Differential Roles of TwoN-Acetylgalactosaminyltransferases

THEJOURNALOFBIOLOGICALCHEMISTRYVol.278,No.5,IssueofJanuary31,pp.3063–3071,2003©2003byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.PrintedinU.S.A.DifferentialRolesofTwoN-Acetylgalactosaminyltransferases,

CSGalNAcT-1,andaNovelEnzyme,CSGalNAcT-2

INITIATIONANDELONGATIONINSYNTHESISOFCHONDROITINSULFATE*

Receivedforpublication,August30,2002,andinrevisedform,November19,2002

Published,JBCPapersinPress,November20,2002,DOI10.1074/jbc.M208886200

TakashiSato?,MasanoriGotoh?§,KatsueKiyohara?,TomohiroAkashima?,HirokoIwasaki?§,

AkihikoKameyama?§,HideoMochizuki?¶,ToshikazuYada¶?,NiroInaba?**,AkiraTogayachi?,

TakashiKudo???,MasahiroAsada§§,HidetoWatanabe?,ToruImamura§§,KojiKimata?,

andHisashiNarimatsu?¶¶

Fromthe?GlycogeneFunctionTeam,ResearchCenterforGlycoscience,NationalInstituteofAdvancedIndustrialScienceand

Technology(AIST),OpenSpaceLaboratoryC-2,1-1-1Umezono,Tsukuba,Ibaraki305-8568,Japan,§AmershamBiosciences

KK,3-25-1,Hyakunincho,Shinjuku-ku,Tokyo169-0073,Japan,the¶SeikagakuCorporation,1253Tateno3-Chome,

Higashi-yamato,Tokyo207-0021,Japan,the?InstituteforMolecularScienceofMedicine,AichiMedicalUniversity,

Nagaute,Aichi480-1195,Japan,**JGSJapanGenomeSolutions,Inc.,51Kamiyacho,Hachioji,Tokyo192-0031,

Japan,the§§CellRegulationTeam,AgeDimensionResearchCenter,AIST,1-1-1Higashi,Tsukuba,Ibaraki305-8566,

Japan,the??NewEnergyandIndustrialTechnologyDevelopmentOrganization(NEDO),Sunshine60Bldg.,

3-1-1HigashiIkebukuro,Toshima-kuTokyo,170-6028,Japan

Byatblastnsearchwith?1,4-galactosyltransferasesscriptswerehighlyexpressedinthesmallintestine,asquerysequences,wefoundanexpressedsequencetagleukocytes,andspleen,however,bothCSGalNAcTsthatshowedsimilarityin?1,4-glycosyltransferasemo-wereubiquitouslyexpressedinvarioustissues.

tifs.Thefull-lengthcomplementaryDNAwasobtained

byamethodof5?-rapidamplificationofcomplementary

DNAends.Thepredictedopenreadingframeencodesa

typicaltypeIImembraneproteincomprising543aminoProteoglycans(PGs),1moleculesconsistingofacoreproteinacids,thesequenceofwhichwashighlyhomologoustoandatleastoneglycosaminoglycan(GAG)chain,existasoneofchondroitinsulfateN-acetylgalactosaminyltransferasethemajorcomponentsofextracellularmatrixandonthecell(CSGalNAcT-1),andwedesignatedthisnovelenzymesurface.Avarietyofproteoglycanfunctionsareexertedde-CSGalNAcT-2.CSGalNAcT-2showedmuchstrongerN-pendingontheGAGchains.Thesechainsareusuallyhighlyacetylgalactosaminyltransferaseactivitytowardglucu-sulfated,andcanbeclassifiedintoseveralgroupsincludingronicacidofchondroitinpoly-andoligosaccharides,chondroitinsulfate(CS)/dermatansulfate,heparansulfateandchondroitinsulfatepoly-andoligosaccharideswith(HS)/heparin,andkeratansulfatebasedontheGAGcomposi-a?1–4linkage,i.e.elongationactivityforchondroitintion.HShasbeenshowntobeinvolvedinsignaltransductionandchondroitinsulfate,butshowedmuchweakeractiv-anddevelopmenttogetherwithcertaingrowthfactors,cyto-itytowardatetrasaccharideoftheglycosaminoglycankines,andextracellularmatrices.InDrosophilamelanogaster,linkagestructure(GlcA-Gal-Gal-Xyl-O-methoxyphenyl),HSdeficienciescausedbymutationsinthegenesencodingi.e.initiationactivity,thanCSGalNAcT-1.TransfectionenzymesinvolvedinthesynthesisofHSresultinabnormaloftheCSGalNAcT-1geneintoChinesehamsterovarydevelopmentalphenotypes(1).HShasbeendemonstratedtocellsyieldedachangeofglycosaminoglycancomposi-bindtoavarietyofcellgrowthfactorssuchasthefibroblasttion,i.e.thereplacementofheparansulfateonasynde-growthfactor(FGF)familymoleculesandtomodulatetheircan-4/fibroblastgrowthfactor-1chimeraproteinbyactivitiesinvariousways(2).Hepatocytegrowthfactor(HGF)chondroitinsulfate,however,transfectionoftheCSGal-andsomeinterleukins(interleukins3and7)bindtoHSforNAcT-2genedidnot.Theaboveresultsindicatedthatefficientsignaltransduction(3,4).Recentstudieshavedem-CSGalNAcT-1isinvolvedintheinitiationofchondroitin

