B-cell receptor sequencing of anti-citrullinated protein antibody (ACPA) IgG-expressing B cells indicates a selective advantage for the introduction of N-glycosylation sites during somatic hypermutation

The majority of patients with rheumatoid arthritis (RA) harbours IgG antibodies targeting citrullinated protein antigens (ACPA). Recently, we showed that >90% of ACPA-IgG in serum are glycosylated in the variable domain.1  N -linked glycosylation requires a consensus sequence in the protein backbone (N-X-S/T (asparagine-X-serine/threonine), where X is any amino acid except proline), which is scarce in germline-encoded Ig variable region genes.2 3 Accordingly, hyperglycosylation of ACPA-IgG requires either clonal expansion of B cells expressing B-cell receptors (BCR) containing germline-encoded N -glycosylation sites or generation of de novo sites through somatic hypermutation (SHM).4 Here, we analysed the BCR repertoire of ACPA-expressing B cells to understand the molecular basis of this remarkable glycosylation. ACPA-expressing B cells were sorted as pools (10 cells per pool) from peripheral blood mononuclear cells of eight patients with ACPA-positive RA.5 Anchoring reverse transcription of immunoglobulin sequences and amplification by nested (ARTISAN) PCR-based BCR sequencing6 followed by full-length variable region IgG transcript analysis revealed high nucleotide mutation rates in 97 unique ACPA-IgG heavy chains (HC; mean ±SD: 52.86±16.73; figure 1A). 81% of these contained one or more N -glycosylation sites. Figure 1 A high degree of somatic hypermutation in antibodies targeting citrullinated protein antigens (ACPA)-IgG clones which does not correlate with the frequency of N -glycosylation sites. Pool and single cells were sorted as described.5 All independent clones are defined as identical V, D, J genes and …