Rev. Bras. Reumatol. vol.57 número3

w w w . r e u m a t o l o g i a . c o m . b r

REVISTA

BRASILEIRA

DE

REUMATOLOGIA

Original

article

Independence

of

carbohydrate-deficient

isoforms

of

transferrin

and

cyclic

citrullinated

peptides

in

rheumatoid

arthritis

Monika

Gudowska

_,

_Ewa

_{Gindzienska-Sieskiewicz}

_,

_Ewa

_Gruszewska

_,

Bogdan

Cylwik

_,

_Stanislaw

_Sierakowski

_,

_Maciej

_Szmitkowski

_,

_Lech

_Chrostek

a_{MedicalUniversityofBiałystok,DepartmentofBiochemicalDiagnostics,Białystok,Poland}

b_{MedicalUniversityofBiałystok,DepartmentofRheumatologyandInternalDiseases,Białystok,Poland}

c_{MedicalUniversityofBiałystok,DepartmentofPediatricLaboratoryDiagnostics,Białystok,Poland}

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Articlehistory: Received9April2015 Accepted5January2016 Availableonline28March2016

Keywords:

Anti-cycliccitrullinatedpeptide antibodies

Carbohydrate-deficienttransferrin Rheumatoidfactor

Rheumatoidarthritis

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b

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Objective:The aim ofthis studywas toassess therelationship between thetwotypes of posttranslational modifications of proteins in RA: glycosylation on the example of carbohydrate-deficienttransferrinandcitrullinationbymeansofautoantibodiestocyclic citrullinatedpeptides.

Methods:Thestudywascarriedoutin50RApatients.CDTwasmeasuredusingNLatex CDTimmunonephelometrictest,theresultswerepresentedinabsoluteandrelativeunits. Anti-CCPweremeasuredusingthechemiluminescentmethodandrheumatoidfactorby immunoturbidimetricmethod.

Results:80%ofRApatientswerepositiveforanti-CCP,70%forRFand62%forboth, anti-CCPandRF.Thelevelof%CDTwassignificantlyelevated,butabsoluteCDTlevelwasnot changed.ThemeanabsoluteCDTconcentrationwashigherinanti-CCPpositivepatients thanthatinanti-CCPnegative.CDT(absoluteandrelativeconcentration)didnotcorrelate withanti-CCPandRF.However,serumRFsignificantlycorrelatedwithanti-CCP.%CDTdid notcorrelatewithanti-CCP,butabsolutelevelcorrelatedwithanti-CCPonlyinanti-CCP negativeandRFnegativepatients.CDTdidnotcorrelatewithRF,butsolelywithanti-CCPin anti-CCPnegativepatients.Anti-CCPcorrelatedwithDAS28onlyinanti-CCPnegativeRA, butCDT(absoluteandrelativeunits)correlatedwithDAS28inallpatientsandinanti-CCP positiveRA.

Conclusions: TheseresultssuggestthatthechangesinCDTandanti-CCPconcentrations arenot associatedwithoneselfandindicateontheindependenceofthese posttransla-tionalmodificationsinrheumatoidarthritis.Onlythealterationsintransferringlycosylation reflectedtheactivityofRA.

©2016ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:chrostek@umb.edu.pl(L.Chrostek). http://dx.doi.org/10.1016/j.rbre.2016.01.005

Independência

de

isoformas

de

transferrina

deficiente

em

carboidrato

e

peptídeos

citrulinados

cíclicos

na

artrite

reumatoide

Palavras-chave:

Anticorposantipeptídeo citrulinadocíclico Transferrinadeficienteem carboidrato

Fatorreumatoide Artritereumatoide

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Objetivo: Oobjetivodesteestudofoiavaliararelac¸ãoentreosdoistiposdemodificac¸ões pós-translacionaisdeproteínasnaAR:glicosilac¸ãonocasodatransferrinadeficienteem carboidrato(TDC)ecitrulinac¸ãopormeiodosanticorposnocasodoantipeptídeocitrulinado cíclico(anti-CCP).

Métodos: Oestudofoirealizadoem50pacientescomAR.ATDCfoimedidautilizandoo testeimunonefelométricoNLatexCDT,eosresultadosforamapresentadosemunidades absolutaserelativas.Oanti-CCPfoimensuradousandoométodoquimioluminescenteeo fatorreumatoide(FR)pelométodoimunoturbidimétrico.

