生物化学与生物物理进展 ProgressinBiochemistryandBiophysics 2013,40(11):1124~1131 www.pibb.ac.cn BiologicalEffectofTheInteractionBetween MentalRetardationRelatedProtein (FXR1P)andCMAS* MAYun1)**,QINLing-Xue1)**,DONGXiao1),LIBin-Yuan1),WANGChang-Bo1), XUCan2),WANGSan-Hu1),HEShu-Ya1)*** (1) DepartmentofBiochemistry&Biology,UniversityofSouthChina,Hengyang 421001,China; 2) DepartmentofCardiologyofTheFisrtAffiliatedHospitalofUniversityofSouthChina,Hengyang 421001,China) Abstract FragileXsyndrome(FXS)isageneticmentalretardationdisease,withincidencesecondonlytotrisomy21syndrome. FragileXmentalretardationprotein(FMRP),isthecausativefactorofFXSandencodedbytheFragileXmentalretardation1(FMR1) gene,whichiswidelyexpressedincellsofthenerve,muscle,andtestes.FragileXrelatedprotein1(FXR1P)isencodedbya homologousgeneto FMR1———FragileXrelatedgene1(FXR1)andcaninteractwithproteinsandRNAs.Manyillnesseswere involvedinthealteredexpressionof FXR1.TounderstandthebiologicaleffectoftheinteractionbetweenFXR1PandCMAS,we constructedaFXR1 overexpressionvectorandinvestigateditsexpressioninPC12(theratpheochromocytoma)cellsandVSMC (vascularsmoothmusclecell)andtheeffectoftheoverexpressiononcellmorphologyandseveralcellprocessesrelatedto CMP-N-acetylneuraminicacidsynthetase(CMAS)activity.Wedemonstratethattheoverexpressionof FXR1 genecanincreaseactivity ofCMASinPC12cellsandprovideacertaindegreegrowthprotectionforthatcells.Thus,itsuggestsFXR1Pisatissue-specific regulatortoaltertheconcentrationofGM1inPC12cells,butnotinVSMC. Keywords FXR1P,CMAS,GM1,biologicaleffect,PC12,VSMC DOI:10.3724/SP.J.1206.2013.00004 FragileXsyndrome(FXS),alsocalledMartin- studydemonstratedthatboth FMR1 and FXR1 gene Bellsyndrome,isaninheritedmentalretardation wereinvolvedindevelopmentoftheeyesandneural diseaseassociatedwiththelowexpressionofFragileX crest.Thereducingexpressionofthesetwogenescan mentalretardation1(FMR1)geneandsubsequentlythe leadtotheabnormaldevelopmentofeyesandcranial reducingormissingofFragileXmentalretardation cartilage,andtheuncoordinateactioninmuscleand protein(FMRP)[1].FragileXrelatedgene1(FXR1)is thegrowthabnormalityofnervetissue[6]. anautosomalhomologueofthe FMR1 andlocatesat FXR1Pisalsoexpressedinmatureoocyteand 3q28 [2].FragileXrelatedprotein1(FXR1P),encoded earlyvitro-fertilizedembryos [7].Itwasconfirmedby by FXR1,sharesapproximately60%ofitsaminoacid vitroRNAimmunoprecipitationassaysinmyocardial sequencewiththeN-terminalofFMRPthatincludes cellsthatFXR1Pboundto dsp(desmoplakin)and tln2 twoKHdomain,aRGGbox,aNuclearlocalization signal(NLS)andaNuclearexportsignal(NES)[3]. ThepreviousfindingsthatFXR1Pwasnormally *ThisworkwassupportedbygrantsfromTheNationalNaturalScience expressedintheFXSpatinentslackingFMRP FoundationofChina(81200881)andHunanProvincialNaturalScience indicatedanindependentfunctionofFXR1Pin FoundationofChina(12JJ6073). **Theseauthorscontributedequallytothiswork. development [4].ThechangeofFXR1Pexpressionand ***Correspondingauthor. thereducedstabilityof mRNAcanresultin FXR1 Tel:86-734-8281620,E-mail:[email protected] [5] facioscapulohumeralmusculardystrophy .Theanimal Received:January4,2013Accepted:April10,2013 马云, 等：智力低下相关蛋白(FXR1P)与 CMAS 相互作用的生物学效应研究 ·1125· 