sulfatesynthesis,whereasCSGalNAcT-2participatesonstratedthatCSalsoplaysvariousimportantrolesincellmainlyintheelongation,notinitiation.Quantitativeadhesion,migration,andrecognition,especiallyofneuronalreal-timePCRanalysisrevealedthatCSGalNAcT-2tran-cells(5–7).ThesulfationprofilesofCSvarywithagingin

thecartilage(8,9).Disulfateddisaccharideunits,CS-D

*ThisworkwasperformedaspartoftheR&DProjectofIndustrial1Theabbreviationsusedare:PG,proteoglycan;GAG,glycosamino-ScienceandTechnologyFrontierProgram(R&DforEstablishmentandglycan;CS,chondroitinsulfate;HS,heparansulfate;FGF,fibroblastUtilizationofaTechnicalInfrastructureforJapaneseIndustry)sup-growthfactor;GlcA,glucuronicacid;Gal,galactose;Xyl,xylose;Glc-portedbytheNewEnergyandIndustrialTechnologyDevelopmentNAc,N-acetylglucosamine;GalNAc,N-acetylgalactosamine;GT,glyco-Organization(NEDO).Thecostsofpublicationofthisarticleweresyltransferase;(?4,?3,orCS)GlcAT,(?1,4-,?1,3-orCS)glucuronyl-defrayedinpartbythepaymentofpagecharges.Thisarticlemusttransferase;?4GlcNAcT,?1,4-N-acetylglucosaminyltransferase;(?4thereforebeherebymarked“advertisement”inaccordancewith18orCS)GalNAcT,(?1,4-orCS)N-acetylgalactosaminyltransferase;U.S.C.Section1734solelytoindicatethisfact.(?4)GalT,(?1,4-)galactosyltransferase;CSS,chondroitinsynthase;Thenucleotidesequence(s)reportedinthispaperhasbeensubmittedORF,openreadingframe;UDP,uridinediphosphate;pNP,para-nitro-totheGenBankTM/EBIDataBankwithaccessionnumber(s)AB079252.phenyl;MES,2-morpholinoethanesulfonicacid;ESI,electrosprayioni-¶¶Towhomcorrespondenceshouldbeaddressed:GlycogeneFunc-zation;MS,massspectrometry;MALDI-TOF,matrix-assistedlaserde-tionTeam,ResearchCenterforGlycoscience(RCG),NationalInstitutesorptionionization-timeofflight;HSase,heparitinase;HPase,ofAdvancedIndustrialScienceandTechnology(AIST),Central-2C-2,heparanase;CSase,chondroitinase;GAPDH,glyceraldehyde-3-phos-1-1-1Umezono,Tsukuba,Ibaraki305-8568,Japan.Tel.:81-298-61-phatedehydrogenase;CHO,Chinesehamsterovary;mAb,monoclonal3200;Fax:81-298-61-3201;E-mail:h.narimatsu@aist.go.jp.antibody;C4ST,chondroitin-4-O-sulfotransferase.

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3064DifferentialRolesofTwoCSGalNAcTs

(GlcA(2S)?1–3GalNAc(6S))andCS-E(GlcA?1–3GalNAc-(4S,6S)),promotetheoutgrowthofneuritesinratbrain(10).ThesereportssuggestthatHSandCShavedifferentfunctions.Thus,itisofinteresttoclarifythemechanismforthebiosyn-thesisofHSandCS,themoleculesresponsibleforthesedi-versebiologicalphenotypes.

TheinitialstageinthebiosynthesisofbothCSandHSinvolvesalinkagetetrasaccharidestructure(GlcA?1–4Gal?1–3Gal?1–4Xyl?1-),whichbindscovalentlytoserineresiduesofcoreproteins.ToinitiatethesynthesisofHS,aGlcNAcresidueistransferredtoGlcAofthelinkagetetrasaccharidewithan?1–4linkage.Ontheotherhand,GalNAcistransferredtothesameacceptorwitha?1–4linkagefortheinitiationofCSsynthesis.Thus,becausetheacceptorsubstrateisidentical,itispossiblethattheinitiationenzymeforHSorCSsynthesiscompetewiththeacceptorsubstrate,i.e.thelinkagetetrasac-charideboundtocoreproteins,inthecells.Ifthisisthecase,theinitiationenzymesmayplayakeyroleindeterminingthespeciesoftheGAGchain,HSorCS,onthecoreproteins.Aftertheinitiationreaction,theadditionofdisaccharideunitsofGlcNAc?1–4GlcA?1–4arerepeatedforelongationoftheHSchain,whereasGalNAc?1–4GlcA?1–3unitsarerepeatedlyaddedforpolymerizationoftheCSchain.

