Background Fab-glycosylation is found in ~15%–25% serum IgG and while its exact consequence remains unknown, it may alter IgG functionality. Recent data revealed extensive Fab-glycosylation in polyclonal anti-citrullinated protein autoantibodies (ACPA) from rheumatoid arthritis (RA) patients. Herein, we characterise the Fab-glycan profile of monoclonal ACPA.

Materials and methods Among 200 single-cell isolated and recombinantly expressed monoclonal antibodies (mAbs) from RA patients, we selected 12 identified ACPA mAbs, four derived from synovial antibody secreting cells (ASC), and eight from peripheral antigen-tetramer sorted memory B cells. The ACPA had high somatic hypermutation (SHM) levels with an average of 49 VH and 35 VL mutations. For comparison, we selected 14 ASC synovial antibodies with mutation rate >20 in VH or VL, and we also searched the literature for 19 highly-mutated broadly-neutralising HIV-derived antibodies (bnAbs). N-linked glycan motifs were identified using the NetNGlyc server. Glycosylation was verified in five ACPA IgG by enzymatic digestion with PNGase-F or Endo-S followed by SDS-PAGE. Antigen binding was investigated by CCP3 ELISA. VH-VL structure models were generated using the online tool PIGS and visualised by Jmol, and the GlyProt server was utilised for in silico glycosylation.

Results The majority of ACPA exhibited variable region N-linked motifs (83.8%), compared to 14.3% of non-ACPA RA mAbs and 63.2% of bnAbs, featured in both framework and CDRs. When adjusted for SHM, N-linked motifs were significantly increased in ACPA compared to non-ACPA RA mAbs (p=0.001) or bnAbs (p=0.002). VH region motif rates were increased in ACPA compared to non-ACPA mAbs (p=0.002) and bnAbs (p=0.0004), while VL region motifs were only higher compared to non-ACPA (p=0.002). Deglycosylation revealed that N-linked motifs were indeed glycosylated, although preliminary data suggests glycan removal had no striking effect on antigen-binding. Homology-based structures predicted glycans to be primarily positioned outside of the potential antigen-binding site.

Conclusions The results support that variable region glycosylation is a key feature of ACPA. Significant increases in N-linked motifs in ACPA compared to other highly-mutated antibodies signifies that this is not solely linked to hypermutation. Future studies are merited to further investigate the selection mechanisms and functional role of Fab-glycosylated autoantibodies.