,an d33,respectively)[23]. TIF1 b /TRIM28,alsoknownasKruppel-associatedbox (KRAB)-associatedprotein1(KAP1),isamulti-functional proteininvolvedingenesilencing,cellgrowthanddifferentiation,pluripotency,neoplastictransformation,apoptosis, DNArepairandthemaintenanceofgenomicintegrity [25].Astrikingassociationofanti-p155/140withcancerassociatedDMhasbeendescribedinseveralreportsfrom theUS[5],UK[7],Spain[9],Japan[6,8,10,11],andKorea [26](reviewedin[9]),however,theirassociationdoesnot seemtoapplytochildren[7]oryoungadults[11].A JapanesePM/DMstudythathasjustbeenpublishedidentifiedp140asTIF1 a andconfirmedthep155asTIF1 g [11].Inaddition,sevencaseswithanti-TIF1 b ,fourwith anti-TIF1 a and g andtwowithanti-TIF1 g havebeen reported.Itshouldbenotedthatincontrasttoaprevious studylimitedtoPM/DM[11],anti-TIF1 b antibodieswere screenedin2,356patientsw ithvariousdiagnosesinthe presentstudy,yetitwasprimarilyassociatedwithDM. Bothstudiessuggestthatanti-TIF1 b ismuchlessprevalent thananti-p155/140.Sixoftheir77anti-p155/140positive Figure2 Characterizationofhumananti-TIF1 b positivesera A .IP-westernblot.Extractfrom5106K562cellswasimmunoprecipitatedby mouseanti-TIF1 b mAb,humananti-TIF1 b positivesera(lanes1to3correspondtocase1to3,case3isaweaklypositivesample),humanantip155/140(TIF1g / a )positiveserum,ornormalhumanserum(NHS).Purifiedproteinswerefractionatedby8%SDS-PAGE,transferredto nitrocellulosefilterandprobedwithmouseanti-TIF1 b mAb.Only1/10amountofimmunoprecipitateswasloadedformAb,case1,and2in ordertoobtainmorecomparablesignalstocase3. B .Anti-TIF1 b antigen-captureELISA.Fourhumananti-TIF1 b positivesera(left)andcontrols (right,3anti-TIF1g ,3anti-Mi-2,and2normalhumansera,NHS)wereseriallydilutedfrom1:500andtestedbyantigen-captureELISA. C.Western blot. D .ImmunofluorescencestainingofHEp-2cells.HEp-2slideswerestainedwithanti-TIF1 b mousemonoclonalantibodies(a),anti-TIF1 b antibodypositivehumanautoimmunesera(b-e),ornormalhumanserum(f).Serumdilution1:80;anti-TIF1 b mAb,1 g/ml.ELISA,enzymelinkedimmunosorbentassay;NHS,normalhumanserum;TIF,transcriptionintermediaryfactor. Satoh etal ArthritisResearch&Therapy 2012, 14 :R79 http://arthritis-research.com/content/14/2/R79 Page5of8

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serawerealsopositiveforanti-TIF1 b [11]whereasnoneof our23anti-p155/140wereanti-TIF1 b positive(datanot shown).Althoughthedifferenceobservedbetweenthetwo studiesisnotstatisticallysignificant,itispossiblethat coexistingpatternsofautoantibodiestoTIFfamilyproteins areaffectedbygeneticand/orenvironmentalfactors,selectionbias(dermatologyversusrheumatologyclinic,for example)andotherfactors. IthasbeenshownthattheseTIF1familyproteinsinteractandworksynergisticallytosuppressthedevelopment ofmalignancyinhumanandmousemodels[23,27].TIF1 b isoverexpressedinvarioustypesofcancertissues[28]and isassociatedwithprogressionormetastasisofthecancer [29,30].Itwouldnotbesurprisingiftheproductionof anti-TIF1 b isalsolinkedwithcancer-associatedDMas overexpressionandmodificationofself-proteinmaytriggeranautoimmuneresponse.Inthepresentstudy,one casewithanti-TIF1 b hadahistoryofcoloncancer,squamousmetaplasiainacervicalsmearandfibroidtumorsof theuterus,butmalignancywasnotrecordedintheother threecases,possiblydueinparttoashortfollow-upperiod.Arecentstudy[11]showedtwoofsevencaseswith anti-TIF1 b hadmalignancy,possiblylessfrequentlythan anti-p155/140positivecases. Inthecaseoftheclassictumorsuppressorgenep53, mutationandaccumulationofp53leadstothedevelopmentofcanceraswellasautoantibodiestop53,whichis consideredaresultofbrokenimmunologicaltolerance dueinparttomutatedp53[31].AsTIFfamilyproteins areknowntumorsuppressors[23,27]andareoverexpressedincertaincancertissues[28-30],apossiblerelationshipbetweenthedevelopmentofautoantibodiesto TIFandcancermaybeexplainedbyasimilarmechanism. Inthisscenario,amutationofTIFoccursasaprimary eventandtriggersanautoimmuneresponseagainstTIF, whilecancerwoulddevelopduetothefailedtumorsuppressiveactivityofTIF.Furtherstudiesarenecessaryto determinewhetheranti-TIF1b hasastrongassociation withcancersimilartoanti-TIFg andwhetherTIFmutationsaretheprimaryevent. Alternativeoradditionalmechanismsofanti-TIF1 b productionmaybehypothesizedbasedontheroleofTIF1 b inviralinfections.TIF1 b /KAP1hasbeenshowntoplaya criticalroleincontrollingreplicationofEpstein-Barrvirus [32],expressionofmurine endogenousretroviruses [33,34],latencyregulationofKaposi ssarcoma-associated herpesvirus(KSHV)[35],andreplicationofhumanpapillomaviruses[36].Inthisscenario,TIF1 b wouldinteract withviralproteinsandtheputativeviral-selfproteincomplexmaytriggerautoantibodiestoTIF1 b ,similartothe inductionofanti-p53byacomplexofviralsimianvirus 40Tproteinandselfp53protein[37].Associationof virusandmyositisautoantibodyproductionhasbeensuggestedtooccurbyvariouspathways,includingviralRNA interactionwithaminoacyltRNAsynthetaseandmolecularmimicryofviralproteinandautoantigens[38,39]. Morerecently,atargetantigenofanti-CADM140was identifiedasanintracellularviralRNAreceptor,melanomadifferentiation-associatedgene5(MDA5)[13]. Thus,anyofthesemechanismsmayalsobeinvolved.ConclusionsInsummary,anti-TIF1 b hasbeencharacterizedasautoantibodiesassociatedwithDMwithamildformofmyopathywithoutlunginvolvement.TIF1 b isamultifunctional proteincloselyrelatedtotheknowncancer-associated DMautoantigenp155/140(TIF1 g/ a )involvedinsuppressionofmalignancyaswellasviralreplication[23,27].Both mutationofTIF1 b anditsinteractionwithvirusesmake anattractivehypothesisforthemechanismofproduction ofautoantibodiestoTIF1 b .Futurestudiesshouldverifyan associationofanti-TIF1 b withmalignancyandclarify mechanismsofitsproduction.Abbreviations ACR:AmericanCollegeofRheumatology;ANA:antinuclearantibody;CADM: clinicallyamyopathicdermatomyositis;CPK:creatinephosphokinase;DM: dermatomyositis;ELISA:enzyme-linkedimmunosorbentassay;EMG: electromyography;IgG:immunoglobulinG;IIF:indirectimmunofluorescence; HRP:horseradishperoxidase;ILD:interstitiallungdisease;IP: immunoprecipitation;IP-WB:immunoprecipitationfollowedbywesternblot; KAP1:Kruppel-associatedbox(KRAB)-associatedprotein1;KSHV:Kaposi s sarcoma-associatedherpesvirus;mAb:monoclonalantibody;MDA5: melanomadifferentiationassociatedantigen5;MSA:myositis-specific autoantibodies;OD:opticaldensity;PAPS:primaryanti-phospholipid syndrome;PM:polymyositis;RA:rheumatoidarthritis;SAE:small-ubiquitin-like modifier-1activatingenzyme;SD:standarddeviation;SLE:systemiclupus erythematosus;SSc:scleroderma;TIF:transcriptionintermediaryfactor;TRIM: tripartitemotif;UCTD:undifferentiatedconnectivetissuedisease. Acknowledgements SupportedinpartbyagrantfromtheLupusResearchInstituteandthe NationalInstitutesofHealthgrantAI47859andbygenerousgiftsfrom LupusLink,Inc.(DaytonaBeach,FL)andMr.LewisM.Schotttothe UniversityofFloridaCenterforAutoimmuneDiseases.Publicationofthis articlewasfundedinpartbytheUniversityofFloridaOpen-Access PublishingFund.WewouldliketothankMarleneSarmiento,AnnieChan, andUFGCRCstaffforassistancewithclinicaldatacollectionandUFICBR ProteinCoreformassspectrometricanalysisoftheprotein. Authordetails1DivisionofRheumatologyandClinicalImmunology,Departmentof Medicine,UniversityofFlorida,P.O.Box100221,1600SWArcherRd, Gainesville,FL32610-0221,USA.2DepartmentofPathology,Immunology, andLaboratoryMedicine,UniversityofFlorida,P.O.Box100221,1600SW ArcherRd,Gainesville,FL32610-0221,USA.3DepartmentofOralBiology, UniversityofFlorida,P.O.Box100424,1600SWArcherRd,Gainesville,FL 32610-0424,USA.4DivisionofRheumatology,DepartmentofInternal Medicine,St.MariannaUniversitySchoolofMedicine,2-16-1Sugao, Miyamae-ku,Kawasaki,Kanagawa,216-8511,Japan.5Departamentode BiologaMolecularyGenmica,InstitutodeInvestigacinenReumatologay delSistemaMsculoEsqueltico,CentroUniversitariodeCienciasdela Salud,UniversidaddeGuadalajara,SierraMojada950,Guadalajara,Jalisco,CP 44340,Mxico.6DivisindeMedicinaInterna,Departamentode Reumatologa,HospitalCivil Dr.JuanI.Menchaca ,SalvadordeQuevedoy ZubietaN750,CP44340,Guadalajara,Jalisco,Mxico.7Directionof EducationandResearch,HospitaldeEspecialidades Dr.AntonioFraga Mouret ,CentroMdicoNacional LaRaza ,IMSS,Seris/Zaachilas/n,ColoniaSatoh etal ArthritisResearch&Therapy 2012, 14 :R79 http://arthritis-research.com/content/14/2/R79 Page6of8

