OP0333 RISING ACPA IGG VARIABLE DOMAIN GLYCOSYLATION PRE-DISEASE ASSOCIATES WITH AN INCREASE IN AUTOANTIBODY LEVELS AND THE DEVELOPMENT OF RHEUMATOID ARTHRITIS
Authors: Theresa Kissel, Karin van Schie, Lise Hafkenscheid, Anders Lundquist, Hans Ulrich Scherer, Heidi Kokkonen, Rene Toes, Solbritt Rantapää Dahlqvist
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Anti-citrullinated protein antibodies (ACPA) are present in the majority of rheumatoid arthritis (RA) patients (60-70%) and play a pivotal role in disease development<sup>1</sup>. ACPA IgGs are unique in a way that they are abundantly N-glycosylated within their variable regions<sup>2</sup>. These variable domain (V-domain) glycans are found on over 90% of ACPAs present in sera from RA-patients<sup>3</sup>. To undergo N-linked glycosylation, a consensus sequence in the protein backbone is required (N-X-S/T, where X ≠ P)<sup>4</sup>. Recently, it was shown that the N-linked glycosylation sites in ACPA-IgG V-domains are introduced during somatic hypermutation and that the introduction of such sites likely conveys a selective advantage to ACPA expressing B-cells<sup>5</sup>. However, it is currently unknown, whether ACPA-expressing B-cells already introduced glycosylation-sites into their V-domains before disease onset or when ACPA-V-domain glycosylation occurs. <h3>Objectives</h3> Investigate the appearance of ACPA V-domain glycosylation in pre-symptomatic individuals and RA patients. <h3>Methods</h3> In a case-control study, individuals (n=201) from the Medical Biobank, who were sampled before onset of symptoms (mean±SEM predating time; 5.8±0.3 years) (140 aCCP+ and 61 aCCP-), and after diagnosis of RA (n=99, 94 aCCP+ and 5 aCCP-) and randomly selected control samples (n=43, 3 aCCP+ and 40 aCCP-) were analyzed for their ACPA IgG V-domain glycosylation levels. ACPA IgGs were affinity purified, N-linked glycans released, and 2-AA labeled for further analysis using UHPLC<sup>6</sup>. Data calibration and integration was performed and a cut-off defined. Samples below the cut-off were determined as non-detectable and excluded from the analysis. The percentage V-domain glycosylation was calculated as described previously<sup>3</sup>. Statistical calculations were performed using SPSS. <h3>Results</h3> Our data indicated that ACPA IgG V-domain glycans are already present years before symptom onset, in pre-symptomatic individuals who subsequently will develop RA. Analysis of matched pairs showed a significant increase of ACPA V-domain glycosylation in RA patients compared to individuals pre-disease (p&lt;0.001). The results showed that ACPA N-glycosylation was correlated with anti-CCP concentrations pre-disease (r<sub>s</sub> =0.504, p&lt;0.001), while no such association can be found after RA onset. Further, V-domain glycosylation levels increase closer to symptom onset. <h3>Conclusion</h3> Our analysis revealed that ACPA IgG molecules can harbor V-domain glycans already relatively long time before disease onset. An increase in ACPA V-domain glycosylation, presumably due to the generation of de-novo N-glycosylation sites or the expansion of N-glycosylation site-bearing clones was associated with an increase in ACPA levels. These results suggest that ACPA-expressing B cells gain a selective advantage through the generation V-domain N-glycosylation in parallel with rising ACPA-levels in serum in the phase prior to the development of arthritis. <h3>References</h3> [1] https://doi.org/10.1038/nrrheum.2011.204 [2] https://doi.org/10.1136/annrheumdis-2014-206598 [3] https://doi.org/10.1074/mcp.M116.062919 [4] J. Biol. Chem. 1996, 271 (11), 6363–6366, PMID: 8626433 [5] https://doi.org/10.1136/annrheumdis-2017-212052 [6] https//doi.org/10.1371/journal.pone.0200280 <h3>Disclosure of Interests</h3> Theresa Kissel: None declared, Karin van Schie: None declared, Lise Hafkenscheid: None declared, Anders Lundquist: None declared, Hans Ulrich Scherer Grant/research support from: Sanofi, BMS, Heidi Kokkonen: None declared, Rene Toes Grant/research support from: Sanofi, Solbritt Rantapää Dahlqvist Consultant for: Member of the advisory board, Lipum AB, Umeå, Sweden.