Elsevier

Journal of Autoimmunity

Volume 23, Issue 2, September 2004, Pages 141-150
Journal of Autoimmunity

Influence of arginine deimination on antigenicity of fibrinogen

https://doi.org/10.1016/j.jaut.2004.06.002Get rights and content

Abstract

Autoreactivity is controlled at various steps by numerous mechanisms and is a key to understanding and treating autoimmune disease. Recently, an antibody against deiminated fibrinogen (DI-FBG) was detected in patients with rheumatoid arthritis (RA) with high specificity and sensitivity. DI-FBG converted enzymically by peptidyl arginine deiminase, was also detected in synovial membrane. In the present study, we investigated whether antibody to DI-FBG is produced in mice immunized with DI-FBG. Mice were immunized with DI-FBG in the presence or absence of adjuvant. Production of the specific antibody was only induced with adjuvant. The resulting antibody was specific for DI-FBG and did not react with intact/native fibrinogen. Furthermore, it recognized deiminated human fibrinogen and cyclic citrullinated peptide (CCP). These results suggested that mouse fibrinogen acquires antigenicity in mice through deimination and therefore, autoantibody such as that detected in RA patients specifically may be induced.

Introduction

Rheumatoid arthritis (RA) is one of the most common systemic autoimmune diseases. It is characterized by chronic joint inflammation, the formation of a rheumatoid pannus, and eventually, tissue degradation and joint destruction. Recently, therapy for RA has become more effective. For example, methotrexate therapy using low dose, newer NSAIDs with greater COX2 selectivity, and biological therapies such as treatment with chimeric monoclonal antibodies to tumor necrosis factor alpha (TNF-α), recombinant human TNF-α (p75)-Fc fusion protein, humanized antihuman interleukin-6 receptor antibody, and recombinant interleukin-1 receptor antagonist have emerged [1], [2], [3], [4], [5], [6], [7], [8], [9]. The etiology of the disease, however, is unknown.

Several autoantibodies have been detected clinically and experimentally in the sera of RA patients, including rheumatoid factor (RF), anti-perinuclear factor (APF), anti-keratin antibodies (AKA), and anti-filaggrin antibodies (AFA) [10], [11], [12], [13], [25], [26]. RF is routinely measured in the diagnosis of RA. However, RF is also present in other autoimmune diseases, infections and even healthy individuals. Recently, it was reported that the antigen recognized by APF, AKA, and AFA is a protein containing peptide in which arginine residues are converted into citrulline residues following deimination mediated by a peptidyl arginine deiminase (PAD) [14], [15], [16]. The antibodies recognizing the citrullinated proteins were detected in over 80% of RA sera with high specificity [16]. Furthermore, an assay of antibodies to cyclic citrullinated peptide (CCP) synthesized with relatively high sensitivity and reliable in a convenient test format, has proved a valuable diagnostic test [17], [18]. In additional research into epitopes of the antibodies recognizing citrullinated proteins, these antibodies were highly reactive to the A-alpha- and B-beta-chains of human fibrinogen only after deimination by PAD [15]. Genetic susceptibility to RA is associated with the presence of certain subsets of HLA class II DR1 and DR4 [24]. Furthermore, PADI4, which encodes a peptidyl arginine deiminase, was identified as a susceptibility gene for rheumatoid arthritis in a case–control study with SNPs and affected the stability of mRNA for peptidyl arginine deiminase [20]. The above points imply that deimination is involved in the pathophysiology of RA.

Deimination is mediated by a peptidyl arginine deiminase which is a Ca2+-dependent enzyme, and catalyzes the posttranslational conversion of arginine residues in proteins to citrulline. Deimination in proteins can cause changes in secondary structure, protein–protein interaction, and protein unfolding due to a decrease in net charge, loss of potential ionic bonds, and interference with hydrogen bonds, and results in alterations in biological function [19]. Such changes may create a new epitope in the body. It is assumed that self-proteins acquire antigenicity by deimination, consequently, antibodies recognizing citrullinated proteins are produced as in RA patient sera.

In the present study, we investigated the deimination of fibrinogen on autoantibody production in mice and found that the reaction clearly enhanced antigenicity.

Section snippets

Mice

Male Balb/c mice (5–6 weeks old) were purchased from Japan SLC, Inc., Shizuoka, Japan, and housed in a specific pathogen-free facility.

Materials

Mouse fibrinogen (mFBG), rabbit skeletal muscle peptidyl arginine deiminase (PAD), bovine serum albumin (BSA), polyarginine, and ProteinG agarose were purchased from Sigma Chemical Co. Human fibrinogen (hFBG) was obtained from Calbiochemicals.

Procedure used for protein deimination

mFBG, hFBG, and BSA were incubated at 1 mg/mL with rabbit skeletal muscle PAD (3.5 U/mg fibrinogen) in 0.1 M Tris–HCl (pH

Deimination of mouse fibrinogen produces autoantibodies to deiminated fibrinogen

To examine whether the antigenicity of DI-mFBG was greater than that of mFBG, Balb/c mice were immunized weekly with DI-mFBG. After 10 weeks, sera were collected, and the antibodies to DI-mFBG or mFBG were detected by ELISA with plates coated with DI-mFBG or mFBG. In sera from all immunized mice, no antibodies to DI-mFBG and mFBG were detected (Fig. 1A, B). It seems that the antigenicity of DI-mFBG was very low.

To acquire autoantigenecity for fibrinogen, human fibrinogen (hFBG) and deiminated

Discussion

Recently, autoantibodies to deiminated proteins have been detected clinically in RA patients with high specificity [10], [11], [12], [13], [16], [17], [18]. This suggests that the deimination of arginine residues in proteins is involved in autoantigenicity in autoimmune disease. One of the features of a posttranslational modification, such as oxidation, lipidation, phosphorylation, glycosylation, and deimination, is the acquisition of autoantigenicity. Several years ago, it was reported that

Acknowledgements

The authors thank Miss Satoko Ishikawa and Miss Misato Ebara for their technical assistance.

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