Positions of arginine residues within the three-dimensional structure of the human G1 domain and detection of citrullinated rhG1.

<p>(<b>A</b>) 3-D images of the A, B, and B’ loops of the G1 domain illustrate the location of arginine (R) residues (red-yellow balls with numbers) and the two previously reported immunodominant epitopes (sequences highlighted), both of which contain arginine residues that may become citrulline (R/Cit) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150784#pone.0150784.ref011" target="_blank">11</a>][<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150784#pone.0150784.ref012" target="_blank">12</a>][<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150784#pone.0150784.ref013" target="_blank">13</a>]. The three loops of G1 were rotated relative to each other using RasMol software in order to expose the R-rich surfaces. The amino acid sequence is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150784#pone.0150784.s001" target="_blank">S1 Fig</a>. (<b>B</b>) <i>In vitro</i> citrullination of rhG1 was performed using PAD4 enzyme. The native (lanes 1) and PAD4-treated (lanes 2) rhG1 proteins were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes. Two of the membranes were probed with either anti-hG1 mAb: (first panel) or with ACPA+ RA serum (second panel). The citrulline residues present in the same proteins on the third membrane were subjected to chemical modification and then probed with an Ab specific for chemically-modified citrulline (anti-modif. Cit Ab, third panel). Native and PAD4-treated rhG1 proteins were also reacted with citrulline-specific phenylglyoxal conjugated with rhodamine (Rhod-Phe-Gly) and subjected to SDS-PAGE (fourth panel). While the anti-hG1 mAb reacted with both the native and citrullinated forms of the protein, only the citrullinated hG1 was detected by ACPA, the anti-modified citrulline Ab, and the phenylglyoxal probe.</p

Western blots to identify PG domains and fragments of OA cartilage extract recognized by ACPA-positive serum.

<p>The crude extract of OA cartilage (shown as sample 4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150784#pone.0150784.g002" target="_blank">Fig 2</a>) was loaded onto 8% SGS-PAGE gel and stained with toluidine blue (TB) to visualize PG GAG chains. Essentially all of the TB-positive PG material remained in the stacking gel. To facilitate resolution, chondroitin sulfate chains were removed by digestion with chondroitinase ABC, and aliquots of the deglycosylated extract was loaded onto 6 lanes of a SDS-PAGE gel. Coomassie blue (CB) staining of the gel (lane 1) showed good resolution of the proteins of the OA cartilage extract after deglycosylation. Following transfer onto a nitrocellulose membrane, vertical strips of the membrane were probed with human sera or PG-specific antibodies (lanes 2–6). Immunostaining with ACPA- (normal) serum followed by anti-human IgG-HRP revealed a single protein band most likely corresponding to the heavy chain of contaminating IgG (lane 2). The ACPA+ serum detected several additional bands (lane 3). To identify these bands, replicate strips of the membrane were probed with antibodies against the G1 or G3 domain of PG and a pair of antibodies recognizing protease-generated PG neoepitopes. The respective antibodies showed reactions with the G1 (lane 4), G3 (lane 5) as well as with the neoepitopes -NITEGE and -VDIPEN (lane 6). There were additional bands above 55 kDa (depicted with asterisks in lane 3) that could not be identified as PG fragments. One representative sample of over 10 Western blots (using different crude extracts and ACPA+ sera) is shown.</p

Immunohistochemical localization of ACPA-reactive (citrullinated) epitopes in OA and RA cartilage sections.

<p>(<b>A-D</b>) Frozen sections of OA knee (tibial plateau) cartilage were immunostained with (<b>A</b>) Texas red-labeled anti-human IgG (anti-hIgG-TR), (<b>B</b>) normal (ACPA-negative) human serum followed by anti-hIgG-TR, (<b>C</b>) ACPA+ serum (RA#9) followed by anti-hIgG-TR, or (<b>D</b>) a biotinylated anti-human G1 antibody followed by Alexa Fluor 488-labeled streptavidin (SA-AF488). (<b>E-H</b>) Frozen sections prepared from the RA tibial plateau cartilage were immunostained with the same sera and antibodies as listed for <b>A-D</b>. Cell nuclei in all sections were visualized by DAPI staining. (<b>A</b> and <b>E</b>) Anti-hIgG-TR alone did not stain the sections, and (<b>B</b> and <b>F</b>) negligible reaction (red fluorescence) was observed when the tissues were first stained with ACPA- serum. (<b>C</b>) The ACPA+ serum primarily stained the chondrocyte pericellular matrix in the OA cartilage, but (<b>G</b>) it diffusely stained the entire matrix of the RA cartilage. Similar staining patters to those with ACPA+ serum were observed when (<b>D</b>) the OA and (<b>H</b>) RA cartilage sections were incubated with biotinylated anti-hG1 mAb (green fluorescence), suggesting at least partial co-localization of PG G1 and citrullinated epitopes in both OA and RA cartilage.</p

Recognition of PG in crude extracts of osteoarthritis (OA) and rheumatoid arthritis (RA) cartilage specimens by ACPA and PG-specific antibodies.

