title: High-Titer Rheumatoid Arthritis Antibodies Preferentially Bind Fibrinogen Citrullinated by Peptidylarginine Deiminase 4.
abstract: Most patients with rheumatoid arthritis (RA) harbor antibodies to citrullinated autoantigens such as citrullinated fibrinogen. Two isoforms of peptidylarginine deiminase (PAD), PAD type 2 (PAD2) and PAD4, which catalyze citrullination with different substrate specificities, can be detected in the synovium of RA patients. This study was undertaken to determine whether RA antibodies preferentially bind PAD2- or PAD4-citrullinated fibrinogen.
  
  
  
  RA patient and normal donor plasma specimens were tested for binding to PAD2- or PAD4-citrullinated fibrinogen, native fibrinogen, or citrullinated fibrinogen peptides in various dilutions by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Bands corresponding to masses demonstrating RA antibody reactivity by Western blotting were excised and analyzed by mass spectrometry.
  
  
  
  At low antibody titers (1:40 and 1:100), there was no significant difference between RA antibody reactivity to PAD2- and PAD4-citrullinated fibrinogen. When plasma was further diluted to 1:250 and 1:1,000, RA patient plasma bound PAD4-citrullinated fibrinogen significantly more than PAD2-citrullinated fibrinogen, as measured by ELISA and Western blotting. An increased antibody titer was associated with increased avidity for both PAD2- and PAD4-citrullinated fibrinogen. Both enzymes hypercitrullinated fibrinogen, but PAD4 citrullinated arginines more intermittently, generating a mix of citrullinated and noncitrullinated arginines. Peptide ELISA and preadsorption assays confirmed that the region of intermittent citrullination accounts for the majority of RA antibody binding to the β-chain of citrullinated fibrinogen.
  
  
  
  At high titers, RA antibodies preferentially bind fibrinogen modified by PAD4, because intermittent citrullination offers a more diverse assortment of citrullinated epitopes.
authors.affiliation: Rockefeller University and Howard Hughes Medical Institute, New York, New York.
authors.forename: Nathalie E
authors.initials: NE
authors.lastname: Blachère
authors.affiliation: Rockefeller University, New York, New York.
authors.forename: Salina
authors.initials: S
authors.lastname: Parveen
authors.affiliation: Rockefeller University and New York Genome Center, New York, New York.
authors.forename: Mayu O
authors.initials: MO
authors.lastname: Frank
authors.affiliation: Rockefeller University, New York, New York.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Dill
authors.affiliation: Rockefeller University, New York, New York.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.affiliation: Rockefeller University, Hospital for Special Surgery, and New York Genome Center, New York, New York.
authors.forename: Dana E
authors.initials: DE
authors.lastname: Orange
mesh_terms: D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D015153:Blotting, Western; D016022:Case-Control Studies; D002853:Chromatography, Liquid; D002956:Citrulline / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D013058:Mass Spectrometry; D010456:Peptides, Cyclic / Q000276:immunology; D000080064:Protein-Arginine Deiminase Type 2; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases
chemical_list: D001323:Autoantibodies; D010456:Peptides, Cyclic; C487763:cyclic citrullinated peptide; D002956:Citrulline; D005340:Fibrinogen; D006867:Hydrolases; C000620352:PADI2 protein, human; C000629235:PADI4 protein, human; D000080064:Protein-Arginine Deiminase Type 2; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases