1. Carbamylation reduces the capacity of IgG for hexamerization and complement activation.
- Author
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Lubbers R, Oostindie SC, Dijkstra DJ, Parren PWHI, Verheul MK, Abendstein L, Sharp TH, de Ru A, Janssen GMC, van Veelen PA, van den Bremer ETJ, Bleijlevens B, de Kreuk BJ, Beurskens FJ, and Trouw LA
- Subjects
- Aged, Amino Acid Sequence, Antibodies, Monoclonal immunology, Antibodies, Monoclonal metabolism, Arthritis, Rheumatoid metabolism, Cell Line, Tumor, Complement C1q metabolism, Cytotoxicity Tests, Immunologic, Enzyme-Linked Immunosorbent Assay, Humans, Immunoglobulin G chemistry, Immunoglobulin G metabolism, Male, Mass Spectrometry, Middle Aged, Protein Carbamylation immunology, Protein Multimerization immunology, Synovial Fluid immunology, Synovial Fluid metabolism, Synovial Membrane immunology, Synovial Membrane metabolism, Arthritis, Rheumatoid immunology, Complement Activation immunology, Complement C1q immunology, Immunoglobulin G immunology
- Abstract
Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers., (© 2019 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)
- Published
- 2020
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