Resultados: 80%dospacientescomARforampositivosparaanti-CCP,70%paraFRe62% paraambos(anti-CCPeFR).Apercentagemdetransferrinatotal(%TDC)esteve significati-vamenteelevada,masonívelabsolutodeTDCnãoestevealterado.Aconcentrac¸ãomédia deTDCabsolutafoimaiornospacientesanti-CCPpositivosdoquenaquelesanti-CCP nega-tivos.ATDC(concentrac¸ãoabsolutaerelativa)nãosecorrelacionoucomoanti-CCPeoFR. Noentanto,oFRséricosecorrelacionousignificativamentecomoanti-CCP.Opercentual deTDCnãosecorrelacionoucomoanti-CCP,masseunívelabsolutosecorrelacionoucom oanti-CCPapenasempacientesFRnegativoseanti-CCPnegativos.ATDCnãose correla-cionoucomoFR,somentecomoanti-CCPempacientesanti-CCPnegativos.Oanti-CCPse correlacionoucomoDAS28apenasnospacientescomARanti-CCPnegativos,masaTDC (unidadesabsolutaserelativas)secorrelacionoucomoDAS28quandoconsideradostodos ospacientescomAReempacientescomARanti-CCPpositivos.

Conclusões: Estesresultadossugeremqueasalterac¸õesnaTDCeasconcentrac¸õesde anti-CCPnãoestãoassociadasentresieindicamaindependênciadestasmodificac¸ões pós-translacionaisnaartritereumatoide.Apenasasalterac¸õesnaglicosilac¸ãodatransferrina refletemaatividadedaAR.

©2016ElsevierEditoraLtda.Este ´eumartigoOpenAccesssobumalicenc¸aCC BY-NC-ND(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Rheumatoid arthritis (RA) is a chronic, autoimmune dis-ease ofconnective tissues, withthe appearance ofseveral typesofautoantibodies.1_{Thespecificautoantibodies} gener-atedinRAhavebeenassociatedwiththeposttranslational modificationsofproteinsandpeptides.2_{Thesemodification} involveglycosylation,citrullination,methylation,acetylation andubiquitinationoccurinphysiologicalconditionsandhave an important role in the normal function of the immune system.3 _{The first type of autoantibodies founded in RA} patientswasrheumatoidfactor(RF),whichisanautoantibody to Fc domain of IgG. Thechanges in glycosylation (galac-tosylation/sialylation)IgGhavebeenfoundtobeassociated with a pathogenesis of RA and can be diagnostically and therapeuticallyuseful.4_{Theseveralstudieswereconducted} torecognizethealterationsinglycosylationofothers glyco-proteinssuchasIgA,alpha1-acidglycoprotein,fibronectin, haptoglobinandtransferrin.Humantransferrinexistsasan heterogeneouspopulationglycosylatedvariantsdiffering in carbohydratecomposition.5 _{Inhealthypeople mostlyoccur} tetrasialylatedglycoformsandglycoformswhichlackoneor bothofcompleteN-glycansarecalledcarbohydrate-deficient transferrin(CDT).5_{IthasbeenassumedthatCDTisthesum} ofthreeisoforms:asialo-,monosialo-anddisialotransferrin.

TheglycosylationofplasmatransferrinchangesinRAandcan leadtoincreasetheCDTlevel.6_{Thenewlyknown} posttransla-tionalmodificationinRAiscitrullination.7_{Itisanenzymatic} conversionofargininetocitrullinecatalysedby peptidylargi-ninedeiminases(PAD).InpatientswithRA,PADmayleakout ofthe dying cells inthe synovial jointswhich can citrulli-nateofarginineandgenerateancycliccitrullinatedpeptides (CCP).8,9 _{AutoantibodiesagainstCCPareconsideredasearly} diagnosticandprognosticbiomarkerofRA.10_{Theirpresence} inRA-negativepatientstestifiesfortheearlyRAwithworse prognosis.8_{InRApatientsmany citrullinatedproteinshave} been detected,asfibrinogen, vimentin,enolaseand typeII collagen.3_{Theaimofthisstudywastoassesstherelationship} betweendifferenttypesofposttranslationalmodifications– glycosylationandcitrullination–inRAbymeansofthe con-centrationofCDTandanti-CCP.

Material

and

methods

Table1–Theresultsoflaboratorytestsinrheumatoidarthritis(RA)patientsandcontrols.

CRP(mg/L) RF(IU/mL) IgG(g/L) CDT(mg/L) %CDT Anti-CCP(U/L)

RA(n=50) 22.96±25.54 p=0.000a

172.23±229.37

p<0.01a

11.79±3.06

p=0.904

42.70±69.40

p=0.564

2.03±0.24

p<0.01a

185.42±325.02

p=0.000a

Controls(n=33) 1.04±0.78 21.58±1.23 11.43±1.42 42.52±9.23 1.77±0.22 0.57±0.21

Dataaremeanandstandarddeviation.

a _{p<0.05thedifferencesbetweenRApatientsandcontrolgroup(Mann–WhitneyUtest).}

Controlgroupconsistedof33healthysubjects(meanage 41)recruited from hospital workers.Blood fasting samples weretakenbyveinpunctureafter12hoffasting.All(healthy andsick)subjectsgavetheirinformedconsenttoparticipate inthestudies.Thisstudywasinaccordancewiththe Bioethi-calCommitteeattheMedicalUniversityinBialystokandwith theHelsinkiDeclaration.

