CH2 domain of mouse IgG3 governs antibody oligomerization, increases functional affinity to multivalent antigens and enhances hemagglutination

FrontiersinImmunology|www.frontiersin.org 1 May2018|Volume9|Article1096 Originalresearch published:23May2018do i:10.3389/fimmu.2018.01096

ch2D o m a i n o f M o u s e i g g 3 gover nsantibodyOligomerization,increa sesFunctionalaffinity

toMultivalentantigens

andenhanceshemagglutination

TomaszKlaus^1,2and

JoannaBereta²*

1Laborato

ryofMonoclonalAntibodies,MałopolskaCentreofBiotechnology,JagiellonianUniversity,Kraków,Pol and,²Department

ofCellBiochemistry,FacultyofBiochemistry,BiophysicsandBiotechnology,JagiellonianUniver sity,Kraków,P o l an d

Editedb y : HarryW.Schroeder,Unive rsityofAlabamaatBirmingha m,Unit edS ta t es Reviewedb y : NeilS.Greenspan,Case WesternReserveUnivers ity,UnitedStates MasakiHikida, AkitaUniverity,Japan

*Correspondence:

JoannaBeretajoanna.b ereta@uj.edu.pl

Specialtysection:

Thisarticlewassubmittedto BCellBiology, asectionofthejournalFr ontiersinImmunology Received:21February2018 Accepted:02May2018 Published:23May2018 Citation:

KlausTandBeretaJ(2018)CH2Dom ainofMouseIgG3GovernsAntibody Oligomerization,IncreasesFuncti onalAffinitytoMultivalentAntigensan dEnhancesHemagglutination.

Front.Immunol.9:1096.doi:

10.3389/fimmu.2018.01096

MouseI g G 3 i s h i g h l y p rotectivea g a i n s t s e v e r a l l i f e -

t h reateningb a c t e r i a . T h i s i s o t y p e ist h e o n l y o n e a mo n g m o u s e I g G s t h a t f o r m s n o n -

c o v a l e n t o li g o m e rs , h a s in c reasedfunctionala ffinityt o poly valen t an t ig en s , ande fficientlyag glutinate s er y th rocytes.Ig G3 alsot r i g g e r s t h e c o m p l e m e n t c a s c a d e . T h e h i g h e fficacyo f p rotectiona f t e r p a s s i v e immunizationwithIgG3iscorrel atedwiththeuniquepropertiesofthisisotype.AlthoughthefeaturesofIgG3arewelldocume nted,theirmolecularbasisremainselusive.BasedonfunctionalanalysesofIgG1/IgG3 hybridmoleculeswithswappedconstantdomains, wei d e n t i f i e d I g G 3 -

d e r i v e d C H 2 d o m a i n a s a m a j o r d e t e r m i n a n t o f a n t i b o d y o l i g o m e r-

izationandinc reasedfunctionalaffinitytoamultivalent antigen. TheCH2domain w as alsoc ruc ialfor e fficienthe ma g g lu ti na ti on trigge redby Ig G 3 an dwas i nd is pe n s ab le for complementc a s c a d e a c t i v a t i o n . T h i s d o m a i n i s g l y c o s y l a t e d a n d a t y p i c a l l y c h a r g e d . Am u t a t i o n a l a n a l y s i s b a s e d o n m o l e c u l a r m o d e l s o f C H 2 d o m a i n c h a r g e d i s t r i b u t i o n indicatedt h a t t h e f u n c t i o n a l a ffinityw a s i n f l u e n c e d b y t h e s p e c i f i c c h a r g e l o c a t i o n . N-

glycansw e ree s s e n t i a l f o r C H 2 -

d e p e n d e n t e n h a n c e m e n t o f h e m a g g l u t i n a t i o n a n d complementactivat ion.OligomerizationwasindependentofCH2chargeandglycosyla-

tion.Wealsoverified thatknown C1q-

bindingmotifs a refunc tion alin mouseIg G3 butnotinIgG1framework.Wegenerat edforthefirsttimeagain-of-

functionantibodywithpropertiestransferredfromIgG3intoIgG1byreplacingtheCH2domai n.FindingthattheCH2domainofIgG3governsuniquepropertiesofthisisotypeislikelytoopenana venuetowardthegenerationofIgG3-

inspiredantibodiesthatwillbeprotectiveagainstexistingoremerginglethalpathogens.

Keywords:igg3,oligomerization,multivalentantigen,polyvalentantigen,hemagglutination,igconstantregion

inTrODUcTiOn

TherearefoursubclassesofmouseIgGs:IgG1,IgG2a,IgG2b,andIgG3.Althoughstructurallyverysimila r,theysig nificantlydifferint heirfunctions(1).MouseIgG3sareparticularlyinteresting,becausethey

FrontiersinImmunology|www.frontiersin.org 2 May2018|Volume9|Article1096

areabletoformoligomers,w hichstronglyinfluencesthei rbiologicalactivities(2).

MouseIgG3wasdescrib edforthefirsttimealmost5 0yearsago(3)anddifferent aspectsofitsbiologyhavebe eninvestigatedbyseveralgr oups.ThepropensityofIgG 3oligomerizationwasnotic edalreadybyitsdiscoverers(

3).Then,otherresearchersre portedcooperativebinding ofIgG3toa multivalentanti gen(4,5).A lthought hei n i tialreportonIgG3oligomeri zationc oncernedmolecule sinsolution(3),laterstudiesr evealedthatbindingtomulti valentantigenspromotedIg G3

KlausandBereta CH2GovernsMouseIgG3Properties

intermolecularinteractions,whichinturnresultedinitsincreasedfun ctionalaffinitytotheantigen(4).ThisphenomenondependedonFc,b ecauseIgG3F(ab’)2fragmentsdidnotbindtotheantigencooperativel y(4).However,theexactmolecularmechanismofIgG3oligomeriz ationremainsunknown.

IgG3isamajorcomponentofcryoglobulinsinmice(2).Cryo- globulinsareplasmaproteinst hatreversiblyprecipitateatl owtempe raturesorathighconcentrations(6).CryogenicactivityofIgG3wass howntocorrelatewithitsabilitytooligomerize,withthepresenceofc hargedresiduesinthevariableregionandthelevelofsialylation(7).

Also,IgG3wasreportedasexceptionallyeffectiveinpreventingo rfightingseverallife-

threateningmicrobialinfections,e.g.,withNeisseriameningitidis(8 )orBacillusanthracis(9).AcomparisonbetweenthefourmouseIgG subclasseswiththesamevariableregionspecifictoB.anthraciscapsu leprovedthatonlyIgG3isprotectiveagainstpulmonaryanthraxinam ousemodel(9).Importantly,mouse–

humanchimericantibodiescontainingacon-

stantregionofanyhumanIgGsubclasswerenoteffective,althoughth eyhadthesamevariableregionastheprotectivemouseIgG3(10).The sereportsindicatedthatmouseIgG3constantregionhasuniqueprope rties,buttheauthorsonlyspeculatedaboutpossiblemolecularmecha nismsbehindtheobservedphenomenon.

Thee xceptionalcharacteristicofIgG3w asa lsoc onfirmedbyou rpreviousreportt hatamongmouseIgGsrecognizinga surfaceantig enoferythrocytesonlyIgG3sareabletoagglutinatethec ells(11).W erejectedt hehypothesist hatIgG3-

mediatedhemagglutinationresultsfromoligomerizationoftheantib odies,becauseIgG3F(ab’)2wassufficienttotriggerhemagglutinatio n.MolecularmodelingindicatedthatIgG3hasalargerspanofFabarm sthanotherIgGsubclasses.IgG3hasalong-

upperhingethatmayextendtheFabrange,butwhetherthismayaccou ntfortheabilitytohemagglutinatewasnotverifiedexperimentally.

