Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

INTRODUCTION: Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients. METHODS: An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined. RESULTS: Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur. CONCLUSIONS: We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients

Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase

BACKGROUND: Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs. METHODS: Activity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen. RESULTS: No activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10–15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity. CONCLUSIONS: Catalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space

Identification of Novel Native Autoantigens in Rheumatoid Arthritis

The majority of patients diagnosed with rheumatoid arthritis (RA) have developed autoantibodies against neoepitopes in proteins that have undergone post-translational modification, e.g., citrullination or carbamylation. There is growing evidence of their molecular relevance and their potential utility to improve diagnosis, patient stratification, and prognosis for precision medicine. Autoantibodies reacting to native proteins may also have a role in RA pathogenesis, however, their reactivity patterns remain much less studied. We hypothesized that a high-density protein array technology could shed light onto the normal and disease-related autoantibodies produced in healthy and RA patient subgroups. In an exploratory study, we investigated the global reactivity of autoantibodies in plasma pools from 15 anti-cyclic citrullinated peptide (CCP)-positive and 10 anti-CCP-negative RA patients and 10 healthy donors against more than 1600 native and unmodified human proteins using a high-density protein array. A total of 102 proteins recognized by IgG autoantibodies were identified, hereof 86 were recognized by antibodies from CCP-positive RA patients and 76 from anti-CCP-negative RA patients, but not by antibodies from healthy donors. Twenty-four of the identified autoantigens have previously been identified in synovial fluid. Multiple human proteins in their native conformation are recognized by autoantibodies from anti-CCP-positive as well as anti-CCP-negative RA patients

Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays

Abstract The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients

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

OBJECTIVE:Peptidylarginine 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. METHODS:Synovial 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. RESULTS:ACPAs 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. CONCLUSION:In 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

Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase

<p>Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H₂O₂ and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H₂O₂ at concentrations above 40 µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H₂O₂ at concentrations up to 1000 µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.</p