生物化学英文版课件：Chapter 5-3 Protein Function,modulation and evolution-immunoglobulins

Chapter5-3

Proteinfunction,modulationandevolution:Immunoglobulins

Anenormouslydiversecollectionof108relatedproteinsfoundinthebloodofvertebrates,specificallybindsuchforeigninvaders(antigens)asbacteria,virusesorlargemolecules,andtagthemfordestruction(neutralization).

Kd=10-4-10-10MProducedbyplasmaBcells.BiochemistryLecture(Oct.11,2012)ExtremespecificityGreatdiversity2012NobelPrizeinChemistry"forstudiesofG-protein–coupledreceptors"RobertJ.LefkowitzBrianK.KobilkaAnoverviewoftheAdaptiveImmuneResponseComplementsystemOrPhagocytosisFivedifferenttypesofimmunoglobulinsarecommonlyfoundintheserumofvertebratesFoundinsalivatearsandmilkThefirstmadeinprimaryimmuneresponse;OfhighavidityIgG:

dominantinSecondaryimmuneresponse;Mostabundantinblood.ThedivalentbindingofIgGallowsantibodiestocross-linkantigenstoformanextendedlattice,whichhastenstheremovaloftheantigensandtriggersBcellproliferationOnlypolyclonalantibodies,notmonoclonalantibodies,willleadtoprecipitateformationAntitoxinproposedtobepresentinbloodserumofinfectedanimals(1890)vonBehring(1854-1917)

NobelPrizein1901“forhisworkonserumtherapy,especiallyitsapplicationagainstdiphtheria,bywhichhehasopenedanewroadinthedomainofmedicalscienceandtherebyplacedinthehandsofthephysicianavictoriousweaponagainstillnessanddeaths.”

Theside-chaintheoryontheimmuneresponseproposed(Ehrlich,1897)Sidechainsintheformofreceptorsexistinginthe“protoplasm”,whichwereabletobinddistincttoxinswithstrictspecificity,like“akeyinalock”.Releasedintotheblood,thesereceptorsrepresentedantitoxins(antibodies).Dr.Ehrlich'sMagicBullet(an1940movie)PaulEhrlich(1854–1915)NobelPrizein1908"inrecognitionoftheirworkonimmunity"

Pneumococcuspolysaccharidesareantigens

Heidelberger&Avery.(1923)Thesolublespecificsubstanceofpneumococcus.JExpMed.38:73-9.Culturefluidsofpneumococcicontainedasubstancethatprecipitatesspecificallywithanti-pneumococciserum.Thesubstancefoundnottobeprotein(containednonitrogen)butapolysaccharide.Thisallowedtheidentificationoftheantibodypresentinanimmunoprecipitateasprotein.HeidelbergerAveryAntibodiesfoundtobeproteinsFeltonLD.(1934)Pneumococcusantibodies–whatarethey?Science79:277-9.

Pepsinandtrypsindigest:lossofimmunologicalcharacteristics.Antibodieswerefoundtobethe

g-globulinintheserum(1938)TiseliusA,KabatEA.(1938)Electrophoresisofimmuneserum.Science.87:416-7.TiseliusA,KabatEA.(1939)Anelectrophoreticstudyofseraandpurifiedantibodypreparation.JExpMed.69:119-31.Electrophoresisofhorseanti-pneumococcusserumbefore(A)andafter(B)absorptionoftheantibodybyantigen:onlyoneproteinbanddisappeared.ABAntigenabsorptionexperimentKabatTiseliusggRabbitanti-eggalbuminserumunabsorbedabsorbedHorseantipneumoccusserumThemechanismofantibodyformationwasspeculated(1940)PaulingL.(1940)Atheoryofthestructureandprocessofformationofantibodies,J.Am.Chem.Soc.62:2643-2657.Heterogeneityofsera;Complementariness(specificity);Weakbonding(H-bonds);Bivalentnatureofantibodies(precipitinreaction;ruleofparsimony)Onesinglechain(ofdifferentconfiguration);MWofAb:160kD;Antigen:bebigenough.Pauling(1901-1994)B:stableA&C:foldintomanyConfigurationsandPro-richTheclonalselectiontheoryFrankM.Burnet(NobelPrize,1960)

AntigenstimulatestheamplificationofthetypeofBcellthatproducesonespecificantibody.(Polyclonalantibodies)(monoclonalantibodies)BurnetFM(1957)AmodificationofJern’stheoryofantibodyproductionusingtheconceptofclonalselection,Austr.J.sci.,20:67-69Papaincleavesg-globulinintofragmentsPorterRR.(1958)Separationandisolationoffractionsofrabbitg-globulincontainingtheantibodyandantigeniccombiningsites.Nature.182:670-1.