Here,wepresenttheresultsofourattemptstofindmoleculardeter minantsofthepropertiesofmouseIgG3.Ourexperimentalmodelco mprisedtwoantibodies(M18andO10)specifictoantigenBoftheAB Obloodgroupsystem(11).AntigenBisapentasaccharideO- glycanattachedtonumerousproteinsandlipidsontheerythrocytesur face.Thelargequantityandhighdensityoftheantigen,aswellasastro ngnegativechargeoftheerythrocytesurface,couldbeconsideredasa safeandeasyto-

handlemodelofapathogensurface.WegeneratedmanymuteinsofIg G3moleculesandanalyzedtheirfunctionalaffinitytotheantigenasw ellastheirabilitytohemagglutinateandoligomerize.T heresultssho wedthatIgG3derivedCH2domaindeterminesantibodyoligomeri- zationandincreasesitsaffinitytotheantigen.Thisdomainalsostrong lyenhancesagglutinationoferythrocytesbearingBantigen.Moreov er,weinvestigatedcomplementactivationbythemuteins,andwecon firmedthatknownC1q-

bindingmotifsarefunctionalinmouseIgG3butnotinthemouseIgG1 framework.

MaTerialsanDMeThODsgener ationofVectorscodingfor

antibodyMuteins

Expressionvectorscodingforheavychainmuteinswithswappeddo mainsormutatedCH2weregeneratedusingsyntheticnucleic

acids(GeneArt,Germany)clonedi ntopFU SEssCHIg- mG1_M18(Addgene#82357)orpFUSEssCHIg- mG3_M18(Addgene

#82356)plasmids(11).Onlyendogenousrestrictionsitespresentint heORFswereusedforcloning.Vectorsc odingforothermuteinswer epreparedwithQ5-

basedsitedirectedmutagenesiskit(NEB).Allplasmidscodingforhe avychainvariantsofM18antibodyareavailableviaAddgenereposit oryalongwiththeirfullsequencesandmaps,accessionnumbers:1 05849–

105863. PlasmidsencodingO10antibodyvariantswereobtainedb yreplacingthesequencecodingforM18variablefragmentwithacorr espondingO10derivedcDNAusingEcoRIandAfeIrestric-

tionsitesinthevectorscodingforM18heavychainvariants.Theseque nceofO10antibodyisproprietaryandcannotbedisclosed.Allplasmi dswereverifiedusingSangersequencing(Genomed,Poland)orNG S(Addgene).

Productionofantibodies

TherecombinantantibodiesweretransientlyexpressedinHEK293 Tcellsculturedi nDMEMwith4.5g/lglucose(Lonza)supplemente dwith10%FBS(Biowest).Thecellswereco-

transfectedwithp lasmidscodingfora h eavyc hainanda cognatelig htchainusingLipofectamine2000(Thermo)orPEIMAX(Polyscie nces,MW40,000).InthecaseofM18variants,theplasmidpFUSE2s sCLIgmK_M18(Addgene,#82358)

(11)codingforM18lightchainwasused.Asimilarplasmidcodingfo rO10lightc hainwasu sedforexpressionofO10variants.Hybridom aderivedM18IgG3wasproducedasdescribedpre-

viously(11).TheantibodieswerepurifiedusingCaptureSelectLC- kappa(mur)affinitymatrix(Thermo)accordingtoinstruc- tionsofthesupplier.Glycine–

HCl(100mM,pH2.0)wasusedforelution.

Productionofigg3F(ab’)

Asequencee n codingIgG2bc oreh i nge,HAt aganda stopcodonw asclonedintopFUSEssCHIg-

mG3_M18downstreamofthesequencecodingfortheupperhingeo fM18.Sequencesoftheupperandcorehingesoftheantibodiesarede scribedbyDangletal(12).MouseIgG2bcorehingecontainsfourcyst eineresiduesthatallowanefficientassociationofFab’fragmentsinto F(ab’)2(13).RecombinantF(ab’)2wasexpressedasdescribedforother antibodies.Alternatively,F(ab’)2wasproducedbyenzy -

maticdigestionwithIdeZ(NEB)accordingtothemanufacturer’sprot ocol.

MeasurementsofantibodyconcentrationA

mouseFabp olyclonalantibody(JacksonLaboratory,c at.

#515005072,lot#105461).Goatanti-

mousekappapolyclonalantibody(1:3,000,BioRad,cat.#105008,ba tch#160617),HRP-

labeledstreptavidin(1:40,000, Sigma),andtheTMBsubstratefor H R P ( BDBioscience)wereusedford e tection.T heH R P- dependentc olorogenicreactionw asstoppedw ith1 M HCl,andt h eabsorbanceat4 5 0 n m w asreadusingt hem i c roplatespectrophot ometerSynergyH1operatedbyGen52.00Software(BioTek).Purifi

KlausandBereta CH2GovernsMouseIgG3Properties

edM18(IgG3)andMCP21(IgG1,Sigma)wereusedasstandards.C oncentrationsofmuteinswerec alculated

basedontheirFabtype(IgG1orIgG3-

typeFab).BCAassay(Sigma)wasusedformeasurementsofpurified antibodyconcentrationswithbovineγ-

globulin(Thermo)asareference.

antibodyBindingtoimmobilizede rythrocytes

Polystyreneplateswerecoatedwith50µg/mlpolylLys(Sigma) for1hatroomtemperature(RT).Then,100µlof0.1%

(hematocrit)suspensionofredbloodcellsinPBSwereaddedtothewe llsandthecellswereallowedtosettlefor1hatRT.Aftergentleaspirati onofthesolution,thecellswerefixedusing0.025%glutaraldehydefo r40min.Endogenousperoxidaseactivitywasblockedwith3%H2O2fo r1h.Theplateswereblockedovernightwith0.2%gelatininPBScontai ning0.05%Tween-

20at4°C.Then,thecellswereincubatedwithserialdilutionsofcellc ulturemediacontaininganalyzedantibodiesfollowedbydetectionw ithanti-

mousekappapolyclonalantibodyc onjugatedw ithbiotin( 1 : 3 , 0 0 0 , BioRad)andH R P-

labeledstreptavidin( 1 : 4 0 , 0 0 0 , Sigma).A llreagentswered i lut edi n t heblockingbuffer.T hec olorogenicreactionwasperformed, andtheabsorbanceat450nmwasmeasuredasdescribedabove.

c1qBindin g

Duplicatesofpolystyreneplateswerecoatedwith6µg/mlofBSAco njugatedwiththediscriminatingtrisaccharideoftheBantigen(Dext raLaboratories,cat#NGP6323,batch

#ATDX232-

039)overnightat4°C,blockedwith1%BSAinPBS(1h)andincubate dwithserialdilutionsofcellculturemediacontainingO10muteins(

2h).Then,onesetofplateswasusedforevaluationofC1qbindingby analyzedantibodies;andtheplateswereincubatedwith2µg/mlofC1 qpurifiedfromhumanserum(Biorad,cat.#22215504,batch#13 0815;2h)andnextwithHRPlabeledanti-

humanC1qpolyclonalantibody(1:400,Abcam,cat.#ab46191,lot

#GR205436-

5;1h).Thesecondsetofplateswasusedtoanalyzequantitiesofthem uteinsboundtotheimmobilizedantigen;andtheplateswereincubat edwithanti-

mousek appapolyclonalantibodyc onjugatedwithbiotin(1:3,000, BioRad,2h)andHRPlabeledstreptavidin(1:40,000,

Sigma;1h).

AbsorbanceofHRPproductwasmeasuredasdescribedabove.E achincubationstepwasprecededbyextensivewashingwith0.05%T ween-

20inPBS.Theantibodies,streptavidin,andC1qweredilutedin0.1

%BSAinPBS.Allincubations,exceptplatecoating,wereatRT.