ForthesynthesisofHS,fiveglycosyltransferasegenes,EXT1,EXT2,EXTL1,EXTL2,andEXTL3,wereclonedandtheirproductscharacterized.TheyretainconservedmotifsofshortaminoacidstretchesintheirCOOHterminusandbelongtoonefamily.TheEXT1andEXT2genes,whichhavebeenidentifiedastumorsuppressorgenesandwereimplicatedinhereditarymultipleexostoses,werefoundtoencodeHSpolym-eraseshavingtheactivityofboth?1,4-glucuronyltransferase(?4GlcAT)and?1,4-N-acetylglucosaminyltransferase(?4Glc-NAcT)(11).BothenzymesareresponsiblefortheelongationofHSchains.Threeenzymes,EXTL1–3,exhibitonly?4GlcNAcTactivity,buttheirsubstratespecificitiesweredifferent.EXTL1showed?4GlcNAcTactivitytowardGlcAinelongation.EXTL2showedactivityininitiation.EXTL3showedactivityinbothinitiationandelongationforthesynthesisofHS(12,13).

Veryrecently,threeglycosyltransferases,chondroitinsyn-thase(CSS)(14),CSglucuronyltransferase(CSGlcAT)(15)andCSGalNAcT(16,17),whichareinvolvedinthesynthesisofCS,havebeenreported.CSSwasfoundtobeaCSpolymerasehavingtheactivityofboth?1,3-glucuronyltransferase(?3Glc-AT)and?4GalNAcT.CSGlcATorCSGalNAcTexhibitstheactivityofonlyoneglycosyltransferase,?3GlcATand?4GalN-AcT,respectively.Furthermore,CSGalNAcTwasfoundtoex-hibitapparent?4GalNAcTactivitytowardthelinkagetet-rasaccharidefortheinitiationofCSsynthesis.GiventhemanyenzymesinvolvedinthesynthesisofHS,itiseasytospeculatethatmultipleglycosyltransferaseswouldalsoparticipateinthesynthesisofCS.

Inthisstudy,wereportthecloningandcharacterizationofanovelN-acetylgalactosaminyltransferase,CSGalNAcT-2,thatisthefourthmemberparticipatinginthesynthesisofCS.InconsideringthesubstratespecificitiesofthesetwoCSGal-NAcTsinvitro,wesuggesttheirdifferentialrolesinvivo.

EXPERIMENTALPROCEDURES

IsolationofHumanCSGalNAcT-2cDNA—WeperformedatblastnsearchoftheGenBankTMdatabaseusing?1,4-glycosyltransferasemo-tifsasqueriesandidentifiedanexpressedsequencetagwithGen-BankTMaccessionnumberNM_018590,whichcontainedapartialopenreadingframe(ORF),butshowedhighhomologytothecarboxyl-termi-nalregionofCSGalNAcT-1.AnadditionalsearchoftheHumanGe-nomeProjectdatabaserevealedthatthegenomesequencewithGen-BankTMaccessionnumberNT_008776wasidenticaltotheexpressedsequencetag.ToobtainthecompleteORF,the5?-rapidamplificationofcomplementaryDNA(cDNA)endsmethodwasemployedusingaMar-