RAactivitywasdeterminedbydiseaseactivityscore(DAS 28)accordingtotheformula:

DAS28=0.56∗sqrt(t28)+0.28∗sqrt(s28)+0.7∗ln(ESR)

+0.014∗VAS

The number of tender joints (t28), swollen joints (s28), erythrocyte sedimentation rate (ESR) and visual analogue scale (VAS) were used inthat formula. Low activity of RA (DAS28between2.6and3.2)hadnoneofpatients,moderate activity(DAS28intherangeof3.2–5.1)had27.08%patients andhighactivity(DAS28above5.1)had72.92%ofpatients. CDTwasmeasuredusingNLatexCDT immunonephelomet-ricteston BNII analyser(SiemensHealthcareDiagnostics, Somerville, USA). The results were given as absolute CDT units (mg/L) and percentage of total transferrin (%CDT). Theantibodiesagainstcycliccitrullinatedpeptide(anti-CCP) weremeasuredontheArchitecti2000analyser.Theresults above 5U/L are considered negativeand ≥5.0U/L positive. RF (normal values<30IU/mL), C-reactive protein(CRP; nor-malrange:0.8–1.2mg/L)andimmunoglobulinG(IgG;normal range:5.52–16.31g/L)weredeterminedusing immunoturbidi-metricmethodsontheArchitectc8000(AbbottLaboratories, AbbottPark,IL,USA).ESR(mm/h)wasmeasuredby Wester-grenmethodontheSediplusS2000(Sarstedt,Germany).

Statisticalanalysis

Thedifferencesbetweentestedandcontrolgroupwere eval-uatedbyMann–WhitneyUtest.Weconsideredp-values<0.05 asstatisticallysignificant.Thecorrelationbetweenvariables wasassessedusingSpearman’srankcorrelationcoefficient.

Results

TheresultsoflaboratorytestsinRApatientsandcontrolsare presentedinTable1.ThemeanlevelsofCRP,RF,%CDTand anti-CCPweresignificantlyhigherinRApatientsin compari-sontothecontrolgroup.80%ofRApatientswerepositivefor anti-CCP,70%forRFand62%forboth,anti-CCPandRF.

Table2–CorrelationofCDTwithanti-CCP.

Test R p

AllRA CDT_%CDT 0.088_0.033 0.547_0.821

Anti-CCPpositive CDT −0.212 0.195 %CDT −0.069 0.677

Anti-CCPnegative CDT 0.696 0.025a

%CDT 0.364 0.301

RFpositive CDT −0.063 0.741

%CDT 0.223 0.235

RFnegative CDT 0.629 0.012a

%CDT −0.016 0.954

a _{Significantcorrelation.}

CDTconcentrationdidnotcorrelatewithanti-CCPinall RApatients(Table2),butonlyinanti-CCPnegative(r=0.696) andRF negative(r=0.629)subjects. ThecomparisonofCDT levels(absoluteunits)inrelationtoanti-CCPandRF concen-trationsexhibitedthesignificantdifferenceonlyconsidering theanti-CCPlevels.Intheanti-CCPpositivepatients,CDT con-centration (43.1±7.4mg/L) was significantly higherthan in anti-CCPnegativesubjects(38.9±2.4mg/L)(p=0.022)(Fig.1).

CDTconcentration(absoluteandrelativeunits)correlated withRAactivity,itmeanswithDAS28inallpatients(Table3). ThehighRAactivityisassociatedbythelowabsoluteCDT andhighrelativeCDTconcentration.CDT(expressedinboth units)correlatedwithDAS28regardlessofanti-CCPlevelsbut %CDTinRFpositivepatients.Anti-CCPconcentrationdidnot

Negative Positive

Anti-CCP 25

30 35 40 45 50 55 60 65 70 75

CDT [mg/L]

P=.022

Mean Mean±SE Mean±SD

Table3–Correlationofdiseaseactivityscore(DAS)28 withanti-CCPandCDT.

Test R p

CorrelationbetweenDAS28andCDT

AllRApatients CDT −0.350 0.014 a

%CDT 0.321 0.026a

Anti-CCPpositive CDT −0.446 0.003

a

%CDT 0.324 0.047a

Anti-CCPnegative CDT 0.567 0.112

%CDT 0.627 0.070

RFpositive CDT −0.306 0.094

%CDT 0.399 0.026a

RFnegative CDT −0.428 0.144

%CDT 0.319 0.288

CorrelationbetweenDAS28andanti-CCP

AllRApatients 0.050 0.739

Anti-CCPpositive 0.193 0.246

Anti-CCPnegative 0.748 0.020a

RFpositive 0.113 0.550

RFnegative −0.198 0.517

a _{Significantcorrelation.}

correlatewithDAS28inallRApatients,butonlyinanti-CCP negativesubjects.

CDTdidnotcorrelatewithRFinallRApatientsbut consid-eringtheanti-CCPandRFvalues(positiveornegative)(p>0.05 foreachcomparisons).Incontrary,anti-CCPcorrelatedwith RFinallRAsubjects(r=0.348;p=0.019),andintheanti-CCP negativesubjects(r=0.750;p=0.032).

DAS 28 did not differ between anti-CCP positive (5.63±1.37U/L) and anti-CCP negative patients (6.14± 1.20U/L)(p=0.337)and betweenRF positive(5.84±1.22U/L) and RF negative subjects (5.06±1.41U/L) (p=0.113). RF value was significantly higher in anti-CCP positive RA (176±216IU/mL) than that in anti-CCP negative (142 ±307IU/mL)(p=0.047),butanti-CCP didnotdifferbetween RFpositive (222±378U/L)incomparisontoRF negativeRA (117±224U/L)(p=0.126).

Discussion

The present study demonstrated the lack of association between concentration of carbohydrate-deficient isoforms of transferrin (CDT) and the level ofcitrullinated proteins and peptides measuredby autoantibodies(anti-CCP)in RA patients.Takinginto considerationtherelationshipof anti-CCP or CDT with DAS 28,there were only the association withCDTconcentration.Ourresultsareinaccordancewith the study of Serdaro ˘glu et al.11 _{and Arido ˘ganet al.}12 _who provednosignificantcorrelationbetweenanti-CCPandDAS 28.Likewise,theydidnotfoundasignificantdifferenceinDAS 28betweenanti-CCPnegativeandanti-CCPpositivepatients andbetweenRFpositiveandRFnegativesubjects.Similarly, theyfoundthesignificantdifferencesbetweenanti-CCP pos-itiveandanti-CCPnegativeRApatientsforRF.Theonlyone study,Önderandco-workers,revealedthatanti-CCPwere pos-itivelyassociatedwithhigherscoresofDAS-28,whilethese werenotassociatedwithESRandCRP.13_{Intheotherstudy,a} weakcorrelationbetweenanti-CCPandDAS28wasobserved,

but all RA patientswere positive foranti-CCP.14 _Although, therewasnocorrelation betweenCDT andanti-CCPinRA, there was association between two serological markersof RA,itmeansRFandanti-CCP.CDTcorrelatedwithanti-CCP onlyinanti-CCPnegativeand RFnegativepatients,thusin seronegativeRA.Thisphenomenamayberesponsiblefor sig-nificantlyhigherCDTconcentrationinanti-CCP-positivethan inanti-CCP-negativeRApatients.Although,asdemonstrated Arido ˘ganetal.,12_{antibodiesagainstCCPweremorespecific} forRAthan RF(100%vs.96.8%), anti-CCPdidnotcorrelate withdiseaseactivity(DAS28)butwithRF.Thediagnostic accu-racy(areaundertheROCcurves)ofanti-CCPwasalsohigher than thatofRF.15 _{Thelackofcorrelationofdisease activity} withanti-CCPanditsexistencewithCDTmaybeindicatedon thetime-dependentdifferenceintheactivityofthesetwo dif-ferentposttranslationalmodificationsoftheproteins.DAS28 indicatesthecurrentdiseaseactivity,buttheactivationofPAD and citrullination ofvariousproteinsovertakesthe clinical symptomsofdisease,but thealterationsintransferrin gly-cosylationundergoparallelwiththedevelopmentofdisease. Hamadetal.alsofoundthatthepresenceofhighanti-CCP levelisnotassociatedwithhigherdiseaseactivity.16_However, anti-CCPpositivityandtitrecorrelatedwithdiseaseduration, whatcanenforceourhypothesisabouttime-dependentshift incitrullinationtowardsglycosylationinRA.Anti-CCPcanbe foundearlyinthecourseofRA,evenyearsbeforetheonset ofclinical symptoms.17 _{Itisvery interestingthat the} anti-CCP statusmaychange (fromnegativetopositiveand vice versa)after3yearsfromtheonsetofRAandthemeanserum anti-CCPleveldeclinesduringtheyears.18

Amajor conclusionofthisstudy isthat, inRApatients thechangesintransferringlycosylationmeasuredbymeans ofcarbohydrate-deficientisoforms(CDT)arenotrelatedwith citrullination,othertypeofposttranslationalmodificationin RA.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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