化学生物学基础ilecture综合

••••••OverviewofBasicsOverviewofBasicsofCellularorganizaConofglycosylaConsN-­­O-­­linkedO-­­ChemicalI.I.@Nature,@Nature,ANDREWThe“darkThe“darkma=er”ofDefinedDefinedinthebroadestsense,glycobiologythestudyofthestructure,biosynthesis,biology,andevoluConofsaccharides(sugarchainsorglycans)thatarewidelydistributedinnature,andtheproteinsthatrecognizethem.Organism’sComplexity&GenomeOrganism’sComplexity&GenomeYeast~6,000Fruitfly~16,000C.elegans~20,000Human~20,000betweenaleaderandaSteve“…..The“…..Thefindingthatthehumancontainsfewergenesthanpreviouslypredictedmightbecompensatedforbycombinatorialdiversitygeneratedatthelevelof...pos=ranslaBonalmodificaBonofproteins….”J.C.Venter,etal.,Science(2001),291,BeyondtheCentralDNAepigenetics:BeyondtheCentralDNAepigenetics:RNAPosttranslationalCell-surfaceglycans:thesugarGlycanssialicCell-surfaceglycans:thesugarGlycanssialicembryonicmolecularsignalimmune~50~5DynamicglycomereflectsDynamicglycomereflectscellularstemhealthydifferentiateddiseasedcelldiseaseGlycansareextremelyGlycansareextremely•••••Non-­­geneBcallyBranchedstructuresScience,2001,Science,2001,291,GlycanAdding“sweet”totheAdding“sweet”tothe@Chem.Soc.Rev.MonosaccharidebuildingblocksinvertebrateOOOGlucoseGalactoseMannoseOOOON-AcetylglucosamineacidN-AcetylgalactosamineN­­乙酰葡萄NMonosaccharidebuildingblocksinvertebrateOOOGlucoseGalactoseMannoseOOOON-AcetylglucosamineacidN-AcetylgalactosamineN­­乙酰葡萄N­­乙酰半乳OHOOOSialicacidFucoseXyloseEssen%alsofEssen%alsofChapter1,FigureBiosynthesis,utilization,andturnoverofacommonGlycosylatedGlycosylGlycosylaBonGlycosylatedGlycosylGlycosylaBonGlycosylationEssen%alsGlycosylationEssen%alsofGlycobiology,Chapter5,FigureNucleotideSugarsasGlycosylationsugar•MostreacConsuseacCvatedformsofmonosaccharidesNucleotideSugarsasGlycosylationsugar•MostreacConsuseacCvatedformsofmonosaccharides：NucleoCdesugars（核苷酸糖Essen%alsofGlycobiology.2nded.,Ch4,TableAcBvated]UDP--­]GDP--­]CMP--­YOOOONO O ZOOONONNOOOOO X=OH,Y=H,Z=OH:UDP-GlucoseX=H,Y=OH,Z=OH:UDP-GalactoseX=OH,Y=H,Z=NHAc:UDP-GlcNAcX=H,Y=OH,Z=NHAc:UDP-GDP-ONOOOOONNOOOOOONO YOOOONO O ZOOONONNOOOOO X=OH,Y=H,Z=OH:UDP-GlucoseX=H,Y=OH,Z=OH:UDP-GalactoseX=OH,Y=H,Z=NHAc:UDP-GlcNAcX=H,Y=OH,Z=NHAc:UDP-GDP-ONOOOOONNOOOOOONO GDP-O NUDP-GlucuronicO OONOO–2OHCMP-SialicStructuresofnucleotidesugarOCMP-Sialic2,3-O–OOGDP-1,3-OO–OOOCMP-Sialic2,3-O–OOGDP-1,3-OO–OOOOExamplesofGlycanMonosaccharideMonosaccharideMonosaccharidesarelinkedtoMonosaccharidesarelinkedtomorecomplexLactose–mike“OligosaccharideorCellulose–plantcellRegiochemistryandSterochemistryof-2OOOOOOO3ReducingNRegiochemistryandSterochemistryof-2OOOOOOO3ReducingNn-­1OSia2,3Gal1,4[Fuc1,3]GlcNAc--­Sialyllewis@springerlab@springerlabsLeXmediatesleukocytesLeXmediatessLeXmediatesleukocyteExamplesofExamplesoftypicalN-andO-linkedabcEssen%alsofSecondabcEssen%alsofSecondChapter1,FigureMonosaccharidesymbolEssen%alsofEssen%alsofGlycobiologyChapter1,FigureCommonclassesofanimalBiosynthesisandBiosynthesisandinterconversionofChapter4,FigureEssen%alsofHumanbloodHumanbloodgroupsaredefinedbyOO OOOOOOOOOOOOOROOOROOH3OABHumanbloodgroupsaredefinedbyOO