athonReadyTMcDNAAmplificationKit(Clontech,PaloAlto,CA).Tworeverseprimersweredesignedforthe1stPCR:GP245,5?-GTCAG-GAAATCTGAACGATGCTGA-3?,andforthenestedPCR,GP244,5?-GCAGCTGTTAAGGAATTCGGCTGA-3?.ThesequenceoftheDNAfragmentobtainedbythe5?-rapidamplificationofcomplementaryDNAmethodwasdeterminedusingaDYEnamicETTerminatorCycleSe-quencingKit(Amershamplc,AmershamPlace,UK).Finally,acDNAsequenceencodingtheORFwasobtainedbyPCRusingtheMarathonReadyTMcDNAofhumanbonemarrowtissue(Clontech)asatemplate.ConstructionandPurificationofCSGalNAcTProteinsFusedwithFLAGPeptide—TheputativecatalyticdomainofCSGalNAcT-2(aminoacids37to542)wasexpressedasasecretedproteinfusedwithaFLAGpeptideininsectcellsaccordingtotheinstructionmanualofGATE-WAYTMCloningTechnology(Invitrogen,Groningen,Netherlands).An?1.6-kbDNAfragmentwasamplifiedbyPCRusingtheMarathonReadyTMcDNAderivedfromhumanbonemarrow(Clontech)asatemplate,andtwoprimers,5?-GGGGACAAGTTTGTACAAAAAAGCA-GGCTTCAAAGGTGACGAGGAGCAGCTGGCAC-3?and5?-GGGGAC-CACTTTGTACAAGAAAGCTGGGTCTCATGTTTTTTTGCTACTTGT-CTTCTGT-3?.TheamplifiedfragmentwasinsertedintothevectorpDONRTM201(Invitrogen),thentransferredintotheexpressionvectorpFBIFtoconstructpFBIF-CSGalNAcT-2asdescribedpreviously(18).ACSGalNAcT-1expressionvector,pFBIF-CSGalNAcT-1,wasalsocon-structedasreportedelsewhere(17).ThecatalyticdomainsofCSGal-NAcT-1andCSGalNAcT-2wereexpressedinSf21insectcells.A50-mlvolumeofculturemediumwasmixedandincubatedwithanti-FLAGM1antibodyresin(SIGMA).Theresinwaswashedtwicewith50mMTris-bufferedsaline(50mMTris-HCl,pH7.4,and150mMNaCl)con-taining1mMCaCl2andsuspendedin100?lofeachassaybufferdescribedbelow.

Constructionof?4GalT-7,?3GalT-6,andGlcAT-IFusedwithFLAGPeptides—Theputativecatalyticdomainof?4GalT-7,?3GalT-6,orGlcAT-IwasexpressedassecretedproteinfusedwithFLAGpeptideininsectcellsorCOS-1cellsasdescribedindetailpreviously(17).Eachenzymewaspurifiedasdescribed(17)andsuspendedin100?loftheglycosylationbufferdescribedbelow.

AssayofCSGalNAcTActivity—Todeterminetheenzymaticactivity,UDP-galactose(UDP-Gal)andUDP-N-acetylgalactosamine(UDP-Gal-NAc)(Sigma)wereutilizedasdonorsubstrates.UDP-[3H]GalNAc(7.0Ci/mmol)waspurchasedfromPerkinElmerLifeSciences.Foracceptorsubstrates,Gal-?-para-nitrophenyl(pNP),Gal-?-pNP,GalNAc-?-ben-zyl,N-acetylglucosamine(GlcNAc)-?-pNP,GlcNAc-?-pNP,glucose-?-pNP,glucose-?-pNP,GlcA-?-pNP,fucose-?-pNP,mannose-?-pNP,xy-lose(Xyl)-?-pNP,andXyl-?-pNPwereobtainedfromCalbiochem(LaJolla,CA)andSigma.ChondroitinandCS-A,-B,-C,and-DwerepurchasedfromSeikagakuCorporation(Tokyo,Japan).Chondroitinhexasaccharideandmethoxyphenyl-linkagetetrasaccharideswerekindlydonatedbySeikagakuCorporation.OligosaccharidesofCSandchondroitinwerepreparedaspreviouslydescribed(19).

FortheGalTassay,14mMHepesbuffer(pH7.4)containing250?MUDP-Gal,12.5mMMnCl2,and500?Mofeachacceptorsubstratewasused.FortheGalNAcTassay,50mMMESbuffer(pH6.5)containing0.1%TritonX-100,1mMUDP-GalNAc,10mMMnCl2,and500?Mofeachacceptorsubstratewasused.A10-?lvolumeofenzymesolutionfor20?lofeachreactionmixturewasaddedandincubatedat37°Cfor2hfortheGalTand16hfortheGalNAcTassay.Afterincubation,themixturewasfiltratedwithanUltrafree-MCcolumn(Millipore,Bedford,MA)anda10-?laliquotwassubjectedtoreversed-phasehighperform-anceliquidchromatographyonanODS-80TsQAcolumn(4.6?250mm;TOSOH,Tokyo,Japan).0.1%Trifluoroaceticacid/H2Owasusedasarunningsolutionandtheproductswereelutedwitha0–15(forGlcA-?-pNP)or7–10%(forlinkagetetrasaccharide-para-methoxy-pheny)acetonitrilegradientataflowrateof1.0ml/minat50°C.Forglycosylatedpeptides,H2Ocontaining0.1%trifluoroaceticacidand21%acetonitrilewasutilizedastherunningsolution.Anultravioletspec-trophotometer(absorbanceat210nm),SPD-10AVP(Shimadzu,Kyoto,Japan)wasusedfordetectionofthepeaks.Intheanalysisofglycosy-latedpeptide,labelingwasdonewithCy5(AmershamBiosciences)andfluorescencewasdetectedwithafluorescencedetector,RF-10AXL(Shi-madzu).Fortheanalysisofelongationactivity,theCSGalNAcTsreac-tionmixturecontaining100?gofchondroitinorCSand40,000–55,500dpmofUDP-[14C]GalNAcwasused.Aftera1-hincubationat37°C,thereactionmixturewasfiltratedandfractionatedwithaG2500PWcol-umn(TOSOH,Tokyo,Japan)orSuperdex30pgcolumn(AmershamBiosciences).Theradioactivityofeachfractionwasmonitoredbyliquidscintillationspectrophotometry.

