Antibodies to carbamylated α-enolase epitopes in rheumatoid arthritis also bind citrullinated epitopes and are largely indistinct from anti-citrullinated protein antibodies

Abstract

BackgroundIn addition to anti-citrullinated protein antibodies (ACPAs), antibodies targeting carbamylated (i.e., homocitrullinated) proteins (anti-CarP antibodies) have been described in rheumatoid arthritis (RA). However, the extent to which anti-CarP antibodies are truly distinct from ACPA remains unclear, and few studies have focused on specific autoantigens. Here, we examine cross-reactivity between ACPA and anti-CarP antibodies, in the context of the candidate autoantigen α-enolase.MethodsCross-reactivity was examined by immunoblotting of citrullinated and carbamylated proteins using purified ACPA; and by peptide absorption experiments, using the citrullinated α-enolase peptide CEP-1 and a homocitrulline-containing version (carb-CEP-1) in ELISA. The population-based case-control cohort EIRA (n = 2836 RA; 373 controls) was screened for reactivity with CEP-1 and carb-CEP-1, using the ISAC multiplex array. Associations between anti-CarP antibodies, smoking and genetic risk factors were analysed using unconditional logistic regression models. Differences in antibody levels were investigated using the Mann-Whitney U test.ResultsAffinity-purified ACPA was found to bind carbamylated proteins and homocitrulline-containing peptides, demonstrating definitive cross-reactivity between ACPA and anti-CarP antibodies. Anti-carb-CEP-1 reactivity in EIRA was almost exclusively confined to the CEP-1-positive subset, and this group of RA patients (21 %) displayed a particularly strong ACPA response with marked epitope spreading. The small RA subset (3 %) with homocitrulline reactivity in the absence of citrulline reactivity did not associate with smoking or risk genes, and importantly had significantly lower anti-carb-CEP-1 antibody levels.ConclusionOur data presented herein cast doubt on the specificity of anti-CarP antibodies in RA, which we posit may be a subset of cross-reactive ACPA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1001-6) contains supplementary material, which is available to authorized users.

Brief Report: Anti–Carbamylated Protein Antibodies in Rheumatoid Arthritis Patients Are Reactive With Specific Epitopes of the Human Fibrinogen β‐Chain

“…Given the similarity of the posttranslational modification, with the ureido functional group added to the primary amine group of a basic (lysine versus arginine) amino acid, it would not be surprising to observe that anti-CarP antibodies and ACPAs cross-react (12). However, advancement in the understanding of anti-CarP antibodies has been limited by the lack of understanding of their cross-reactivity with ACPAs.…”

Autoantibodies and B Cells: The ABC of rheumatoid arthritis pathophysiology

“…Interestingly, mice immunized with a carbamylated protein develop antibodies recognizing both carbamylated and acetylated (self) proteins; the same can be observed when mice are immunized with an acetylated protein . This cross‐reactivity is also detected in patients in which anti‐CarP and ACPA responses show cross‐reactivity to varying degrees . Furthermore, preliminary data illustrated that an ACPA/AAPA monoclonal antibody can be cross‐reactive, and commercially available (polyclonal) antibodies are often unable to distinguish between different citrullinated and carbamylated residues .…”

“…148 This cross-reactivity is also detected in patients in which anti-CarP and ACPA responses show crossreactivity to varying degrees. [148][149][150] Furthermore, preliminary data illustrated that an ACPA/AAPA monoclonal antibody can be crossreactive, 151 and commercially available (polyclonal) antibodies are often unable to distinguish between different citrullinated and carbamylated residues. 152 Together, these observations hint toward a cross-reactive antibody response targeting citrullinated, carbamylated, and acetylated proteins.…”

Clinical and etiological meaning of anti-carbamylated protein antibodies in rheumatoid arthritis

“…Shi et al selected FCS because it contains many types of proteins and, thus, was considered to be suitable for screening [1]. After this study, fibrinogen, vimentin, alpha-enolase and 78-kDa glucoseregulated protein (GRP78/BIP) were identified as target antigens of anti-CarP Ab [1,3,10,[12][13][14]. However, they are also target antigens of ACPA.…”

“…Therefore, the possibility of a cross-reaction is a cause of great concern. Previous studies reported that anti-CarP Ab and ACPA are cross-reactive to some extent [9][10][11]. However, since many patients who have only anti-CarP Ab or ACPA [1], or both without showing cross-reactivity [9], they are regarded as similar but different antibodies.…”