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Defining the role of Porphyromonas gingivalispeptidylarginine deiminase (PPAD) in rheumatoidarthritis through the study of PPAD biologyMaximilian F Konig,1 Alizay S Paracha,2 Malini Moni,1 Clifton O Bingham III,1

Felipe Andrade1

Handling editor Tore K Kvien

▸ Additional material ispublished online only. To viewplease visit the journal online(http://dx.doi.org/10.1136/annrheumdis-2014-205385).1Division of Rheumatology,The Johns Hopkins UniversitySchool of Medicine, Baltimore,Maryland, USA2The University of Oklahoma,Norman, Oklahoma, USA

Correspondence toDr Felipe Andrade, Division ofRheumatology, Johns HopkinsUniversity School of Medicine,Mason F Lord Building CenterTower, Suite 6000, 5200Eastern Avenue, Baltimore,MD 21224, USA;andrade@jhmi.edu

Received 10 February 2014Revised 28 April 2014Accepted 1 May 2014

To cite: Konig MF,Paracha AS, Moni M, et al.Ann Rheum Dis PublishedOnline First: [please includeDay Month Year]doi:10.1136/annrheumdis-2014-205385

ABSTRACTBackground Antibodies to citrullinated proteins are ahallmark of rheumatoid arthritis (RA). Porphyromonasgingivalis peptidylarginine deiminase (PPAD) has beenimplicated in the initiation of RA by generating citrullinatedneoantigens and due to its ability to autocitrullinate.Objectives To define the citrullination status and biologyof PPAD in P gingivalis and to characterise the anti-PPADantibody response in RA and associated periodontal disease(PD).Methods PPAD in P gingivalis cells and culturesupernatant were analysed by immunoblotting and massspectrometry to detect citrullination. Recombinant PPAD(rPPAD), inactive mutant PPAD (rPPADC351S), and N-terminaltruncated PPAD (rPPADNtx) were cloned and expressed inEscherichia coli. Patients with RA and healthy controls wereassayed for IgG antibodies to citrullinated rPPAD andunmodified rPPADC351S by ELISA. Anti-PPAD antibodieswere correlated with anti-cyclic citrullinated peptide (third-generation) antibody levels, RA disease activity and PDstatus.Results PPAD from P gingivalis is truncated at theN-terminal and C-terminal domains and not citrullinated.Only when artificially expressed in E coli, full-length rPPAD,but not truncated (fully active) rPPADNtx, is autocitrullinated.Anti-PPAD antibodies show no heightened reactivity tocitrullinated rPPAD, but are exclusively directed against theunmodified enzyme. Antibodies against PPAD do notcorrelate with anti-cyclic citrullinated peptide levels anddisease activity in RA. By contrast, anti-PPAD antibody levelsare significantly decreased in RA patients with PD.Conclusions PPAD autocitrullination is not the underlyingmechanism linking PD and RA. N-terminal processingprotects PPAD from autocitrullination and enhances enzymeactivity. Anti-PPAD antibodies may have a protective role forthe development of PD in patients with RA.

INTRODUCTIONRheumatoid arthritis (RA) is a systemic autoimmunedisease of unknown aetiology characterised by syn-ovial inflammation and joint destruction.1 2 Loss oftolerance to citrullinated proteins is a hallmark ofRA pathogenesis.3 Autoantibodies to citrullinatedproteins (ACPA) are highly specific for RA andprecede the onset of clinical disease by years.4–6

Peptidylarginine deiminases (PAD) are enzymes thatcatalyse the post-translational modification reactionof arginine residues to citrulline.7 8 By modifyingautoantigens in synovial tissue and alternative sitesof inflammation, PAD activity may be a sine qua non

in initiating and sustaining autoimmunity inACPA-positive RA. Porphyromonas gingivalis pepti-dylarginine deiminase (PPAD), a bacterial deiminaseevolutionarily unrelated to human PAD,8 9 hasreceived considerable attention by investigatorstrying to identify a mechanism linking periodontaldisease (PD), bacterial citrullination and RA.10–13

PD is a chronic inflammatory disease caused byinfection of the supporting tissues of the teeth, ultim-ately resulting in alveolar bone destruction and toothloss.14 An increased prevalence of PD has repeatedlybeen reported in RA.15–19 Among the periodontalpathogens, P gingivalis, an anaerobic bacteriumstrongly associated with chronic periodontitis,20–22 isunique in its expression of a bacterial PAD.9 PPAD isbelieved to be a major virulence factor of P gingivalisdue to its capacity to generate ammonia in the deimi-nation reaction of arginine to citrulline.8 Ammoniamay protect P gingivalis during acidic cleansing cyclesin the mouth,9 23 24 and promote periodontal infec-tion via inhibitory effects on neutrophil function.25 26

PPAD is almost exclusively detected in outer mem-brane fractions of P gingivalis,12 and as a secretedenzyme.9 Interestingly, outer membrane-associatedPPAD and secreted PPAD were only recently shownto be truncated at the C-terminal and N-terminaldomains.9 27 While C-terminal cleavage is requiredfor cell surface translocation of PPAD,27–29

N-terminal processing appears to maintain enzymeactivity and stability.30

By citrullinating C-terminal peptidylarginine in thecontext of periodontal infection, P gingivalis hasbeen hypothesised to play a primary role in RApathogenesis.11 31 32 Moreover, the recent findingthat recombinant PPAD (rPPAD) is autocitrullinatedand preferentially recognised by antibodies in RAsuggests that loss of tolerance to citrullinated proteinsin RA may originate from an antimicrobial immuneresponse directed against citrullinated PPAD.12

This study defines the structure and citrullinationstatus of the cellular and secreted forms of PPAD(cPPAD and sPPAD, respectively) in P gingivalis.The relevance of the anti-PPAD antibody responsefor RA and associated PD is examined in thecontext of PPAD biology.

