Cell-surface engineering with

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1、:-Cell-surfaceengineeringwithGPI-anchoredproteinsM.EDWARDMEDOF,’SHANMUGAMNAGARAJAN,ANDMARKLTYKOCINSKIInstituteofPathology,CaseWesternReserveUniversity,Cleveland,Ohio44106,USAABSTRACTProteinengineeringofcellsurfacesislinkedtoaninositolphospholipid(seeFig.1).Theyareapotentiallypowerfultechnologythr

2、oughwhichtheprefabricatedintheendoplasmicreticulum(ER)andaresurfaceproteincompositionofcellscanbemampu-addedenbloctoprimarytranslationproductsatthetimelatedwithoutgenetransfer.ThistechnologyexploitsoftheirtranslocationacrosstheERmembrane.TheGPI-thefactthatproteinsthatareanchoredbyglycoi-modifiedp

3、roductsthenareglycosylatedintheERandnositolphospholipids(GPIs),whenpurifiedandGolgiandtransportedtothecellsurface.addedtocellsinvitro,incorporateintotheirsurfaceWhycertainproteinsareexpressedwithGPIanchorsmembranesandarefullyfunctional.Bysubstitutingisunknown.Photobleachingexperimentshaveshowntha

4、t3’-mRNAendsequenceofnaturallyGPI-anchoredGPIanchoringprovidesforrapidlocomotioninthesur-proteins(i.e.,asequencethatcontainsthesignalsfacemembrane(4).ConventionallyanchoredproteinsthatdirectGPIanchormg)forendogenous3-mRNAwithshortortruncatedcytoplasmicsequences,however,endsequence,virtuallyanypro

5、teinofinterestcanbeexhibitclosetothesamemobility;whentheseanchorsexpressedasaGPI-anchoredderivative.TheGPI-andGPIsareshuffled,thesizeoftheectodomainappearsanchoredproductthencanbepurifiedfromtranfec-tobeamoreimportantdeterminant(5).GPI-anchoredtantsandthepurifiedproteinusedto“paint”anyproteinscan

6、becleavedfromcellsbycertainphospholi-targetcell.Suchproteinengineeringor“painting”ofpases-e.g.,phosphatidylinositol-specificphospholipasethecellsurfaceoffersseveraladvantagesovercon-C(PI-PLC)-andconsequentlycanbereleasedashydro-ventionalgenetransfer.Amongtheseadvantagesarephilicderivatives(1-3).N

7、ophysiologicalfunction,how-that1)GPI-anchoredproteinscanbepaintedontoever,isknowntobeassociatedwiththisreleasereaction.cellsthataredifficulttotransfect,2)cellscanbeBecauseGPIanchorsareattachedtotheoutermembranealteredi