<p>Dots of crude extracts of cartilage from OA donors (row A, samples 1–5) and RA donors (samples 6–10) were applied to a nitrocellulose membrane strip. Purified human PG and CII (far left and far right dots, respectively) served as controls. Blotting with the secondary antibody (anti-human IgG-HRP) revealed positive reactions (row A) with all crude extracts, but not with purified hPG or hCII. The “positive” reaction disappeared after the contaminating IgG was removed from the crude extracts (row B). These IgG-free cartilage extracts were used for subsequent dot blots. The ACPA+#20 serum (row A) reacted with all cartilage extracts and purified PG (row C). Similarly, the G1 domain-specific mAb recognized purified PG and PG in the crude extracts (row D), but no reaction was detected when the G1 domain was removed by immune absorption (row E). The reactivity of the cartilage extracts with ACPA+#20 serum (row F) was nearly completely lost after G1 domain immunodepletion (row G). As demonstrated by the anti-chondroitin 4-sulfate (C4S)-specific antibody, PG in all extracts and in the purified PG sample were glycosylated (row H). Some extracts and the purified PG contained small amounts of the PG G3 domain (row I), and all crude extracts contained cartilage-specific CII (row J).</p

Cit:R ratios of IL-17, IFNγ, IL-6, and IL-10 concentrations in peptide-stimulated culture supernatants of spleen cells from mice with PGIA or G1 domain-induced arthritis (GIA) or from naïve mice.

<p>Data are shown as the mean of Cit:R ratios±SEM for the concentrations of (A) IL-17, (B), IFNγ, (C) IL-6, and (D) IL-10 in peptide-treated spleen cell cultures from mice with PGIA (red bars) or GIA (blue bars) or from non-arthritic naïve mice (gray bars) (PGIA n = 10 mice; GIA n = 5 mice; Naïve n = 3 mice). Cit:R ratio of 1.0 is indicated by a dotted line in each graph. Wilcoxon signed rank test was used to identify Cit:R ratios significantly higher than 1.0 (*p = <0.05: Cit:R ratio vs 1.0). Cit:R ratios of cytokines induced by the P13 and P49 pairs of peptides in cells from the 3 groups of mice were compared using Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: PGIA or GIA vs naïve mice). Average net concentrations of IL-17 in the cell cultures stimulated with the Cit version of P13 (Cit13) were as follows: 86.4 pg/ml in the PGIA group, 70.5 pg/ml in the GIA group, and 76.8 pg/ml in the naïve group (graphs not shown). In the Cit49-stimulated cultures, the average net amounts of IL-17 were: 136.9 pg/ml in the PGIA group, 60.8 pg/ml in the GIA group, and 29.8 pg/ml in the naïve group (graphs not shown).</p

Cit:R ratios of IL-17, IFNγ, and IL-6, induced in human PBMC cultures by Cit and R pairs of P13 and P49.

<p>(A) Cit:R ratios of IL-17 induced by the P13 and P49 peptide pairs in PBMC cultures of anti-citrullinated protein antibody positive (ACPA+) and ACPA negative (ACPA-) RA patients and HC subjects. Data are expressed as the mean±SEM of Cit:R ratios of peptide-induced IL-17 in PBMC of RA all (purple bars), RA ACPA+ (red bars), RA ACPA- (blue bars) and HC (green bars) groups. (B) Net concentrations of IL-17 in the same PBMC cultures produced in the absence of a peptide (no peptide) or in the presence of peptide Cit13 or Cit49. Data shown are the mean±SEM of IL-17 (pg/ml). Cit:R ratios of (C) IFNγ and (D) IL-6 in the P13 and P49 peptide pair-stimulated PBMC cultures were determined as described for IL-17 in panel A above. Sample numbers for IL-17 (RA all n = 42; RA ACPA+ n = 32; RA ACPA- n = 10; HC n = 8), for IFNγ (RA all n = 28; RA ACPA+ n = 22; RA ACPA- n = 6; HC n = 8), and for IL-6 (RA all n = 41; RA ACPA+ n = 31; RA ACPA- n = 10; HC n = 7). Cit:R ratio of 1.0 (in panels A, C and D) is indicated by a dotted line. Cit:R ratios of cytokines (in A, C and D) were analyzed using Wilcoxon signed rank test (*p<0.05: Cit:R ratio vs 1.0) and Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: RA groups vs HC group). For the data in panel B, inter-group and inter-peptide comparisons were made using two-way ANOVA followed by Tukey’s test (#p<0.05: RA groups vs HC group) and Shidak’s test (^Xp<0.05: Cit49 vs Cit13 or no peptide).</p

Recognition of proteoglycan (PG) aggrecan purified from normal human cartilage by ACPA-positive human sera.