AssayofCSGalNAcTActivitywithaTetrasaccharide-bikuninPep-tide—AXyl-bikuninpeptide(VLPQEEEGS(-Xyl)GGGQLVT)waspur-

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DifferentialRolesofTwoCSGalNAcTs

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chasedfromthePeptideInstituteInc.(Osaka,Japan).TheCy5(Am-ershamBiosciences)-labeledXyl-peptidewasincubatedwith5?lofthreeglycosyltransferases,?4GalT-7,?3GalT-6,andGlcAT-I,and1mMdonorsubstrates,UDP-GalandUDP-GlcA,at37°°Cfor16hasde-scribedindetailpreviously(17).A50mMMESbuffer(pH6.5)contain-ing0.1%TritonX-100,1mMUDP-Gal,1mMUDP-GlcA,10mMMnCl2,and500?MXyl-bikuninpeptidewasusedforthereaction.Theglyco-syltransferasesforthesynthesisofthetetrasaccharide-bikuninpeptidewereinactivatedbyheatingat95°Cfor5min.Then,thereactionmixturewasfiltratedwithanUltrafree-MCcolumn(Millipore),anda10-?laliquotwasincubatedwith1mMdonorsubstrate,UDP-GalNAc,andeachCSGalNAcTat37°Cfor8hfortheassayofinitiationactivityofeachCSGalNAcT.ThereactionproductsofCSGalNAcTswerefil-tratedwithanUltrafree-MCcolumnanda10-?laliquotwassubjectedtoreversed-phasehighperformanceliquidchromatographyonanODS-80TsQAcolumnasdescribedabove.

DeterminationofProductsbyCSGalNAcT-2withMassSpectrometry(MS)—Anadditionalpeakdetectedbyreversed-phasechromatographywasisolatedandanalyzedbyanelectrosprayionization(ESI)orma-trix-assistedlaserdesorption/ionization-timeofflight(MALDI-TOF)MS(ESI-MS,esquire3000plus,andMALDI-TOF-MS,ReflexIV;BrukerDaltonics,Billerica,MA).Then,25pmolofproductwasdissolvedin5?lofH2O,and45?lof0.1%formicacidand50?lofmethanolwereadded.Theproductsolutionwasinfusedatarateof3?l/minwithacapillaryvoltageof3kV.ThehypotheticalproductpeaksobtainedbyESI-MSwereanalyzedbyESItandemmassspectrometry(ESI-MS/MS).Theseanalysesweredoneinbothpositive-ionandnegative-ionESImodes.ForMALDI-TOFMSanalysis,10pmolofproductwasdried,dissolvedin1?lofH2O,andapplied.

InVivoCS/HSSynthesisonaSyndecan-4/FGF-1ChimeraPro-tein—FortheexpressioninCHO-K1cells,thefull-lengthcDNAsofCSGalNAcT-1andCSGalNAcT-2wereamplifiedbyPCRwithprimersCSGalNAc-T1,5?-CCCAAGCTTATGATGATGGTTCGCCGGGGGCT-3?and5?-GCTCTAGATCATGTTTTTTTGCTACTTGTCTTCTGT-3?,andCSGalNAc-T2,5?-CCCAAGCTTATAAGAATGCCTAGAAGAGGACTG-A-3?and5?-GCTCTAGATCAACCAACAGCTTCACTGTTTGTC-3?.TheamplifiedfragmentswereinsertedintopcDNA3.1/hygromycin(?)(pcDNA3.1/Hygro,Invitrogen)afterHindIIIandXbaIdigestion.Theresultingplasmid,pcDNA3.1/HygroCSGalNAcT-1,-CSGalNAcT-2,andthepcDNA3.1/HygroexpressionvectorweretransfectedintoCHO-K1cellsharboringpMEXneo-PG-FGF-1thatwereexpressingthesynde-can-4/FGF-1chimeraprotein(PG-FGF-1)usingLipofectAMINE2000(Invitrogen)accordingtothemanufacturer’sinstructions.Mediumcon-ditionedbytheCHOtransfectantcontainingthesecretedPG-FGF-1proteinwithGAGswascollected.FractionationofPG-FGF-1usingaDEAE-Sepharosecolumn(AmershamBiosciences)andendoglycosi-dases:heparitinase(HSase),heparanase(HPase),andchondroitinaseABC(CSase)(SeikagakuCorp.)digestionandWesternblottingwithmonoclonalantibodyagainstFGF-1(mAb1)(20)wereperformedasdescribedpreviously(21).ThePG-FGF-1bandsdigestedbyglycosi-daseswerequantifiedbydensitometricscanningofthedigitizedimageusingNIHImage(version1.60)software(NationalInstitutesofHealth,Bethesda,MD).

