Defining the role of Porphyromonas gingivalis peptidylarginine deiminase (PPAD) in rheumatoid arthritis through the study of PPAD biology

Abstract

Background: Antibodies to citrullinated proteins are a hallmark of rheumatoid arthritis (RA). Porphyromonas gingivalis peptidylarginine deiminase (PPAD) has been implicated in the initiation of RA by generating citrullinated neoantigens and due to its ability to autocitrullinate.

Objectives: To define the citrullination status and biology of PPAD in P gingivalis and to characterise the anti-PPAD antibody response in RA and associated periodontal disease (PD).

Methods: PPAD in P gingivalis cells and culture supernatant were analysed by immunoblotting and mass spectrometry to detect citrullination. Recombinant PPAD (rPPAD), inactive mutant PPAD (rPPAD(C351S)), and N-terminal truncated PPAD (rPPAD(Ntx)) were cloned and expressed in Escherichia coli. Patients with RA and healthy controls were assayed for IgG antibodies to citrullinated rPPAD and unmodified rPPAD(C351S) by ELISA. Anti-PPAD antibodies were correlated with anti-cyclic citrullinated peptide (third-generation) antibody levels, RA disease activity and PD status.

Results: PPAD from P gingivalis is truncated at the N-terminal and C-terminal domains and not citrullinated. Only when artificially expressed in E coli, full-length rPPAD, but not truncated (fully active) rPPAD(Ntx), is autocitrullinated. Anti-PPAD antibodies show no heightened reactivity to citrullinated rPPAD, but are exclusively directed against the unmodified enzyme. Antibodies against PPAD do not correlate with anti-cyclic citrullinated peptide levels and disease activity in RA. By contrast, anti-PPAD antibody levels are significantly decreased in RA patients with PD.

Conclusions: PPAD autocitrullination is not the underlying mechanism linking PD and RA. N-terminal processing protects PPAD from autocitrullination and enhances enzyme activity. Anti-PPAD antibodies may have a protective role for the development of PD in patients with RA.

Keywords: Ant-CCP; Autoimmune Diseases; Rheumatoid Arthritis.

Figure 1

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

Figure 2

Citrullination of rPPAD expressed in E coli is dependent on the N-terminal domain. (A) Purified rPPAD, mutant rPPAD^C351S (C351S) and truncated rPPAD missing the N-terminal domain (Ntx) were analyzed by SDS-PAGE and visualized by anti-PPAD immunoblotting (top panel). AMC immunoblotting (bottom panel) was used to detect citrullination. (B) Enzymatic activity of rPPAD, rPPAD^C351S and rPPAD^Ntx was measured in the BAEE assay. Average citrulline concentrations (in μM) from two independent experiments are shown (error bars indicate standard deviation). Enzyme activities were analyzed by two-way ANOVA (rPPAD^Ntx vs. rPPAD; *p<0.0001). (C) Mass spectrometry sequence coverage map of full-length rPPAD expressed in E coli. Peptide sequences identified by MS are shown in blue; confirmed citrullination sites in pink. Arginine residues are underlined. (D) Localization probabilities for individual arginine deimination sites in rPPAD were calculated using the Ascore algorithm. An Ascore of 20 (99% certainty; p=0.01) was considered significant.

Figure 3

Antibodies to citrullinated rPPAD (cit-rPPAD) and uncitrullinated PPAD (rPPAD^C351S) in serum of patients with RA and controls. (A) RA patients (n=83) and controls (n=39) were assayed for antibodies to cit-rPPAD (full circles) and rPPAD^C351S (empty circles) by ELISA. Antibody reactivity is expressed as arbitrary units (AU). Statistical analysis was performed using the Wilcoxon matched-pairs signed-rank test (anti-citPPAD vs. anti-PPAD^C351S) and Mann-Whitney test (RA vs. control subjects). The red line represents the median reactivity for individual groups. ns: not significant. (B) Correlation of anti-cit-rPPAD and anti-rPPAD^C351S antibody levels in patients with RA (r=0.99, p<0.0001). (C) ³⁵S-methionine-labeled PPAD (top panel), PPAD^C351S (middle panel) and truncated PPAD^Ntx (bottom panel) generated by IVTT were immunoprecipitated using RA patient serum. Immunoprecipitates were electrophoresed on SDS-PAGE and visualized by fluorography. Representative results for anti-rPPAD positive (anti-PPAD+) and anti-rPPAD negative (anti-PPAD-) patients, as determined by ELISA, are shown.

Figure 4

Anti-PPAD antibodies in RA are exclusively directed against uncitrullinated PPAD. (A) Antibody reactivity against cit-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 rPPAD^C351S (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: not significant. (B) AMC immunoblotting to demonstrate the presence or absence of citrullination in proteins used in A. Purified cit-rPPAD, rPPAD^C351S, enolase and cit-enolase were resolved by SDS-PAGE, transferred onto nitrocellulose and visualized by Ponceau staining as a loading control (top panel). Protein citrullination in the same membrane was detected by AMC immunoblotting (bottom panel).

Figure 5

Anti-PPAD levels do not correlate with anti-CCP antibodies or 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 to CCP3 were measured by ELISA (QUANTA Lite IgG, INOVA). (B) Correlation of anti-PPAD levels with RA disease activity as measured using DAS28-CRP. ns: not significant. (C) Anti-PPAD antibody levels in RA patients with and without periodontal disease (RA PD and RA No PD, respectively; full circles) compared to periodontally healthy controls (empty circles). Antibody levels are expressed in arbitrary units (red line shows median reactivity; *p<0.05). (D) Antibody levels in PD-positive RA patients as a function of RA disease duration in years.