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OP0177 A High Frequency of N-Glycans in the Acpa-Igg Variable Domain Modulates Reactivity to Citrullinated Antigens
  1. H.U. Scherer1,
  2. Y. Rombouts1,2,
  3. A. Willemze1,
  4. J. van Beers3,
  5. J. Shi1,
  6. P. Kerkman1,
  7. G. Janssen4,
  8. P. van Veelen4,
  9. A. Zaldumbide5,
  10. R. Hoeben5,
  11. G. Pruijn3,
  12. A. Deelder2,
  13. G.-J. Wolbink6,7,
  14. T. Rispens6,
  15. T.W. Huizinga1,
  16. M. Wuhrer2,
  17. L. Trouw1,
  18. R. Toes1
  1. 1Department of Rheumatology
  2. 2Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden
  3. 3Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen
  4. 4Department of Immunohematology and Blood Transfusion
  5. 5Department of Molecular Cell Biology, Leiden University Medical Center, Leiden
  6. 6Sanquin Research and Landsteiner Laboratorium, Academic Medical Center
  7. 7Jan van Breemen Research Institute | Reade, Amsterdam, Netherlands

Abstract

Background Antibodies against citrullinated proteins antigens (ACPA) are the most relevant prognostic and diagnostic biomarker for rheumatoid arthritis (RA). ACPA positive patients are characterized by progressive disease, a high rate of joint destruction, and a low chance to achieve remission. Despite these strong and well-defined associations with clinical phenotype, however, molecular mechanisms underlying the citrulline-specific immune response are incompletely understood. Of note, this immune response is highly polyclonal with considerable cross-reactivity, develops and matures prior to disease onset, and is of remarkably low avidity. These features indicate that the underlying B cell response is different from “conventional” antibody responses, but also different from other autoantibody responses such as the high affinity double-stranded DNA antibody response in SLE.

Objectives To study molecular characteristics of ACPA in order to obtain insight into the properties of the underlying B cell response.

Methods Serum of ACPA positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. In addition, ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum and synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and subsequently analysed by HPLC and mass-spectrometry. Recombinant monoclonal ACPA were used to study binding affinity for multiple antigens by surface plasmon resonance.

Results In almost all donors studied (23 out of 24), ACPA-IgG molecules were found to have a higher molecular weight than non auto-reactive IgG. Structural analysis pinpointed this observation to a high frequency of N-glycans in the ACPA variable regions, which was found on up to 90% of all ACPA. These Fab-glycans were linked to N-glycosylation sites generated by somatic hypermutation, were absent from other disease-specific autoantibodies, were characterized by a high degree of sialylation, and clearly differentiated ACPA from “conventional” IgG molecules. Moreover, ACPA Fab glycans modulated reactivity to citrullinated antigens.

Conclusions These data describe a unique and completely novel feature of the citrulline-specific immune response in RA, with important implications for ACPA pathogenicity and disease prognostication. The high frequency of variable domain Fab glycans points to specific developmental abnormalities of the underlying B cell response.

Disclosure of Interest None declared

DOI 10.1136/annrheumdis-2014-eular.4717

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