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Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis
  1. Yoann Rombouts1,2,
  2. Annemiek Willemze1,
  3. Joyce J B C van Beers3,
  4. Jing Shi1,
  5. Priscilla F Kerkman1,
  6. Linda van Toorn1,
  7. George M C Janssen4,5,
  8. Arnaud Zaldumbide6,
  9. Rob C Hoeben6,
  10. Ger J M Pruijn3,
  11. André M Deelder2,
  12. Gertjan Wolbink7,8,
  13. Theo Rispens7,
  14. Peter A van Veelen4,
  15. Tom W J Huizinga1,
  16. Manfred Wuhrer2,
  17. Leendert A Trouw1,
  18. Hans U Scherer1,
  19. René E M Toes1
  1. 1Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands
  2. 2Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands
  3. 3Radboud Institute for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University, Nijmegen, the Netherlands
  4. 4Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands
  5. 5Netherlands Proteomics Centre, Utrecht, the Netherlands
  6. 6Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands
  7. 7Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Amsterdam, the Netherlands
  8. 8Jan van Breemen Research Institute Reade, Amsterdam, the Netherlands
  1. Correspondence to Dr Yoann Rombouts, Department of Rheumatology, C-05-61, Leiden University Medical Center, Postbus 9600, Leiden 2300 RC, The Netherlands; y.j.p.c.rombouts{at}lumc.nl

Abstract

Objectives 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.

  • Rheumatoid Arthritis
  • Autoantibodies
  • Ant-CCP

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