title: Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis.
abstract: OBJECTIVE
  To understand the molecular features distinguishing anti-citrullinated protein antibodies (ACPA) from 'conventional' antibodies in rheumatoid arthritis (RA).
  
  
  METHODS
  Serum of ACPA-positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum, plasma and/or synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and analysed by mass spectrometry. Binding affinity to citrullinated antigens was measured by ELISA and imaging surface plasmon resonance using recombinant monoclonal ACPA with molecular modifications.
  
  
  RESULTS
  In all donor samples studied (n=24), ACPA-IgG exhibited a 10-20 kDa higher molecular weight compared with non-autoreactive IgG. This feature also distinguished ACPA-IgG from antibodies against recall antigens or other disease-specific autoantibodies. Structural analysis revealed that a high frequency of N-glycans in the (hyper)variable domains of ACPA is responsible for this observation. In line with their localisation, these N-glycans were found to modulate binding avidity of ACPA to citrullinated antigens.
  
  
  CONCLUSIONS
  The vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the 'germline-counterparts' of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Yoann
authors.initials: Y
authors.lastname: Rombouts
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Annemiek
authors.initials: A
authors.lastname: Willemze
authors.affiliation: Radboud Institute for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University, Nijmegen, the Netherlands.
authors.forename: Joyce J B C
authors.initials: JJ
authors.lastname: van Beers
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Jing
authors.initials: J
authors.lastname: Shi
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Priscilla F
authors.initials: PF
authors.lastname: Kerkman
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Linda
authors.initials: L
authors.lastname: van Toorn
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands Netherlands Proteomics Centre, Utrecht, the Netherlands.
authors.forename: George M C
authors.initials: GM
authors.lastname: Janssen
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Zaldumbide
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Rob C
authors.initials: RC
authors.lastname: Hoeben
authors.affiliation: Radboud Institute for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University, Nijmegen, the Netherlands.
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Pruijn
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.affiliation: Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Amsterdam, the Netherlands Jan van Breemen Research Institute Reade, Amsterdam, the Netherlands.
authors.forename: Gertjan
authors.initials: G
authors.lastname: Wolbink
authors.affiliation: Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Amsterdam, the Netherlands.
authors.forename: Theo
authors.initials: T
authors.lastname: Rispens
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Peter A
authors.initials: PA
authors.lastname: van Veelen
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Tom W J
authors.initials: TW
authors.lastname: Huizinga
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Leendert A
authors.initials: LA
authors.lastname: Trouw
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Hans U
authors.initials: HU
authors.lastname: Scherer
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: René E M
authors.initials: RE
authors.lastname: Toes
mesh_terms: D000328:Adult; D000368:Aged; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000737:chemistry / Q000378:metabolism*; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D002850:Chromatography, Gel; D002956:Citrulline / Q000378:metabolism*; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology* / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D008970:Molecular Weight; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013582:Synovial Fluid / Q000276:immunology
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D007074:Immunoglobulin G; D011134:Polysaccharides; D002956:Citrulline
keywords: Ant-CCP; Autoantibodies; Rheumatoid Arthritis