Relative efficiencies of peptidylarginine deiminase 2 and 4 in generating target sites for anti-citrullinated protein antibodies in fibrinogen, alpha-enolase and histone H3

Abstract

ObjectivePeptidylarginine deiminase 2 (PAD2) and PAD4 are expressed in the synovium of rheumatoid arthritis (RA) patients and catalyze citrullination of arginine residues in proteins targeted by anti-citrullinated protein antibodies (ACPAs). Little is known about the relative importance of PAD2 and PAD4 in generating citrullinated self-antigens. Here we investigate the ability of PAD2 and PAD4 to generate citrullinated targets for ACPAs in four human proteins.MethodsSynovial fluid (SF) and plasma were collected from 42 RA patients. Human fibrinogen, human alpha-enolase (ENO1), human histone H3, and human serum albumin (HSA) were citrullinated in vitro by PAD2 or PAD4. The total degree of citrullination was determined using the anti-modified citrulline approach. Antibody binding to native and citrullinated proteins was measured by ELISA.ResultsACPAs within pooled SF from multiple RA patients reacted equally well with, and cross-reacted with, PAD2- and PAD4-citrullinated fibrinogen. ACPAs from most individual patient SF and plasma samples bound equally well to PAD2- and PAD4-citrullinated fibrinogen or ENO1. When histone H3 was used as target, PAD4 was generally superior in generating epitopes recognized by ACPAs. No binding to citrullinated HSA was observed.ConclusionIn most patients, PAD2 and PAD4 are equally efficient in generating citrullinated target sites for ACPAs in fibrinogen and ENO1. The binding of autoantibodies to histone H3 was generally higher after citrullination with PAD4 than with PAD2. Citrullinated HSA is not a target for ACPAs.

The Atypical Fibrin Fibre Network in Rheumatoid Arthritis and its Relation to Autoimmunity, Inflammation and Thrombosis

“…Two isoforms of PAD (PAD2 and PAD4) are primarily responsible for generating citrullinated antigens that incite the generation of anti-citrullinated protein antibodies (ACPAs), a hallmark serological feature of RA (128). Fibrinogen and fibrin are prominent substrates for PAD2 and PAD4 and autoantibodies targeting citrullinated fibrin(ogen) have been identified (40,43,68,(129)(130)(131). The pathogenicity of citrullinated fibrin(ogen) immune complexes have been demonstrated both in vitro (33) and in vivo (132,133).…”

The Central Role of Acute Phase Proteins in Rheumatoid Arthritis: Involvement in Disease Autoimmunity, Inflammatory Responses, and the Heightened Risk of Cardiovascular Disease

“…4,71,73,74 PAD2 and PAD4 exhibit different subcellular localization and antigen target specificities in RA and are associated with the presence of inflammatory markers. 75,76 The most prominent protein targets for PAD-driven citrullination are human fibrin(ogen), 57 histones, 58 vimentin, 59 α-enolase, 60 and types I-and II collagen. 61,62 The primary environmental risk factors associated with RA development are infection 76 and smoking.…”

“…91 The presence of ACPAs represent a specified subgroup that constitutes 70 to 80% of all RA patients. 74,75 These observations indicate a strong role of ACPAs in development of clinical RA, ACPAs are therefore listed as an important criterion for RA disease classification in American College of Rheumatology/ European League against Rheumatism (ACR-EULAR) guidelines. 92 The discovery of the first citrullinated antigens (filaggrin) and ACPAs (antikeratin factor and antiperinuclear factor) in RA 93,94 led to the development of enzyme-linked immunosorbent assay (ELISA)-based methods for detecting ACPAs.…”

“…The enhanced efficiency of PAD2 for deaminating arginine residues is due to the enzyme being less restricted by the amino acid composition surrounding acceptor arginine residues in fibrinogen and fibrin compared with PAD4. 75 There is no significant discrimination of ACF-directed reactivity toward citrullinated peptides generated by either PAD2 or PAD4, as there is a great degree of overlap in the citrullination sites targeted by both PAD2 and PAD4. 75 The mapping of citrullinated epitopes in fibrinogen and fibrin has been conducted by a large number of studies.…”

“…75 There is no significant discrimination of ACF-directed reactivity toward citrullinated peptides generated by either PAD2 or PAD4, as there is a great degree of overlap in the citrullination sites targeted by both PAD2 and PAD4. 75 The mapping of citrullinated epitopes in fibrinogen and fibrin has been conducted by a large number of studies. The identification of 42 citrullinated residues in fibrinogen (27 in the α-chain, 11 in the β-chain and 4 in the γ-chain) currently represents the most comprehensive epitope list for citrullinated fibrinogen compiled thus far.…”