QuantitativeAnalysisofCSGalNAcT-1andCSGalNAcT-2Tran-scriptsinHumanTissuesbyReal-timePCR—ForquantificationofthetwoCSGalNAcTtranscripts,weemployedthereal-timePCRmethod,asdescribedindetailpreviously(18,22,23).MarathonReadyTMcDNAsderivedfromvarioushumantissuesandcellswerepurchasedfromClontech.Standardcurvesfortheendogenouscontrol,glyceraldehyde-3-phosphatedehydrogenase(GAPDH)cDNAs,weregeneratedbyserialdilutionofapCR2.1(Invitrogen)DNAcontainingtheGAPDHcDNA.TheprimersetsandtheprobesforCSGalNAcT-1andCSGalNAcT-2wereasfollows.TheforwardprimerforCSGalNAcT-1was5?-GACT-TCATCAATATAGGTGGGTTTGAT-3?,thereverseprimer,5?-GTCCG-TACCACTATGAGGTTGCT-3?,andtheprobe,5?-ACCTTTATCG-CAAGTATCT-3?withaminorgroovebinder(24).TheforwardprimerforCSGalNAc-T2was5?-CTGACCATTGGTGGATTTGACAT-3?,there-verseprimer,5?-AACCGGAGTCCGAATCACAA-3?,andtheprobe,5?-CATCTTTATCGAAAATACTTACATGG-3?withaminorgroovebinder.PCRproductswerecontinuouslymeasuredwithanABIPRISM7700SequenceDetectionSystem(AppliedBiosystems,FosterCity,CA).TherelativeamountofeachCSGalNAcTtranscriptwasnormalizedtotheamountofGAPDHtranscriptinthesamecDNA.

RESULTS

DeterminationofNucleotideandAminoAcidSequenceofCSGalNAcT-2—Wedeterminedanovelfull-lengthcDNAse-

quencebythe5?-rapidamplificationofcDNAendsmethodandregistereditintheGenBankTMdatabasewithaccessionnum-berAB079252.ThenucleotidesequenceandtheputativeaminoacidsequenceareshowninFig.1.ThiscDNAsequenceconsistedofa254-bp5?-untranslatedregion,1629-bpcodingregion,and1791-bp3?-untranslatedregionthatcontainedapoly-Atail(Fig.1).AlthoughtheoriginalexpressedsequencetagwasobtainedinaGenBankTMdatabasesearchwith?1,4-glycosyltransferasemotifsasquerysequences,thefull-lengthORFsequencewasidentifiedashighlyhomologoustoCSGal-NAcTaspreviouslyreportedbyus(17).WedesignatedthisnovelcDNAasCSGalNAcT-2,andrenamedthepreviousCS-GalNAcT,CSGalNAcT-1.AnalignmentofCSGalNAcT-1andCSGalNAcT-2isshowninFig.2.AhydropathyprofileoftheputativeaminoacidsequencebasedonKyteandDoolittlehydrophobicityplotsindicatesthattheORFofCSGalNAcT-2encodesatypicaltypeIImembraneprotein,whichisconsistentwiththetopologyofotherglycosyltransferases,withacytoplas-mictailof14aminoacids,atransmembranedomainof20aminoacids,andalargecatalyticportionof508aminoacids.CSGalNAcT-2containsaDXDmotif,whichisconservedinmanyglycosyltransferasesandfunctionsasakeysequencefordivalentcationbinding,andaGWGGEDmotif,whichishighlyconservedamongsomeofthe?4GalTfamily.

AgenomesequenceidenticaltothatoftheCSGalNAcT-2cDNAwasfoundinagenomeclone(GenBankTMAC011890),whichislocalizedonchromosome10.ThegenomicstructureoftheCSGalNAcT-2genewasdeterminedtobecomposedofatleast7exonsbycomparisonbetweenthecDNAandgenomesequences(Fig.1).Theexon/intronjunctionsofCSGalNAcT-2wereidenticaltothoseofCSGalNAcT-1(datanotshown).DeterminationofCSGalNAcT-2ActivityinElongationandInitiationofChondroitinPoly-andOligosaccharides—ThetruncatedsolubleformsofCSGalNAcT-1andCSGalNAcT-2wereexpressedininsectcellsandemployedforallexperi-ments.OnWesternblottingusinganti-FLAGantibody,eachenzymewasdetectedasasinglebandcorrespondingtothepredictedsize(Fig.3B).AnadditionalbandappearedbyCoo-massiestainingat?50kDasharedbyalllanes(Fig.3A),however,theproteinrecoveredfrommocktransfectantsshowednoactivityforanydonorsandacceptorsubstrates(datanotshown).Atfirst,wescreenedthetransferactivityofCS-GalNAcT-2usingninedonorsubstratesandmultiplemonosac-charide-pNPacceptors.Inscreeningofdonorandacceptorsub-strates,CSGalNAcT-2transferredaGalNAcresiduetoGlcA-pNP,however,noactivitywasobservedwithothercombinationsofdonorandacceptorsubstrates(datanotshown).ItwasidentifiedinapreviousreportthatCSGalN-AcT-1is?4GalNAcT,whichtransfersGalNActoGlcAforini-tiationandelongationinthesynthesisofCS(17).Sofar,thelinkageofGalNAcwithGlcAhasbeenidentifiedonlyinCS.WeconsideredthatCSGalNAcT-2wasalsoaGalNAcTinvolvedinthesynthesisofCS,andperformedfurtheranalysisusingCS-relatedsubstratesasacceptors.