ThenormalizedC1qbinding(a)wascalculatedbydividingthesi gnalcorrespondingtoC1qbinding(b)bythesignalcor-

respondingtoboundantibody(c).

C1qbindingsignal

(

)

ormalizedC1qbinding

(

)

boundantibodysignal

(

)

Uncertaintyofa ( Δa)w asc alculatedbye x a ctd i f ferential.Unce rtaintiesofbandc(ΔbandΔc)equaledtostandarddevia-

tionsoftheabsorbancemeasurements.

complementcascadeactivatio n

Washedhumanredbloodcellssuspendedtoahematocritof2%were coatedwith3µg/mlor1.5µg/mloftheanalyzedantibodiesfor1.5hat RT,washedtwicewithPBSandresuspendedinPBSwithCa^2+andM g²⁺.Then,thesamevolumeofhumancomple-

mentserum(Sigma,cat.#S1764,lot#SLBS5471V,#SLBQ0752V or#SLBP0461V)dilutedto7CH50U/mlinPBSwithCa²⁺andMg^2+was addedtothecoatedredbloodcells.Thesampleswerecentrifugedaft er2hofincubationat37°C,andtheabsorbanceofreleasedhemoglob inwasmeasuredinthesupernatantsat540nm.

redBloodcellsandagglutination

StandardhumanredbloodcellswerepurchasedfromRegionalCen treofBloodDonationandBloodTreatmenti n Katowice,Poland.A gglutinationtestswerep erformedi n 9 6 -

flatbottomplates.Seriallydilutedsolutionsofanalyzedantibodies (100µl)weregentlym i xedw ith0 . 4 5 %

( hematocrit)suspensionofredbloodcells.Thelevelofagglutinatio nwasanalyzedusingaphase-

contrastmicroscopeafter20minofmoderateshaking.Asix- pointscalewasusedforevaluationofagglutinationintensity:from4 +(completecellaggregation),to3+,2+,1+,

±toanegativescore.Theagglutinationscorereflectsboththesizeof aggregatesandquantityofnonagglutinatedcells.

igg3self-

associationassay

OligomerizationofIgG3wasanalyzedsimilarlytothemethoddes cribedbyAbdelmoulaetal(2).Threedifferentconcentrationsofthe

purifieddomainmuteinswithM18variableregion(150,100,and2 0 µ g / m l)werei ncubatedw ithnonmutated,bioti-

nylatedIgG3M18(100ng/ml)for72–

96hat4°Cinthepresenceof4%BSA(Sigma,cat.#A9576).Then,thean tibodycomplexeswereprecipitatedbyadding50%PEG-

6000(Sigma)tothefinalconcentrationof7.5%.After1hofincubation onice,thesampleswerecentrifuged(30min,3,000×g,4°C).Thesupern atantswerepreservedforfurtheranalysis,andtheprecipitateswerew ashedwithi c e cold7 . 5 % PEG-

6000i n PBSandc e ntrifugedagain(30min,3,000×g,4°C).Then, theprecipitatesweredissolvedinPBSwith0.1%BSAbypipettingat3 7°C.BiotinylatedIgG3intheprecipitatesandsupernatantswasquanti fiedusingELISAonpolystyreneplatescoatedwithstreptavidin(8µg/

ml,Thermo,cat.

#434301,lot#RB233354).BoundbiotinylatedIgG3wasdetectedusin grabbitmonoclonalantibodyM1112(1:1,000,Abcam,cat.

#ab125904,lot#C050311,#GR1570921)andHRPlabeledgoatanti- rabbitpolyclonalantibody(1:3,000,Sigma,cat#A6667).Theabsorbance ofHRPproductwasmeasuredasdescribedabove.

sDs-PageandWesternBlotting

Sampleswereresolvedin8or12%polyacrylamidegelsundernon- reducingorreducingc onditionsa c c ordingtot heprotocolofLaem mli(14).AfterwetelectrotransferontoP V DFmem-

braneandblockingwith4%skimmilkinPBS,thesampleswereprobe dw ithanti-

mousek appap olyclonalantibodyc onjugatedwithbiotin(1:3,000, BioRad)andHRP-

labeledstreptavidin(1:40,000,Sigma).Alternatively,rabbitanti- HAtagpolyclonal

uncertaintyofa

(

)

∂b ∂c c c² antirabbitF ( ab’)2polyclonalantibody( 1 : 1 0 , 0 0 0 , Sigma,c at.

#A6667,lot#SLBG3029)wereused.MouseIgG3heavychainwasd etectedusinggoatantiserumtomouseIgG3(1:500,Sigma,cat.

#ISO2)andrabbitanti-

goatpolyclonalantibody(1:5,000,Sigma,cat.#A4174).Bandswere visualizedusingImmobilonWesternChemiluminescentSubstratef orH R P ( Millipore).T hei mageswerecapturedandanalyzedusing FusionFxapparatuswiththeFusionCaptAdvanceFx5program(Vil bertLourmat,France).

resUlTs

comparisonofhemagglutinationinducedb yigg3andigg3F(ab’)

Inourpreviouswork,wereportedt hatF ( ab’)2obtainedfromIgG3i n ducesagglutinationofe rythrocytesbearinga c o g nate

antigen(11).However,asshownbelow,completeIgG3aggluti- nateserythrocytesmoreefficientlythanitsF(ab’)2,i.e.,amuchhigh erconcentrationofF(ab’)2thanthatoftheintactmoleculeisrequired foragglutination.Wec omparedhemagglutinationtriggeredby:

(i)native,purified,fulllengthIgG3,anditsF(ab’)2

obtainedbyIdeZproteasedigestionand(ii)culturemediaofcellsprod ucingrecombinantIgG3orrecombinantF(ab’)2(Table1).Concentr ationsofIgG3andF(ab’)2i nthemediaweremeasuredusingELISAan dequalizedforthecomparativetests.Weverifiedthequalityofanalyz edproteinsandconfirmedthatrecombinantIdeZprotease,whichcle avesIgG3atas i nglesitei n itsh ingeregion,generateshomogeneou sF(ab’)2(Figures1A,B).Basedonthisanalysis,weestimatedthatIgG 3wasabout32to64-

timesmorepotentthanitsF(ab’)^{2i n}hemagglutination(Table1;Fig ure1 B ).T heresultsi ndicatet hatt heFcofIgG3stronglyenhancesh emagglutinationinducedbythisisotype.

Thech2DomainDerivedFromigg3e nhancedh emagglutinatione fficacyof anantibody

OurpreviousattemptstoexplainthemechanismofIgG3dependent hemagglutinationbroughtustothehypothesisthattheelongatedhing eofIgG3determinesitshemagglutinationability(11).Inlightofthen ewresults,thehypothesisrequiredrevision.Toelucidate

TaBle1|M18full-lengthIgG3 andM18IgG3F (ab’)2inducedhemagglutinationwit hdifferentefficacy.

concentration(nM) scoreofhemagglutination^a concentration(nM) scoreofhemagglutination

nativeigg3 F(ab’)2obtainedusingideZ recombinantigg3 recombinantF ( a b ’

)2

82.5 ++++ + 93 ++++ ±

41.3 ++++ ± 46.5 ++++ –

20.6 ++++ – 23.3 ++++ –

10.3 ++++ – 11.6 +++ –

5.2 +++ – 5.8 +++ –

2.6 +++ – 2.9 + –

1.3 ++ – 1.5 ± –

0.6 ± – 0.7 – –

0.3 – – 0.4 – –

aRepresentativeresultsofthreeindependentexperiments.

