Rheumatoid arthritis–specific autoantibodies to peptidyl arginine deiminase type 4 inhibit citrullination of fibrinogen

Abstract

Objective. Autoantibodies to citrullinated proteins are specific for rheumatoid arthritis (RA) and recognize epitopes centered by citrulline, a posttranslationally modified form of arginine. Peptidyl arginine deiminase type 4 (PAD-4), the enzyme that converts arginine into citrulline, is in itself a target for RAspecific autoantibodies. This study was undertaken to assess whether anti-PAD-4 autoantibodies interfere with citrullination in vitro in patients with RA, and to identify peptide targets of anti-PAD-4 antibodies that can activate or inhibit citrullination.Methods. To test whether autoantibodies to PAD-4 influence citrullination, an in-house citrullination assay was developed using purified autoantibodies to PAD-4. To map B cell epitopes on PAD-4, 65 overlapping 20-mer peptides encompassing the entire PAD-4 were analyzed for their reactivity in RA sera.Results

Fine specificity of anti-citrullinated peptide antibodies discloses a heterogeneous antibody population in rheumatoid arthritis

“…Recently, Van Beers et al have shown limited serum reactivity against specific citrullinated epitopes compared to the extended repertoire of citrullinated polypeptides contained in synovial fluid of RA patients, including vimentin and fibrinogen, implying that the formation of in-situ immune complexes might contribute to disease onset [31]. Interestingly, we also included citrullinated PAD-4 peptide, as anti-PAD4 antibodies have been detected in the serum of RA patients before disease onset and have been implicated in disease pathogenesis [16]. An approach based on citrullinated epitopes rather than whole proteins has been selected to achieve optimal reactivity.…”

“…Linear citrullinated (Cit) peptides derived from the sequence of human proteins were as follows: PAD 211-30 (VCitVFQATCitGKLSSKCSVVLG) derived from PAD, vim 60-75 (VYATCitSSAVCitLCitSSVP) derived from vimentin, enol [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] (KIHACitEIFDSCitGNPTVE) derived from alpha-enolase, fibrin 617-31 (HSTKCitGHAKSCitPVCitG) derived from the alpha chain of fibrinogen, col-II 358-75 (GACitGLTGCitPGDAGPPGPP) from type II collagen and fillaggrin [SHQEST(Cit)G(Cit)SRGRSGRSG] derived from filaggrin. A non-citrullinated peptide, ctrlPAD (EPLGLQCTFINDFFTYHIRH) derived from PAD was used as control.…”

“…No clinical differences were recorded among SLE and SS patients with and without reactivity against the citrullinated peptides. SLE patients were found positive for antibodies against PAD 211-30 , vim 60-75 , enol [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] , fibrin 617-31 , col-II 358-75 and filaggrin 306-24 in 13·33, 6·67, 1·67, 21·67 and 1·67%, respectively and SS patients in 14·81, 12·96, 7·41, 3·70, 5·56 and 1·85%, respectively. Specificity of antibodies against PAD 211-30 , vim 60-75 , enol [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] , fibrin 617-31 , col-II 358-75 and filaggrin 306-24 among SLE patients ranged from 14·58 to 20·68%, respectively, and for SS patients from 6·67 to 22·75%, respectively.…”

“…The percentages of inhibition of purified anti-peptide antibodies of the two prototype sera after incubation with the homologous peptides were 83·09% for PAD 211-30 , 62·60% for vim 60-75 , 87·15% for enol [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] …”

“…However, it has been demonstrated previously that B cell epitopes within the parent proteins vimentin, fibrin, filaggrin [15], PAD [16] and alpha-enolase [12] are recognized consistently by ACPA. Specifically, the vimentin and fibrinogen epitopes are the most frequently recognized by sera from ACPA-positive patients with long-standing RA [17].…”