TwokindsofGalNAc-GlcAlinkagesareknowninCS,oneinitspolymerstructure(3GalNAc?1–4GlcA?1-)nandtheotherbetweenthepolymerCSandthelinkagetetrasaccharide(GlcA?1–3Gal?1–3Gal?1–4Xyl).Atfirst,chondroitinwasuti-lizedasanacceptorsubstratetoexaminetheelongationactiv-ityofCSGalNAcT-2.AsshowninFig.3C,CSGalNAcT-2ap-parentlytransferredGalNActochondroitintoproduceanadditionalpeak(indicatedbyanarrowinFig.3C)asareactionproduct.ThispeakwasisolatedandidentifiedtohaveGalNAconitsnonreducingterminuswithchondroitinaseACIItreat-ment(datanotshown),themethodusedhavingbeendescribedinapreviousstudy(17).Second,alinkagetetrasaccharide

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3066DifferentialRolesofTwoCSGalNAcTs

FIG.1.NucleotideandaminoacidsequencesofCSGalNAcT-2.Thepredictedstartandstopcodonsareunderlined.Thesplicingjunctionsareindicatedwitharrowheads.Apoly(A)signalisdoubleunderlined.

(GlcA?1–3Gal?1–3Gal?1–4Xyl?-O-methoxyphenyl)synthe-phenylwithNa?orwithK?,respectively.Moreover,thepeakPsizedchemicallywasutilizedasanacceptorsubstratetoiden-wasidentifiedtohaveGalNAcatitsnonreducingterminuswithtifytheinitiationactivityofCSGalNAcT-2.AsshowninFig.chondroitinaseACIItreatment(datanotshown).Theseresults3D,thepeakPappearedata30.3-minretentiontimeinaddi-suggestedthatCSGalNAcT-2hastwoGalNAcTactivities,i.e.tiontotheacceptorsubstratepeak(peakS)http://wendang.chazidian.comparisonofAcceptorSubstrateSpecificitybetweenCSGal-PeakSgaveamolecularmassof779.14m/z,thesameasthatNAcT-1andCSGalNAcT-2—Inviewoftheseresults,CSGalN-ofthelinkagetetrasaccharidewithNa?(Fig.3E).PeakPgaveAcT-2wassuggestedtobeGalNAcTinvolvedinthesynthesistwopeaksof982.28and998.25m/zasshowninFig.3F.TheofCSaswellasCSGalNAcT-1.Todistinguishthefunctionsofmolecularmassof982.28or998.25m/zwasthesamemolecularthetwoCSGalNAcTs,wecomparedGalNAcTactivitytowardweightasaGalNAc-addedlinkagetetrasaccharide-O-methoxy-GlcAfoundatthenonreducingterminusofvariouskinds

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DifferentialRolesofTwoCSGalNAcTs3067

FIG.2.MultiplealignmentofaminoacidsequencesofCSGal-NAcT-2andCSGalNAcT-1byGENETYX.Introducedgapsareshownwithhyphens.Theputativetransmembranedomainsareunder-lined.TheDXDmotifandthe?1,4-glycosyltransferasemotifareboxed.Identicalaminoacidsareshownwithasterisks.PossibleN-glycosyla-tionsitesareindicatedbyarrowheads

.

