Structural Analysis of Variable Domain Glycosylation of Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Reveals the Presence of Highly Sialylated Glycans*
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.
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Author contributions: L.H. and Y.R. carried out the experiments. A.B. assisted with data analysis. H.U.S., T.W.J.H., M.W., R.E.M.T., and Y.R. designed the study. L.H., R.E.M.T., and Y.R. interpreted the data and wrote the paper. All authors reviewed the manuscript.
The authors declare no competing financial interests.
This work was supported by The Netherlands Organization for Scientific Research (NWO) (project 435000033) and the IMI funded project BeTheCure (contract 1151422). L.H. was supported by the Dutch Arthritis Foundation (NR 12-2-403). H.U.S. is the recipient of an NWO-ZonMW clinical fellowship (project 90714509). Y.R. was supported by a Boehringer Ingelheim funded project within BeTheCure and by NWO (435000033). A.B. and M.W. were supported by funding from the European Union's Seventh Framework Programme (FP7-Health-F5-2011) under Grant Agreement no. 278535 (HighGlycan).
