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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMTMVOLUME125GlycoproteinGlycoproteinMethodsMethodsandProtocolsandProtocolsTheMucinsTheMucinsEditedbyEditedbyAnthonyP.CorfieldAnthonyP.CorfieldHUMANAPRESSHUMANAPRESSIsolationofLargeGel-FormingMucins31IsolationofLarge
2、Gel-FormingMucinsJuliaR.DaviesandIngemarCarlstedt1.IntroductionThelargegel-formingmucins,whichformthemajormacromolecularcomponentsofmucoussecretions,aremembersofthemucin“superfamily.”Ninemucingenes(MUC1–MUC4,MUC5AC,MUC5B,andMUC6–MUC8)havebeenidentified(forreviewssee
3、refs.1and2),witheachgeneshowingexpressioninseveraltissues.OnlytheMUC1,MUC2,MUC4,MUC5,andMUC7mucinshavebeensequencedcom-pletely(3–11)althoughlargestretchesofMUC5AC(12–15)aswellastheC-terminalsequencesofMUC3(16)andMUC6(17)arenowknown.Acharacteristicfeatureofmucinsisth
4、epresenceofoneormoredomainsrichinserineand/orthreonineresiduesthat,owingtoahighdegreeofoligosaccharidesubsti-tution,areresistanttoproteolysis.Mucinscomprisecell-associated,usuallymono-mericspecies,aswellasthosethataresecreted;thelattercanbesubdividedintolarge,gel-fo
5、rmingglycoproteinsandsmaller,monomericones.Thegel-formingmucins(Mr=10–30millionDalton)areoligomersformedbysubunits(monomers)joinedviadisulfidebonds(forareviewseeref.18),andtreatmentwithreducingagentswillreleasethesubunitsandcauseunfoldingofregionsstabilizedbyintramo
6、leculardisul-fidebonds.Thus,afterreduction,wetermthemonomersreducedsubunits.Reducedsubunitsaremoresensitivetoproteasedigestionthantheintactmucinmolecules.Theisolationproceduresthatweuseforthelargeoligomericmucinsdependontheirsource.Insecretionssuchasrespiratorytract
7、sputum,tracheallavagefluid,andsaliva,thematerialiscentrifugedtoseparatethegelfromthesolphase,allowingtheidentificationofthegel-formingmucins.Repeatedextractionofthegelphasesolubi-lizesthe“soluble”gel-formingspecies,leavingthe“insoluble”mucincomplexintheextractionres
8、idue.Mucinsubunitsmaybeisolatedfromthe“insoluble”glycoproteincomplexfollowingreductionofdisulfidebonds.Whenmucinsareisolatedfromtissuesamp
