SAT0053 GLYCOSYLATION IN MAMMALS PROTECTS CITRULLINATED CHEMOKINES FROM PARTIAL DEGRADATION
Authors: Olexandr Korchynskyi
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Citrullination is a postranslational modification of specific proteins by peptidylarginine deiminase (PAD) activities. Nowadays citrullination is recognized as a hallmark of rheutamoid arthritis and other autoimmune diseases. In our recent study we have shown a presence of citrullinated chemokines epithelial-derived monocyte chemotactic protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1α (MIP-1α/CCL3) and neutrophil-activating peptide 78 (ENA-78/CXCL5) in biological fluids collected from patients suffering from rheumatoid arthritis (RA) versus non-autoimmune arthritis diseases and demonstrated citrullinated ENA-78/CXCL5 as an efficient macrophage chemoattractant in a contrast with non-citrullinated ENA-78/CXCL5. In our current work we found that citrullinated <i>in vitro</i> bacterially produced chemokines cannot be efficiently used as the standards in modified enzyme linked immunosorbent assay (ELISA) assays designed to detect citrullinated chemokines. <h3>Objectives:</h3> In our work we aimed generate a procedure for preparation of stable citrullinated chemokines suitable for research aplications and to validate a<i> hypothesis that posttranslational modifications occurring in mammalian cells that can stabilize chemokines can also protect citrullinated chemokines from quick degradation</i>. <h3>Methods:</h3> MCP-1/CCL2 and MIP-1α/CCL3 were cloned from total RNA isolated from synovial fibroblasts obtained from RA patient. Both bacterially-produced and mammalian cells-produced recombinant human chemokines were citrullinated by commercial rabbit PAD2. Success in citrullination was confirmed with Western blotting and mass-spectrometry. Citrullinated chemokine concentrations were measured by modified sandwich ELISA assays. <h3>Results:</h3> Both commercially available and self-made bacterially produced chemokines MCP-1/CCL2, MIP-1α/CCL3 and ENA-78/CXCL5 undergo quick partial degradation upon their <i>in vitro</i> citrullination by PAD2 and cannot be detected with either Western blotting or mass-spectrometry. At the same time mammalian cells-produced properly glycosylated MCP-1/CCL2, MIP-1α/CCL3 and ENA-78/CXCL5 can be efficiently citrullinated and successfully used as the standards in modified ELISA assays as well as in bioassays. <h3>Conclusion:</h3> Glycosylation that is lucking in bacterially-produced proteins but occurs in mammalian cells stabilizes citrullinated chemokines thus protecting them from quickly ongoing partial degradation. <h3>BioEthics Committee Approval:</h3> All human subject samples were collected after approval by the Institutional Review Board of the Academic Medical Center/University of Amsterdam, Amsterdam, The Netherlands (Protocol MEC 07/079 #10.17.0708) and provision of informed consent by the patients. <h3>Reference</h3> [1] Yoshida K., Korchynskyi O., Tak P.-P., Isozaki T., Ruth J.H., Campbell P.L., Baeten D.L., Gerlag D.M., Amin M.A., and. Koch A.E. (2014) Arthritis and Rheumat 66, No 10, P.2716-2727. <h3>Disclosure of Interests:</h3> None declared