RunningHead:IGGCITRULLINATION

CitrullinationofImmunoglobulinGbyPeptidylarginineDeiminase4
byMatthewLim

Advisor:
WilsonMeng,Ph.D.

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Abstract
Citrullinationisabiochemicalreactioninvolvingaclassofenzymescalledpeptidylarginine
deiminases(PADs)thatconvertarginineresiduestocitrulline.Citrullinationreactionsare
thoughttoplayanimportantroleinthemechanismsofcertainautoimmunediseases.
ImmunoglobulinG(IgG)isacommonantibodythatisoneofthetargetsofautoantibodiesin
rheumatoidarthritis(RA).ItishypothesizedthatcitrullinationisthemechanismbywhichIgG
antibodiesaretaggedfordestructioninRApatients.Thepresentedexperimentaimedtomeasure
theextenttowhichIgGundergoescitrullinationinthepresenceofPAD4.Twoexperimental
controlswerealsomeasured:untreatedIgGandNalphabenzoylLarginineethylestertreated
withPAD4.Eachsamplewaslabelledusingrhodaminephenylglyoxal(RhPG),a
citrullinespecificfluorescentprobe.Theresultsfromtheinitialexperimentindicatedthatno
amountofcitrullinationtookplaceinanyofthethreesamples,suggestingthataproceduralerror
musthaveoccurred.However,asecondexperimentconductedbyotherstudentssuggestedthe
presenceofcitrullineresiduesinthesamplescontainingIgGandPAD4.Whilenoconclusions
couldbedrawnfromthepresentedexperiment,valuableinformationwasdiscoveredabout
modificationsthatcouldbemadetoensuremoretellingresultsinfuturetrials.

Introduction
Inadditiontothetwentystandardaminoacids,thereexistavarietyofnonstandard
aminoacidsthatarenotdirectlytranslatedfromcodons.Manyofthesenonstandardamino
acidsareproducedasaresultofposttranslationalproteinmodifications.Citrullinationisthe
posttranslationalprocessbywhichthestandardaminoacidarginineisconvertedtothe

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nonstandardaminoacidcitrulline.Thereactioninvolvestheconversionofthepositive
guanidinogroupinargininetotheneutralketonegroupincitrulline,resultinginashiftinthe
chargeoftheaminoacid.Therefore,aproteinsfoldingpatternmaysignificantlychangeafter
undergoingcitrullination.Citrullinationreactionsoccurinthepresenceofcalciumionsanda
classofenzymescalledpeptidylargininedeiminases(PADs).Onememberofthisclass,PAD4,
isthoughttoplayaroleinthedevelopmentofwhitebloodcells.Consequently,PAD4is
commonlyfoundnearinflamedareaswherethewhitebloodcellcountisrelativelyhigh.
Citrullinationisthoughttoplayanimportantroleincertainautoimmunediseases,
includingrheumatoidarthritis(RA).Itisbelievedthattheimmunesystemusescitrullineasa
waytolabelcertainproteinsfordestructionbycitrullinespecificantibodies.Anticitrullinated
proteinantibodies(ACPAs)havebeenfoundintheserumofamajorityofRApatientsandhave
beenidentifiedasapossiblebiomarkerforRA.
ImmunoglobulinG(IgG)isthemostabundantantibodyinhumanserum.IgGisalsoa
commontargetforautoantibodiesinRApatients.ThehypothesizedmechanismofRAproposes
thatPAD4citrullinatesIgG,therebydecreasingtheactivityoftheantibodyandmakingitmore
accessiblefordestructionbyACPAs.Inaddition,IgGisthechemicalbasisofactive
pharmaceuticalingredientsindrugproducts.CitrullinationofIgGdrugsmayrenderthe
medicineineffective.Theexperimentisdesignedtotesttheextenttowhichpeptidylarginine
deiminase4(PAD4)isabletocitrullinateIgG.

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Methods
TheexperimentalsampleispreparedtocontainIgGtreatedwithPAD4ata1:10enzyme
tosubstrateratio.Inadditiontotheexperimentalsample,anegativecontrolsamplecontaining
untreatedIgGandapositivecontrolsamplecontainingNalphabenzoylLarginineethylester
treatedwithPAD4arealsoprepared.Eachsampleisincubatedovernightat25Cinareaction
mixturecontainingTrisHClbuffer(pH7.4),5mMdithioerythritol(DTE),and8mMcalcium
chloride.
Afluorescentprobecontainingrhodaminephenylglyoxal(RhPG)isusedtodetectthe
presenceofcitrullineinthesamples.Gelelectrophoresisisperformedonthelabelledsamples,
andtheresultinggelisobservedunderfluorescentlight.ThisstepisneededtoresolvePAD4
fromIgGinthereactionmixture.Thelabelledsamplesarealsoanalyzedusingspectroscopy.

Figure1Experimentalscheme

ResultsandDiscussion
IntheinitialexperimenttheRhPGprobedidnotdetectthepresenceofcitrullineinany
ofthethreesamples.Becausethepositivecontrolresultedinanegativeresult,noconclusions

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canbedrawnabouttheoriginalhypothesis.Weconcludedthattherewasanerrorinthe
procedurethatledtofaultyresults.
Weareabletopointtoseveralfactorsthatmayhaveinfluencedtheunusualresultsofthe
experiment.Onefactorthatstandsoutasapossiblecauseisthelabellingprocess.Theprobes
manufacturernotesthattheprobeisactiveinveryacidicenvironmentsandsuggeststhatthe
labellingtakeplacein20%trichloroaceticacid(TCA).Weconductedthelabellingin2N
sulfuricacid.WhilebothenvironmentshaveasimilarpH,itispossiblethattheactivityofthe
probewasisdependentonsomethingotherthanpH.Thisdetailwillbeconsideredforfuture
iterationsoftheexperiment.Inthesecondattempt,theproteinsrecoveredshowedsomedegree
ofrhodaminefluorescence,suggestingthatthePAD4mediatedreactionmighthavetakenplace.
Additionalexperimentationisneededtodrawconclusions.

Figure2Initialexperiment:proteinswerenotisolatedfromthereactionmixture

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Rhfluorescence

Figure3Secondattemptperformedbyotherstudentsshowing
separationofproteinsinthemixtureanddifferentialRhfluorescence

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