Glycan profiling of anti–citrullinated protein antibodies isolated from human serum and synovial fluid

Abstract

Objective. Anti-citrullinated protein antibodies (ACPA) exhibit unique specificity for rheumatoid arthritis. However, it is incompletely understood whether and how ACPA contribute to disease pathogenesis. The Fc part of human IgG carries 2 N-linked glycan moieties that are crucial for the structural stability of the antibody and that modulate both its binding affinity to Fc␥ receptors and its ability to activate complement. We undertook this study to analyze Fc glycosylation of IgG1 ACPA in serum and synovial fluid (SF) in order to further characterize the immune response to citrullinated antigens.Methods. ACPA were isolated by affinity purification using cyclic citrullinated peptides as antigen. IgG1 Fc glycosylation was analyzed by mass spectrometry. ACPA IgG1 glycan profiles were compared with glycan profiles of total serum IgG1 obtained from 85 wellcharacterized patients. Glycan profiles of paired SF and serum samples were available from 11 additional patients.Results. Compared with the pool of serum IgG1, ACPA IgG1 lacked terminal sialic acid residues. In SF, ACPA were highly agalactosylated and lacked sialic acid residues, a feature that was not detected for total SF IgG1. Moreover, differential ACPA glycan profiles were detected in rheumatoid factor (RF)-positive and RFnegative patients.Conclusion. ACPA IgG1 exhibit a specific Fclinked glycan profile that is distinct from that of total serum IgG1. Moreover, Fc glycosylation of ACPA differs markedly between SF and serum. Since Fc glycosylation directly affects the recruitment of Fc-mediated effector mechanisms, these data could further our understanding of the contribution of ACPA to disease pathogenesis.Antibodies relevant to tissue pathology in autoimmune diseases are identified based on antigen-binding specificity of the variable region. Only very few autoantibodies, however, mediate pathology by direct interaction with the antigen. In most other cases, the constant region (Fc part) determines antibody-mediated effector functions, such as complement activation, antibodydependent cell-mediated cytotoxicity (ADCC), and engagement of activating or inhibitory Fc receptors (FcR). These Fc-mediated effects are influenced by the amino acid sequence of the Fc part (i.e., antibody isotype and subclass) and by Fc-linked carbohydrate structures. The latter are located in the C␥2 domain of the heavy chain in close proximity to amino acids that interact with FcR and the complement system. Accordingly, Fc-linked carbohydrate structures have recently received increasing attention, since modification of Fc-linked glycan residues of therapeutic antibodies has been shown to strongly influence the therapeutic potential of the antibodies (1-6).

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).…”

“…(ACPA)-IgG Derived from Plasma and Synovial Fluid Display Different Fab Glycosylation Profiles-We previously demonstrated that ACPA-IgG derived from the synovial fluid display a more proinflammatory Fc glycosylation profile than ACPAIgG purified from serum (28). Given this observation, we hypothesized that differences may also occur in the Fab-linked glycan structures and/or Fab-glycosylation levels of ACPAIgG and control IgG.…”

Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity

“…For example, rheumatoid arthritis and juvenile idiopathic arthritis are associated with decreased levels of IgG sialylation (15,16). Additional studies have shown that this translates particularly to the pathogenic antibodies in inflamed joints of arthritis patients (17). It has also been shown that IgG sialylation increases during pregnancy and that this increase may be associated with the remission of rheumatoid arthritis during pregnancy (18,19).…”

IgM and IgA Rheumatoid Factors Purified from Rheumatoid Arthritis Sera Boost the Fc Receptor– and Complement-Dependent Effector Functions of the Disease-Specific Anti–Citrullinated Protein Autoantibodies

“…Moreover, RF seem to exhibit enhanced avidity toward agalactosylated forms of IgG (30), which are elevated in RA patients with high disease activity (31). In RA patients, it was reported that ACPA IgG1 tended to be more agalactosylated compared with total serum IgG1, and that, in the joints, ACPA IgG1 was more heavily agalactosylated than in the general circulation (32). Within RA joints, ACPA IgG and citrullinated autoantigenic peptides likely form IC.…”