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LaRaza,DelegacinAzcapotzalco,CP02990,MexicoCity,Mxico.8UniversidadNacionalAutnomadeMxico,AvenidaUniversidad3000, DelegacinCoyoacn,CP04510,MexicoCity,Mxico.9Departmentof Rheumatology,HospitaldeEspecialidades Dr.AntonioFragaMouret ,Centro MdicoNacional LaRaza ,IMSS,Seris/Zaachilas/n,ColoniaLaRaza,CP 02990,MexicoCity,Mxico. Authors contributions MS,JYFC,SJR,YL,andYYcarriedouttheimmunoassays.MSandEKLC designedthestudy.MSperformedthestatisticalanalysis.YY,HY,MVM,MP, LJJ,MAS,CCR,ESS,WHR,andACenrolledpatientsforthestudy,collected informationandmaintaineddatabases.MS,AC,andEKLCdraftedthe manuscript.Allauthorsreadandapprovedthefinalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:9January2012Revised:12March2012 Accepted:18April2012Published:18April2012 References1.SatohM,ChanEKL,SobelES,KimpelDL,YamasakiY,NarainS,MansoorR, ReevesWH: Clinicalimplicationofautoantibodiesinpatientswith systemicrheumaticdiseases. 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title p Autoantibodies to transcription intermediary factor (TIF)1β associated with dermatomyositis
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au id A1 ca yes snm Satohfnm Minoruinsr iid I1 I2 email minoru.satoh@medicine.ufl.edu
A2 Chanmi YFJasonjchan89@ufl.edu
A3 RossJStevenI3 stevenr966@gmail.com
A4 LiYiyi.li@medicine.ufl.edu
A5 YamasakiYoshiokiI4 yamasakiy@mac.com
A6 YamadaHidehirosoramameyamada@marianna-u.ac.jp
A7 Mercadomnm Vazquez-delMonicaI5 I6 dravme@hotmail.com
A8 PetriHMarcelomarcelomedicina@yahoo.com
A9 JaraJLuisI7 I8 luis_jara_quezada@hotmail.com
A10 SaavedraAMiguelI9 miansaavsa@gmail.com
A11 Cruz-ReyesClaudiadracruz90@hotmail.com
A12 SobelSEriceric.sobel@medicine.ufl.edu
A13 ReevesHWestleywestley.reeves@medicine.ufl.edu
A14 CeribelliAngeladott.ceribelli@libero.it
A15 ChanKLEdwardechan@ufl.edu
insg
ins Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Florida, P.O.Box 100221, 1600 SW Archer Rd, Gainesville, FL 32610-0221, USA
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, P.O.Box 100221, 1600 SW Archer Rd, Gainesville, FL 32610-0221, USA
Department of Oral Biology, University of Florida, P.O.Box 100424, 1600 SW Archer Rd, Gainesville, FL 32610-0424, USA
Division of Rheumatology, Department of Internal Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa, 216-8511, Japan
Departamento de Biología Molecular y Genómica, Instituto de Investigación en Reumatología y del Sistema Músculo Esquelético, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada 950, Guadalajara, Jalisco, CP 44340, México
División de Medicina Interna, Departamento de Reumatología, Hospital Civil 'Dr. Juan I. Menchaca', Salvador de Quevedo y Zubieta N° 750, CP 44340, Guadalajara, Jalisco, México
Direction of Education and Research, Hospital de Especialidades 'Dr. Antonio Fraga Mouret', Centro Médico Nacional 'La Raza', IMSS, Seris/Zaachila s/n, Colonia La Raza, Delegación Azcapotzalco, CP 02990, Mexico City, México
Universidad Nacional Autónoma de México, Avenida Universidad 3000, Delegación Coyoacán, CP 04510, Mexico City, México
Department of Rheumatology, Hospital de Especialidades 'Dr. Antonio Fraga Mouret', Centro Médico Nacional 'La Raza', IMSS, Seris/Zaachila s/n, Colonia La Raza, CP 02990, Mexico City, México
source Arthritis Research & Therapy
issn 1478-6354
pubdate 2012
volume 14
issue 2
fpage R79
url http://arthritis-research.com/content/14/2/R79
xrefbib pubidlist pubid idtype doi 10.1186/ar3802pmpid 22513056
history rec date day 9month 1year 2012revrec 1232012acc 1842012pub 1842012
cpyrt 2012collab Satoh et al.; licensee BioMed Central Ltd.note This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec st Abstract
Introduction
Myositis specific autoantibodies are associated with unique clinical subsets and are useful biomarkers in polymyositis/dermatomyositis (PM/DM). A 120 kD protein recognized by certain patients with DM was identified and clinical features of patients with this specificity were characterized.
Methods
The 120 kD protein recognized by a prototype serum was purified and identified by mass spectrometry and immunological methods. Autoantibody to this 120 kD protein was screened in sera from 2,356 patients with various diagnoses from four countries, including 254 PM/DM, by immunoprecipitation of sup 35S-methionine labeled K562 cell extracts. Clinical information of patients with this specificity was collected.
Results
The 120 kD protein, which exactly comigrated with PL-12, was identified as transcription intermediary factor TIF1β (TRIM28) by mass spectrometry and validated by immunoassays. By immunofluorescence, anti-TIF1β positivity showed a fine-speckled nuclear staining pattern. Four cases of anti-TIF1β were identified; all are women, one each in a Japanese, African American, Caucasian, and Mexican individual. Three had a diagnosis of DM and one case was classified as having an undifferentiated connective tissue disease with an elevated CPK but without significant muscle symptoms. This individual also had a history of colon cancer, cervical squamous metaplasia and fibroid tumors of the uterus. Myopathy was mild in all cases and resolved without treatment in one case. The anti-TIF1β specificity was not found in other conditions.
Conclusions
Anti-TIF1β is a new DM autoantibody associated with a mild form of myopathy. Whether it has an association with malignancy, as in the case of anti-TIF1γ, or other unique features will need to be evaluated in future studies.
bdy
Introduction
Autoantibodies to cellular constituents are clinically important biomarkers associated with particular diagnoses, specific clinical features or subsets of disease, helping to establish a diagnosis, and/or predicting organ involvement and prognosis abbrgrp abbr bid B1 1B2 2. In particular, in polymyositis/dermatomyositis (PM/DM) and scleroderma (systemic sclerosis, SSc) patients can be classified into several subsets associated with characteristic clinical features based on specific autoantibodies, since coexistence of other disease-specific autoantibodies is uncommon B3 3. Each myositis specific antibody (MSA) is associated with a unique clinical subset. For example, the anti-synthetase syndrome was named for the presence of anti-Jo-1 and other autoantibodies to aminoacyl tRNA synthetases found in a subset of patients with PM/DM whose clinical presentation was dominated by interstitial lung disease (ILD), Raynaud's phenomenon, arthritis, fever, and mechanic's hands 3B4 4. Although new autoantibody specificities have been reported, approximately 40% to 50% of patients with PM/DM are still without a known MSA compared with only approximately 15% in SSc without association to known SSc antibodies 2. Thus, identifying new MSA may help in monitoring PM/DM patients and several new clinically significant autoantibodies associated with DM including anti-p155/140 B5 5B6 6B7 7B8 8B9 9B10 10B11 11, anti-CADM (clinically amyopathic DM) 140/MDA5 (melanoma differentiation associated antigen 5) 10B12 12B13 13B14 14, anti-SAE (small ubiquitin-like molecule activating enzyme) and anti-MJ/NXP-2 have been reported recently B15 15B16 16. Among these, anti-p155/140 has been studied extensively in a very short period of time due to its strong association with malignancy 5678911 which was confirmed by a recent meta-analysis B17 17. However, this association does not appear to apply to children 7 or young adults 11. p155 was identified as transcription intermediary factor1γ, (TIF1γ, also known as tripartite motif (TRIM) 33) B18 18. A recent study in Japanese patients has identified the p140 as TIF1α and another related molecule TIF1β has also been identified as a target of autoantibodies in DM 11. In the present study, we have independently identified the approximately 120 kD autoantigenic protein as TIF1β by mass spectrometry. The presence of anti-TIF1β and clinical features of American, Mexican, and Japanese patients with this specificity were characterized.
Materials and methods
Patients
A total of 2,356 sera, including 1,966 subjects enrolled in the University of Florida Center for Autoimmune Diseases (UFCAD) registry from 2000 to 2010, were studied. Diagnoses of the UFCAD patients include 434 systemic lupus erythematosus (SLE), 86 PM/DM (51 PM including 12 PM-SSc overlap, 35 DM), 121 SSc, and 122 rheumatoid arthritis (RA). Additionally, sera from 36 PM/DM (13 PM, 20 DM, 3 amyopathic DM) from St. Marianna University Hospital (Kawasaki, Japan), 74 PM/DM (18 PM, 56 DM) sera from Guadalajara and Mexico City (Mexico), 58 PM/DM (25 PM, 27 DM, 6 overlap: 4 PM-SSc, 1 DM-SLE, 1 PM-RA), 57 SSc, and 113 SLE, and 52 primary anti-phospholipid syndrome (PAPS) from Spedali Civili di Brescia (Brescia, Italy) were also screened. Diagnosis of PM/DM is by physician's assessment based on Bohan's criteria (PM/DM). Other diagnoses were established by the American College of Rheumatology (ACR) (SLE, SSc, RA) or European criteria (Sjögren's syndrome). Clinical information was from database and medical records. The protocol was approved by the Institutional Review Board (IRB). This study meets and is in compliance with all ethical standards in medicine, and informed consent was obtained from all patients according to the Declaration of Helsinki.