chondroitin-relatedacceptorsubstrates.TheamountofenzymewasestimatedbyWesternblotting,andapproximatelyequalamountsofenzymewereusedfortheGalNAcTreaction.TheresultsaresummarizedinTableI.Regardingtheelongationactivity,CSGalNAcT-2utilizedchondroitinpolysaccharideasanacceptormorethananyothersubstrateexamined,andshowedhigherlevelsofenzymaticactivitythanCSGalNAcT-1towardallsubstratesexceptforthelinkagetetrasaccharide(TableI).Furthermore,CSGalNAcT-2showedremarkablystrongactivity,comparedwithCSGalNAcT-1,towardsulfatedsubstratessuchasCSpoly-andoligosaccharides(TableI).CS-AandCS-B,bothofwhicharesulfatedatpositionC-4ofGalNAc,werebettersubstratesforCSGalNAcT-2thanCS-C,whichissulfatedatpositionC-6ofGalNAcandCS-D,whichissulfatedatC-6ofGalNAcandC-2ofGlcA(Fig.4).CSGal-NAcT-2exhibitedmuchstrongeractivitytowardthelongeroligosaccharides,preparedfromchondroitinandCS,thanto-wardtheshorterones.TheseresultsstronglyindicatedthatCSGalNAcT-2ismuchmoreactiveintheelongationofchon-droitinandCSthanisCSGalNAcT-1.

Regardingtheinitiationactivity,CSGalNAcT-1preferredthelinkagetetrasaccharideassubstrateandshowedamuchhigherlevelofactivityforthelinkagetetrasaccharidethandidCSGalNAcT-2.ThisindicatedthatCSGalNAcT-1istheen-zymemainlyresponsiblefortheinitiationofchondroitinandCSsynthesis,notforelongation.ToexaminewhethertwoGalNAcTsshowasynergisticeffectforinitiationandelonga-tionactivities,twoenzymesweremixedintheenzymereactiontowardthelinkagetetrasaccharideorchondroitin.Weob-servedadditionaleffectsofGalNAcTactivity,butnotsynergis-ticeffect,toanyacceptorsubstrates(datanotshown).

ComparisonofInitiationActivitywithLinkageTetrasaccha-ridePeptidebetweenTwoCSGalNAcTs—Weperformedaninvitroenzymaticsynthesisofachondroitinpentasaccharide-bikuninpeptide(VLPQEEEGS*GGGQLVT;thepeptidese-quenceencodestheNH2-terminalsequenceofbikuninandtheserineresiduewithanasteriskisattachedwithasaccharidechain)fordeterminationoftheinitiationactivitybyCSGalN-AcTs.PeakSinFig.5A,whichwasidentifiedtopossessthelinkagetetrasaccharide(GlcA-Gal-Gal-Xyl-bikuninpeptide)inapreviousstudy,wasusedastheacceptorsubstrate.Toobtaintherelativeactivityofeachenzyme,equalamountsofenzyme

FIG.3.DeterminationofCSelongationandinitiationactivi-tiesofCSGalNAcT-2.CSGalNAcT-1and-2werepurifiedwithanti-FLAGM1antibodyresinandwereappliedtoSDS-PAGE.ThedetectionofproteinswasperformedwithCoomassiestaining(A)andWesternblottingusinganti-FLAGantibody(B).C,chondroitinwasusedasanacceptorsubstrateforCSGalNAcT-2.ThepurifiedreactionmixtureswereappliedtoaG2500PWcolumnwith0.2MNaClasarunningbufferataflowrateof0.6ml/min.Theradioactivityofeachfraction(340?leach)wasquantifiedbyliquidscintillationspectrophotometry.Thepeakofthereactionproductisindicatedwithanarrow.D,linkagetetrasaccharide-O-methoxyphenylwasusedasanacceptor,andtheproductwasanalyzedbyreversed-phasechromatography.Eachpeakofsubstrate(peakS)andreactionproduct(peakP)wasisolatedandsubjectedtoMALDI-TOF-MSanalysis(EandF).

wereemployed,andtheincubationwasterminatedbeforealargeamountofacceptorsubstrateremained.AsseeninFig.5,BandC,thereactionproduct(PeakP)appearedat30.1minwithCSGalNAcT-1or-2,respectively.PeakPwasidentifiedasGalNAc-GlcA-Gal-Gal-Xyl-bikuninpeptideinapreviousstudy(17).ThepeakareaoftheCSGalNAcT-1product(Fig.5C)was3-foldlargerthanthatoftheCSGalNAcT-2product(Fig.5B).Thevalueofthisrelativeactivitywasconsistentwiththattowardthemethoxyphenyl-linkagetetrasaccharideinTableI,i.e.theactivityofCSGalNAcT-1was2.3-foldthatofCSGalN-AcT-2.TheseresultsindicatedthatthepresenceofbikuninpeptidedoesnotinfluencetherelativeactivityofthetwoCS-GalNAcTsinvitro.

ChangeofGAGCompositionofPG-FGF-1byCSGalNAcT-1orCSGalNAcT-2Transfection—Previously,thegeneconstructencodingachimeraprotein(PG-FGF-1)consistingofsynde-can-4andFGF-1wastransfectedintoCHO-K1cellstoexpressthechimeraprotein(21).Syndecan-4isawellanalyzedproteo-glycanpossessingchainsofHSand/orCS.TheGAG

composi-

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