FigUre1|HemagglutinationinducedbyM18IgG3anditsF(ab’)2.(a)IntegrityofgeneratedF(ab’)2wasverifiedusingSDS-

PAGE.InthecaseofthepurifiedantibodydigestedwithIdeZ,thegelswerestainedwithCoomassieBrilliantBlue(CBB).RecombinantF(ab’)2wasequippedwithH AtaganditsintegritywasconfirmedusingWesternblottingwithanti-HAtagantibody.Themolecularmassofnon-

reducedF(ab’)2isabout120kDa.HC,heavychain;HC’,heavychainfragmentsgeneratedafterIdeZcleavage;LC,lightchain;

(B)MicroscopicimagesoferythrocytesagglutinatedbyequalmolarconcentrationsofIgG3antibodyanditsF(ab’)2.Theantibodyfragmentwasobtainedfromna tiveIgG3usingIdeZdigestion.

whichdomainsofIgG3arecrucialforitshemagglutinationabil- ity,wegeneratedapanelofdomainmuteinsofagglutinatingIgG3and non-

agglutinatingIgG1isotypes(Figure2A).WegeneratedpairsofIgG1 andIgG3moleculeswiththesamevariableregionsandwithswapped:

(i)hingeregions;(ii)hingeregions+ CH1domains;

(iii)CH2domains,and(iv)CH3domainsandsearchedformuteinsoft wotypes:lossoffunctioninthecaseofIgG3andgainof-

functioninthecaseofIgG1.

PreliminaryexperimentsshowedthathingeswappingbetweenIg G1andIgG3hindersdisulfideb ondsformationb etweenchainsoft h emuteins( F i g u reS 1 i n SupplementaryMaterial).However,theI gG1_h3andIgG3_h-

1variantswerefunctionalandtheiraffinitytotheantigenwassimilart othatoftheparentalmolecules(showninFigure3).Asdemonstrated byDall’Acqua

eta l.,i m munoglobulinsw ithmodifiedh i ngesfrequentlyformfunc tionalheterotetramers( HC)2(LC)2d espitet hel ackofdisulfidebond sbetweenthechains(15).GelfiltrationconfirmedthatIgG1_h- 3andIgG3_h-

1havemolecularmassgreaterthan150kDaandformstable(HC)2(L C)2heterotetramers(datanotshown).Tomakesurethattheresultsofth efollowingexperimentsarenott heconsequenceofincorrectassemb liesoft hehinge-

swappedmuteins,wealsogeneratedmuteinswithswappedfrag- mentscomprisinghingeregionsandCH1domains.Allmuteinswere successfullyexpressedandtheirintegritywasverifiedusingSDS- PAGEandWesternblotting(FigureS1inSupplementaryMaterial).

Then,wecomparedhemagglutinationinducedbythemuteins(Ta bles2and3;Figures2B,C).Theresultsshowedthatneither

FigUre2|Hemagglut inationinducedbyIgG1andIgG3mut eins. (a)Generated domainmuteinsandtheirnom encl atu re.

(B) Microscopicimages ofhemagglutinationinducedbythedomainmuteins.Allantibodieswe reusedataconcentrationof1.5µg/ml,exceptofO10I gG1_CH2-3thatwasusedat3µg/ml.Scalebar—100µm;(c)HemagglutinationinducedbyselectedO10muteinsusedat10µg/ml.

FigUre3|Antigenbindingbythedomainmuteins.M18andO10antibodiesarespecifictoB-antigenpresentonhumanerythrocytes.B- anti genisapentasaccharideO-glycan.O10antibody,butnotM18,bindsterminalfragmentoftheantigen,calledB-trisaccharide.Antigen–

antibodyinteractionwasanalyzedusingELISAonimmobilizederythrocytes.InthecaseofO10,platescoatedwithBSAconjugatedwiththesyntheticB- trisaccharidewerealsoused.Theplotspresentmeanvaluesfromtwoindependentexperimentsperformedinduplicates.ResultsobtainedforIgG1andIgG3,the parentalmolecules,arepresentedoneachplottoallowconvenientcomparisons.

TaBle2 | S c o reso f h e m a g g l ut i n a t i o n i n d u c e d b y M 1 8 v a r i a n t s .

conc.(μg/ml) Parentaliggs swapofhingeregions swapofch1+hingedo mains

swapofch2 domains

swapofch3 domains

igg1 igg3 igg1_ igg3_ igg1_ igg3_ igg1_ igg3_ igg1_ igg3_

h-3 h-1 ch1h-3 ch1h-1 ch2-3 ch2-1 ch3-3 ch3-1

i^a ii i ii i ii i ii i ii i ii i ii i ii i ii i ii

1.500 – – +++ ++ – – +++ +++ – – +++ +++ + + – + – ± ++ +++

0.750 – – +++ +++ – – + +++ – – ++ +++ – ± – – – – + ++

0.375 – – ++ +++ – – ± + – – + ++ – – – – – – ± ++

0.188 – – + ++ – – – – – – ± + – – – – – – – –

0.094 – – ± ± – – – – – – – – – – – – – – – –

0.047 – – – – – – – – – – – – – – – – – – – –

aResultsoftwoindependentexperimentsdesignatedasIandII.

TaBle3| S c o resof h e m a g g l u t i n at i o n i n duc e db y O 10 v ar i a nt s .

conc.(μg/ml) Parentaliggs swapofhingeregions swapofhinge+ch1do mains

swapofch2 domains

swapofch3 domains

igg1 igg3 igg1_h-3 igg3_h-1 igg1_ igg3_ igg1_ igg3_ igg1_ igg3_

ch1h-3 ch1h-1 ch2-3 ch2-1 ch3-3 ch3-1

i^a ii i ii i ii i ii i ii i ii i ii i ii i ii i ii

3.000 – – ++ ++ – – +++ ++ – – +++ ++ + + – ± – + +++ +++

1.500 – – +++ +++ – – +++ ++ – – +++ ++ ± – – – – – + ++

0.750 – – ++ ++ – – ++ ++ – – + ++ – – – – – – ± ±

0.375 – – + + – – + + – – ± ± – – – – – – – –

0.188 – – ± ± – – ± ± – – – + – – – – – – – –

0.094 – – – – – – – – – – – – – – – – – – – –

aResultsoftwoindependentexperimentsdesignatedasIandII.

theC H 1 d omainnort heh i nged e terminedt heagglutinationabil ityofIgG3.TheintroductionoftheCH3domainfromIgG1intoIgG3r esultedinamoleculewithslightlyreducedhemag-

glutinationscorebutCH3fromIgG3didnottranslateintoIgG1abilit yofhemagglutination.Incontrast,CH2swappingledtothegeneratio nofIgG1mutein(IgG1_CH2-

3)thatgainedtheabilityofhemagglutination(Tables2and3).Moreo ver,thepairedIgG3mutein(IgG3_CH21)hadabout16-

timesreducedhemaggluti-

nationscoreincomparisontotheparentalIgG3.

IgG1_CH23asa gainof-

functionmuteinwasparticularlyinteresting,becauseitindicatedthat theCH2domainofIgG3istheonecriticalforhemagglutination.How ever,IgG1_CH23aggluti-

natederythrocyteswithconsiderablylowerscorethannativeIgG3.

WealsocomparedhemagglutinationefficacyofnativeM18Ig G3anditsdeglycosylatedform.DeglycosylatedIgG3agglu- tinatederythrocytesabout16-

timesweakerthanthenativemolecule(TableS1inSupplementaryM aterial).