Materials and methods
Immunoprecipitation
Autoantibodies in sera were screened by immunoprecipitation using 35S-methionine labeled K562 cell extracts B19 19. Specificity of autoantibodies was determined using previously described reference sera. Analysis of RNA components of autoantigens was by urea-PAGE and silver staining (Silver Stain Plus, Bio-Rad, Hercules, CA, USA) B20 20.
Identification of proteins by LC-MS/MS
The new unidentified autoantigen of 120 kD protein was purified using serum from the prototype patient. Immunoglobulin G (IgG) from 20 μl of serum was crosslinked to protein A Sepharose beads using dimethylpimerimidate. Protein was then affinity-purified from a cell extract of 4 × 108 K562 cells and fractionated by SDS-PAGE. The 120 kD protein band was cut following silver staining of the gel, trypsin-digested and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis on a hybrid quadrupole-TOF mass spectrometer (QSTAR elite, Applied Biosystems) at UF ICBR Protein Core. Tandem mass spectra were extracted by ABI Analyst version 2.0. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2.2). Scaffold (version Scaffold-02-03-01, Proteome Software Inc.) was used to validate MS/MS based peptide (> 95.0% probability) and protein identifications (> 99.0% probability and at least two identified unique peptides).
Affinity purification of the TIF1β and western blot
Identity of the approximately 120 kD protein as TIF1β was verified by immunoprecipitation followed by western blot (IP-WB) using an extract from 5 × 106 K562 cells and 2 μl of human serum. Purified proteins were fractionated in 8% acrylamide SDS-PAGE, transferred to nitrocellulose filter and probed with mouse anti-TIF1β monoclonal antibodies (mAb) (EMD Millipore, Billerica, MA, USA), followed by horseradish peroxidase-conjugated (HRP) goat anti-mouse Ig light-chain antibodies (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) and developed with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
In another experiment, TIF1β was affinity purified from an extract of 2 × 108 K562 cells using 10 μg of anti-TIF1β mAb, and purified proteins were fractionated by SDS-PAGE and transferred to nitrocellulose filter. Strips (2 mm width) of nitrocellulose filter were probed with mouse mAb and human autoimmune sera. Strips incubated with mouse mAb were then incubated with HRP goat anti-mouse Ig light-chain antibodies and developed, while samples probed with human sera were incubated with HRP-donkey IgG F(ab)'2 anti-human IgG (γ-chain specific) antibodies (Jackson ImmunoResearch Laboratories, Inc.) and developed.
Immunofluorescent antinuclear antibodies
Immunofluorescent antinuclear/cytoplasmic antibodies (HEp-2 ANA slides; INOVA Diagnostics, San Diego, CA, USA) were tested using a 1:80-diluted human serum or 2 μg/ml mouse mAb to TIF1β. Secondary antibodies were DyLight 488 donkey IgG F(ab)'2 anti-human or -mouse IgG (1:200 dilution, γ-chain-specific, Jackson ImmunoResearch Laboratories).
ELISA
Antigen-capture ELISA for anti-TIF1β was performed using 2 μg/ml mouse mAb to TIF1β (Millipore), following a protocol that was used for other autoantibody systems B21 21. Antibodies to TIF1α (p140 of p155/140), TIF1β, and TIF1γ (p155) were also tested using full-length recombinant proteins from Abnova (TIF1α and β, Taipei, Taiwan) and OriGene Technologies (TIF1γ, Rockville, MD, USA), respectively. Wells of microtiter plates were coated using 0.5 μg/ml of protein and ELISA was performed following standard protocol as described B22 22.
Results
First, the 120 kD protein was affinity purified using the human prototype serum and the identity of the protein was determined by LC-MS/MS. Sixteen unique (total of 18) peptides that contained sequences identical to TIF1β were isolated as follows: aa128-136 DIVENYFMR (it n = 2), aa254-261 KLLASLVK, aa283-290 QVSDVQKR, aa297-304 MAILQIMK, aa310-319 GRVLVNDAQK, aa312-319 VLVNDAQK, aa312-327 VLVNDAQKVTEGQQER, aa331-337 QHWTMTK, aa408-427 IVAERPGTNSTGPAPMAPPR, aa473-483 SGEGEVSGLMR (n = 2), aa493-507 LDLDLTADSQPPVFK, aa508-524 VFPGSTTEDYNLIVIER, aa751-767 LSPPYSSPQEFAQDVGR, aa775-790 LTEDKADVQSIIGLQR, aa 780-790 ADVQSIIGLQR, and aa796-804 MNEAFGDTK. These peptides covered 19.5% (163/835 amino acids) of the sequence of TIF1β. Since the reported molecular weight of TIF1β is approximately 120 kD, it was considered a good candidate and its identity was validated using mAb to TIF1β.
Immunoprecipitation
The 120 kD proteins immunoprecipitated by four sera (Figure figr fid F1 1A, lanes 1-4), both in size and appearance, appeared identical to the TIF1β protein immunoprecipitated by the mAb, consistent with the identity of the 120 kD proteins as TIF1β. TIF1β showed a migration pattern similar to the known myositis autoantigen PL-12 but had a broader band in contrast to the sharp band of PL-12 (Figure 1A). Two sera (lanes 1-2) had strong reactivity, while the other two (lanes 3-4) were weak. When 20% of the sample of case two was run (lane 2), it showed an appearance that was very similar to the weak ones in lanes 3-4, suggesting that they are the same proteins. Although TIF1β/TRIM28 and TIF1γ/TRIM33 are in the same family of related proteins and their interactions have been reported B23 23, anti-TIF1β mAb did not immunoprecipitate TIF1γ/p155 (Figure 1A, lane TIF1β mAb). Also, anti-TIF1γ (p155/140) positive serum did not immunoprecipitate TIF1β (Figure 1A, lane p155/140), consistent with a lack of crossreactivity and interactions between TIF1β and TIF1γ under the conditions used for immunoprecipitation. None of these four human sera with anti-TIF1β antibodies clearly immunoprecipitated TIF1γ/α (p155/140) (Figure 1A). However, the serum from case three was positive for anti-TIF1α by ELISA using recombinant protein (Table tblr tid T1 1). Case four was negative for antibodies to ecombinant TIF1γ and α by ELISA. Also, none of the 23 anti-p155/140 positive sera in our cohort immunoprecipitated TIF1β (data not shown). Case four had coexisting anti-Mi-2 antibodies (Figure 1A, lane Mi-2), cases two and four had anti-Su/Ago2, and case two also had anti-Ro and U1RNP. Sequential serum samples available for case two were tested by immunoprecipitation (Figure 1B). The levels of anti-U1RNP antibodies that were weakly positive at the initial visit increased followed by the development of additional autoantibodies to an unidentified protein of approximately 160 kD (white arrowhead).
fig Figure 1caption Immunoprecipitation using 35S-methionine labeled K562 cell extracttext
b Immunoprecipitation using 35S-methionine labeled K562 cell extract. A. 8% SDS-PAGE. 35S-methionine labeled K562 cell extract was immunoprecipitated as follows: Anti-TIF1β mAb; lanes 1 to 4, anti-TIF1β positive human sera; 20% 2, 20% loading of prototype serum in lane 2; PL-12, p155/140, NHS, normal human serum; Mi-2, reference serum for each specificity. Positions of TIF1β, Su/Ago2, Ro 60, U1snRNP A (U1-A), and molecular weight markers are shown on the left. White arrowheads indicate PL-12 (lane PL-12) and p155 (TIF1γ) and p140 (TIF1α) (lane p155/140). B. 12.5% SDS-PAGE. Sequential sera from case two were tested by immunoprecipitation. Positions of components of UsnRNPs, TIF1β, and Ro-60 and molecular weight markers are shown. NHS, normal human serum; Sm, U1, anti-Sm and anti-U1RNP reference serum, respectively; TIF, transcription intermediary factor.
graphic file ar3802-1 hint_layout double
tbl Table 1Clinical features of patients with anti-TIF1β autoantibodies.tblbdy cols 5
r
c
center
1
2
3
4
cspan
hr
left
Diagnosis
DM
UCTD
DM
DM
Symmetrical muscle weakness
P
N
P
Y
Muscle biopsy
Y
ND
ND
NA
Elevated muscle enzyme
Y
Y
Y
Y
EMG
myopathic pattern
myopathic pattern
myopathic pattern
NA
Dermatologic features
Y (G)
N
P (S)
Y (G, H, S)
Malignancy
N
Y
N
N
Interstitial lung disease
N
N
N
N
Dysphagia
Y
N
N
N
Raynaud's phenomenon
N
N
N
N
Arthritis
N
Y
N
N
CPK (U/L) initial
/after Tx (lowest)
654
167
341
239
314
2414
Initial Tx
none
PSL 10 mg
PSL 40 mg
PSL 50 mg
HCQ 200 mg
NSAIDs
HCQ 200 mg
MTX 20 mg/w
HCQ 150 mg
Response to Tx
NA
good
good
good
Other autoantibodies
Ro, Su, U1RNP (Sm)
Mi-2, Su
ELISA (RP) TIF1α
-
-
+
-
TIF1β
+
+
-
-
TIF1γ
-
-
-
-
antigen-capture ELISA
TIF1α
-
-
-
-
TIF1β
+
+
weak +
weak +
tblfn
DM, dermatomyositis; ELISA, enzyme-linked immunosorbent assay; EMG, electromyogram; G, Gottron; H, heliotrope; HCQ, hydroxychloroquine; MTX, methotrexate; N, no; NA, not available; ND, not done; NSAIDs, non-steroid anti-inflammatory drugs; P, possible; PSL, prednisolone; RP, recombinant protein; S, shawl sign; TIF, transcription intermediary factor; Tx, treatment; UCTD, undifferentiated connective tissue disease; Y, yes;
Since many autoantigens in PM/DM are RNA-protein complexes, RNA components in immunoprecipitates were also analyzed by silver staining. No common RNA component was detected, suggesting TIF1β is not complexed with specific RNA (data not shown).
