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

• Published in: Molecular & cellular proteomics Vol. 16; no. 2; pp. 278 - 287
• Main Authors: Hafkenscheid, Lise, Bondt, Albert, Scherer, Hans U., Huizinga, Tom W.J., Wuhrer, Manfred, Toes, René E.M., Rombouts, Yoann
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2017; American Society for Biochemistry and Molecular Biology; The American Society for Biochemistry and Molecular Biology
• Subjects: Antibodies; Arthritis; Arthritis, Rheumatoid - immunology; Autoantibodies - chemistry; Autoantibodies - immunology; Autoantibodies - metabolism; Biological effects; Chromatography, High Pressure Liquid; Citrulline; Fab; Glycoproteins; Glycosylation; Humans; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin G; Immunoglobulin G - analysis; Immunoglobulin G - blood; Immunoglobulin G - immunology; Immunology; Lectins; Light chains; Mass spectrometry; Mass spectroscopy; N-glycans; Patients; Peripheral blood; Polysaccharides; Polysaccharides - chemistry; Rheumatoid arthritis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structural analysis; Synovial fluid; Synovial Fluid - chemistry; Synovial Fluid - immunology
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M116.062919

Recently, we showed the unexpectedly high abundance of N-linked glycans on the Fab-domain of Anti-Citrullinated Protein Antibodies (ACPA). As N-linked glycans can mediate a variety of biological functions, we now aimed at investigating the structural composition of the Fab-glycans of ACPA-IgG to better understand their mediated biological effects. ACPA-IgG and noncitrulline specific (control) IgG from plasma and/or synovial fluid of nine ACPA positive rheumatoid arthritis patients were affinity purified. The N-linked glycosylation of total, Fc and F(ab′)2 fragments, as well as heavy and light chains of ACPA-IgG and control IgG were analyzed by UHPLC and MALDI-TOF mass spectrometry. The Fc-glycosylation of ACPA-IgG and IgG was analyzed at the glycopeptide level using LC-MS. The structural analyses revealed that ACPA-IgG molecules contain highly sialylated glycans in their Fab-domain. Importantly, Fab-glycans were estimated to be present on over 90% of ACPA-IgG, which is five times higher than in control IgG isolated from the same patients. This feature was more prominent on ACPA isolated from synovial fluid compared with peripheral blood. These observations provide the first evidence pointing to the ability of ACPA-IgG to mediate novel immunological activities, for example through binding specific lectins via hyper-sialylated Fab-glycans.