Tosumup,theabilityofIgG3toagglutinateerythrocytesresultsfro mitsuniquestructure,inwhichtheCH2domainisespeciallyimporta ntandstronglye n hancest hee f f i c acyoft heprocess.Althoughth eIgG3F(ab’)2issufficienttotriggerhemagglutina-

tion,itsefficacyismuchlowerincomparisontofull-

lengthIgG3,probablyjustduetothelackoftheCH2domain.Thehinge regionseemstohavel ittlei n f luenceonagglutinationability,becaus eIgG3withtheIgG1-

derivedhingeagglutinatederythrocytesonlyslightlylesseffectively thantheparentalmolecule.

igg3constantDomainsModifyFunctional affinitytoanantigen

Thereisageneralagreementthattheincreasedfunctionalaffinity ofIgG3resultsfromanavidityeffectcausedbytheinteractionsbetwe entheFcfragmentsofthemolecules(16).Inlinewiththat,weobserve dt hatIgG3bindstoe rythrocytesmuchmoree f f i -

cientlythanIgG1withthesamevariableregionandmuchmoreefficie ntlythanIgG3-

derivedF(ab’)2(FigureS2inSupplementaryMaterial).Someauthorsd i scusseda lsot hep otentialroleofN-

glycansi n IgG3u n i queproperties(17,1 8),butwed i d notobserve

anydifferencesinantigenbindingbetweencontrolanddeglycosylate dantibody(FigureS2inSupplementaryMaterial).

Aimingt ounderstandwhyIgG3h asincreasedfunctionalaffinity, w ea nalyzeda ntigenb indingbythedomainmuteins

(Figure3).TheresultsshowedthatthehingeregionofIgG3doesn otinfluencethefunctionalaffinityoftheantibody,butmuteinswit htheswappedCH1+ hinge,CH2,o rCH3domainshadchangedfu nctionalaffinity.TheintroductionofIgG3-

derivedCH1+ hingeo rCH2domainintotheIgG1frameworkenh ancedantigenbindingincomparisontotheparentalIgG1.Conver sely,thepairedIgG3muteinswithIgG1-

derivedCH1+hingeorCH2hadreducedfunctionalaffinity.Thes wappingoftheCH3domainsresultedintheIgG3muteinwithdecr easedaffinity,butinthepairedIgG1mutein,theeffectwasnotsubs tantial.ThecalculatedEC50valuesofantigenbind-

ingforIgG3muteinsindicatedthattheCH2domainhadthestrong estinfluenceonIgG3-

antigeninteraction(Table4).CH2swappingresultedinIgG3mut einswith3–12timesdecreasedfunctionalaffinity.

Overall,theresultsindicatethatthehigher(incomparisontoIg G1)functionalaffinityofIgG3toitsantigendoesnotdependona se paratec onstantd omainoft hisisotype,butr atherisanadditiveresu ltofd iscretepropertiesoft hea llt hreec onstantdomainsCH1,C H2,andCH3,butnotthehingeregion.Ofallthec onstantd omain s,C H 2 c ontributest hemosttot heh i ghfunctionalaffinityofIg G3.

Fc-

DependentOligomerizationoftheDo mainMuteins

ThehallmarkofmouseIgG3isitsabilitytooligomerize.The processdependsonFcfragment,butitsexactmolecularmecha- nismisu n known.Weanalyzedwhethert hed omainmuteinsform non-

covalentcomplexesusingpolyethyleneglycol(PEG)precipitatio nwithalabeledIgG3probe(2).Incomparisontotheoriginalmethod ,weusedbiotinylatedIgG3insteadofradiolabeledIgG3.TheIgG3- biotininteractedwitholigomerizingmuteinsandthecomplexesc omprisedt hemuteinandt heprobe.Thecom-

plexeswereprecipitatedusingPEG,andthen,IgG3-

biotinwasquantifiedinprecipitatesandsupernatantsusingELISA .Ahighprecipitate/supernatantr atioofIgG3-

biotinquantitiesi ndicatesthatthemuteinformsoligomers.

Theexperimentshowedthat5outof10analyzedmoleculesfor mPEGprecipitableoligomers—

IgG3(control)andallIgG3muteinsbuttheonecontainingIgG1- derivedCH2domainandnoneofIgG1muteinsbuttheonewithIgG 3derivedCH2domain

TaBle4|EC50ofmuteinbindingtotheantigencalculatedusingdatafromFigure3.

Variableregionofthemuteinandtypeoftheantigen ec50o f muteinbinding(nM)

igg3 igg3_h-1 igg3_ch1h-1 igg3_ch2-1 igg3_ch3-1

M18(erythrocytes) 0.24±0.02 0.38±0.01 0.61±0.03 0.86±0.04 0.87±0.07

O10(erythrocytes) 0.81±0.01 0.88±0.02 2.05±0.06 9.75±2.04 3.60±0.29

O10(B-trisaccharideconjugatedtoBSA) 0.50±0.04 0.38±0.02 1.15±0.06 1.40±0.03 0.79±0.06

FigUre4 |

Oli gomeriz atio nof the dom ai n m ut ei ns . T he ant i b odi es (M 18 variants, 150µg/ml)wereincubatedat4°CandoligomerswereprecipitatedusingPEG.

BiotinylatedIgG3wasusedasaprobethatoligomerizedwiththemuteinsandb ecameapartofthecomplexes.Thechartspresentresultsfromtwoindepende ntexperiments.Theresultsobtainedfor100and20µg/mlofthemuteinsares howninFigureS3inSupplementaryMaterial.ApercentageofthetotalIgG3- biotindetectedintheprecipitatesandthesupernatantsispresentedinFigureS4 inSupplementaryMaterial.

(Figure4).OligomerizationdidnotdependonCH2glycosyla- tion(FigureS5inSupplementaryMaterial).Theresultsindicatethatt heCH2domainiscrucialforoligomerizationofIgG3.

complementactivationbytheig g1andigg3Muteins

Similartohumanantibodies,t herearepronouncedd i f ferences betweenmouseIgGsubclassesintheirabilitytotriggercomple- mentc ascade.MouseIgG3a ctivatesc omplemente f f i c i e ntly, whereasmouseIgG1doesnot.Althoughtherearemanyexcel- lentreportsconcerningcorrelationbetweenhumanantibodystructu reanditsabilityofcomplementactivation,thestructural

determinantsofmouseantibodiesthatallowtotriggerthecas- cadearenotpreciselyknown.

ThebestcharacterizedIgwithrespecttocomplementactiva- tionishumanIgG1,inwhichseveralaminoacidresidueswereidentif iedascrucialfortheinitiationofthecomplementcascade(FigureS6i nSupplementaryMaterial)(19–

21).ThesequencealignmentofhumanIgG1,mouseIgG1,andmous eIgG3indicatedthatthemajorityofhumanIgG1aminoacid residue sinvolvedincomplementactivationareconservedinbothmouseiso types(FigureS6inSupplementaryMaterial).However,itrevealedt wodifferencesbetweenmouseIgG1andIgG3withintheregionscor- respondingtothoseinvolvedinC1qbindingbyhumanIgG1—

intheNterminalfragmentoftheCH2domain(Val231- Ser238inIgG1andIle234-

Pro238inIgG3,EUnumbering(22))andintheresidue322(FigureS6in SupplementaryMaterial).

Toverifyw hethert hesemotifsareinvolvedincomplementactiva tionbymouseIgG3,wegeneratedadditionalmuteinsinwhichweswa ppedthembetweenIgG1andIgG3—

IgG1_ILGGP(Val231IlePro232LeuGlu236GlyVal237GlySer23 8Pro);IgG3_VPEVS(Ile234ValLeu235ProGly236GluGly237Va lPro238Ser);IgG1_Arg322Lys;IgG3_Lys322Arg,andadoublemu teinIgG1_ILGGP_Arg322Lys.TheIgG3heavychaincontainingV PEVSdidnotassociatewithalightchainandwasnotsecreted(Figure 5A).Lys322Argreplacementcompletelyabolishedcomplementact i-

vationbyIgG3indicatingthatLys322iscrucialforthisprocess(Figu res5B,C).ThethreemuteinsofIgG1didnotbindC1qnoractivatedco mplementcascadeindicatingthattheIgG1frameworkpreventsactiv ationofcomplement(Figures5B,C).Theresultsshowedthatthekno wnC1q-

bindingmotifsarefunctionalinthemouseIgG3butnotinthemouseIg G1framework.