Confirmation of the 120 kD protein as TIF1β
The identity of the 120 kD protein immunoprecipitated by human sera as TIF1β was confirmed by IP-WB (Figure F2 2A). The proteins immunoprecipitated by human autoimmune sera were recognized by mAb to TIF1β, confirming the identity of the protein.
Figure 2Characterization of human anti-TIF1β positive sera
Characterization of human anti-TIF1β positive sera. A. IP-western blot. Extract from 5 × 106 K562 cells was immunoprecipitated by mouse anti-TIF1β mAb, human anti-TIF1β positive sera (lanes 1 to 3 correspond to case 1 to 3, case 3 is a weakly positive sample), human anti-p155/140 (TIF1-γ/α) positive serum, or normal human serum (NHS). Purified proteins were fractionated by 8% SDS-PAGE, transferred to nitrocellulose filter and probed with mouse anti-TIF1β mAb. Only 1/10 amount of immunoprecipitates was loaded for mAb, case 1, and 2 in order to obtain more comparable signals to case 3. B. Anti-TIF1β antigen-capture ELISA. Four human anti-TIF1β positive sera (left) and controls (right, 3 anti-TIF1-γ, 3 anti-Mi-2, and 2 normal human sera, NHS) were serially diluted from 1:500 and tested by antigen-capture ELISA. C. Western blot. D. Immunofluorescence staining of HEp-2 cells. HEp-2 slides were stained with anti-TIF1β mouse monoclonal antibodies (a), anti-TIF1β antibody positive human autoimmune sera (b-e), or normal human serum (f). Serum dilution 1: 80; anti-TIF1β mAb, 1 μg/ml. ELISA, enzyme-linked immunosorbent assay; NHS, normal human serum; TIF, transcription intermediary factor.
ar3802-2
Antigen-capture ELISA using mouse anti TIF1β mAb was also performed using serially diluted anti-TIF1β positive sera (Figure 2B). All four anti-TIF1β positive sera were positive in ELISA in titers up to 1:12,500 to 1: 312,500. Control sera including anti-TIF1γ or anti-Mi-2 positive sera and NHS, were all negative.
Western blot using affinity purified TIF1β
Strips of nitrocellulose filter with TIF1β affinity-purified by mAb were probed with mouse mAb anti-TIF1β and human autoimmune sera. Mouse mAb strongly reacted with the purified TIF1β protein; however, human autoimmune sera were negative when the western blot was developed using SuperSignal West Pico. Only the serum from case one was very weakly positive when developed using more sensitive SuperSignal West Femto (Figure 2C).
Immunofluorescence staining of anti-TIF1β positive sera
HEp-2 ANA slides were stained with mouse anti-TIF1β mAb or human sera (Figure 2D). Mouse mAb showed a fine nuclear speckled pattern sparing the nucleoli and without chromosomal staining (panel a). Relatively monospecific serum from a Japanese patient (case one) showed a pattern very similar to that of mAb (panel b). Other sera (cases two to four) also showed a fine speckled nuclear staining pattern (panels c-e); however, one serum (case three) also had large nuclear speckles (panel d). A serum with coexisting anti-Mi-2 (case four) also had fine speckled nuclear staining (panel e).
Clinical manifestations of patients with anti-TIF1β antibodies
Clinical features of anti-TIF1β antibody positive cases are summarized in Table 1. All four were women, three with a diagnosis of DM and one classified as undifferentiated connective tissue disease (UCTD) with elevated creatine phosphokinase (CPK) but without significant muscle symptoms. Thus, anti-TIF1β was found in 3/130 DM (1/35 Americans, 1/23 Japanese, 1/56 Mexicans) but 0/92 PM and none in SLE, SSc, or other conditions, suggesting that this specificity may be closely associated with DM, similar to antibodies to p155/140 (TIF1γ/α). The Japanese DM patient (case one) had elevated CPK (654 IU/L), mild muscle weakness, myalgia, a positive muscle biopsy and electromyogram (EMG); however, her myopathy resolved without treatment. The UCTD case (case two) also had elevated muscle enzymes, but muscle symptoms were not clear. Moreover, she had leukopenia, lymphopenia and autoimmune hemolytic anemia. This case also had a history of colon cancer (at age 41), cervical squamous metaplasia and fibroid tumors of the uterus. Two American patients (cases two and three) had mildly elevated levels of CPK (approximately 300 IU/L) that were controlled with low to moderate doses of steroids. The Mexican case also had high CPK but responded well to initial treatment.
Discussion
In the present study, a 120 kD protein recognized by sera from four patients with DM or UCTD was identified as TIF1β based on mass spectrometric analysis and immunological confirmation, and it is closely related to a known cancer-associated DM autoantigen p155/140 (TIF1γ/α). Since the mobility of TIF1β in SDS-PAGE is identical to that of PL-12 (Figure 1), approximately 120 kD protein bands seen in IP will need to be cautiously interpreted. However, TIF1β and PL-12 have distinctive immunofluorescence patterns, which should help to differentiate the specificities. Anti-TIF1β produces a fine nuclear speckled pattern (Figure 2D) while anti-PL-12 gives a cytoplasmic pattern B24 24.
TIF1α, TIF1β, and TIF1γ belong to the TIF family of transcription cofactors and are part of a tripartite motif superfamily (TRIM24, 28, and 33, respectively) 23. TIF1β/TRIM28, also known as Kruppel-associated box (KRAB)-associated protein 1 (KAP1), is a multi-functional protein involved in gene silencing, cell growth and differentiation, pluripotency, neoplastic transformation, apoptosis, DNA repair and the maintenance of genomic integrity B25 25. A striking association of anti-p155/140 with cancer-associated DM has been described in several reports from the US 5, UK 7, Spain 9, Japan 681011, and Korea B26 26 (reviewed in 9), however, their association does not seem to apply to children 7 or young adults 11. A Japanese PM/DM study that has just been published identified p140 as TIF1α and confirmed the p155 as TIF1γ 11. In addition, seven cases with anti-TIF1β, four with anti-TIF1α and γ and two with anti-TIF1γ have been reported. It should be noted that in contrast to a previous study limited to PM/DM 11, anti-TIF1β antibodies were screened in 2,356 patients with various diagnoses in the present study, yet it was primarily associated with DM. Both studies suggest that anti-TIF1β is much less prevalent than anti-p155/140. Six of their 77 anti-p155/140 positive sera were also positive for anti-TIF1β 11 whereas none of our 23 anti-p155/140 were anti-TIF1β positive (data not shown). Although the difference observed between the two studies is not statistically significant, it is possible that coexisting patterns of autoantibodies to TIF family proteins are affected by genetic and/or environmental factors, selection bias (dermatology versus rheumatology clinic, for example) and other factors.
It has been shown that these TIF1 family proteins interact and work synergistically to suppress the development of malignancy in human and mouse models 23B27 27. TIF1β is overexpressed in various types of cancer tissues B28 28 and is associated with progression or metastasis of the cancer B29 29B30 30. It would not be surprising if the production of anti-TIF1β is also linked with cancer-associated DM as overexpression and modification of self-protein may trigger an autoimmune response. In the present study, one case with anti-TIF1β had a history of colon cancer, squamous metaplasia in a cervical smear and fibroid tumors of the uterus, but malignancy was not recorded in the other three cases, possibly due in part to a short follow-up period. A recent study 11 showed two of seven cases with anti-TIF1β had malignancy, possibly less frequently than anti-p155/140 positive cases.
In the case of the classic tumor suppressor gene p53, mutation and accumulation of p53 leads to the development of cancer as well as autoantibodies to p53, which is considered a result of broken immunological tolerance due in part to mutated p53 B31 31. As TIF family proteins are known tumor suppressors 2327 and are overexpressed in certain cancer tissues 282930, a possible relationship between the development of autoantibodies to TIF and cancer may be explained by a similar mechanism. In this scenario, a mutation of TIF occurs as a primary event and triggers an autoimmune response against TIF, while cancer would develop due to the failed tumor suppressive activity of TIF. Further studies are necessary to determine whether anti-TIF1β has a strong association with cancer similar to anti-TIF-γ and whether TIF mutations are the primary event.
Alternative or additional mechanisms of anti-TIF1β production may be hypothesized based on the role of TIF1β in viral infections. TIF1β/KAP1 has been shown to play a critical role in controlling replication of Epstein-Barr virus B32 32, expression of murine endogenous retroviruses B33 33B34 34, latency regulation of Kaposi's sarcoma-associated herpesvirus (KSHV) B35 35, and replication of human papillomaviruses B36 36. In this scenario, TIF1β would interact with viral proteins and the putative viral-self protein complex may trigger autoantibodies to TIF1β, similar to the induction of anti-p53 by a complex of viral simian virus 40 T protein and self p53 protein B37 37. Association of virus and myositis autoantibody production has been suggested to occur by various pathways, including viral RNA interaction with aminoacyl tRNA synthetase and molecular mimicry of viral protein and autoantigens B38 38B39 39. More recently, a target antigen of anti-CADM140 was identified as an intracellular viral RNA receptor, melanoma differentiation-associated gene 5 (MDA5) 13. Thus, any of these mechanisms may also be involved.
Conclusions
In summary, anti-TIF1β has been characterized as autoantibodies associated with DM with a mild form of myopathy without lung involvement. TIF1β is a multifunctional protein closely related to the known cancer-associated DM autoantigen p155/140 (TIF1γ/α) involved in suppression of malignancy as well as viral replication 2327. Both mutation of TIF1β and its interaction with viruses make an attractive hypothesis for the mechanism of production of autoantibodies to TIF1β. Future studies should verify an association of anti-TIF1β with malignancy and clarify mechanisms of its production.
