LAUSR

Background: 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 in vitro bacterially produced chemokines cannot be efficiently used as the standards in modified enzyme linked immunosorbent assay (ELISA) assays designed to detect citrullinated chemokines. Objectives: In our work we aimed generate a procedure for preparation of stable citrullinated chemokines suitable for research aplications and to validate a hypothesis that posttranslational modifications occurring in mammalian cells that can stabilize chemokines can also protect citrullinated chemokines from quick degradation. Methods: 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. Results: 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 in vitro 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. Conclusion: Glycosylation that is lucking in bacterially-produced proteins but occurs in mammalian cells stabilizes citrullinated chemokines thus protecting them from quickly ongoing partial degradation. BioEthics Committee Approval: 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. Reference [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. Disclosure of Interests: None declared