Yoann Rombouts1, Annemiek Willemze2, Joyce J B C van Beers3, Jing Shi2, Priscilla F Kerkman2, Linda van Toorn2, George M C Janssen4, Arnaud Zaldumbide5, Rob C Hoeben5, Ger J M Pruijn3, André M Deelder6, Gertjan Wolbink7, Theo Rispens8, Peter A van Veelen9, Tom W J Huizinga2, Manfred Wuhrer6, Leendert A Trouw2, Hans U Scherer2, René E M Toes2. 1. Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands. 2. Department of Rheumatology, Leiden University Medical Center, Leiden, the Netherlands. 3. Radboud Institute for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University, Nijmegen, the Netherlands. 4. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands Netherlands Proteomics Centre, Utrecht, the Netherlands. 5. Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands. 6. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands. 7. Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Amsterdam, the Netherlands Jan van Breemen Research Institute Reade, Amsterdam, the Netherlands. 8. Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Amsterdam, the Netherlands. 9. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands.
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. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/
OBJECTIVES: To understand the molecular features distinguishing anti-citrullinated protein antibodies (ACPA) from 'conventional' antibodies in rheumatoid arthritis (RA). METHODS: Serum of ACPA-positive RApatients 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. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/
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