Someauthorsobservedacorrelationbetweenhingedepend- entsegmentalflexibilityofanantibodyanditsabilitytoactivatecomp lement(12).Thus,thedifferencesbetweenactivityofmouseIgG1and IgG3arefrequentlyexplainedonthebasisofthelengthoftheirhinges.

Wedecidedtoempiricallyverifythishypothesisusingthedomainm uteins.

First,weanalyzedthebindingofthecomplementcascadeini- tiator(C1q)tothemuteins(Figure6A).Theresultsshowedthatthehi ngemodificationdoesnotaffectC1qbinding.TheswappingoftheCH 2domainfromIgG1intoIgG3abolishedC1qbindingbythelatter.Int erestingly,thepairedmutein(IgG1_CH2-

3)didnotgaintheabilitytostronglyinteractwithC1q;itsbindingof C1qreached~12%ofthatcharacteristicfornativeIgG3.Swappingoft heCH1+hingedomainsortheCH3domainsbetweenIgG1andIgG3 moderatelydiminishedC1qbindingbyIgG3anddidnotincreaseits bindingbyIgG1.

Wealsoanalyzedcomplementactivationinserumtriggeredby e rythrocytesc o atedw itht hed omainmuteins( Figure6 B ).

FigUre5|FunctionalityofknownC1q-biningmotifsinmouseIgG1andmouseIgG3frameworks.

(a)IgG3_VPEVSdidnotassociatewithalightchainandwasnotsecretedbytheproducingcells.

(B)C1qbindingbythemuteins.PlatescoatedwithBSAconjugatedwiththeantigen,B-

trisaccharide,wereincubatedwiththemuteinsat3µg/ml(O10variants).ThenpurifiedC1qwasadded.Themuteinsbindtheantigenwithdifferentfunctionalaffinity.Th us,theC1qsignalwasnormalizedtothequantityoftheboundantibody.DatausedforcalculationofthenormalizedbindingareshowninFigureS7inSupplementaryMa terial.ErrorbarscorrespondtouncertaintycalculatedaspresentedinSection“MaterialsandMethods.”(c)Complementcascadeactivationbythemuteins(3µ g/ml).Erythrocytescoatedwiththeantibodies wereincubatedwithcomplementserum.Completelysis(100%)correspondstowater-inducedlysis.In(a–

c)representativeresultsoftwoindependentexperimentsareshown.

Thelevelsoferythrocytelysisindicatedthatallmuteinscontain- ingt heC H 2 d omaind e rivedfromIgG3a ctivatec omplementcasc ade.T heobservedd i f ferencesi n C 1 q bindingwerenotreflected bythedifferentefficacyofthecascadetriggering.Themuteinsw ithl ow( IgG1_CH23)ormoderate( IgG3_CH1h1,IgG3_CH3- 1)abilityofC 1 q -

bindinga ctivatedc omplementcascadew ithe f f i c acys i m i lartot hatoft hep arentalIgG3.Itseemsthatinthecaseoftheantibodiescom prisingIgG3-

derivedCH2domain,evenweakinteractionwithC1qwassufficient toeffectivelyactivatethewholecomplementcascade.

TheresultsshowedthatbothIgG1andIgG3-

derivedCH1,hinge,andC H 3 d omainsarep ermissiveforC 1 q bin dingandcomplementa ctivation.T heC H 2 d omainofIgG1isa no npermissiveframeworkfortheknownC1qbindingmotifs.

Overall,theresultspointedtotheCH2domainasthemajordeter minantofmouseIgG3functionsanduniquepropertiesofthisisotype .Inthelastpartofourwork,wesoughtforfeaturesoft heIgG3- derivedC H 2 d omaint hatmaya c c ountforIgG3distinctivecharac teristic.

PropertiesofMuteinsWithreversed chargeof t h e c h2D o m a i n s

ThemoststrikingdifferencebetweenmouseIgG3derivedCH2 andCH2domainsofotherIgGsubclassesistheircharges;onlythefor merhasastrongpositivecharge.Forexample,atpH7.0,thenetcharge oftheCH2domainofIgG1is−2.6andofIgG3is+2.6(cal-

culatedusinghttp://protcalc.sourceforge.net/).Hovendenetal.

(9)foundacorrelationbetweenthechargeofCH2domainsofmouseI gGsubclassesandtheiraffinitytoanegativelychargedpolyvalentant igen(polyglutamicacid,poly-

GA);andthehighaffinityofIgG3topoly- GAwasattributedtothechargeofitsCH2domain.

WeanalyzedspatialdistributionofchargedresiduesontheCH2sur faceofIgG1andIgG3usingpreviouslyobtainedmolecularmodels(1 1)anddatadepositedinPDBrecord1IGY(Figure7A).Weidentified 29residuesthatdifferb etweenCH2domainsofmouseIgG1andIgG3 ,9ofwhichhavedifferentcharge(FigureS9inSupplementaryMateri al).Basedonthemodels,weselectedfourbasicresidues(His274,Lys 282,Arg315,andLys326)thatareregularlyspacedontheoutersurfac eoftheCH2domainofIgG3

FigUre6|Complement activationinducedbythedomainmuteins.

(a)C1qbindingtothedomainmuteins(O10variants).Thedataused forcalculationsarepresentedinFigureS7inSupplementaryMaterial.Errorbarsc orrespondtouncertaintycalculatedasdescribedinSection“MaterialsandMethods.”

(B)Complementcascadeactivationbythedomainmuteins.Erythrocytescoatedwith3µg/mlofthemuteinswereincubatedwithcomplementseru m.100%lysiscorrespondstowater-

inducedlysis.Thebarspresentmeanvaluesandstandarddeviationofduplicatesfromoneexperiment.Resultsobtainedwith1.5µg/mlofthemuteinsarepr esentedinFigureS8inSupplementaryMaterial.(a,B)Representativeresultsoftwoindependentexperiments.

(Figure7A;FigureS9inSupplementaryMaterial).Thesameresi- duesinIgG1arenotcharged.ToverifywhetherCH2chargeinflu- encesIgG3properties,wegeneratedtwomuteinsinwhichthefourresi dueswereswapped—

IgG3_CH2charge(His274GlnLys282ValArg315AsnLys326Ala) andIgG1_CH2charge(Gln274HisVal282LysAsn315ArgAla326 Lys).Thesemuteinswereexpressed,correctlyassembled,ands olub le(FigureS1inSupplementaryMaterial).T heintroducedmutationsr eversedthechargeoftheCH2domains.Itwas0.6and−0.7atpH7.0for theCH2domainofIgG1_CH2chargeandIgG3_CH3charge,respect ively.

Wec omparedpropertiesoft hep arentalmoleculesandt hemutei nswithmodifiedCH2charge.Weobservedthatthechargeinfluenced bindingtoerythrocytes(Figure7B).However,hemag-

glutination,oligomerization,C 1 q binding,andc omplementactiva tionwerenotaffectedbythischargemodification(Table5;Figures7 C–E).Theresultsindicatethatthefouranalyzedresi-

dueshaveonlylimitedimpactontheIgG3properties.Wecannotexcl udethatotherchargedresidueswithintheCH2domainofIgG3mayi nfluenceordeterminepropertiesofthisisotype.

DiscUssiOn

Wesummarizedtheresultsoftheex perimentsinTable6.Weobser vedthatmoleculardeterminantsoftheuniquefeaturesofIgG3arepr esentintheCH2domain.However,themodificationsofC H 2 d i f fe

rentlyaffectedt hefeaturessuggestingt hatt heirmolecularbasesaredi fferent.

