Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis

Abstract

The vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the 'germline-counterparts' of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.

Structural Analysis of Variable Domain Glycosylation of Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Reveals the Presence of Highly Sialylated Glycans

“…The supplier's protocol was adjusted to simplify the procedure as previously described (13). Briefly, for each sample, 30 g of (ACPA)-IgG antibodies were dried under centrifugal evaporator and digested by adding 200 l digestion buffer (50 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA) containing 30U of IdeS followed by incubation at 37°C for overnight.…”

“…Plasma and SF samples were then centrifuged at 3000 rpm for 10 min and the resulting supernatants were filtered using 0.4 m filters (Millipore). ACPA-IgG and IgG were purified on fast protein liquid chromatography (Ä KTA, GE Healthcare) as previously described (13). Briefly, samples were loaded on a biotinylated CCP2-arginine-HiTrapstreptavidin column (GE Healthcare) followed by a biotinylated CCP2-citrulline-HiTrap-streptavidin column connected in series.…”

“…ACPA-IgG-We have previously demonstrated that ACPAIgG produced by RA patients are extensively N-glycosylated in the variable region if compared with other IgG (auto)antibodies (13). Here we performed a comprehensive quantitative and qualitative analysis of the glycosylation of ACPA-IgG and its fragments and compared it to that of noncitrulline specific IgG (i.e.…”

“…To undergo N-linked glycosylation, proteins need to express an N-linked glycosylation consensus sequence (N-X-S/T, where X P). Importantly, we previously showed that N-linked glycosylation consensus sites in ACPA-IgG were not germ line-encoded but introduced after somatic hypermutation (13). This suggests that ACPA-producing B-cells with an N-glycosylation site in the BCR variable domain might have a selective advantage compared with other ACPA-producing B-cells.…”

“…Fab-glycans can also modulate cellular function and have been implicated in the emergence of lymphoma's such as follicular lymphoma, diffuse large B-cell lymphoma and Burkitt's lymphoma B-cells, presumably through the provision of aberrant Fab-glycosylated B-cell receptor cross-linking via the glycan to lectins (9 -12). Recently, we made the intriguing observation that anti-citrullinated protein antibodies (ACPA) 1 isolated from rheumatoid arthritis (RA) patients are extensively Fab-glycosylated (13). ACPA are highly specific for RA and have been implicated in disease pathogenesis, as their presence associates with disease severity and predicts the development of RA in subjects at risk (14,15).…”