OOOOOOOOOOOOOROOOROOH3OABHumanbloodgroupsaredefinedbyGlycanshavemorecomplexstructuresGlycanshavemorecomplexstructuresthanDNAandHeparansulfateSchematicrepresentationSchematicrepresentationoftheThy-1Essen%alsofSecondChapter1,FigureerythropoieBnerythropoieBnerythropoieBnerythropoieBnFigureadoptedfromAngew.Chem.2013,52,BiosynthesisofextracellularcellBiosynthesisofextracellularcellnucleotideScience2001,291,CellularOrganizaBonofCellularOrganizaBonof•EvoluConhasrepeatedlyselectedforglycansbeingthemostdiverseandflexiblemoleculestoposiConattheinterfacebetweencellsandtheextracellularmilieu;Everyfree-­­livingcellandeverycelltypewithinmulCcellularorganismsiscoveredwithadenseandcomplexlayerofglycans;GlycoproteinspassthroughanendoplasmicreCculum(ER)-­­Golgipathway,thecellularsysteminwhichmanymajorglycosylaConrxnsoccur.••InitiationandInitiationandmaturationofthemajortypesofeukaryoticglycoconjugatesinrelationtosubcellulartraffickingintheER-Golgi–plasmamembraneEssen%alsof•Notall•NotallglycansandglycoconjugatesassembletheER-­­Golgipathway.E.g.O-­­MostglycosylaConreacConsuseacCvatedformsofmonosaccharides,nucleoCdesugarsGlycosylaConsarecatalyzedbyenzymescalledGlycanscanbefurtherThedonorsforglycosylaConreacConsandglycanmodificaConsaremostlysynthesizedwithinthecytoplasmiccompartment.(excepConforCMP-­­Sia)••••GlycotransferasesGlycotransferasesarephysicallylinedupalongthispathwaytheprecisesequenceinwhichtheyactuallyTypicaltransmembraneTypicaltransmembranetopologyandproteolyticprocessingofGolgiEssen%alsofChapter3,FigureSomefactsaboutSomefactsabout••GlycotransferasesarealsoafewstudiesindicatethatglycosyltransferasesaresubjectBiochemicalandultrastructuralstudiesindicatethatglycosyltransferasesparCallysegregateintodisCnctcompartmentswithinthesecretorythesolubleformswereactuallyderivedfromtheirmembrane-­­associatedformsbyvirtueofoneormoreproteolyCccleavageeventsthatoccurredashortdistanceawayfromthetransmembranesegmentwithinthestemthestemregioncontributesliHletothecatalyCcfuncConofa“oneenzyme–onelinkage”;Thereare••••StrictacceptorStrictacceptorsubstratespecificityofEssen%alsofChapter5,Figure知识点知识点N-­­•••RefersN-­­•••ReferstoGlcNAcβ1-­­AsnN--­MembraneproteinsandsecretedRegulateproteinqualitycontrol,ligandbinding,tranlocaBon,TheAsnresiduestowhichglycansaretobeattachedmust:–Befoundinthesequence:Asn-X-Ser/Thr(XcannotbePro)–Belocatedonthesufaceofthe–Befoundinsecretory/membraneMajorstructuralMajorstructuralclassesandCorestructure:Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAcβ1--Asn-­­X-­­•N-linkedglycanscanbe•N-linkedglycanscanbereleasedbydigestionwithpeptide-N-glycosidaseF(PNGaseF)•EndoglycosidaseHandendoglycosidaseFcleavebetweentwocoreGlcNAcresiduesN-linkedglycanscanbereleasedbyCo-translationalN-glycosylationCo-translationalN-glycosylationofproteinsinthesecretorypathwayOT:OligosaccharylFirststepofFirststepofglycosylaBononSynthesisofSynthesisofdolichol-P-P-Essen%alsofChapter8,FigureDolichol••FormationDolichol••Formationofalipid-linkedprecursorN-GlycansynthesisbeginsbythetransferofGlcNAc-1-PfromUDP-GlcNActoDol-PtogeneratedolicholpyrophosphateN-acetylglucosamine(Dol-P-P-Transferoftheoligosaccharidetothetargetpolypeptidebyoligosaccharyltransferase(OT)Processingofthe••TunicamycinisTunicamycinisaninhibitorofN-linked•TunicamycinblocksthetransferofGlcNAc-1-PfromUDP-GlcNActoDol-PtogeneratedolicholpyrophosphateN-acetylglucosamine(Dol-P-P-GlcNAc)Essen%alsofEssen%alsofProcessingandmaturationofanN-BranchingBranchingofcomplexN-ModificationsModificationsofthecoreofN-TypicalcomplexN-glycanTypicalcomplexN-glycanstructuresfoundonmatureEssen%alsofSecondChapter8FigurecargoFigureCompetitionbetweenfolding,aggregation,anddegradation.ProteinsenterthesecretorypathwaycargoFigureCompetitionbetweenfolding,aggregation,anddegradation.ProteinsenterthesecretorypathwayviaSec61(bluearrowhead)andbegintofoldwiththehelpofendoplasmicreticulum(ER)foldingfactors(FFs).Proteinsthatdonotfoldcorrectlyself-associatetoformaggregatesandintheprocesssequesterfoldingfactors.Nonnativefoldingintermediatesmaybetargetedfordegradationiftheirfoldingtothenativestructureisdelayedorhampered.Degradationtakesplaceinthecytosol,sotargetedpolypeptidesmustbetranslocatedoutoftheER.Aggregatedproteinsdonotenterthispathway,althoughtheymaybecomedegradedfollowingautophagy.CorrectlyfoldedproteinsexittheER,usuallyfollowingrecognitionbycargoreceptors,whichconcentrateatERexitsitesandformCOPII-coatedtransportvesiclesfordeliveryofthecargototheER-to-GolgiintermediatecompartmentandtheGolgiapparatus.ProteinFoldingandModificationintheInekeBraakman1andNeilJ.1CellularProteinChemistry,FacultyofScience,UtrechtUniversity,3584CHUtrecht,TheNetherlands;email:i.braakman@uu.nl2CollegeofMedical,VeterinaryandLifeSciences,UniversityofGlasgow,Glasgow,G128QQScotland,UnitedKingdom;email:neil.bulleid@glasgow.ac.uk Targetedfor FFinthe EnsembleofintermediatesbyfoldingFoldedcorrectly:readyforexport•N-­­glycans•N-­­glycanshelpglycoproteinsfoldbycreaCngseriesofcheckpointsthatdictatethelifeorthedeathofnewlymademembraneandsecretedproteins.ModelproposedModelproposedforthequalitycontrolofglycoproteinEssen%alsofDegradationofDegradationofpolymannosylglycansintheER,cytoplasm,andEssen%alsofGlycobiology,Chapter36,Figure•••Thediscoveryofmucin-typeO-linkedglycoproteinsStructural•••Thediscoveryofmucin-typeO-linkedglycoproteinsStructuralfeaturesofmucin-typeO-linkedglycoproteinsTherolesofmucin-typeO-linkedglycoproteinsinphysiologyanddiseaseBiosynthesisofmucin-typeO-linkedglycansStructuralandmechanisticstudiesofppGalNAcTsSubstratespecificityoftheppGalNAcTsGeneticapproachestowardunderstandingmucin-typeO-linkedglycosylation••••Mucin-typeO-linkedThediscoveryofmucin-typeO-linked•Thediscoveryofmucin-typeO-linked•“Mucin”wasfirstusedbyNicolasTdeSaussuretodescribesubstancesisolatedfrommucus•Eichwalddiscoveredthatmucinsareconjugatedto•Hoppe-Seylerdiscoveredthatmucinsbytheepithelialcellsofmucousmembranescontainanacidiccarbohydrate-GalNAclinkagetoThr/SerwasdeterminedbyWeissmanwithapurified-N-acetylgalactos-aminidasefrombovineliver•Structuralfeaturesofmucin-typeStructuralfeaturesofmucin-typeO-linked•Sugarcomponent>50%••Thisstructuralfeatureisconferredby-GalNAclinkageInsteadof-GalNAcHilightsHilightsofmucin-typeO-linkedglycoproteinsinAAsimplifiedmodelofalargesecretedMucin-typeO-linkedglycosylationdeterminesMucin-typeO-linkedglycosylationdeterminestheaggregationstateofCD45BiosynthesisBiosynthesisofmucin-typeO-linkedStructureStructureofcoreO-linkedComplexO-GalNAcComplexO-GalNAcglycanswithdifferentcoreEssen%alsofChapter9,FigureBiosynthesisBiosynthesisofmucin-typeO-linkedglycansintheGolgiBiosynthesisBiosynthesisofcore1and2O-GalNAcBiosynthesisBiosynthesisofcore3andcore4O-GalNAcStructuralfeaturesof~Structuralfeaturesof~24putativeppGalNAcTsinhumans,21ofthemhavebeenclonedandbiochemicallychar