IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses, Fc-Glycans and a Fab-Peptide Sequence

Abstract

The Fc-glycan profile of IgG1 anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG1, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG4 peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG4, the ACPA-IgG4 Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG1 compared to FT-IgG1 were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG1 relative to FT-IgG1 as well as lower content of IgG2 (p = 0.0000001) and elevated content of IgG4 (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG1 and ACPA-IgG4 are distinct. Given that IgG1 and IgG4 have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.

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

“…Interestingly, the Fab-glycosylation pattern of ACPA-IgG showed a significantly higher frequency of galactose, sialic acid and fucose residues as compared with that of control IgG depleted of ACPA. These high galactosylation and sialylation levels of the Fab-linked glycans are in clear contrast to the lower level of Structural Analysis of ACPA-IgG Fab-glycosylation galactosylation and sialylation previously detected in the Fcpart of ACPA-IgG (23,28,29). Therefore, our data show that the changes in antibody glycosylation occurring during RA are not only (auto)antibody-specific, but also site-specific.…”

“…ACPA-positive RA is characterized by a high rate of joint erosions and a low chance to achieve remission when left untreated (15). Earlier, we and others reported that the galactosylation and sialylation levels of Fc-glycans from ACPA-IgG is lower compared with control IgG, and this is more pronounced in ACPA-IgG isolated from SF (28,29). In addition, we observed that the changes of ACPA-IgG Fcglycosylation already occur a few months before of the diagnosis of RA (23).…”

The perfect storm: HLA antibodies, complement, FcγRs, and endothelium in transplant rejection

“…Future work detailing glycan profiles of DSA would determine if antibody glycosylation status correlates with severity of AMR. Additionally, new methodology described to simultaneously measure both the subclass and glycosylation of antigen-specific IgG [94] may be adapted to transplantation.…”

Microscale purification of antigen-specific antibodies

“…Among these, glycoprofile changes among antigen-specific antibodies over time following vaccination have been observed (Selman et al, 2012), and a role for T cell help in modulating IgG glycosylation defined (Hess et al, 2013; Oefner et al, 2012). In addition, altered glycosylation of virus-specific IgG has been associated with altered antiviral antibody function among HIV-infected subjects (Ackerman et al, 2013), anti-citrullinated peptide/protein antibodies (ACPA) isolated from subjects with rheumatoid arthritis have been observed to posses altered glycosylation that can precede disease onset and differ between serum and within synovial fluid (Lundstrom et al, 2014; Rombouts et al, 2015a; Scherer et al, 2010), and glycovariation among anti-proteinase 3 antibodies has been found to correlate with disease activity as determined by clinical vasculitis score (Wuhrer et al, 2015). Successful allergy immunotherapy has been associated with the induction of sialylated allergen-specific antibodies (Oefner et al, 2012).…”