MATERIAL AND METHODSPatients and controlsSerum was obtained from 83 patients with RA(1987 revised American Rheumatism Association(ARA) criteria)33 and 39 healthy controls recruited

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under the protocol of the Comprehensive Oral HealthAssessment in Patients with Arthritis and AutoimmuneInflammatory Diseases. The protocol was approved by theJohns Hopkins Medicine Institutional Review Board, andinformed consent was obtained from all participants. Study par-ticipants underwent a comprehensive dental evaluation. PD wasdefined using the 2007 Centers for Disease Control (CDC)/American Academy of Periodontology (AAP) standard cases defi-nitions for surveillance of moderate and severe periodontitisbased on assessments of pocket depth and attachment loss14 34;subjects not fulfilling these definitions were considered period-ontally healthy.

Bacterial samples and immunoblottingP gingivalis strain W83 was obtained from the University ofMaryland (courtesy of Mark A Reynolds). Bacterial cells werepelleted by centrifugation, lysed in ice-cold NP-40 lysis buffer(20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40Alternative), sonicated, and boiled in sodium dodecyl sulfate(SDS) sample buffer. P gingivalis culture supernatant was concen-trated using Amicon Ultra-15 units (EMD Millipore) and bufferexchanged with 20 mM Tris pH 7.6, 150 mM NaCl, 10% gly-cerol. Samples were resolved by SDS-PAGE and analysed by anti-modified citrulline immunoblotting,35 or using a polyclonal anti-body raised in rabbits by immunisation with recombinant PPAD(Covance).

Cloning and purification of recombinant PPAD andα-enolaseThe full-length PPAD coding sequence was amplified from P gin-givalis W83 DNA (ATCC) and cloned into pET-28a(+)(Novagen), generating a fusion protein with N-terminal His-tag.Enzymatically inactive PPAD was generated by site-directedmutagenesis to replace cysteine 351 at the active site of theprotein with serine (PPADC351S).36 N-terminal truncated PPAD(rPPADNtx) was generated by amplifying the coding sequence foramino acids 44 to 556 of full-length PPAD with primers contain-ing a 50 Kozak sequence. The PCR product was cloned intopET-28a(+) to encode for a C-terminal His-tagged fusionprotein. α-Enolase encoding cDNAwas cloned into pET-28a(+).Recombinant PPAD, PPADC351S, PPADNtx and enolase wereexpressed in Escherichia coli BL21 (DE3) (Agilent), and purifiedfrom the soluble fraction of cell lysates prepared in 20 mMTris pH 7.6, 400 mM NaCl, 5 mM imidazole, 20 mMβ-mercaptoethanol, 1% Triton X by Ni-NTA affinity chromatog-raphy (Qiagen). Purity of rPPAD and rPPADC351S used in ELISAassays exceeded 95% (see online supplementary figure S1A).Enolase was citrullinated in vitro using human rPAD4 as previ-ously described.37

PPAD ELISAPolystyrene plates (Costar) were coated with 100 ng/well citrulli-nated rPPAD (cit-rPPAD), rPPADC351S or phosphate bufferedsaline (PBS) pH 7.4 alone (used as a negative control). Coatedplates were washed with PBS 0.05% Tween 20 (PBS-T), andunoccupied binding sites blocked with PBS-T 3% non-fat drymilk (PBS-TM). Sera were diluted at 1:1000 in PBS-TM 1% andassayed in duplicate. Horseradish peroxidase (HRP)-conjugatedantihuman IgG ( Jackson ImmunoResearch) was used as a sec-ondary antibody. A serial dilution of rabbit anti-PPAD was usedas a standard. Arbitrary units were calculated from standard dilu-tions, and individual values corrected for background by sub-tracting the reactivity of PBS-coated wells.

RESULTSPPAD from P gingivalis is truncated and not citrullinatedCell lysates and supernatant from P gingivalis were initiallystudied by immunoblotting to characterise bacterial PPAD. Twodistinct patterns of PPAD were identified. In bacterial cells,anti-PPAD immunoblotting detected bands of approximately75–85 kDa (cPPAD), while a single band of 47 kDa (sPPAD)was identified in P gingivalis supernatant (figure 1A, middlepanel). The observed size of sPPAD is consistent with thetruncated form of PPAD.9 27 In contrast to a previous report,11

only low levels of protein citrullination were detected by anti-modified citrulline immunoblotting in bacteria lysed in NP-40buffer (figure 1A, right panel), and no citrullination was foundwhen P gingivalis was directly lysed and boiled in SDS samplebuffer (see online supplementary figure S1B). Bacterial citrullina-tion may therefore occur in vitro during cell lysis. Importantly,cPPAD and sPPAD are not citrullinated in bacterial samples(figure 1A, right panel and online supplementary figure S1B).

