Abstract
IntroductionFibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA.MethodsCitrullinated residues in fibronectin isolated from RA patient synovial fluid were identified by mass spectrometry. The corresponding citrullinated and non-citrullinated peptides were synthesized and used to analyze the presence of autoantibodies to these peptides in RA sera and sera from other diseases and healthy controls by ELISA. The data were compared with risk factors like shared epitope HLA alleles and smoking, and with clinical features.ResultsFive citrullinated residues were identified in fibronectin from RA synovial fluid. RA sera reacted in a citrulline-dependent manner with two out of four citrullinated fibronectin peptides, one of which contains two adjacent citrulline residues, in contrast to non-RA sera, which were not reactive. The most frequently recognized peptide (FN-Cit1035,1036, LTVGLTXXGQPRQY, in which × represents citrulline) was primarily targeted by anti-CCP (cyclic citrullinated peptide) 2-positive RA patients. Anti-FN-Cit1035,1036 autoantibodies were detected in 50% of established anti-CCP2-positive RA patients and in 45% of such patients from a early arthritis clinic. These antibodies appeared to be predominantly of the immunoglobulin G (IgG) isotype and to be associated with HLA shared epitope alleles (odds ratio = 2.11).ConclusionsFibronectin in the inflamed synovia of RA patients can be citrullinated at least at five positions. Together with the flanking amino acids, three of these citrullinated residues comprise two epitopes recognized by RA autoantibodies. Anti-citrullinated fibronectin peptide antibodies are associated with HLA shared epitope alleles.
Highlights
Fibronectin is one of the most abundant proteins present in the inflamed joint
Citrullinated fibronectin in synovial fluid samples of rheumatoid arthritis (RA) patients To study the immune response to citrullinated FN in RA, first the positions of the citrulline residues in FN isolated from the inflamed joints of RA patients were mapped
One supernatant fraction and one pellet fraction from synovial fluid (SF) samples obtained from two different RA patients (RA1 and RA2, respectively) were depleted of albumin by a differential precipitation procedure as described previously [49]
Summary
Fibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA. Citrullinated proteins occur at inflamed sites in healthy individuals as well as. It is still not fully understood how RA originates and develops, there is experimental evidence for several steps in this process [7]. Other environmental risk factors that have been suggested to enhance the chance of developing RA include the exposure to mineral oil, diet restrictions and coffee intake [18,19,20].
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