Porter,R.R.,(1959)Thehydrolysisofrabbitg-globulinandantibodieswithcrystallinepapain,Biochem.J.,78:119-127.AntigenBinding(Fab)Crystallizes(Fc)50kD53kD80kDFractionsIandII,nolongerformprecipitatewithantigen,butinhibitstheantibody-antigenprecipitateformationRodneyPorter(FredSanger’sFirstPh.D.student)“Rabbity-globulinisformedoftwopieceswithverysimilarstructurejoinedtoathirdpieceofquitedifferentcharacter.”g-globulinwasfoundtobemadeofheavy(H)andlight(L)chainslinkedbydisulfidebonds(1959)Edelman,G.M.,(1959)Dissociationofg-globulin,J.Am.Chem.Soc.,,81:3155.Edelman,GM,Poulik,MD.(1961)Studiesonstructuralunitsoftheg-globulins.JExpMed.113:861-84.

Treatmentwithreducingagentmercaptoethanolanddenaturanturea.ReducedNon-reducedBenceJonesproteinfromurineLightchainfromserumGeraldM.EdelmanFromthesamepatientAfour-chainstructureproposed

(Porter,1962)PorterRR.(1973)Structuralstudiesofimmunoglobulins.Science.180:713-6.(review)Reductionwithoutdenaturant.TheLchaindoesnotreactwithantiserumtoFc.

TheNobelPrizeinPhysiologyorMedicine1972"fortheirdiscoveriesconcerningthechemicalstructureofantibodies"GeraldM.Edelman

RockefellerUniversity

USAb.1929(studiedconsciousnessInhislaterlife)RodneyR.PorterUniversityofOxfordUnitedKingdomB.1917-1985

Y-shapedIgGobserved(1965)Feinstein,A.andRowe,A.J.(1965)Molecularmechanismofformationofanantigen-antibodycomplex.Nature.205:147-9.A“click-open”theoryproposed:Acompactantibodyopensuponbindingtotwoantigens.AminoacidsequencesoftheBenceJonesproteinsrevealedtheuniquesequencefeaturesofIgG(byEdelman)(HchainofIgG)N-term.Hypervariable(HV)regionsontheHchainCDR1CDR2CDR3ElvinKabat1970sComparisonoftheaminoacidsequencesoftheVHandVLregionsComparisonoftheaminoacidsequencesofCL,CH1,CH2andCH3regions:homologoustoeachother

ThechemicalstructureofIgGasrevealedbyPorterandEdelman(1950Sand1960S).2The3-Dstructureofantibodiesdetermined(1990s)Huberetal.(1976)CrystallographicstructurestudiesofanIgGmoleculeandanFcfragment.Nature.264:415-20.

Harrisetal.(1992)Thethree-dimensionalstructureofanintactmonoclonalantibodyforcaninelymphoma.Nature.360:369-72.Onlymonoclonalantibodiesandmonospecificantibodiesisolatedfrompatientswithmultiplemyelomacouldbeusedforcrystallization.TheIgGmolecule(H2L2)containssixdomains,eachhascharacteristicimmunoglobulinfolds.Eachhypervariableregion(i.e.,theCDRs)oftheHandLchainsexistasaloop,withthesixloopsclosetoeachotherinspace.StructureofanintactIgG(ofb-sheets!)VLVHCH1CLCH2CH3CH2CH3VLCLVHCH1HarrisLJ.,1992,Nature,360:369-72.The“hinge”region(extendedandflexible)Anasymmetricconformation!ThethreehypervariableRegionsexistasthreeloopsthatareclosetoeachotherinspace.Manyproteinspossesstheimmunoglobulindomain(fold)Formingtheimmunoglobulinsuperfamilyofhundredsofmembers,containingdomainsofasandwichoftwoantiparallelb-sheetsstabilizedbyadisulfidebond.Include:cellsurfaceantigenreceptors,co-receptorsandco-stimulatorymoleculesoftheimmunesystem,moleculesinvolvedinantigenpresentationtolymphocytes,celladhesionmolecules,certaincytokinereceptors,intracellularmuscleproteins(titin),etc.Thedeterminedstructuresofantibody-antigencomplexesrevealthebindingnatureTheantigenbindingsitesareindeedmadeofthehypervariableregions(CDRs)ofboththeHandLchains(aswashypothesized).Conformationalchangesintheantibodyand/ortheantigenoccur,allowingacomplementarytightbindingbetweenaspecificantigenanditsspecificantibody.Noncovalentinteractionsareusedfortheantibody-antigeninteraction.TheKdforantibody-antigeninteractioncanbeaslowas10-10M.

Inthisview,theHVregionsoftheFabhavebeendeleted:thereisnocontactbetweenantibodyandantigen.Thisribbonstructureshowstheantibody'sHV(purple)regionoftheFab,andtheirinteractionwithanepitopeoftheantigen.

FabAntigenHVAntigenAninducedfitconformationalchangeoccursinIgGuponbindingtoitsantigenTheantibody-antigeninteractionisthebasisforavarietyofimportantanalyticaltechniquesAffinitychromatography:one-stepproteinpurificationusingaspecificantibodythatiscovalentlyattachedtoaresin.Immunoblottingassay:

includingWesternblottingandEnzyme-linkedimmunosorbentassay(ELISA);qualitativeandquantitativedetectionofaspecificantigenpresentinamixture,whichcouldbeasolution,agel,oralivingcell,usingitsspecifica