Mass spectrometry (MS) analysis of cPPAD and sPPAD invari-ably identified a truncated protein missing the first 43N-terminal and 97 C-terminal amino acids of full-length PPAD(figure 1B, C, respectively). Peptides outside these terminaldomains showed sequence coverage of 82.9% (cPPAD) and92.5% (sPPAD). In contrast to a previous study of full-lengthrPPAD expressed in E coli,12 we found that PPAD in P gingivalisis not citrullinated (figure 1B,C). Lack of autocitrullination wasnot attributable to loss of enzymatic function as confirmed usingthe BAEE assay (data not shown).

N-terminal truncation prevents PPAD autocitrullination andamplifies enzyme functionPrevious studies of recombinant full-length PPAD andN-terminal truncated PPAD have shown conflicting results withregards to enzyme autocitrullination.12 30 While full-lengthPPAD is strongly citrullinated during expression in E coli,12 thetruncated enzyme is not autocitrullinated.30 We hypothesisedthat N-terminal processing of PPAD may protect the enzymefrom autocitrullination in P gingivalis. Indeed, when weexpressed full-length PPAD (rPPAD) and N-terminal truncatedPPAD (rPPADNtx) in E coli, only the full-length enzyme wascitrullinated (figure 2A). Analogously, enzymatically inactivefull-length PPAD (PPADC351S) was not citrullinated whenexpressed in E coli (figure 2A, lower panel), confirming thatPPAD autocitrullination is dependent on the catalytic activity ofthe enzyme and its N-terminal domain. Importantly, we con-firmed that the absence of autocitrullination in rPPADNtx wasnot due to loss of enzymatic function. Indeed, N-terminal trun-cated PPAD showed a significant increase in enzyme activity ascompared with full-length rPPAD (mean citrulline production inμM: 191.9 vs 88.9; *p<0.0001) (figure 2B).

MS of full-length rPPAD achieved 96% protein sequencecoverage, which included 17 of the 18 arginines found in theenzyme. Among these, eight arginine residues were citrullinated(figure 2C). The probability of correct citrullination site localisa-tion approached 100% for all eight sites (figure 2D). These find-ings are in general accordance with the citrullination sitespreviously reported for rPPAD,12 with two additional sites iden-tified at R129 and R286. In peptides covering R63 and R70,citrullination was located to R63, but not confirmed at R70 aspreviously reported (figure 2D).12 While this data confirms thatrPPAD is autocitrullinated in E coli, this process appears to beunique to artificially expressed PPAD in the absence of physio-logical enzyme processing and regulators found in P gingivalis.

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Figure 2 Citrullination of rPPAD expressed in E coli is dependent on the N-terminal domain. (A) Purified rPPAD, mutant rPPADC351S (C351S) andtruncated rPPAD missing the N-terminal domain (Ntx) were analysed by SDS-PAGE and visualised by anti-PPAD immunoblotting (top panel).Anti-modified citrulline (AMC) immunoblotting (bottom panel) was used to detect citrullination. (B) Enzymatic activity of rPPAD, rPPADC351S andrPPADNtx was measured in the BAEE assay. Average citrulline concentrations (in μM) from two independent experiments are shown (error barsindicate SD). Enzyme activities were analysed by two-way Analysis of variance (ANOVA) (rPPADNtx vs rPPAD; *p<0.0001). (C) Mass spectrometrysequence coverage map of full-length rPPAD expressed in E coli. Peptide sequences identified by mass spectrometry are shown in blue; confirmedcitrullination sites in pink. Arginine residues are underlined. (D) Localisation probabilities for individual arginine deimination sites in rPPAD werecalculated using the Ascore algorithm. An Ascore of 20 (99% certainty; p=0.01) was considered statistically significant.38

Figure 1 Cellular PPAD (cPPAD) and secreted PPAD (sPPAD) from Porphyromonas gingivalis are truncated and not citrullinated. (A) P gingivaliscell lysate (CL) and culture supernatant (CS) were analysed by SDS-PAGE. Ponceau staining before antibody probing is shown to visualise loading(left panel). Anti-PPAD immunoblotting shows cPPAD (black arrowhead) and sPPAD (white arrowhead) in P gingivalis CL and CS, respectively(middle panel). Anti-modified citrulline (AMC) immunoblotting was used to detect citrullination in bacterial samples (right panel). (B and C) Massspectrometry sequence coverage map of P gingivalis cPPAD (B) and sPPAD (C). Peptide sequences identified are shown in blue; the N-terminal andC-terminal domains missing in cPPAD and sPPAD are shown in grey. Arginine residues are underlined. Numbered arrows indicate possible PPADtruncation sites at R43 and K459.

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Anti-PPAD antibodies in RA are not directed againstcitrullinated PPADWhile we found no evidence that PPAD is citrullinated in P gin-givalis, antibodies specific for citrullinated rPPAD have previ-ously been reported in patients with RA.12 To dissect the role ofcitrullination for anti-PPAD antibody recognition, we assayedserum samples of patients with RA and healthy controls for anti-bodies to cit-rPPAD and uncitrullinated rPPADC351S by ELISA.IgG anti-PPAD antibodies were common in patients with RAand in healthy controls (figure 3A). Strikingly, citrullination didnot heighten antibody reactivity to PPAD. Antibody levels tocit-rPPAD versus uncitrullinated rPPADC351S did not differ sig-nificantly among patients with RA (median 0.069 vs 0.070;median of differences: 0.0; p=0.14) or controls (median 0.165vs 0.170; median of differences: −0.001; p=0.87) (figure 3Aand online supplementary figure S2). Indeed, anti-cit-rPPADand anti-rPPADC351S antibody levels in RA were highly corre-lated (r=0.99, *p<0.0001) (figure 3B). Control subjects demon-strated a similar degree of correlation (r=0.99, *p<0.0001).