2013;40(11) (talin2)mRNAandinhibitedtheirtranslation,which withoutfetalcalfserumorantibioticsand twocognateproteinswerelocalizedtothedesmosomes cotransfectedwiththemammalianexpression andcostameres,respectively[8].Inaddition,FXR1Pcan plasmidspcDNA3.1(-)/FXR1 orpcDNA3.1(-)using alsobindtopre-miR-9andpre-miR-124andregulate Lipofectamine2000,thenormalcellascontrol.Then, theexpressionlevelofmiR-124byeffectingthe thetransfectedcellswereincubatedabout5h,after forming [9].ThecompartmentalizedAGO2-FXR1- whichthemediumwasreplacedwithcomplete isoform-acomplexwasreportedtobeselectively DMEMcontaining10%fetalcalfserum.At48hafter contributedtomicroRNA-mediatedupregulationof transfection,thecellswereharvested. somespecificmRNAstranscriptioninquiescent(G0) 1.4 Westernblot mammaliancellsandimmature Xenopus laevis Harvestedcellswerelysedbystrengthenlysate. oocytes[10]. BCAProteinAssayKit(Beyotime)wasusedto Wehavepreviouslyscreenedsomeproteinsby quantifytheproteinasmanufacturer.Subpackage yeast-2-hybridsandidentifiedsomeproteinthatcan protein,accordingto50 gtotalprotein,preparedto interactwithFXR1PsuchasCMP-N-acetylneuraminic workassample,andadd5滋 SDS-PAGEloadingbuffer 伊 acidsynthetase(CMAS), Homosapiens ferritin,heavy andrunon120V.Theprimaryantibodyandsecondary polypeptide1(FTH1)[11]. antibodywasRIPAb TM FXR1andanti-GAPDH, CMASisanenzymethatcancatalyzethe respectively. + activationofsialic,whichisinvolvedinthe 1.5 Observationofcellmorphology sialoglycoconjugatesynethsisinthemembrane [12].In Cellsinthelogarithmicphasewerecollectedon thisstudy,weperformedan invitro studyofFXR1 batchcultureplate,andculturedinahumidified5% expressionintwocelllines,thePC12(rat CO2 incubatorat37℃ for24～ 48h.Then,cellswere pheochromocytoma)cellsandVSMC(thevascular fixedwithparaformaldehydeandstainedwithDAPI smoothmusclecell),usingrecombinantvectorand (KeyGENBioTECH).Theobservationwasperformed emptyvector,thewild-typecellsascontrol,and underconfocallaserscanningmicroscope(Nikon). investigatedtheeffectofoverexpressionofFXR1on 1.6 AssayofconcentrationofGM1 cellmorphologyandseveralcelleventsmediatedby ELISAassaywasperformedbytheHuman CMASsuchastheconcentrationofGM1,thetotal anti-gangliosideantibody(GM1)ELISAKit ATPaseactivityandtheapoptosisrate.Wewantto (BiocalvinBioTechCo.Ltd)asmanufacturer. determinethebiologicaleffectoftheinteraction 1.7 AssayofactivityoftotalATPase betweenFXR1PandCMASusingthesemethods. Digesttheculturedcelltoacquirecell precipitation.Thentheprecipitationweretreatedwith 1 Materialsandmethods iso-osmiaphysiologicalsaline.Transferthesuspension 1.1 Strainsandcelllines to2mlglasshomogenatetubeinordertogetcell The Escherichiacoli (E.coli)strains,DH5 suspendingliquidwhichwillbedetectedthetotal 琢 (supE44 lacU169hsdR17recA1endA1gyrA96 activityofATPasebythesupermicroscaleATPase 驻 thi-1relA1),wasculturedinLBmedium(1%tryptone, testKit. 0.5%yeastextract,1%NaCl,pH7.0)at37℃ .Two 1.8 Assayofaopotosisrate celllines,PC12andVSMC,wereculturedin Preparetheanalytesampletosinglecell Dulbecco'smodifiedEagle'smedium(DMEM) suspensionandfixeditwith70%coldethanolinoder supplementedwith10%fetalcalfserumina tosuspendthecell.Thendeliverthesampleto humidified5%CO2 incubatorat37℃ . JunhongBio(Changsha)Co.Ltdtodetectthe 1.2 Plasmid apoptosisrate. Thefullopenreadingframeofthehuman FXR1 genewasinsertedintothepcDNA3.1(-)vectorto 2 Results generaterecombinantvectorpcDNA3.1(-)/FXR1. 