Title: Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: implications for B-cell selection,
Abstract: 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 decreased 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 also through non-antigen driven
mechanisms.