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1、MAMMALIANCELLSURFACEDISPLAYOFFUNCTIONALALPHA1-PROTEASEINHIBITOR:BUILDING“DESIGNER”SERPINSByRICHARDFRANKGIERCZAKAThesisSubmittedtotheSchoolofGraduateStudiesinPartialFulfilmentoftheRequirementsfortheDegreeDoctorofPhilosophyMcMasterUniversity©CopyrightbyRicha
2、rdFrankGierczak,April2015135135DOCTOROFPHILOSOPHY(2015)(MedicalSciencesProgram,BloodandVasculature)McMasterUniversityHamilton,OntarioTITLE:MammalianCellSurfaceDisplayofFunctionalAlpha1-ProteaseInhibitor:Building“Designer”SerpinsAUTHOR:RichardFrankGierczak,
3、B.Sc.,M.Sc.(McMasterUniversity),M.Sc(UniversityofWaterloo).SUPERVISORDr.WilliamP.SheffieldNUMBEROFPAGESix,135135ABSTRACTHumanserpinsbelongtoasuperfamilyofserineproteaseinhibitorsinvolvedintheregulationofessentialphysiologicalprocesses,includingcoagulationv
4、iathrombininhibitionbyAT.Inhibitoryserpinsundergoaremarkablefoldingmechanismtoathermodynamicallyunstable(i.e.metastable)conformation,highlightedbyalongandflexibleRCL,priortosecretionassolubleproteinsintocirculation.Theserpinalpha-1-proteinaseinhibitor(API)
5、normallyprotectstissuesfromproteasesreleasedfrominflammatorycells(e.g.,neutrophilelastase).Importantly,avariantofAPI(i.e.API-PittsburghorAPI-M358R)wasreportedtobethecauseofafatalbleedingdisorderinayoungpatientinthelate1970`s;thepointmutationM358RatP1oftheR
6、CLresultedinadramaticshiftinfunctiontowardthrombininhibition.Thisdissertationsummarizestheresultsfromexperimentsperformedwithserpinsexpressedasmembraneproteinstetheredtothesurfaceofmammaliancells.SerpinsAPI-M358R,AT,HCII,andthenon-inhibitorydoublemutantAPI
7、-M358R/T345Rwereanchoredto293andCOScellplasmamembraneswithN-terminalnon-cleavableproteinsequencesderivedfromeitherthehumanasialoglycoprotein(AR)ortransferrin(TF)receptors.Sub-cellularfractionation(withorwithoutmonolayerexposuretothrombin)immunoblotsconfirm
8、edserpinlocalizationtotheintegralmembranefractionusingeitheranchoringapproachorcelltype.TheblotsalsorevealedthattetheredAPI-Pittsburghinparticular,andATtoalesserextentformedserpin-enzymecomplex(SEC)withthromb