Abbreviations
ACR: American College of Rheumatology; ANA: antinuclear antibody; CADM: clinically amyopathic dermatomyositis; CPK: creatine phosphokinase; DM: dermatomyositis; ELISA: enzyme-linked immunosorbent assay; EMG: electromyography; IgG: immunoglobulin G; IIF: indirect immunofluorescence; HRP: horseradish peroxidase; ILD: interstitial lung disease; IP: immunoprecipitation; IP-WB: immunoprecipitation followed by western blot; KAP1: Kruppel-associated box (KRAB)-associated protein 1; KSHV: Kaposi's sarcoma-associated herpesvirus; mAb: monoclonal antibody; MDA5: melanoma differentiation associated antigen 5; MSA: myositis-specific autoantibodies; OD: optical density; PAPS: primary anti-phospholipid syndrome; PM: polymyositis; RA: rheumatoid arthritis; SAE: small-ubiquitin-like modifier-1 activating enzyme; SD: standard deviation; SLE: systemic lupus erythematosus; SSc: scleroderma; TIF: transcription intermediary factor; TRIM: tripartite motif; UCTD: undifferentiated connective tissue disease.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MS, JYFC, SJR, YL, and YY carried out the immunoassays. MS and EKLC designed the study. MS performed the statistical analysis. YY, HY, MVM, MP, LJJ, MAS, CCR, ESS, WHR, and AC enrolled patients for the study, collected information and maintained databases. MS, AC, and EKLC drafted the manuscript. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
Supported in part by a grant from the Lupus Research Institute and the National Institutes of Health grant AI47859 and by generous gifts from Lupus Link, Inc. (Daytona Beach, FL) and Mr. Lewis M. Schott to the University of Florida Center for Autoimmune Diseases. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund. We would like to thank Marlene Sarmiento, Annie Chan, and UF GCRC staff for assistance with clinical data collection and UF ICBR Protein Core for mass spectrometric analysis of the protein.
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patients with dermatomyositis: an association with malignancyKajiKFujimotoMHasegawaMKondoMSaitoYKomuraKMatsushitaTOritoHHamaguchiYYanabaKItohMAsanoYSeishimaMOgawaFSatoSTakeharaKRheumatology (Oxford)200746252810.1093/rheumatology/kel161Clinical associations of autoantibodies to a p155/140 kDa doublet protein in juvenile dermatomyositisGunawardenaHWedderburnLRNorthJBetteridgeZDunphyJChinoyHDavidsonJECooperRGMcHughNJRheumatology (Oxford)200847324328Association of distinct clinical subsets with myositis-specific autoantibodies towards anti-155/140-kDa polypeptides, anti-140-kDa polypeptides, and anti-aminoacyl tRNA synthetases in Japanese patients with dermatomyositis: a single-centre, cross-sectional studyFujikawaKKawakamiAKajiKFujimotoMKawashiriSIwamotoNAramakiTIchinoseKTamaiMKamachiMNakamuraHIdaHOriguchiTIshimotoHMukaeHKuwanaMKohnoSTakeharaKSatoSEguchiKScand J Rheumatol20093826326710.1080/0300974080268745519444719Cancer-associated myositis and anti-p155 autoantibody in a series of 85 patients with idiopathic inflammatory myopathyTrallero-AraguasELabrador-HorrilloMSelva-O'CallaghanAMartinezMAMartinez-GomezXPalouERodriguez-SanchezJLVilardell-TarresMMedicine (Baltimore)201089475210.1097/MD.0b013e3181ca14ffAnti-MDA5 and anti-TIF1-{gamma} antibodies have clinical significance for patients with dermatomyositisHoshinoKMuroYSugiuraKTomitaYNakashimaRMimoriTRheumatology (Oxford)2010491726173310.1093/rheumatology/keq153Myositis-specific anti-155/140 autoantibodies target transcriptional intermediary factor 1 family proteinsFujimotoMHamaguchiYKajiKMatsushitaTIchimuraYKoderaMIshiguroNUeda-HayakawaIAsanoYOgawaFFujikawaKMiyagiTMabuchiEHiroseKAkimotoNHattaNTsutsuiKHigashiAIgarashiASeishimaMHasegawaMTakeharaKArthritis Rheum20126451352210.1002/art.3340321987216Autoantibodies to a 140-kd polypeptide, CADM-140, in Japanese patients with clinically amyopathic dermatomyositisSatoSHirakataMKuwanaMSuwaAInadaSMimoriTNishikawaTOddisCVIkedaYArthritis Rheum2005521571157610.1002/art.2102315880816RNA helicase encoded by melanoma differentiation-associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: association with rapidly progressive interstitial lung diseaseSatoSHoshinoKSatohTFujitaTKawakamiYKuwanaMArthritis Rheum2009602193220010.1002/art.2462119565506The RIG-I-like receptor IFIH1/MDA5 is a dermatomyositis-specific autoantigen identified by the anti-CADM-140 antibodyNakashimaRImuraYKobayashiSYukawaNYoshifujiHNojimaTKawabataDOhmuraKUsuiTFujiiTOkawaKMimoriTRheumatology (Oxford)20104943344010.1093/rheumatology/kep375Myositis-specific autoantibodies: their clinical and pathogenic significance in disease expressionGunawardenaHBetteridgeZEMcHughNJRheumatology (Oxford)20094860761210.1093/rheumatology/kep078Novel autoantibodies and clinical phenotypes in adult and juvenile myositisBetteridgeZEGunawardenaHMcHughNJArthritis Res Ther20111320910.1186/ar3275313202121457520Usefulness of anti-p155 autoantibody for diagnosing cancer-associated dermatomyositis: A systematic review and meta-analysisTrallero-AraguasERodrigo-PendasJASelva-O'CallaghanAMartinez-GomezXBoschXLabrador-HorrilloMGrau-JunyentJMVilardell-TarresMArthritis Rheum20126452353210.1002/art.3337921953614Autoantibodies to transcriptional intermediary factor 1-gamma (TIF1-γ) in dermatomyositisTargoffINTrieuEPLevy-NetoMPrasertsuntarasaiTMillerFWArthritis Rheum200654SupplS518Distinctive immune response patterns of human and murine autoimmune sera to U1 small nuclear ribonucleoprotein C proteinSatohMLangdonJJHamiltonKJRichardsHBPankaDEisenbergRAReevesWHJ Clin Invest1996972619262610.1172/JCI1187115073498647956Unusually high frequency of autoantibodies to PL-7 associated with milder muscle disease in Japanese patients with polymyositis/dermatomyositisYamasakiYYamadaHNozakiTAkaogiJNicholsCLyonsRChin LoyAChanEKReevesWHSatohMArthritis Rheum2006542004200910.1002/art.2188316732549Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseasesYamasakiYNarainSHernandezLBarkerTIkedaKSegalMSRichardsHBChanEKReevesWHSatohMArthritis Res Ther20068R11112010.1186/ar2000177942216846524Reduced IgG anti-small nuclear ribonucleoproteins autoantibody production in systemic lupus erythematosus patients with positive IgM anti-cytomegalovirus antibodiesAzucena Palafox-SanchezCSatohMChanEKCarcamoWCFrancisco Munoz-ValleJOrozco-BarocioGOregon RomeroEElena Navarro HernandezRSalazar-ParamoMCabral CastanedaAVazquez-Del MercadoMArthritis Res Ther200911R2710.1186/ar2621268826119232124Transcription cofactors TRIM24, TRIM28, and TRIM33 associate to form regulatory complexes that suppress murine hepatocellular carcinomaHerquelBOuararhniKKhetchoumianKIgnatMTeletinMMarkMBechadeGVan DorsselaerASanglier-CianferaniSHamicheACammasFDavidsonILossonRProc Natl Acad Sci USA20111088212821710.1073/pnas.1101544108310098221531907Clinical manifestations in patients with antibody to PL-12 antigen (alanyl-tRNA synthetase)TargoffINArnettFCAm J Med19908824125110.1016/0002-9343(90)90149-82178410KAP1 protein: an enigmatic master regulator of the genomeIyengarSFarnhamPJJ Biol Chem2011286262672627610.1074/jbc.R111.252569314358921652716Myositis autoantibodies in Korean patients with inflammatory myositis: anti-140-kDa polypeptide antibody is primarily associated with rapidly progressive interstitial lung disease independent of clinically amyopathic dermatomyositisKangEHNakashimaRMimoriTKimJLeeYJLeeEBSongYWBMC Musculoskelet Disord20101122310.1186/1471-2474-11-223295499020875136Transcription intermediary factor 1gamma is a tumor suppressor in mouse and human chronic myelomonocytic leukemiaAucagneRDroinNPaggettiJLagrangeBLargeotAHammannABatailleAMartinLYanKPFenauxPLossonRSolaryEBastieJNDelvaLJ Clin Invest20111212361237010.1172/JCI45213310475321537084Human IgG antibody profiles differentiate between symptomatic patients with and without colorectal 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Med19941791243125210.1084/jem.179.4.124321914308145041Myositis autoantibody inhibits histidyl-tRNA synthetase: a model for autoimmunityMathewsMBBernsteinRMNature198330417717910.1038/304177a06866113Polymyositis-dermatomyositis and infectionsZampieriSGhirardelloAIaccarinoLBrianiCSarzi-PuttiniPAtzeniFArientiSTodescoSDoriaAAutoimmunity20063919119610.1080/0891693060062234816769652


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ui ar3802
ji 1478-6354
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dochead Research article
bibl
title p Autoantibodies to transcription intermediary factor (TIF)1β associated with dermatomyositis
aug
au id A1 ca yes snm Satohfnm Minoruinsr iid I1 I2 email minoru.satoh@medicine.ufl.edu
A2 Chanmi YFJasonjchan89@ufl.edu
A3 RossJStevenI3 stevenr966@gmail.com
A4 LiYiyi.li@medicine.ufl.edu
A5 YamasakiYoshiokiI4 yamasakiy@mac.com
A6 YamadaHidehirosoramameyamada@marianna-u.ac.jp
A7 Mercadomnm Vazquez-delMonicaI5 I6 dravme@hotmail.com
A8 PetriHMarcelomarcelomedicina@yahoo.com
A9 JaraJLuisI7 I8 luis_jara_quezada@hotmail.com
A10 SaavedraAMiguelI9 miansaavsa@gmail.com
