Variable fab domain N-glycosylation patterns in the B cell receptor repertoires of healthy individuals and patients with rheumatoid arthritis

Abstract

Abstract N-linked glycosylation (N-glyc) sites (N-X-S/T, X≠P) can be introduced by somatic hypermutation in immunoglobulin Fab regions. In patients with rheumatoid arthritis (RA), anti-citrullinated protein antibodies have a striking overrepresentation of Fab N-glycosylation. To further explore this, we sequenced B cell receptors (BCRs) from peripheral blood of 13 RA patients and 6 healthy control subjects, analyzing in total >250,000 heavy chain (VH) and >100,000 light chain sequences from both total B cells and citrullinated fibrinogen–reactive (Cit-Fib+) cells. Distribution of variable VH genes in VDJ DNA, and transcripts of unmutated IgM and class-switched BCR, revealed transcript gene-usage bias and higher VH4 in natural rearrangements by out-of-frame VDJ DNA in RA patients compared with control subjects. IgG Fab N-glyc sites were slightly more prominent in RA than control subjects (14.9% versus 12.1%; P = 0.048) with certain VH genes (e.g. VH1-18, VH1-69, VH3-9) displaying enriched N-glyc cumulative frequencies by somatic hypermutation. VH gene N-glyc hotspots were identified, explained by a lower threshold for codon conversion (i.e. K/S/T-X-S/T) and especially frequent in VH4s. Yet, RA patients had significantly more N-glyc in complementarity-determining region (CDR) 1 and 3 compared with control subjects. Expanded clonotypes with somatic hypermutation–induced N-glyc sites were delineated by network analysis in both the RA patients and control group, but patients with RA displayed more highly mutated class-switched members. Furthermore, Cit-Fib+ BCR-expanded clonotypes could be traced in the total B cell repertoire and exhibited increased frequency of N-glyc in mutated IgG/IgA subsets. Our findings highlight how Fab N-glyc sites can be linked to biased clonotype evolution and B cell selection in chronic responses.