Citrulline Not a Major Determinant in the Recognition of Peptidylarginine Deiminase 2 and 4 by Autoantibodies in Rheumatoid Arthritis

Abstract

Objective: Citrullinated proteins are hallmark targets of autoantibodies in rheumatoid arthritis (RA). Our study was undertaken to determine the effect of autocitrullination on the recognition of peptidylarginine deiminases (PADs) 2 and 4 by autoantibodies in RA.

Methods: Autocitrullination sites in PAD2 and PAD4 were determined by mass spectrometry and literature review. Antibodies against native and autocitrullinated PADs in 184 patients with RA were detected by enzyme-linked immunosorbent assay. Linear regression analysis, outlier calculations, and competition assays were performed to evaluate antibody reactivity to native and citrullinated PADs.

Results: Autocitrullination of PAD2 and PAD4 was detected in 16 (48%) of 33 arginine residues and 7 (26%) of 27 arginine residues, respectively. Despite robust autocitrullination, autoantibodies bound similarly to native and citrullinated PAD2 or PAD4 (ρ = 0.927 and ρ = 0.903, respectively; each P < 0.0001). Although subsets of anti-PAD-positive sera were identified as exhibiting preferential recognition of native or citrullinated PAD2 (40.5% or 4.8%, respectively) or PAD4 (11.7% or 10.4%, respectively), competition assays confirmed that the majority of anti-PAD reactivity was attributed to a pool of autoantibodies that bound irrespective of citrullination status.

Conclusion: Autocitrullination does not affect autoantibody reactivity to PADs in the majority of patients with RA, demonstrating that anti-PAD antibodies are distinct from anti-citrullinated protein antibodies in their dependence on citrullination for binding.

Figure 1.. Citrullination sites in PAD2 and PAD4.

(A) PAD2 and PAD4 autocitrullinate in a calcium-dependent manner as detected by anti-modified citrulline (AMC) immunoblotting. PAD detection by rabbit anti-PAD2 or anti-PAD4 antibodies was included as a loading control. Citrullination sites in autocitrullinated PAD2 (B) and PAD4 (C) were identified by mass spectrometry (orange). (C) Citrullination sites in autocitrullinated PAD4 that have been identified in previously published studies(–12) are also indicated (gray).

Figure 2.. Anti-PAD antibodies bind similarly to native and citrullinated PADs.

Linear regression was used to determine the relationship between RA sera reactivity (n=184) to native and citrullinated PAD2 (A) or PAD4 (B). The trendline (solid line), 90% confidence intervals (dotted lines), Spearman’s rho, and p-values for each fit are indicated. (C and D) The difference in antibody reactivity to native PAD and citrullinated (cit) PAD was plotted on the y-axis (“cit-native PAD reactivity”) versus the average recognition of native and citrullinated PAD2 (C) and PAD4 (D) for each patient. A value of zero (solid line) represents equal binding to native and citrullinated PAD, and the 95% confidence interval is shown (dotted lines). (E and F) Sera that demonstrated a statistical preference for the native (negative values, blue) or citrullinated (positive values, orange) form of PAD2 or PAD4 are indicated. RA sera were grouped into three groups according to their binding to native (open circles, “Nat”) and citrullinated (open diamonds, “Cit”) PAD2 (E) or PAD4 (F) with Nat<Cit (orange), Nat>Cit (blue), and Nat=Cit (gray) binding indicated. Immunoreactivity of healthy control sera (HC, purple) to native or citrullinated PAD2 (n=37) or PAD4 (n=39) is also shown, and the >95^th percentile is indicated (dotted line at 3.83 and 0.658, respectively). AU=arbitrary units

Figure 3.. Anti-PAD autoantibodies bind PADs irrespective of citrullination status.

Two hypothetical scenarios are shown that could explain the observed binding of sera to both the native and citrullinated forms of PAD2 or PAD4. (A) Depicts the co-existence of two distinct autoantibody subsets, with unique specificities for either the native or the citrullinated form of the PAD. (B) Depicts a single group of anti-PAD autoantibodies that bind to epitopes irrespective of citrullination status, and can therefore bind both native and citrullinated forms of the PAD. A subset of anti-PAD2 (n=12, C) or PAD4 (n=15, D) antibody positive sera were pre-incubated with buffer alone (unblocked) or blocked with either native or citrullinated PAD prior to performing ELISAs with the reciprocally modified PAD. The “% PAD2 (or PAD4) reactivity” of each serum with specificity to native PAD (blue), citrullinated PAD (orange), or to bind both forms irrespective of citrullination status (gray) is shown on the y-axis. Each RA sera is indicated with a unique ID#, and they are color coded according to the binding preferences identified in Figure 2C.