Variable domain N‐linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B‐cell selection

Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti‐citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable‐region glycosylation in single‐cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N‐linked glycosylation motifs in silico, and compared to 452 highly‐mutated mAbs from RA patients and controls. Variable region N‐linked motifs (N‐X‐S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N‐linked motifs compared to all studied mAbs including highly mutated HIV broadly‐neutralizing and malaria‐associated mAbs. The Fab glycans of ACPA‐mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B‐cell selection pressure and SHM‐mediated decrease in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti‐citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly through non‐antigen driven mechanisms.

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