2.1 ConstructionandidentificationofpcDNA3.1(鄄)/ ThenthatwastransfectedintoPC12andVSMCcells. FXR1 1.3 Celltransfection Inordertostudythebiologicaleffectofthe Whenthecellsreached80%confluence,they interactionbetweenFXR1PandCMAS,wedesigneda wereplacedinto6-wellplateswithbasalDMEM pairofprimerstoamplifythefullopenreadingframe ·1126· 生物化学与生物物理进展 Prog.Biochem.Biophys. 2013;40(11) ofhuman FXR1 about1900bpbyPCR,theninserted thisamplifiedfragmentintothepcDNA3.1(-)plasmid (b) 0.8 (a) toobtainarecombinantpcDNA3.1(-)/FXR1 vector. * 0.6 Then,therecombinantvectorpcDNA3.1(-)/FXR1 was 1 2 3 FXR1P confirmedtohavefullcodingsequenceofhuman 0.4 FXR1 bydirectsequencingandrestrictionendonuclease GAPDH analysis( Xho EcoR ). 0.2 玉+ 玉 2.2 Detection of the recombinant vector 0 pcDNA3.1(鄄)/FXR1transfectionbyWesternblot 1 2 3 Weselectedtwocelllines,PC12(Rat pheochromocytoma)andVSMC(vasularsmooth Fig.1 Transfectionefficiencyofrecombinantvector musclecell)asthetargetcellstransfectedwith pcDNA3.1(鄄)/FXR1 inPC12cellsbyWesternblot (a)WesternblottingofpcDNA3.1(-)/FXR1 inPC12cells.(b)Grayvalue recombinantvectorpcDNA3.1(-)/ duetohigher FXR1 analysisofWesternblotting. 1:Normalgroup; 2:Emptyvectorgroup; expressionof FXR1 intheneuralandmusclecells. 3:Overexpressionof FXR1 group.*Comparedwithnormalcellgroup, Thetransfectionefficiencywasdeterminedbythe P <0.05, n=3. expressionamountofFXR1.Todeterminerelative levelofFXR1expressionaftertransfectionwe (b) 1.0 (a) designedtwonegativecontrolgroups,thevector-free * 0.8 andtheemptyvector,besidesthegroupwith FXR1 1 2 3 0.6 vectortransfection.Westernblottinganalysisshowed FXR1P that FXR1 expressionaftertransfectionwasincreased 0.4 by36%inPC12cells(Figure1)andby97.1%in GAPDH 0.2 VSMCs(Figure2)againstthevector-freegroup, whereastherewasnosignificantalterationof FXR1 0 1 2 3 expressionbetweentheemptyvectorandnormal groups.Theresultsrevealedthattwocelllines Fig.2 Transfectionefficiencyofrecombinantvector presentedanover-expressionof after FXR1 FXR1 pcDNA3.1(鄄)/FXR1 inVSMCbyWesternblot vectortransfected. (a)WesternblottingofpcDNA3.1(-)/FXR1 inVSMC.(b)Grayvalue 2.3 Overexpressionof FXR1 altersthemorphology analysisofWesternblotting. 1:Normalgroup; 2:Emptyvectorgroup; ofVSMCbutnotPC12cells 3:Overexpressionof FXR1 group.*Comparedwithnormalcellgroup, ToevaluatetheeffectofoverexpressionofFXR1 P <0.05, n=3. onthecellmorphology,wecomparedthecell morphologybyconfocallaserscanningmicroscope amongthreegroups.Theresultsshowedthatthecell thanthefusiformwhichisthemorphologyofnormal morphologyhasnosignificantdifferenceinthePC12 cellsandotherstransfectedwiththeemptyvector cellsamongthethreegroups(Figure3).However,the (Figure4).Thisindicatedthatoverexpressionof FXR1 VSMCcellstransfectedwiththerecombinantvector canaltercellmorphologyoftheVSMC,butnotthatof pcDNA3.1(-)/FXR1 exhibitedanirregularshaperather PC12cells. (a) (b) (c) Fig.3 Effectofoverexpression FXR1 onthemorphologyofPC12cell (a)Normalgroup.(b)Emptyvectorgroup.(c)Recombinantvectorgroup. 200 伊 马云, 等：智力低下相关蛋白(FXR1P)与 CMAS 相互作用的生物学效应研究 ·1127· 2013;40(11)