A11 Cruz-ReyesClaudiadracruz90@hotmail.com
A12 SobelSEriceric.sobel@medicine.ufl.edu
A13 ReevesHWestleywestley.reeves@medicine.ufl.edu
A14 CeribelliAngeladott.ceribelli@libero.it
A15 ChanKLEdwardechan@ufl.edu
insg
ins Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Florida, P.O.Box 100221, 1600 SW Archer Rd, Gainesville, FL 32610-0221, USA
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, P.O.Box 100221, 1600 SW Archer Rd, Gainesville, FL 32610-0221, USA
Department of Oral Biology, University of Florida, P.O.Box 100424, 1600 SW Archer Rd, Gainesville, FL 32610-0424, USA
Division of Rheumatology, Department of Internal Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa, 216-8511, Japan
Departamento de Biología Molecular y Genómica, Instituto de Investigación en Reumatología y del Sistema Músculo Esquelético, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada 950, Guadalajara, Jalisco, CP 44340, México
División de Medicina Interna, Departamento de Reumatología, Hospital Civil 'Dr. Juan I. Menchaca', Salvador de Quevedo y Zubieta N° 750, CP 44340, Guadalajara, Jalisco, México
Direction of Education and Research, Hospital de Especialidades 'Dr. Antonio Fraga Mouret', Centro Médico Nacional 'La Raza', IMSS, Seris/Zaachila s/n, Colonia La Raza, Delegación Azcapotzalco, CP 02990, Mexico City, México
Universidad Nacional Autónoma de México, Avenida Universidad 3000, Delegación Coyoacán, CP 04510, Mexico City, México
Department of Rheumatology, Hospital de Especialidades 'Dr. Antonio Fraga Mouret', Centro Médico Nacional 'La Raza', IMSS, Seris/Zaachila s/n, Colonia La Raza, CP 02990, Mexico City, México
source Arthritis Research & Therapy
issn 1478-6354
pubdate 2012
volume 14
issue 2
fpage R79
url http://arthritis-research.com/content/14/2/R79
xrefbib pubidlist pubid idtype doi 10.1186/ar3802pmpid 22513056
history rec date day 9month 1year 2012revrec 1232012acc 1842012pub 1842012
cpyrt 2012collab Satoh et al.; licensee BioMed Central Ltd.note This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec st Abstract
Introduction
Myositis specific autoantibodies are associated with unique clinical subsets and are useful biomarkers in polymyositis/dermatomyositis (PM/DM). A 120 kD protein recognized by certain patients with DM was identified and clinical features of patients with this specificity were characterized.
Methods
The 120 kD protein recognized by a prototype serum was purified and identified by mass spectrometry and immunological methods. Autoantibody to this 120 kD protein was screened in sera from 2,356 patients with various diagnoses from four countries, including 254 PM/DM, by immunoprecipitation of sup 35S-methionine labeled K562 cell extracts. Clinical information of patients with this specificity was collected.
Results
The 120 kD protein, which exactly comigrated with PL-12, was identified as transcription intermediary factor TIF1β (TRIM28) by mass spectrometry and validated by immunoassays. By immunofluorescence, anti-TIF1β positivity showed a fine-speckled nuclear staining pattern. Four cases of anti-TIF1β were identified; all are women, one each in a Japanese, African American, Caucasian, and Mexican individual. Three had a diagnosis of DM and one case was classified as having an undifferentiated connective tissue disease with an elevated CPK but without significant muscle symptoms. This individual also had a history of colon cancer, cervical squamous metaplasia and fibroid tumors of the uterus. Myopathy was mild in all cases and resolved without treatment in one case. The anti-TIF1β specificity was not found in other conditions.
Conclusions
Anti-TIF1β is a new DM autoantibody associated with a mild form of myopathy. Whether it has an association with malignancy, as in the case of anti-TIF1γ, or other unique features will need to be evaluated in future studies.
bdy
Introduction
Autoantibodies to cellular constituents are clinically important biomarkers associated with particular diagnoses, specific clinical features or subsets of disease, helping to establish a diagnosis, and/or predicting organ involvement and prognosis abbrgrp abbr bid B1 1B2 2. In particular, in polymyositis/dermatomyositis (PM/DM) and scleroderma (systemic sclerosis, SSc) patients can be classified into several subsets associated with characteristic clinical features based on specific autoantibodies, since coexistence of other disease-specific autoantibodies is uncommon B3 3. Each myositis specific antibody (MSA) is associated with a unique clinical subset. For example, the anti-synthetase syndrome was named for the presence of anti-Jo-1 and other autoantibodies to aminoacyl tRNA synthetases found in a subset of patients with PM/DM whose clinical presentation was dominated by interstitial lung disease (ILD), Raynaud's phenomenon, arthritis, fever, and mechanic's hands 3B4 4. Although new autoantibody specificities have been reported, approximately 40% to 50% of patients with PM/DM are still without a known MSA compared with only approximately 15% in SSc without association to known SSc antibodies 2. Thus, identifying new MSA may help in monitoring PM/DM patients and several new clinically significant autoantibodies associated with DM including anti-p155/140 B5 5B6 6B7 7B8 8B9 9B10 10B11 11, anti-CADM (clinically amyopathic DM) 140/MDA5 (melanoma differentiation associated antigen 5) 10B12 12B13 13B14 14, anti-SAE (small ubiquitin-like molecule activating enzyme) and anti-MJ/NXP-2 have been reported recently B15 15B16 16. Among these, anti-p155/140 has been studied extensively in a very short period of time due to its strong association with malignancy 5678911 which was confirmed by a recent meta-analysis B17 17. However, this association does not appear to apply to children 7 or young adults 11. p155 was identified as transcription intermediary factor1γ, (TIF1γ, also known as tripartite motif (TRIM) 33) B18 18. A recent study in Japanese patients has identified the p140 as TIF1α and another related molecule TIF1β has also been identified as a target of autoantibodies in DM 11. In the present study, we have independently identified the approximately 120 kD autoantigenic protein as TIF1β by mass spectrometry. The presence of anti-TIF1β and clinical features of American, Mexican, and Japanese patients with this specificity were characterized.
Materials and methods
Patients
A total of 2,356 sera, including 1,966 subjects enrolled in the University of Florida Center for Autoimmune Diseases (UFCAD) registry from 2000 to 2010, were studied. Diagnoses of the UFCAD patients include 434 systemic lupus erythematosus (SLE), 86 PM/DM (51 PM including 12 PM-SSc overlap, 35 DM), 121 SSc, and 122 rheumatoid arthritis (RA). Additionally, sera from 36 PM/DM (13 PM, 20 DM, 3 amyopathic DM) from St. Marianna University Hospital (Kawasaki, Japan), 74 PM/DM (18 PM, 56 DM) sera from Guadalajara and Mexico City (Mexico), 58 PM/DM (25 PM, 27 DM, 6 overlap: 4 PM-SSc, 1 DM-SLE, 1 PM-RA), 57 SSc, and 113 SLE, and 52 primary anti-phospholipid syndrome (PAPS) from Spedali Civili di Brescia (Brescia, Italy) were also screened. Diagnosis of PM/DM is by physician's assessment based on Bohan's criteria (PM/DM). Other diagnoses were established by the American College of Rheumatology (ACR) (SLE, SSc, RA) or European criteria (Sjögren's syndrome). Clinical information was from database and medical records. The protocol was approved by the Institutional Review Board (IRB). This study meets and is in compliance with all ethical standards in medicine, and informed consent was obtained from all patients according to the Declaration of Helsinki.
Materials and methods
Immunoprecipitation
Autoantibodies in sera were screened by immunoprecipitation using 35S-methionine labeled K562 cell extracts B19 19. Specificity of autoantibodies was determined using previously described reference sera. Analysis of RNA components of autoantigens was by urea-PAGE and silver staining (Silver Stain Plus, Bio-Rad, Hercules, CA, USA) B20 20.
Identification of proteins by LC-MS/MS
The new unidentified autoantigen of 120 kD protein was purified using serum from the prototype patient. Immunoglobulin G (IgG) from 20 μl of serum was crosslinked to protein A Sepharose beads using dimethylpimerimidate. Protein was then affinity-purified from a cell extract of 4 × 108 K562 cells and fractionated by SDS-PAGE. The 120 kD protein band was cut following silver staining of the gel, trypsin-digested and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis on a hybrid quadrupole-TOF mass spectrometer (QSTAR elite, Applied Biosystems) at UF ICBR Protein Core. Tandem mass spectra were extracted by ABI Analyst version 2.0. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2.2). Scaffold (version Scaffold-02-03-01, Proteome Software Inc.) was used to validate MS/MS based peptide (> 95.0% probability) and protein identifications (> 99.0% probability and at least two identified unique peptides).
