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.

Autocitrullination and Changes in the Activity of Peptidylarginine Deiminase 3 Induced by High Ca2+ Concentrations

“…Autocitrullination has also been observed in PAD2 as well as PAD4, 13 , 28 − 30 and the percentage of autocitrullinated sites in PAD2 and PAD4 accounted for 48% (16/33) and 70% (19/27) of the total arginine residues, respectively ( Table 3 ). The citrullination rate of PAD3 is the lowest among the three isozymes (23%), thus suggesting that this correlates with the higher substrate specificity of PAD3.…”

“…Structures around Arg372, which is citrullinated in PAD2, PAD3, and PAD4, and Arg394, which is citrullinated in PAD3 and PAD4. , The structures of PAD2, PAD3, and PAD4 are all Ca 2+ -bound forms and colored in gray, green, and magenta, respectively. Only Arg372 and Arg394 (PAD3 numbering) are depicted in stick models, in which the N and O atoms are colored in blue and red, respectively.…”

“…It has been reported that PAD2 and PAD4 catalyze autocitrullination reactions in the presence of Ca 2+ . − Recently, autocitrullination of PAD3 was also identified and the sites were reported . Since the “nonproductive” WT PAD3-Ca 2+ was crystallized in the presence of a very high Ca 2+ concentration (260 mM), we also considered the possibility that the obtained structure underwent autocitrullination.…”

“…It has been reported that Arg372 in PAD4 is autocitrullinated (Table 3). [28][29][30]32 In addition, PAD4 whose Arg372 is genetically engineered to citrulline significantly lost its activity. 36 Conversely, the autocitrullination reaction of PAD4 showed small effects on the activity, 31 similar to that reported regarding PAD3 in this study.…”

The Expression of Non-Coding RNAs and Their Target Molecules in Rheumatoid Arthritis: A Molecular Basis for Rheumatoid Pathogenesis and Its Potential Clinical Applications

“…In consideration of PAD4 as an effector and target molecule in RA pathogenesis, Auger et al [ 47 ] further proved that ACPAs are induced after PAD4 is recognized by T cells to facilitate the production of antibodies against “citrullinated peptides” bound by PAD4, mimicking a so-called “hapten-carrier” model. Recently, Darrah et al [ 48 ] investigated the antibodies against native and auto-citrullinated PADs in patients with RA. They found that citrulline was not a major determinant in the recognition of anti-PAD2 or anti-PAD4 in RA patients.…”