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.