Abstract
IntroductionRheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.MethodsHuman fibrinogen was citrullinated in vitro by peptidylarginine deiminases (PAD), subjected to proteolysis and the resulting peptides were fractionated by ion exchange chromatography. The peptide composition of the citrullinated peptide-containing fractions was determined by high resolution tandem mass spectrometry. The recognition of these fractions by patient sera was subsequently analyzed by imaging surface plasmon resonance on microarrays.ResultsIn total about two-thirds of the 81 arginines of human fibrinogen were found to be susceptible to citrullination by the human PAD2, the human PAD4 or the rabbit PAD2 enzymes. Citrullination sites were found in all three polypeptide chains of fibrinogen, although the α-chain appeared to contain most of them. The analysis of 98 anti-citrullinated protein antibody-positive RA sera using the new methodology allowed the identification of three major citrullinated epitope regions in human fibrinogen, two in the α- and one in the β-chain.ConclusionsA comprehensive overview of citrullination sites in human fibrinogen was generated. The multiplex analysis of peptide fractions derived from a post-translationally modified protein, characterized by mass spectrometry, with patient sera provides a versatile system for mapping modified amino acid-containing epitopes. The citrullinated epitopes of human fibrinogen most efficiently recognized by RA autoantibodies are confined to three regions of its polypeptides.
Highlights
Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity
Identification of citrullination sites in human fibrinogen To generate a map of the citrullinated autoantigenic epitopes on human fibrinogen, the protein was first citrullinated in vitro using recombinant human PAD2 (hPAD2) and human PAD4 (hPAD4), as well as rabbit muscle peptidylarginine deiminases (PAD)
To exclude that the lower levels of fibrinogen citrullination by hPAD4 was due to differences in the specific activity of the PAD enzymes, the same amount of fibrinogen was incubated with increasing amounts of hPAD4
Summary
Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullination, the conversion of peptidylarginine into peptidylcitrulline, of the fibrinogen a and b chains generates antigenic targets for autoantibodies present in the serum and synovial fluid of RA patients [1,7]. Ho and others found that mice that were immunized with citrullinated fibrinogen developed arthritis and fibrinogen-reactive T cells which produce the proinflammatory cytokines IL-6, IL-17, TNF-a, and IFN-g and that these mice possess rheumatoid factor, circulating immune complexes and anti-CCP, all of which are characteristics of human RA [11]. Circulating immune complexes containing citrullinated fibrinogen were found in a large subset of ACPA-positive RA patients [14]. These findings suggest a crucial role for fibrinogen in RA pathogenesis. We describe a novel method to map the epitopes of posttranslationally modified proteins and apply this method, which is schematically illustrated in Figure 1, to map the autoepitopes of citrullinated fibrinogen recognized by RA sera
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