Importantly, levels of anti-cit-rPPAD were not significantlydifferent between patients with RA and controls (median 0.069vs 0.165; p=0.085). Although the highest antibody reactivity

was observed in the RA group (5/83 above the 95th centile)compared with controls (1/39), statistical significance to supportthis association was not attained (Fisher’s exact test; p=0.66).In fact, the overall trend observed was towards higheranti-PPAD antibody levels in the control group (figure 3A). Toconfirm the specificity of the ELISA assay for anti-PPAD, anti-bodies were additionally demonstrated by immunoprecipitationusing radiolabelled PPAD generated by in vitro transcription/translation (IVTT) (figure 3C). In agreement with the ELISAassay, sera of patients with RA showed similar reactivity againstPPAD and mutant PPADC351S by immunoprecipitation (figure3C, top and middle panels, respectively). Importantly, only serareactive by ELISA immunoprecipitated radiolabelled PPAD(figure 3C, anti-PPAD+ vs anti-PPAD-). Similarly, radiolabelledtruncated PPADNtx (figure 3C, bottom panel), a protein thatbetter mimics PPAD in P gingivalis, was only precipitated bysera positive by ELISA. These data support the specificity of theELISA assay and strongly suggest that anti-PPAD antibodybinding in RA is independent of PPAD citrullination.

To confirm that citrullination is not a determinant in the anti-body recognition of rPPAD, we further performed competitionexperiments. Antibody binding to cit-rPPAD was completely

Figure 3 Antibodies to citrullinated rPPAD (cit-PPAD) and uncitrullinated rPPAD (PPADC351S) in serum of patients with rheumatoid arthritis (RA)and controls. (A) Patients with RA (n=83) and controls (n=39) were assayed for antibodies to cit-rPPAD (full circles) and rPPADC351S (empty circles)by ELISA. Antibody reactivity is expressed as arbitrary units (AU). Statistical analysis was performed using the Wilcoxon matched-pairs signed-ranktest (anti-cit-rPPAD vs anti-rPPADC351S) and Mann-Whitney U test (RA vs control subjects). The red line represents the median reactivity forindividual groups. ns: not significant. (B) Correlation of anti-cit-rPPAD and anti-rPPADC351S antibody levels in patients with RA (r=0.99, p<0.0001).(C) 35S-methionine-labelled PPAD (top panel), PPADC351S (middle panel) and truncated PPADNtx (bottom panel) generated by IVTT wereimmunoprecipitated using serum of patients with RA. Immunoprecipitates were resolved by SDS-PAGE and visualised by fluorography. Representativeresults for anti-rPPAD positive (anti-PPAD+) and anti-rPPAD negative (anti-PPAD−) patients, as determined by ELISA, are shown.

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abrogated by preincubation of anti-cit-rPPAD positive RA serawith uncitrullinated rPPADC351S, but not by preincubation withunmodified or citrullinated enolase (figure 4A,B). This demon-strates that anti-PPAD antibodies in RA are exclusively directedagainst unmodified PPAD.

Anti-PPAD antibodies do not correlate with anti-CCP levelsor RA disease activityTo further characterise their relevance in RA, we inquired howanti-PPAD antibodies relate to anti-cyclic citrullinated peptide(anti-CCP) levels and disease activity measures. Anti-CCP3 anti-bodies in RA did not correlate with anti-cit-rPPAD oranti-rPPADC351S levels (r=0.07, p=0.56; r=0.09, p=0.44;respectively) (figure 5A). Moreover, we found no significantassociation between anti-rPPAD antibody levels and RA diseaseactivity as measured using DAS28-C reactive protein (r=−0.11,p=0.30) (figure 5B).

Anti-PPAD antibody levels are decreased in patients withRA that have PDTo elucidate the role of anti-PPAD antibodies in RA-associatedPD, we investigated the PD status in RA and healthy controls(clinical characteristics summarised in table 1). PD was morecommon among patients with RA compared with age-matchedand sex-matched controls (60/83; 72.3% vs 9/39; 23.1%).Interestingly, anti-rPPAD antibody levels were significantly lowerin patients with RA that have PD compared with periodontallyhealthy controls (median 0.058 vs 0.162; *p=0.046).Analogously, anti-rPPAD levels were decreased in PD-positivepatients with RA when compared with PD-negative patientswith RA (median 0.058 vs 0.162). However, this analysis wasunderpowered and did not reach statistical significance(p=0.073) (figure 5C). Moreover, a significant decline in anti-body levels with RA disease duration was observed in thePD-positive RA group (r=−0.28, *p=0.03) (figure 5D), but not

in the PD-negative RA group (r=−0.01, p=0.97; data notshown). Although the duration of PD in these groups isunknown, the negative association with RA-associated PD maysuggest a protective role of anti-PPAD antibodies for periodon-tal health.