Affinity purification of the TIF1β and western blot
Identity of the approximately 120 kD protein as TIF1β was verified by immunoprecipitation followed by western blot (IP-WB) using an extract from 5 × 106 K562 cells and 2 μl of human serum. Purified proteins were fractionated in 8% acrylamide SDS-PAGE, transferred to nitrocellulose filter and probed with mouse anti-TIF1β monoclonal antibodies (mAb) (EMD Millipore, Billerica, MA, USA), followed by horseradish peroxidase-conjugated (HRP) goat anti-mouse Ig light-chain antibodies (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) and developed with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
In another experiment, TIF1β was affinity purified from an extract of 2 × 108 K562 cells using 10 μg of anti-TIF1β mAb, and purified proteins were fractionated by SDS-PAGE and transferred to nitrocellulose filter. Strips (2 mm width) of nitrocellulose filter were probed with mouse mAb and human autoimmune sera. Strips incubated with mouse mAb were then incubated with HRP goat anti-mouse Ig light-chain antibodies and developed, while samples probed with human sera were incubated with HRP-donkey IgG F(ab)'2 anti-human IgG (γ-chain specific) antibodies (Jackson ImmunoResearch Laboratories, Inc.) and developed.
Immunofluorescent antinuclear antibodies
Immunofluorescent antinuclear/cytoplasmic antibodies (HEp-2 ANA slides; INOVA Diagnostics, San Diego, CA, USA) were tested using a 1:80-diluted human serum or 2 μg/ml mouse mAb to TIF1β. Secondary antibodies were DyLight 488 donkey IgG F(ab)'2 anti-human or -mouse IgG (1:200 dilution, γ-chain-specific, Jackson ImmunoResearch Laboratories).
ELISA
Antigen-capture ELISA for anti-TIF1β was performed using 2 μg/ml mouse mAb to TIF1β (Millipore), following a protocol that was used for other autoantibody systems B21 21. Antibodies to TIF1α (p140 of p155/140), TIF1β, and TIF1γ (p155) were also tested using full-length recombinant proteins from Abnova (TIF1α and β, Taipei, Taiwan) and OriGene Technologies (TIF1γ, Rockville, MD, USA), respectively. Wells of microtiter plates were coated using 0.5 μg/ml of protein and ELISA was performed following standard protocol as described B22 22.
Results
First, the 120 kD protein was affinity purified using the human prototype serum and the identity of the protein was determined by LC-MS/MS. Sixteen unique (total of 18) peptides that contained sequences identical to TIF1β were isolated as follows: aa128-136 DIVENYFMR (it n = 2), aa254-261 KLLASLVK, aa283-290 QVSDVQKR, aa297-304 MAILQIMK, aa310-319 GRVLVNDAQK, aa312-319 VLVNDAQK, aa312-327 VLVNDAQKVTEGQQER, aa331-337 QHWTMTK, aa408-427 IVAERPGTNSTGPAPMAPPR, aa473-483 SGEGEVSGLMR (n = 2), aa493-507 LDLDLTADSQPPVFK, aa508-524 VFPGSTTEDYNLIVIER, aa751-767 LSPPYSSPQEFAQDVGR, aa775-790 LTEDKADVQSIIGLQR, aa 780-790 ADVQSIIGLQR, and aa796-804 MNEAFGDTK. These peptides covered 19.5% (163/835 amino acids) of the sequence of TIF1β. Since the reported molecular weight of TIF1β is approximately 120 kD, it was considered a good candidate and its identity was validated using mAb to TIF1β.
Immunoprecipitation
The 120 kD proteins immunoprecipitated by four sera (Figure figr fid F1 1A, lanes 1-4), both in size and appearance, appeared identical to the TIF1β protein immunoprecipitated by the mAb, consistent with the identity of the 120 kD proteins as TIF1β. TIF1β showed a migration pattern similar to the known myositis autoantigen PL-12 but had a broader band in contrast to the sharp band of PL-12 (Figure 1A). Two sera (lanes 1-2) had strong reactivity, while the other two (lanes 3-4) were weak. When 20% of the sample of case two was run (lane 2), it showed an appearance that was very similar to the weak ones in lanes 3-4, suggesting that they are the same proteins. Although TIF1β/TRIM28 and TIF1γ/TRIM33 are in the same family of related proteins and their interactions have been reported B23 23, anti-TIF1β mAb did not immunoprecipitate TIF1γ/p155 (Figure 1A, lane TIF1β mAb). Also, anti-TIF1γ (p155/140) positive serum did not immunoprecipitate TIF1β (Figure 1A, lane p155/140), consistent with a lack of crossreactivity and interactions between TIF1β and TIF1γ under the conditions used for immunoprecipitation. None of these four human sera with anti-TIF1β antibodies clearly immunoprecipitated TIF1γ/α (p155/140) (Figure 1A). However, the serum from case three was positive for anti-TIF1α by ELISA using recombinant protein (Table tblr tid T1 1). Case four was negative for antibodies to ecombinant TIF1γ and α by ELISA. Also, none of the 23 anti-p155/140 positive sera in our cohort immunoprecipitated TIF1β (data not shown). Case four had coexisting anti-Mi-2 antibodies (Figure 1A, lane Mi-2), cases two and four had anti-Su/Ago2, and case two also had anti-Ro and U1RNP. Sequential serum samples available for case two were tested by immunoprecipitation (Figure 1B). The levels of anti-U1RNP antibodies that were weakly positive at the initial visit increased followed by the development of additional autoantibodies to an unidentified protein of approximately 160 kD (white arrowhead).
fig Figure 1caption Immunoprecipitation using 35S-methionine labeled K562 cell extracttext
b Immunoprecipitation using 35S-methionine labeled K562 cell extract. A. 8% SDS-PAGE. 35S-methionine labeled K562 cell extract was immunoprecipitated as follows: Anti-TIF1β mAb; lanes 1 to 4, anti-TIF1β positive human sera; 20% 2, 20% loading of prototype serum in lane 2; PL-12, p155/140, NHS, normal human serum; Mi-2, reference serum for each specificity. Positions of TIF1β, Su/Ago2, Ro 60, U1snRNP A (U1-A), and molecular weight markers are shown on the left. White arrowheads indicate PL-12 (lane PL-12) and p155 (TIF1γ) and p140 (TIF1α) (lane p155/140). B. 12.5% SDS-PAGE. Sequential sera from case two were tested by immunoprecipitation. Positions of components of UsnRNPs, TIF1β, and Ro-60 and molecular weight markers are shown. NHS, normal human serum; Sm, U1, anti-Sm and anti-U1RNP reference serum, respectively; TIF, transcription intermediary factor.
graphic file ar3802-1 hint_layout double
tbl Table 1Clinical features of patients with anti-TIF1β autoantibodies.tblbdy cols 5
r
c
center
1
2
3
4
cspan
hr
left
Diagnosis
DM
UCTD
DM
DM
Symmetrical muscle weakness
P
N
P
Y
Muscle biopsy
Y
ND
ND
NA
Elevated muscle enzyme
Y
Y
Y
Y
EMG
myopathic pattern
myopathic pattern
myopathic pattern
NA
Dermatologic features
Y (G)
N
P (S)
Y (G, H, S)
Malignancy
N
Y
N
N
Interstitial lung disease
N
N
N
N
Dysphagia
Y
N
N
N
Raynaud's phenomenon
N
N
N
N
Arthritis
N
Y
N
N
CPK (U/L) initial
/after Tx (lowest)
654
167
341
239
314
2414
Initial Tx
none
PSL 10 mg
PSL 40 mg
PSL 50 mg
HCQ 200 mg
NSAIDs
HCQ 200 mg
MTX 20 mg/w
HCQ 150 mg
Response to Tx
NA
good
good
good
Other autoantibodies
Ro, Su, U1RNP (Sm)
Mi-2, Su
ELISA (RP) TIF1α
-
-
+
-
TIF1β
+
+
-
-
TIF1γ
-
-
-
-
antigen-capture ELISA
TIF1α
-
-
-
-
TIF1β
+
+
weak +
weak +
tblfn
DM, dermatomyositis; ELISA, enzyme-linked immunosorbent assay; EMG, electromyogram; G, Gottron; H, heliotrope; HCQ, hydroxychloroquine; MTX, methotrexate; N, no; NA, not available; ND, not done; NSAIDs, non-steroid anti-inflammatory drugs; P, possible; PSL, prednisolone; RP, recombinant protein; S, shawl sign; TIF, transcription intermediary factor; Tx, treatment; UCTD, undifferentiated connective tissue disease; Y, yes;
Since many autoantigens in PM/DM are RNA-protein complexes, RNA components in immunoprecipitates were also analyzed by silver staining. No common RNA component was detected, suggesting TIF1β is not complexed with specific RNA (data not shown).
Confirmation of the 120 kD protein as TIF1β
The identity of the 120 kD protein immunoprecipitated by human sera as TIF1β was confirmed by IP-WB (Figure F2 2A). The proteins immunoprecipitated by human autoimmune sera were recognized by mAb to TIF1β, confirming the identity of the protein.
Figure 2Characterization of human anti-TIF1β positive sera
Characterization of human anti-TIF1β positive sera. A. IP-western blot. Extract from 5 × 106 K562 cells was immunoprecipitated by mouse anti-TIF1β mAb, human anti-TIF1β positive sera (lanes 1 to 3 correspond to case 1 to 3, case 3 is a weakly positive sample), human anti-p155/140 (TIF1-γ/α) positive serum, or normal human serum (NHS). Purified proteins were fractionated by 8% SDS-PAGE, transferred to nitrocellulose filter and probed with mouse anti-TIF1β mAb. Only 1/10 amount of immunoprecipitates was loaded for mAb, case 1, and 2 in order to obtain more comparable signals to case 3. B. Anti-TIF1β antigen-capture ELISA. Four human anti-TIF1β positive sera (left) and controls (right, 3 anti-TIF1-γ, 3 anti-Mi-2, and 2 normal human sera, NHS) were serially diluted from 1:500 and tested by antigen-capture ELISA. C. Western blot. D. Immunofluorescence staining of HEp-2 cells. HEp-2 slides were stained with anti-TIF1β mouse monoclonal antibodies (a), anti-TIF1β antibody positive human autoimmune sera (b-e), or normal human serum (f). Serum dilution 1: 80; anti-TIF1β mAb, 1 μg/ml. ELISA, enzyme-linked immunosorbent assay; NHS, normal human serum; TIF, transcription intermediary factor.
ar3802-2
Antigen-capture ELISA using mouse anti TIF1β mAb was also performed using serially diluted anti-TIF1β positive sera (Figure 2B). All four anti-TIF1β positive sera were positive in ELISA in titers up to 1:12,500 to 1: 312,500. Control sera including anti-TIF1γ or anti-Mi-2 positive sera and NHS, were all negative.