TheprominentroleoftheCH2domaininIgG3biologywasorigi nallyreportedbyHovendene t a l.(9).T heauthorsi nves-

tigatedh i ghlyprotectiveIgG3antibodiesagainstt hec apsularanti genofB.anthracis.T heygeneratedanIgG3muteinw ithCH2swap pedfromnonprotectiveIgG2b.Themuteinlostpro-

tectiveactivityoftheparentalmoleculeandhadreducedaffinitytot heantigen.Inc ontrasttot heworkofHovendene t a l.,wegenerated, forthefirsttime,anantibodymuteinthatgainedtheuniqueproperti esofIgG3.WeswappedIgG3-

derivedCH2intoIgG1,andtheobtainedmolecule(IgG1_CH2- 3)hadpropertiestypicalforIgG3—

itagglutinatederythrocytes,oligomerized,hadi ncreasedfunction alaffinitytoa p olyvalentantigen,andactivatedthecomplementca scade.Thus,weprovedthatt heseuniquefeaturesofmouseIgG3co uldbetransferredintoanewantibodyframework.

ThemechanismofIgG3-

dependenthemagglutinationisstillnotcompletelyunderstood.We previouslyreportedthatF(ab’)2ofIgG3issufficienttoagglutinateer ythrocytes(11).Here,weshowthatthepresenceoftheCH2domain intheIgG3moleculepro-

foundlydiminishestheantibodyconcentrationrequiredfortheF(

ab’)2mediatedprocess.Moreover,t hei ntroductionofIgG3- derivedCH2intoIgG1frameworkresultedintheIgG1_CH2- 3muteint hatagglutinatese rythrocytes.T heresultsi ndicatet hatef ficienthemagglutinationist riggeredonlybyt heantibodiesequipp edwiththeIgG3derivedCH2domain.

TheCH2domainofIgG3ispositivelychargedatneutralpH.Inco ntrast,theCH2domainsofotherIgGsubclassesarenegatively

FigUre7|PropertiesofthemuteinswithmodifiedchargeoftheCH2domain.

(a)ChargelocationontheCH2domainofIgG1andIgG3.Basicresidues(Arg,His,andLys)arefaintred,acidicresidues(Asp,Glu)areblue,andasiteofCH2N- glycosylation(Asn297)isgreen.His274,Lys282,Arg315,andLys326ofIgG3CH2aredarkred.ThesefourresidueswereswappedbetweenIgG1andIgG3togenerateIgG 1_CH2chargeandIgG3_CH2chargemuteins.Theimagespresentviewsobtainedby90°rotationofthedomainmodels.

(B)Antigenbindingbythemuteins.Thechartspresentrepresentativeresultsoftwoindependentexperimentsperformedinduplicatesortriplicates.Errorbarsequal toSD.

(c)Oligomerizationofthemuteins.Resultsfromtwoindependentexperimentswith100µg/mloftheantibodies(M18variants)areshown.ApercentageofthetotalIg G3-

biotindetectedinprecipitatesandsupernatantsarepresentedinFigureS4inSupplementaryMaterial.ResultsforIgG1andIgG3arethesameasinFigure4becausethed atawerecollectedinthesameexperiments.

(D)C1qbindingbythemuteins(O10variants,3µg/ml).DatausedforcalculationofthenormalizedbindingareshowninFigureS7inSupplementaryMaterial.Thechartpres entsrepresentativeresultsoftwoindependentexperiments.Er rorbarscorrespondtouncertaintycalculatedasp resentedi nSection“MaterialsandMethods.” (e

)Complemen tcascadeactivationbythemuteins(3µg/ml).Erythrocytescoatedwiththeantibodieswereincubatedwithcomplementserum.Completelysis(10 0%)correspondsto

water-inducedlysis.Representativeresultsoftwoindependentexperimentsareshown.

TaBle5|Hemagglut inationi nduc ed byth emut eins w ith modifi edcharge of t heCH2domai n.

M18variants O10variants

conc.(μg/ml) igg1 igg3 igg1_ch2charge igg3_ch2charge conc.(μg/ml) igg1 igg3 igg1_ch2charge igg3_ch2charge

5.00 ±^a ++++ – +++ 2.00 – ++++ ± ++++

2.50 – ++++ – +++ 1.00 – +++ – +++

1.25 – +++ – ++ 0.50 – ++ – ++

0.63 – ++ – ++ 0.25 – ± – ±

0.31 – + – ± 0.13 – – – –

0.16 – ± – – 0.06 – – – –

0.00 – – – – 0.00 – – – –

aRepresentativeresultsoftwoindependentexperiments.

TaBle6 |

S u m m a r y o f e x p e r i m e n t a l results.

igg3feature/functioni nfluencebythech2domain

Presence netcharge^a glycosylation Hemagglutination

Functionalaffinitytop olyvalentantigens Oligomerizationinsolution Activationo f c om p l e m e n t cascade

Strongenhancem ent

Strongenhancem ent

Dependence Dependence

Noeffect Enhancement

Weaktomodera tee ffect

Noeffect

Noeffect Noeffect

Noeffect Dependence^b

aAssociatedwiththepresenceofHis274, Lys282,Arg315, Lys326.

bDatanotshown.

chargedunderthesamecondition.Consideringthaterythrocytesurf acehasa strongnegativechargeandhighzetap otential,itwaslikelyth atapositivechargeoftheIgG3-

derivedCH2domainreducesthezetapotentialandasaconsequencee nhanceshema-

gglutination.Unexpectedly,netchargemodificationoftheCH2dom ainsinIgG1andIgG3didnotchangehemagglutinationpotentialofth eseisotypes,andwehadtorejectthehypothesislinkingtheCH2netch argewiththeefficiencyofhemagglutination.

Alternatively,antibodyoligomerizationmayexplainhemag- glutinationenhancementbytheCH2domainofIgG3.Weshowedtha tthisdomainsolelydeterminedantibodyoligomerizationinsolutio nandthusmostprobablyalsoonamulti-

epitopesurface.Itispossiblethatoligomerizationbetweenantibodie sboundtoseparateerythrocytesoccursparalleltoasensitizationpha seofhemagglutination.T hus,antibodyoligomerizationmayl eadto theformationofzipperlikestructuresthatstabilizecellaggre- gatesandincreaseahemagglutinationscore.Moreover,theCH2dom ainofIgG3increasedfunctionalaffinityofanantibodytoerythrocyte surface.Thus,hemagglutinationenhancementmayatleastpartially dependontheincreasedaffinity.

However,theobservedenhancementofhemagglutinationbythe CH2domainofIgG3wasaffectedbyenzymaticdeglyco-

sylation.Incontrast,oligomerizationin solutionandi ncreasedfunc tionalaffinitytop olyvalentantigenwerei ndependentofC H 2 glyc osylation.T h isd i f ferencei ndicatest hatantibodyoligomerizatio nd oesnotfullya c c ountfort heC H 2 d omain-

mediatedenhancementofhemagglutination.

MouseIgG3hasaputativesiteofN-

glycosylationinitsCH3domainonAsn471.Pankareportedthatthemut ationofthis

Asnresiduei ntoSerd i m i n i shedtheself-

associationofIgG3(17).ThisfindingwaslatercontradictedbyK urokietal.,whoprovidedevidencethatthisputativeN-

glycosylationsiteintheCH3domainisnotoccupiedandthemutati onAsn471ThrdoesnotinfluenceIgG3self-

associationorcryoglobulinactivity(18).Ourobservationsareinl inewiththefindingsofKurokietal.Wedidnotobserveanydiffere ncesbetweenoligomerizationofIgG3anditsenzymaticallydegly cosylatedvariant.Itisimpor-

tantton otethatweandKurokie t al.u sedP EG-

precipitationforoligomerizationanalyses.Pankauseddifferentm ethods,ELISAandnativeelectrophoresis,whichmayaccountfo rthediscrepancies.