DISCUSSIONPPAD autocitrullination has provided an intriguing frameworkto explain the loss of tolerance to citrullinated proteins inRA. In this study, we show that PPAD expressed by P gingivalisis not citrullinated, and demonstrate that PPAD citrullinationis not recognised by anti-PPAD antibodies in RA. Thesefindings have critical implications for the biology of PPADand redefine the relevance of the anti-PPAD response in RA.

Despite the difference in molecular weight, cellular PPAD(75–85 kDa) and secreted PPAD (47 kDa) were indistinguish-able in protein sequence. MS identified both as a truncatedform of PPAD missing the first 43 N-terminal and 97C-terminal amino acids. Full-length PPAD was not detectedin our analysis, suggesting that similar to proforms of other Pgingivalis virulence factors, this proenzyme is rapidly pro-cessed by truncation and concomitantly translocated to thebacterial cell surface.27 39 40 As such, glycosylation of cPPADfor membrane attachment may be sufficient to explain thestriking difference in molecular weight to sPPAD,29 as previ-ously shown for arginine gingipain.41 The truncation sitesidentified in this study may indicate specific processing by Pgingivalis envelope-associated cysteine proteases witharginine-X (at R43) and lysine-X activity (at K459).42

Truncation at R43 has previously been shown for sPPAD byN-terminal sequencing9 and MS.27 While we cannot excludethat truncation at K459 is an artefact of tryptic digestion,semitryptic peptides indicative of alternative cleavage siteswere not detected.

Figure 4 Anti-PPAD antibodies in rheumatoid arthritis (RA) are exclusively directed against uncitrullinated PPAD. (A) Antibody reactivity againstcit-rPPAD was measured after preincubation of anti-cit-rPPAD positive RA sera (n=8) with buffer alone (column 1), purified enolase (column 2),citrullinated enolase (cit-enolase) (column 3) or uncitrullinated rPPADC351S (column 4; ***p<0.001). Comparisons were made using Friedman’s test/Dunn’s post test for matched groups. Column height shows mean antibody reactivity expressed as arbitrary units (AU); bars indicate SEM. ns: notsignificant. (B) Anti-modified citrulline (AMC) immunoblotting to demonstrate the presence or absence of citrullination in proteins used in A. Purifiedcit-rPPAD, rPPADC351S, enolase and cit-enolase were resolved by SDS-PAGE, transferred onto nitrocellulose and visualised by Ponceau staining as aloading control (top panel). Protein citrullination in the same membrane was detected by AMC immunoblotting (bottom panel).

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Considering full-length rPPAD is readily autocitrullinatedwhen expressed in E coli,12 the lack of citrullinated PPAD in Pgingivalis is astounding. Unique strategies, which are absent in Ecoli, may have evolved in P gingivalis to prevent PPAD autoci-trullination. In this study, we provide evidence that N-terminalprocessing may be the primary mechanism by which PPAD isprotected from autocitrullination. Indeed, PPAD citrullinationmay be biologically unfavourable for P gingivalis. Rodríguezet al30 reported that PPAD activity declines with enzyme autoci-trullination, and citrullination of R352 adjacent to the activesite of PPAD (C351) has been hypothesised to explain thisreduction in enzyme activity.12 30 36 Mechanisms that blockPPAD autocitrullination in P gingivalis may therefore conservePPAD activity. The molecular basis by which N-terminal trunca-tion abrogates PPAD autocitrullination is unclear. While PPADis generally restricted to modify only free arginine andC-terminal arginine residues in cleaved proteins,9 11 12 PPADautocitrullination involves citrullination of internal arginine resi-dues in the intact enzyme. The N-terminal domain may there-fore facilitate peptidylarginine autocitrullination by uniquelyexpanding the catalytic function of full-length PPAD. Rapid

removal of the N-terminal domain by P gingivalis may renderautocitrullination insufficient and aid bacterial survival in theperiodontal pocket.

The finding that anti-PPAD antibodies in RA do not targetcitrullinated PPAD is consistent with the biology of PPAD identi-fied in this study. However, our findings markedly contrast theconclusions reached by another study in which antibodies specificfor citrullinated full-length rPPAD were identified in 38% ofpatients with RA.12 The discrepancy between the two studiesmay be explained by differences in ELISA methodology, inpatient cohorts studied, or in the rPPAD fusion proteins used. Wetried to minimise error and exclude plate variability by measuringantibodies to cit-rPPAD, rPPADC351S and background reactivityon the same plate. Using competition assays, we show thatanti-PPAD antibodies in patients with RA are exclusively directedagainst the unmodified enzyme. We cannot fully exclude majordifferences in the populations studied. Lastly, there is a significantdifference in the recombinant proteins used in our study.Previously, glutathione S-transferase (GST)-His-tagged rPPADwas used to screen for anti-PPAD antibodies.12 The GST tag(25 kDa) adds nine additional arginine residues to the fusion

Figure 5 Anti-PPAD levels do not correlate with anti-cyclic citrullinated peptide (anti-CCP) antibodies or rheumatoid arthritis (RA) disease activity,but are negatively associated with periodontal disease in RA. (A) Correlation of anti-PPAD antibodies with anti-CCP3 levels in RA. Antibody levels toCCP3 were measured by ELISA (QUANTA Lite IgG, INOVA). (B) Correlation of anti-PPAD levels with RA disease activity as measured using DAS28-Creactive protein. ns: not significant. (C) Anti-PPAD antibody levels in patients with RA with and without periodontal disease (RA PD and RA No PD,respectively; full circles) compared with periodontally healthy controls (empty circles). Antibody levels are expressed in arbitrary units (red line showsmedian reactivity; *p<0.05). (D) Antibody levels in patients with PD-positive RA as a function of RA disease duration in years.