Western blot using affinity purified TIF1β
Strips of nitrocellulose filter with TIF1β affinity-purified by mAb were probed with mouse mAb anti-TIF1β and human autoimmune sera. Mouse mAb strongly reacted with the purified TIF1β protein; however, human autoimmune sera were negative when the western blot was developed using SuperSignal West Pico. Only the serum from case one was very weakly positive when developed using more sensitive SuperSignal West Femto (Figure 2C).
Immunofluorescence staining of anti-TIF1β positive sera
HEp-2 ANA slides were stained with mouse anti-TIF1β mAb or human sera (Figure 2D). Mouse mAb showed a fine nuclear speckled pattern sparing the nucleoli and without chromosomal staining (panel a). Relatively monospecific serum from a Japanese patient (case one) showed a pattern very similar to that of mAb (panel b). Other sera (cases two to four) also showed a fine speckled nuclear staining pattern (panels c-e); however, one serum (case three) also had large nuclear speckles (panel d). A serum with coexisting anti-Mi-2 (case four) also had fine speckled nuclear staining (panel e).
Clinical manifestations of patients with anti-TIF1β antibodies
Clinical features of anti-TIF1β antibody positive cases are summarized in Table 1. All four were women, three with a diagnosis of DM and one classified as undifferentiated connective tissue disease (UCTD) with elevated creatine phosphokinase (CPK) but without significant muscle symptoms. Thus, anti-TIF1β was found in 3/130 DM (1/35 Americans, 1/23 Japanese, 1/56 Mexicans) but 0/92 PM and none in SLE, SSc, or other conditions, suggesting that this specificity may be closely associated with DM, similar to antibodies to p155/140 (TIF1γ/α). The Japanese DM patient (case one) had elevated CPK (654 IU/L), mild muscle weakness, myalgia, a positive muscle biopsy and electromyogram (EMG); however, her myopathy resolved without treatment. The UCTD case (case two) also had elevated muscle enzymes, but muscle symptoms were not clear. Moreover, she had leukopenia, lymphopenia and autoimmune hemolytic anemia. This case also had a history of colon cancer (at age 41), cervical squamous metaplasia and fibroid tumors of the uterus. Two American patients (cases two and three) had mildly elevated levels of CPK (approximately 300 IU/L) that were controlled with low to moderate doses of steroids. The Mexican case also had high CPK but responded well to initial treatment.
Discussion
In the present study, a 120 kD protein recognized by sera from four patients with DM or UCTD was identified as TIF1β based on mass spectrometric analysis and immunological confirmation, and it is closely related to a known cancer-associated DM autoantigen p155/140 (TIF1γ/α). Since the mobility of TIF1β in SDS-PAGE is identical to that of PL-12 (Figure 1), approximately 120 kD protein bands seen in IP will need to be cautiously interpreted. However, TIF1β and PL-12 have distinctive immunofluorescence patterns, which should help to differentiate the specificities. Anti-TIF1β produces a fine nuclear speckled pattern (Figure 2D) while anti-PL-12 gives a cytoplasmic pattern B24 24.
TIF1α, TIF1β, and TIF1γ belong to the TIF family of transcription cofactors and are part of a tripartite motif superfamily (TRIM24, 28, and 33, respectively) 23. TIF1β/TRIM28, also known as Kruppel-associated box (KRAB)-associated protein 1 (KAP1), is a multi-functional protein involved in gene silencing, cell growth and differentiation, pluripotency, neoplastic transformation, apoptosis, DNA repair and the maintenance of genomic integrity B25 25. A striking association of anti-p155/140 with cancer-associated DM has been described in several reports from the US 5, UK 7, Spain 9, Japan 681011, and Korea B26 26 (reviewed in 9), however, their association does not seem to apply to children 7 or young adults 11. A Japanese PM/DM study that has just been published identified p140 as TIF1α and confirmed the p155 as TIF1γ 11. In addition, seven cases with anti-TIF1β, four with anti-TIF1α and γ and two with anti-TIF1γ have been reported. It should be noted that in contrast to a previous study limited to PM/DM 11, anti-TIF1β antibodies were screened in 2,356 patients with various diagnoses in the present study, yet it was primarily associated with DM. Both studies suggest that anti-TIF1β is much less prevalent than anti-p155/140. Six of their 77 anti-p155/140 positive sera were also positive for anti-TIF1β 11 whereas none of our 23 anti-p155/140 were anti-TIF1β positive (data not shown). Although the difference observed between the two studies is not statistically significant, it is possible that coexisting patterns of autoantibodies to TIF family proteins are affected by genetic and/or environmental factors, selection bias (dermatology versus rheumatology clinic, for example) and other factors.
It has been shown that these TIF1 family proteins interact and work synergistically to suppress the development of malignancy in human and mouse models 23B27 27. TIF1β is overexpressed in various types of cancer tissues B28 28 and is associated with progression or metastasis of the cancer B29 29B30 30. It would not be surprising if the production of anti-TIF1β is also linked with cancer-associated DM as overexpression and modification of self-protein may trigger an autoimmune response. In the present study, one case with anti-TIF1β had a history of colon cancer, squamous metaplasia in a cervical smear and fibroid tumors of the uterus, but malignancy was not recorded in the other three cases, possibly due in part to a short follow-up period. A recent study 11 showed two of seven cases with anti-TIF1β had malignancy, possibly less frequently than anti-p155/140 positive cases.
In the case of the classic tumor suppressor gene p53, mutation and accumulation of p53 leads to the development of cancer as well as autoantibodies to p53, which is considered a result of broken immunological tolerance due in part to mutated p53 B31 31. As TIF family proteins are known tumor suppressors 2327 and are overexpressed in certain cancer tissues 282930, a possible relationship between the development of autoantibodies to TIF and cancer may be explained by a similar mechanism. In this scenario, a mutation of TIF occurs as a primary event and triggers an autoimmune response against TIF, while cancer would develop due to the failed tumor suppressive activity of TIF. Further studies are necessary to determine whether anti-TIF1β has a strong association with cancer similar to anti-TIF-γ and whether TIF mutations are the primary event.
Alternative or additional mechanisms of anti-TIF1β production may be hypothesized based on the role of TIF1β in viral infections. TIF1β/KAP1 has been shown to play a critical role in controlling replication of Epstein-Barr virus B32 32, expression of murine endogenous retroviruses B33 33B34 34, latency regulation of Kaposi's sarcoma-associated herpesvirus (KSHV) B35 35, and replication of human papillomaviruses B36 36. In this scenario, TIF1β would interact with viral proteins and the putative viral-self protein complex may trigger autoantibodies to TIF1β, similar to the induction of anti-p53 by a complex of viral simian virus 40 T protein and self p53 protein B37 37. Association of virus and myositis autoantibody production has been suggested to occur by various pathways, including viral RNA interaction with aminoacyl tRNA synthetase and molecular mimicry of viral protein and autoantigens B38 38B39 39. More recently, a target antigen of anti-CADM140 was identified as an intracellular viral RNA receptor, melanoma differentiation-associated gene 5 (MDA5) 13. Thus, any of these mechanisms may also be involved.
Conclusions
In summary, anti-TIF1β has been characterized as autoantibodies associated with DM with a mild form of myopathy without lung involvement. TIF1β is a multifunctional protein closely related to the known cancer-associated DM autoantigen p155/140 (TIF1γ/α) involved in suppression of malignancy as well as viral replication 2327. Both mutation of TIF1β and its interaction with viruses make an attractive hypothesis for the mechanism of production of autoantibodies to TIF1β. Future studies should verify an association of anti-TIF1β with malignancy and clarify mechanisms of its production.
Abbreviations
ACR: American College of Rheumatology; ANA: antinuclear antibody; CADM: clinically amyopathic dermatomyositis; CPK: creatine phosphokinase; DM: dermatomyositis; ELISA: enzyme-linked immunosorbent assay; EMG: electromyography; IgG: immunoglobulin G; IIF: indirect immunofluorescence; HRP: horseradish peroxidase; ILD: interstitial lung disease; IP: immunoprecipitation; IP-WB: immunoprecipitation followed by western blot; KAP1: Kruppel-associated box (KRAB)-associated protein 1; KSHV: Kaposi's sarcoma-associated herpesvirus; mAb: monoclonal antibody; MDA5: melanoma differentiation associated antigen 5; MSA: myositis-specific autoantibodies; OD: optical density; PAPS: primary anti-phospholipid syndrome; PM: polymyositis; RA: rheumatoid arthritis; SAE: small-ubiquitin-like modifier-1 activating enzyme; SD: standard deviation; SLE: systemic lupus erythematosus; SSc: scleroderma; TIF: transcription intermediary factor; TRIM: tripartite motif; UCTD: undifferentiated connective tissue disease.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MS, JYFC, SJR, YL, and YY carried out the immunoassays. MS and EKLC designed the study. MS performed the statistical analysis. YY, HY, MVM, MP, LJJ, MAS, CCR, ESS, WHR, and AC enrolled patients for the study, collected information and maintained databases. MS, AC, and EKLC drafted the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
Supported in part by a grant from the Lupus Research Institute and the National Institutes of Health grant AI47859 and by generous gifts from Lupus Link, Inc. (Daytona Beach, FL) and Mr. Lewis M. Schott to the University of Florida Center for Autoimmune Diseases. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund. We would like to thank Marlene Sarmiento, Annie Chan, and UF GCRC staff for assistance with clinical data collection and UF ICBR Protein Core for mass spectrometric analysis of the protein.
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