Greenspane t a l.showedt hatFc-

dependentoligomerizationincreasesfunctionalaffinityofIgG3to polyvalentantigens(5).Ourresultsc onfirmt hatf i nding,butwesh owedt hatt herela-

tionbetweenoligomerizationandincreasedfunctionalaffinityism orecomplexthanpreviouslythought.First,functionalaffinityofIg G3wasinfluencednotonlybyFcregion(CH2andCH3 domains )butalsobytheCH1domain.Second,functionalaffinitytothepoly valentantigen(Bantigen)wasmodulatedbytheCH2charge.Incont rast,oligomerizationinsolutionrequiredonlythepresenceoft heC H 2 d omainofIgG3andw asi nsensitivetotheintroducedc harge m odifications.T heresultsshowedthatthemechanismbehindhig hfunctionalaffinitymaydependonmorefactorsthanoligomerizat ioninsolutiondoes.

Theobservedi n f luenceoft heC H 1 d omainonfunctionalaff inityisdifficulttoexplain.TheCH1domainofIgG3hasamorepositiv enetchargethantheCH1domainofIgG1(9).Itislikelythatthenetc hargeoftheCH1domaininfluencesthebindingofthedomainmute instoerythrocytes,whichhaveastrongnegativecharge.However,t heIgG3-

derivedCH1domainalsoenhancedthebindingofIgG1_CH1- 3toasurfacewiththeimmobilizedtrisaccharideB-

BSAconjugate.Thus,theresultssupportpreviousobservations(2 3)thattheCH1domainmayinfluenceavariabledomainandaparat opeofanantibody.

Accordingtothegeneralview,theFabandFcfragmentsareinde pendentpartsofanantibody(24).However,ourresultsdem- onstratethattheFc,particularlyitsCH2domain,mayinfluenceFa b-

mediatedantigenbinding.Therearetwopossiblemechanismsofth isphenomenon—intramolecularsignaling(25)

[calledbysomeauthorsasanintramolecularallostery(16)]orinter molecularcooperativity.

Thereareseveralexamplesofintramolecularsignalingobser vedbydifferentauthorsinvestigatinghowtheisotype

switchingchangesanantibodyaffinitytoitsantigen[reviewedinRef.

(16,26)].TheeffectsoftheCH1domainsorFcfragmentsonvariabler egionsarewelldocumented,butconsideredaratheruniquep henom enon(16).Itism orelikelythattheincreasedaffinityofIgG3toitsanti genresultsfromcooperativityofitsCH2domains.Withinthisdomai n,aspecificsiteofself-

associationmaybepresent,whichgovernsoligomerizationofananti bodyandpre-

determinestheincreasedaffinitytomultivalentantigens.However,w ecannotexcludeotherscenarios—

theinvolvementofboththeCH2andCH3domainsinIgG3intermole cularinteractionsorevensoleCH3-

CH3interactions,assumingthattheCH2domainsinfluencethew hol emoleculestructureandpromotereciprocalinteractionsoftheCH3d omainsofneighboringmolecules.

Otherfactors,e.g.,influenceoftheCH1domainonaparatope,prop ertiesofanantigen(charge),spatialdistributionofepitopes,intermol ecularforcesbetweenepitopeandparatope,oravariabledomainfram eworkmayfurthermodulatefunctionalaffinityofIgG3uponmultiva lentantigenbinding.

Diebolderatal.describedrecentlyaninterestingexampleofFc- dependentantibodyoligomerization.Analysesofantibodybindingt oDNP-

labeledliposomes(amultivalentantigen)revealedthathumanIgGm ayformhexamerst hroughnon-

covalentinteractionsbetweent heirconstantregions(27).Severalm uta-

tionsthatenhancetheseinteractionsandsubsequentcomplementacti vationwerereported(27).TheFc-

interactionspromotingantibodyhexamerizationdidnotchangeaffin itytot hecognateantigen.Thus,thisphenomenonseemstobedifferen tfromIgG3oligomerization,anditisstillanopenquestionwhetherm ouseantibodiesareabletoformsuchhexamers.

Currently,nostructureisavailablefora full-

lengthmouseIgG3oritsFcfragment.Weperformedsomeanalysesu singamolecularmodelofIgG3obtainedbycomparativemodeling,b utitsresolutionisnotsufficientforindepthstudies.IgG3crystal- lizationmightprovideadirectinsightintothemechanismofitsoligo merization,aswasinthecaseofhumanIgG1hexameriza-

tiondescribedinthecitedwork(27).

Complementc ascadeactivation,asaneffectorf unctionofantibo dies,constitutesa first-

lineofdefenseagainstmicrobialinfections.Asthecascadeprogresse s,componentsofthecomple-

mentaredepositedona p athogensurfaceandactasopsoninsforphag ocyticcells.Moreover,thecomplementlysesinvadingpathogensby formingmembraneattackingcomplex.WeconfirmedthatC1q- bindingmotifs,knownfromhumanIgG1,arefunctionalinthemouse IgG3framework.Ontheotherhand,wedidnotobservecomplementa ctivationbymouseIgG1equippedwiththemotifs.Theresultsindicat ethatthepresenceoftheknownC1q-

bindingmotifsisnotsufficientforcomplementactivation

byanantibody.Themotifsmustbesurroundedbyapermissiveframe work,providede.g.,byhumanIgG1ormouseIgG3.

Ourworksuggeststhatanoveltypeofmonoclonalantibod- iesmaybegeneratedbyreplacingtheCH2domainofahumanantibo dywiththehomologsfragmentofmouseIgG3.HumanIgG1subclas sisthemostfeasibletargetframeworkforgenerationofsuchIgG3- inspiredhybridmouse/humanmolecule(28).Ourobservationindic atesthatthegeneratedhybridanti-

bodyshouldp reservetheabilityt oactivatecomplementandmayha veincreasedaffinitytopolyvalentantigens.SincethemouseIgG3s ubclassishighlyprotectiveagainstseverallife-

threateningmicrobialinfections,thehybridmoleculemaybeveryu sefulinpreventingorfightinglethalpathogens.However,thehybrid antibodywiththemouseCH2maybeimmuno-

genic.Todecreasetheriskofanunwantedimmuneresponse,themo usecomponentshouldbereducedtoaminimum.Thus,thep ropertie so ftheCH2domainderivedfrommouseIgG3shouldbefurtherinve stigatedandeffortsshouldbemadeespe-

ciallytoidentifyfragmentsofthisdomainthatdeterminesitspropert ies.

aUThOrcOnTriBUTiOns

TKconceivedanddidallexperiments.TKandJBanalyzedanddiscus sedtheresults.ThemanuscriptwaswrittenbyTKandJB.Theauthors acceptedthefinalversionofthemanuscript.

acKnOWleDgMenTs

WethankDr.PawełM akforanalyticalgelf iltrationo fIgGmuteins.

FUnDing

ThisworkwassupportedbythePreludiumGrantno2015/17/N/NZ1/

00039toTKfundedbytheNationalScienceCentre,Poland.Facultyo fBiochemistry,BiophysicsandBiotechnologyoft heJagiellonianU niversityi n Krakówisa p artneroft heLeadingNationalResearchCe nter(KNOW)supportedbythePolishMinistryofScienceandHigher Education.

sUPPleMenTarYMaTerial

TheSupplementaryMaterialforthisarticlecanbefoundonlineathttp s://www.frontiersin.org/articles/10.3389/fimmu.2018.01096/full#s upplementarymaterial.

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ConflictofInterestStatement:Theauthorsdeclarethattheresearchwascon- ductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstru edasapotentialconflictofinterest.

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