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protein, which may potentially be citrullinated in the process ofGST-His-rPPAD autocitrullination. By contrast, rPPAD in ourstudy was designed without GST tag and only contains one add-itional arginine in the His-tag used (2.5 kDa). Thus, we cannotexclude that the antibodies identified in the previous study mayin fact target citrullinated GST in the fusion protein. Such anti-bodies would not be identified when screening against enzymati-cally inactive GST-His-rPPAD. Nonetheless, the existence ofcitrullinated-PPAD-specific antibodies in RA cannot be sup-ported, and appears implausible in the absence of PPAD citrulli-nation in P gingivalis.

While anti-PPAD antibodies are common in patients with RA(and healthy controls), the significance of the antibody responseagainst this bacterial virulence factor for RA-associated PD is notclear. In our study, anti-PPAD antibodies did not correlate withdisease activity or anti-CCP levels, reinforcing that anti-PPADantibodies are not part of the ACPA-response. Antibodies to Pgingivalis cell extracts have been reported to correlate withanti-CCP antibodies and disease activity measures in RA,19 43–45

suggesting that anti-PPAD antibodies may represent a uniqueantibody population. Despite this association, anti-P gingivalisantibodies do not distinguish RA from controls cases,45 and haveno predictive value for the development of RA in seropositivepatients with arthralgia.46 In this context, it is compelling thatanti-PPAD levels are significantly lower in RAwith PD comparedwith periodontally healthy controls. Anti-PPAD antibodies mayhave a protective role for PD in RA, and clearance of PPAD maydecrease survival of P gingivalis in the periodontal pocket by lim-iting ammonia production. The finding that antibodies againstmajor P gingivalis virulence factors confer protection in murinemodels of periodontal infection and potentially human diseasefurther supports this possibility.18 47–50

Our findings suggest that PPAD autocitrullination is not theunderlying mechanism linking P gingivalis-associated PD andRA. Anti-PPAD antibodies may be useful markers to predict therisk of PD progression in patients with RA.

Acknowledgements The authors thank Lauren DeVine and Robert N Cole (MassSpectrometry and Proteomics Facility, The Johns Hopkins University School of

Medicine) for assistance with the citrullination site analyses, Mark A Reynolds andMark E Shirtliff (University of Maryland School of Dentistry, Baltimore, Maryland,USA) for providing bacterial cultures, and Sharon R Ghazarian (Biostatistics Core,The Johns Hopkins University School of Medicine) and Janelle M Montagne (Divisionof Rheumatology, The Johns Hopkins University School of Medicine) for criticalreview of the manuscript.

Contributors Each author has contributed to one or more of the following aspectsof the manuscript—conception and design, acquisition, analysis and interpretationof data, drafting and revising the article.

Funding The study was supported by Grant Number P30AR053503 from theNational Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS). Thecontent is solely the responsibility of the authors and does not necessarily representthe official views of NIAMS or the National Institutes of Health. Additional fundingwas provided by the Rheumatology Research Foundation (American College ofRheumatology Research and Education Foundation, Within Our Reach), The DonaldB and Dorothy L Stabler Foundation, the Mackley Fund from Sibley MemorialHospital, and The Johns Hopkins Arthritis Center Research Fund.

Competing interests None.

Ethics approval Use of stored/coded serum was approved by the Johns HopkinsMedicine Institutional Review Board.

Provenance and peer review Not commissioned; externally peer reviewed.

Data sharing statement All data relevant for this study have been included inthe manuscript. Any additional data referred to as not shown will be availablethrough direct contact with the corresponding author. Reagents derived from thisproject will be available upon request through material transfer agreements.

REFERENCES1 Imboden JB. The Immunopathogenesis of Rheumatoid Arthritis. Annu Rev Pathol

Mech Dis 2009;4:417–34.2 Aletaha D, Neogi T, Silman AJ, et al. 2010 Rheumatoid arthritis classification

criteria: an American College of Rheumatology/European League AgainstRheumatism collaborative initiative. Arthritis Rheum 2010;62:2569–81.

3 Arend WP, Firestein GS. Pre-rheumatoid arthritis: predisposition and transition toclinical synovitis. Nat Rev Rheumatol 2012;8:573–86.

4 Schellekens GA, de Jong BA, van den Hoogen FH, et al. Citrulline is an essentialconstituent of antigenic determinants recognized by rheumatoid arthritis-specificautoantibodies. J Clin Invest 1998;101:273–81.

5 Nishimura K, Sugiyama D, Kogata Y, et al. Meta-analysis: diagnostic accuracy ofanti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoidarthritis. Ann Intern Med 2007;146:797–808.

6 Van de Stadt LA, de Koning MHMT, van de Stadt RJ, et al. Development of theanti-citrullinated protein antibody repertoire prior to the onset of rheumatoidarthritis. Arthritis Rheum 2011;63:3226–33.

7 Arita K, Hashimoto H, Shimizu T, et al. Structural basis for Ca(2+)-inducedactivation of human PAD4. Nat Struct Mol Biol 2004;11:777–83.

8 Shirai H, Mokrab Y, Mizuguchi K. The guanidino-group modifying enzymes:structural basis for their diversity and commonality. Proteins 2006;64:1010–23.

