title: Identification of carbamylated alpha 1 anti-trypsin (A1AT) as an antigenic target of anti-CarP antibodies in patients with rheumatoid arthritis.
abstract: In 2011 a novel autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, was described in rheumatoid arthritis (RA) patients. Anti-CarP antibody positivity associates with a more severe disease course, is observed years before disease onset, and may predict the development of RA in arthralgia patients. Although many clinical observations have been carried out, information on the antigenic targets of anti-CarP antibodies is limited. Most studies on anti-CarP antibodies utilize an ELISA-based assay with carbamylated fetal calf serum (Ca-FCS) as antigen, a complex mixture of proteins. Therefore, we analysed the molecular identity of proteins within Ca-FCS that are recognized by anti-CarP antibodies. Ca-FCS was fractionated using ion exchange chromatography, selecting one of the fractions for further investigation. Using mass-spectrometry, carbamylated alpha-1-antitrypsin (Ca-A1AT) was identified as a potential antigenic target of anti-CarP antibodies in RA patients. A1AT contains several lysines on the protein surface that can readily be carbamylated. A large proportion of the RA patients harbour antibodies that bind human Ca-A1AT in ELISA, indicating that Ca-A1AT is indeed an autoantigen for anti-CarP antibodies. Next to the Ca-A1AT protein, several homocitrulline-containing peptides of A1AT were recognized by RA sera. Moreover, we identified a carbamylated peptide of A1AT in the synovial fluid of an RA patient using mass spectrometry. We conclude that Ca-A1AT is not only a target of anti-CarP antibodies but is also present in the synovial compartment, suggesting that Ca-A1AT recognized by anti-CarP antibodies in the joint may contribute to synovial inflammation in anti-CarP-positive RA.
authors.affiliation: Department of Rheumatology, Leiden Univeristy Medical Center, Leiden, The Netherlands.
authors.forename: Marije K
authors.initials: MK
authors.lastname: Verheul
authors.affiliation: Inova Diagnostics, Inc., San Diego, CA, USA.
authors.forename: Alvin
authors.initials: A
authors.lastname: Yee
authors.affiliation: Inova Diagnostics, Inc., San Diego, CA, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Seaman
authors.affiliation: Center for Proteomics and Metabolomics, Leiden, The Netherlands.
authors.forename: George M
authors.initials: GM
authors.lastname: Janssen
authors.affiliation: Center for Proteomics and Metabolomics, Leiden, The Netherlands.
authors.forename: Peter A
authors.initials: PA
authors.lastname: van Veelen
authors.affiliation: Center for Proteomics and Metabolomics, Leiden, The Netherlands.
authors.forename: Jan W
authors.initials: JW
authors.lastname: Drijfhout
authors.affiliation: Department of Rheumatology, Leiden Univeristy Medical Center, Leiden, The Netherlands.
authors.forename: Rene E M
authors.initials: REM
authors.lastname: Toes
authors.affiliation: Inova Diagnostics, Inc., San Diego, CA, USA. Electronic address: m.mahler.job@web.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Mahler
authors.affiliation: Department of Rheumatology, Leiden Univeristy Medical Center, Leiden, The Netherlands.
authors.forename: Leendert A
authors.initials: LA
authors.lastname: Trouw
mesh_terms: D018771:Arthralgia / Q000276:immunology*; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000378:metabolism; D001324:Autoantigens / Q000276:immunology* / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D002956:Citrulline / Q000031:analogs & derivatives / Q000276:immunology / Q000302:isolation & purification; D019295:Computational Biology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D013583:Synovial Membrane / Q000276:immunology*; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D010446:Peptide Fragments; D000515:alpha 1-Antitrypsin; C001352:homocitrulline; D002956:Citrulline
keywords: Anti-CarP antibodies; Autoantibodies; Homocitrulline; Rheumatoid arthritis