<p>(<b>A-C</b>) Dots of human PG aggrecan that was purified from 6 normal cartilage samples (1–6) were applied to nitrocellulose membranes (upper dots: 2 μg, lower dots: 0.2 μg PG) along with various control IgGs purified from human (Hu), rabbit (Rb) or mouse (Mo) serum (upper dots: 20 ng, lower dots: 2 ng IgG) and human type II collagen (hCII) purified from normal cartilage (upper dot: 10 μg, lower dot: 1 μg hCII). The membranes were subjected to immunostaining with ACPA-positive sera including the (<b>A</b>) “Calibrator” serum from the anti-CCP3 assay kit and (<b>B, C</b>) sera from two ACPA+ RA patients (#20 and #9), followed by horseradish peroxidase (HRP)-labeled anti-human IgG. (<b>D-G</b>) Specificity controls included blotting with (<b>D</b>) a biotinylated monoclonal antibody (mAb) specific to human PG G1 domain (anti-hG1-biot) followed by streptavidin (SA)-HRP, (<b>E</b>) ACPA- serum followed by anti-human IgG-HRP, (<b>F</b>) anti-rabbit IgG-HRP, and (<b>G</b>) mouse antibody to hCII (anti-hCII) followed by anti-mouse IgG-HRP.</p

Correlations between ACPA of different specificities including citrullinated PG in the sera of RA patients.

<p>(<b>A</b>) Concentrations of antibodies to mutated citrullinated vimentin (MCV) and cyclic citrullinated peptides (CCP) were measured in serum samples from 84 RA patients using commercial ELISA kits. Results were expressed as units/ml (U/ml). Correlation analysis revealed strong positive correlation between anti-MCV and anti-CCP3 levels (r = 0.95, R square = 0.9, p<0.0001). (<b>B</b>) Concentrations of anti-CitPG antibodies were measured by in-house ELISA using <i>in vitro</i> citrullinated rhG1 (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150784#pone.0150784.g005" target="_blank">Fig 5B</a>) as antigen (CitPG). The results were expressed as delta optical density (ΔOD, the OD values of anti-CitPG minus the OD values of anti-PG as described in the Methods). The ΔOD values were correlated with the concentrations (U/ml) of anti-CCP in the same 84 RA serum samples. (<b>C</b>) Purified hCII was also citrullinated by PAD4, and CitCII was used to measure anti-CitCII levels in the 84 RA serum samples by ELISA. The dotted lines indicate the 95% confidence intervals.</p

Proliferation of spleen cells from mice with PG-induced arthritis (PGIA) in response to stimulation with peptide pools and individual peptides.

<p>Spleen cells from mice with PGIA were cultured with (A) Cit and R pairs of PG or OVA peptide pools or (B) Cit and R pairs of selected individual peptides (P) in triplicate wells. The Cit:R ratio of SI was calculated as described in the Methods. Data are expressed as the mean of Cit:R ratio of SI±SEM (n = 12 mice). Equal response to the Cit and R version of a peptide pool or peptide (theoretical Cit:R ratio of 1.0, at which no preference for the Cit or R version is assumed) is indicated with a dotted line. Statistical analysis was performed using Wilcoxon signed rank test (*p<0.05: Cit:R ratio vs 1.0) and Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: Cit:R ratio of PG13 vs Cit:R ratio of any other peptide pool).</p

Antibodies reacting with P13 and P49 peptides in plasma samples from RA patients and HC subjects.

<p>IgG antibodies reacting with Cit or R versions of P13 and P49 in the human plasma samples were detected by ELISA as described in the Methods. Cit:R ratios of (A) anti-P13 and (B) anti-P49 antibodies in plasma samples from RA and HC subjects. ΔOD 450 nm values representing plasma levels of (C) anti-Cit13 and (D) anti-Cit49 antibodies. Data shown are the mean±SEM. Sample numbers for both peptide pairs (RA all n = 46; RA ACPA+ n = 34; RA ACPA- n = 12; HC n = 9). Cit:R ratio of 1.0 is indicated by a dotted line. Statistical analysis was performed using Wilcoxon signed rank test (*p<0.05: Cit:R ratio vs 1.0) and Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: ACPA+ group vs ACPA- group).</p