9 McGraw WT, Potempa J, Farley D, et al. Purification, characterization, and sequenceanalysis of a potential virulence factor from Porphyromonas gingivalis,peptidylarginine deiminase. Infect Immun 1999;67:3248–56.

10 Rosenstein ED, Greenwald RA, Kushner LJ, et al. Hypothesis: the humoral immuneresponse to oral bacteria provides a stimulus for the development of rheumatoidarthritis. Inflammation 2004;28:311–18.

11 Wegner N, Wait R, Sroka A, et al. Peptidylarginine deiminase from Porphyromonasgingivalis citrullinates human fibrinogen and α-enolase: Implications forautoimmunity in rheumatoid arthritis. Arthritis Rheum 2010;62:2662–72.

12 Quirke A-M, Lugli EB, Wegner N, et al. Heightened immune response toautocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potentialmechanism for breaching immunologic tolerance in rheumatoid arthritis. AnnRheum Dis 2014;73:263–9.

13 Bingham CO III, Moni M. Periodontal disease and rheumatoid arthritis: the evidenceaccumulates for complex pathobiologic interactions. Curr Opin Rheumatol2013;25:345–53.

14 Page RC, Eke PI. Case definitions for use in population-based surveillance ofperiodontitis. J Periodontol 2007;78:1387–99.

15 De Pablo P, Dietrich T, McAlindon TE. Association of periodontal disease and toothloss with rheumatoid arthritis in the US population. J Rheumatol 2008;35:70–6.

16 Pischon N, Pischon T, Kröger J, et al. Association among rheumatoid arthritis, oralhygiene, and periodontitis. J Periodontol 2008;79:979–86.

17 Potikuri D, Dannana KC, Kanchinadam S, et al. Periodontal disease is significantlyhigher in non-smoking treatment-naive rheumatoid arthritis patients: results from acase-control study. Ann Rheum Dis 2012;71:1541–4.

18 Scher JU, Ubeda C, Equinda M, et al. Periodontal disease and the oral microbiotain new-onset rheumatoid arthritis. Arthritis Rheum 2012;64:3083–94.

Table 1 Demographic and clinical characteristics of patients withrheumatoid arthritis (RA) and controls

Parameter RA (83) Controls (39) p Value*

Female (%) 79.5 (66) 82.1 (32) 0.74Mean age in years (±SD) 50.7 (±12.9) 48.1 (±13.3) 0.31Race/ethnicity 0.00Caucasian (%) 74.7 (62) 33.3 (13)African-American (%) 16.9 (14) 35.9 (14)

Smoking (%)† 16.9 (14) 12.8 (5) 0.56Periodontal disease (%) 72.3 (60) 23.1 (9) 0.00Mean RA disease duration in years (±SD) 6.3 (±7.9)Seropositive for IgM RF (%) 68.5 (50)Seropositive for IgG anti-CCP3 (%) 75.9 (63)Mean CRP in mg/L (±SD) 6.9 (±11.3)Mean DAS28-CRP (±SD) 3.5 (±1.4)Mean CDAI (±SD) 15.6 (±14.0)

*Comparison of parameters reflected in percentages: χ2 test for proportions,comparison of means: independent-samples t test.†Current cigarette smoking.anti-CCP3, anti-cyclic citrullinated peptide (third-generation), cut-off level for positivity20 units; CDAI, Clinical Disease Activity Index; CRP, C reactive protein; DAS28,Disease Activity Score in 28 joints; RF, rheumatoid factor, cut-off level for positivity20 IU.

Konig MF, et al. Ann Rheum Dis 2014;0:1–8. doi:10.1136/annrheumdis-2014-205385 7

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19 de Smit M, Westra J, Vissink A, et al. Periodontitis in established rheumatoidarthritis patients: a cross-sectional clinical, microbiological and serological study.Arthritis Res Ther 2012;14:R222.

20 Socransky SS, Haffajee AD, Cugini MA, et al. Microbial complexes in subgingivalplaque. J Clin Periodontol 1998;25:134–44.

21 Griffen AL, Lyons SR, Becker MR, et al. Porphyromonas gingivalis Strain Variabilityand Periodontitis. J Clin Microbiol 1999;37:4028–33.

22 Griffen AL, Beall CJ, Campbell JH, et al. Distinct and complex bacterial profiles inhuman periodontitis and health revealed by 16S pyrosequencing. ISME J2012;6:1176–85.

23 Marquis RE, Bender GR, Murray DR, et al. Arginine deiminase system and bacterialadaptation to acid environments. Appl Environ Microbiol 1987;53:198–200.

24 Casiano-Colón A, Marquis RE. Role of the arginine deiminase system in protectingoral bacteria and an enzymatic basis for acid tolerance. Appl Environ Microbiol1988;54:1318–24.

25 Niederman R, Brunkhorst B, Smith S, et al. Ammonia as a potential mediator ofadult human periodontal infection: inhibition of neutrophil function. Arch Oral Biol1990;35(Suppl):205S–9S.

26 Shawcross DL, Wright GAK, Stadlbauer V, et al. Ammonia impairs neutrophilphagocytic function in liver disease. Hepatol Baltim Md 2008;48:1202–12.

27 Sato K, Yukitake H, Narita Y, et al. Identification of Porphyromonas gingivalisproteins secreted by the Por secretion system. FEMS Microbiol Lett2013;338:68–76.

