title: Antibodies to carbamylated α-enolase epitopes in rheumatoid arthritis also bind citrullinated epitopes and are largely indistinct from anti-citrullinated protein antibodies.
abstract: In 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.
  
  
  
  Cross-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.
  
  
  
  Affinity-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.
  
  
  
  Our 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.
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden. evan.reed@ki.se.
authors.forename: Evan
authors.initials: E
authors.lastname: Reed
authors.affiliation: Cardiovascular Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Xia
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Nastya
authors.initials: N
authors.lastname: Kharlamova
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.|Division of Physiological Chemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: A Jimmy
authors.initials: AJ
authors.lastname: Ytterberg
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Anca I
authors.initials: AI
authors.lastname: Catrina
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Israelsson
authors.affiliation: ThermoFisher Scientific, Uppsala, Sweden.|Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
authors.forename: Linda
authors.initials: L
authors.lastname: Mathsson-Alm
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Monika
authors.initials: M
authors.lastname: Hansson
authors.affiliation: Cardiovascular Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.|Centre for Occupational and Environmental Medicine, Stockholm County Council, Stockholm, Sweden.
authors.forename: Lars
authors.initials: L
authors.lastname: Alfredsson
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Rönnelid
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden. Karin.lundberg@ki.se.
authors.forename: Karin
authors.initials: K
authors.lastname: Lundberg
mesh_terms: D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D015153:Blotting, Western; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D006801:Humans; D013058:Mass Spectrometry; D010456:Peptides, Cyclic / Q000276:immunology; D010751:Phosphopyruvate Hydratase / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D018985:Epitopes, B-Lymphocyte; D010456:Peptides, Cyclic; C487763:cyclic citrullinated peptide; D010751:Phosphopyruvate Hydratase
keywords: Anti-carbamylated protein antibodies; Anti-citrullinated protein antibodies; Cross-reactivity; Rheumatoid arthritis