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 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.

Complex Relationships of Smoking, HLA–DRB1 Genes, and Serologic Profiles in Patients With Early Rheumatoid Arthritis: Update From a Swedish Population‐Based Case–Control Study

“…The situation was substantially different in the RF−/anti-CCP2+ RA group, in whom there was a major and gene dose-dependent effect of HLA-DRB1 and a more limited, but still visible, effect of smoking, particularly in heavy smokers. This finding is compatible with prior reports of high numbers of T cell-dependent somatic mutations in genes coding for anticitrulline-reactive antibodies (15)(16)(17).…”

Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell–Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis

“…Peptide ELISAs were performed as previously described (20), with some minor modifications. The ELISAs assessed binding to both citrulline-containing and arginine (Arg)-containing peptides, including filaggrin, α-enolase, vimentin, fibrinogen, and histones H4 , H4 [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] , and H3 . All peptides assessed by ELISA are described in further detail in Supplementary Table 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/ art.40699/abstract.…”

Reply

“…Interestingly, one emerging unifying feature of ACPAs, which was first demonstrated in polyclonal ACPAs purified from RA serum, is the extensive N ‐glycosylation sites in the Fab region . This was verified, in mAb studies, to be a result of the pronounced accumulation of somatic hypermutations ; these results were based on mAb sequences from our recent study that Dr. Holmdahl comments on, as well as the study by Titcombe et al . This feature is also true for the mAb from van de Stadt et al that Dr. Holmdahl used.…”