28 Slakeski N, Seers CA, Ng K, et al. C-Terminal Domain Residues Important forSecretion and Attachment of RgpB in Porphyromonas gingivalis. J Bacteriol2011;193:132–42.

29 Shoji M, Sato K, Yukitake H, et al. Por Secretion System-Dependent Secretion andGlycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35. PLoS ONE2011;6:e21372.

30 Rodríguez SB, Stitt BL, Ash DE. Expression of peptidylarginine deiminase fromPorphyromonas gingivalis in Escherichia coli: Enzyme purification andcharacterization. Arch Biochem Biophys 2009;488:14–22.

31 Lundberg K, Kinloch A, Fisher BA, et al. Antibodies to citrullinated alpha-enolasepeptide 1 are specific for rheumatoid arthritis and cross-react with bacterial enolase.Arthritis Rheum 2008;58:3009–19.

32 Lundberg K, Wegner N, Yucel-Lindberg T, et al. Periodontitis in RA-the citrullinatedenolase connection. Nat Rev Rheumatol 2010;6:727–30.

33 Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum1988;31:315–24.

34 Eke PI, Page RC, Wei L, et al. Update of the case definitions for population-basedsurveillance of periodontitis. J Periodontol 2012;83:1449–54.

35 Senshu T, Sato T, Inoue T, et al. Detection of citrulline residues in deiminatedproteins on polyvinylidene difluoride membrane. Anal Biochem 1992;203:94–100.

36 Rodríguez SB, Stitt BL, Ash DE. Cysteine 351 is an essential nucleophile in catalysisby Porphyromonas gingivalis peptidylarginine deiminase. Arch Biochem Biophys2010;504:190–6.

37 Andrade F, Darrah E, Gucek M, et al. Autocitrullination of human peptidyl argininedeiminase type 4 regulates protein citrullination during cell activation. ArthritisRheum 2010;62:1630–40.

38 Beausoleil SA, Villén J, Gerber SA, et al. A probability-based approach forhigh-throughput protein phosphorylation analysis and site localization. NatBiotechnol 2006;24:1285–92.

39 Glew MD, Veith PD, Peng B, et al. PG0026 Is the C-terminal Signal Peptidase of aNovel Secretion System of Porphyromonas gingivalis. J Biol Chem2012;287:24605–17.

40 Veith P, Talbo G, Slakeski N, et al. Major outer membrane proteins and proteolyticprocessing of RgpA and Kgp of Porphyromonas gingivalis W50. Biochem J2002;363:105–15.

41 Curtis MA, Thickett A, Slaney JM, et al. Variable carbohydrate modifications to thecatalytic chains of the RgpA and RgpB proteases of Porphyromonas gingivalis W50.Infect Immun 1999;67:3816–23.

42 Veith PD, Talbo GH, Slakeski N, et al. Major outer membrane proteins andproteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50. BiochemJ 2002;363:105–15.

43 Mikuls TR, Payne JB, Reinhardt RA, et al. Antibody responses to Porphyromonasgingivalis (P. gingivalis) in subjects with rheumatoid arthritis and periodontitis. IntImmunopharmacol 2009;9:38–42.

44 Arvikar SL, Collier DS, Fisher MC, et al. Clinical correlations with Porphyromonasgingivalis antibody responses in patients with early rheumatoid arthritis. ArthritisRes Ther 2013;15:R109.

45 Mikuls TR, Payne JB, Yu F, et al. Periodontitis and Porphyromonas gingivalis inPatients with Rheumatoid Arthritis. Arthritis Rheum 2014;66:1090–100.

46 de Smit M, van de Stadt LA, Janssen KMJ, et al. Antibodies against Porphyromonasgingivalis in seropositive arthralgia patients do not predict development ofrheumatoid arthritis. Ann Rheum Dis 2014;73:1277–9.

47 Genco CA, Odusanya BM, Potempa J, et al. A peptide domain on gingipain Rwhich confers immunity against Porphyromonas gingivalis infection in mice. InfectImmun 1998;66:4108–14.

48 Kuboniwa M, Amano A, Shizukuishi S, et al. Specific antibodies to Porphyromonasgingivalis Lys-gingipain by DNA vaccination inhibit bacterial binding to hemoglobinand protect mice from infection. Infect Immun 2001;69:2972–9.

49 Gibson FC, Genco CA. Prevention of Porphyromonas gingivalis-Induced Oral BoneLoss following Immunization with Gingipain R1. Infect Immun 2001;69:7959–63.

50 Sweier DG, Shelburne PS, Giannobile WV, et al. Immunoglobulin G (IgG) Class, butNot IgA or IgM, Antibodies to Peptides of the Porphyromonas gingivalis ChaperoneHtpG Predict Health in Subjects with Periodontitis by a Fluorescence Enzyme-LinkedImmunosorbent Assay. Clin Vaccine Immunol CVI 2009;16:1766–73.

8 Konig MF, et al. Ann Rheum Dis 2014;0:1–8. doi:10.1136/annrheumdis-2014-205385

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doi: 10.1136/annrheumdis-2014-205385 published online May 26, 2014Ann Rheum Dis

 Maximilian F Konig, Alizay S Paracha, Malini Moni, et al. study of PPAD biology(PPAD) in rheumatoid arthritis through the

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