Background/Purpose:

The vast majority (>90%) of anti-citrullinated protein antibodies (ACPA) of the IgG isotype in serum and synovial fluid of patients with rheumatoid arthritis carry N-linked glycans in the antibody variable region. This remarkable degree of Fab-glycosylation is absent from ACPA-depleted control IgG and from autoantibodies in other diseases. N-glycosylation requires a specific amino acid consensus sequence in the protein backbone (termed an N-glycosylation site), which is very rare in germline-encoded variable region genes. Here, we analysed the B cell receptor (BCR) repertoire of ACPA-expressing B cells to understand the molecular basis for this remarkable glycosylation.

Methods:

We used anchoring reverse transcription of immunoglobulin (Ig) sequences and amplification by nested PCR (ARTISAN) to obtain full-length rearrangements of ACPA-expressing B cells. ACPA-expressing B cells and non citrulline-reactive control B cells were obtained from peripheral blood of patients with established RA by antigen-specific tetramer staining and fluorescence activated cell sorting. Cells were either sorted in pools, or sorted as single cells. Somatic mutations and N-glycosylation sites were identified in the sequence reads using IMGT (High)V-QUEST. Paired heavy and light chain sequences (HC/LC) were used to model the spatial positioning of N-glycosylation sites.

Results:

Sequence analysis of pools of cells identified 97 unique ACPA-IgG clones derived from n=8 donors. 87 unique ACPA-IgG clones were retrieved from single cell analysis (n=6 donors). In both datasets, over 80% of ACPA-IgG clones contained N-glycosylation sites in both HC and LC. All sites were created by somatic mutations. The mutation rate did not correlate with the number of N-glycosylation sites, and their distribution across the variable region and their preference differed from the pattern seen in controls. Structural modelling predicted N-glycosylation sites on the exterior of the antibody molecule.

Conclusion:

ACPA-expressing B cells generate BCRs with a high frequency of N-glycosylation sites. Frequency and localization of sites suggest that ACPA-expressing B cells gain a selective survival advantage by acquiring glycans in the variable domain, thereby escaping from putative checkpoints in B cell selection.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/b-cell-receptor-sequencing-of-anti-citrullinated-protein-antibody-expressing-b-cells-indicates-a-selective-advantage-for-the-introduction-of-n-glycosylation-sites-during-somatic-hypermutation/