1. Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation
- Author
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Olexandr Korchynskyi, Ken Yoshida, Nataliia Korchynska, Justyna Czarnik-Kwaśniak, Paul P. Tak, Ger J. M. Pruijn, Takeo Isozaki, Jeffrey H. Ruth, Phillip L. Campbell, M. Asif Amin, Alisa E. Koch, Korchynskyi, Olexandr [0000-0002-4557-4003], Tak, Paul P [0000-0002-3532-5409], Pruijn, Ger JM [0000-0001-7208-6200], Isozaki, Takeo [0000-0002-8621-7643], and Apollo - University of Cambridge Repository
- Subjects
partial degradation ,Mammals ,citrullination ,Glycosylation ,Organic Chemistry ,monocyte chemoattractant protein-1 ,Bio-Molecular Chemistry ,Endothelial Cells ,Proteins ,General Medicine ,Catalysis ,Computer Science Applications ,Inorganic Chemistry ,Arthritis, Rheumatoid ,post-translational modifications ,Animals ,Humans ,Citrulline ,glycosylation ,peptidylarginine deiminase ,Physical and Theoretical Chemistry ,Molecular Biology ,Spectroscopy ,Chemokine CCL2 - Abstract
Peer reviewed: True, Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid arthritis (RA), compared to osteoarthritis or other arthritis patients. Several studies suggested an important role for MCP-1 in the massive inflammation at the damaged joint, in part due to its chemotactic and angiogenic effects. It is a known fact that the post-translational modifications (PTMs) of proteins have a significant impact on their properties. In mammals, arginine residues within proteins can be converted into citrulline by peptidylarginine deiminase (PAD) enzymes. Anti-citrullinated protein antibodies (ACPA), recognizing these PTMs, have become a hallmark for rheumatoid arthritis (RA) and other autoimmune diseases and are important in diagnostics and prognosis. In previous studies, we found that citrullination converts the neutrophil attracting chemokine neutrophil-activating peptide 78 (ENA-78) into a potent macrophage chemoattractant. Here we report that both commercially available and recombinant bacterially produced MCP-1/CCL2 are rapidly (partially) degraded upon in vitro citrullination. However, properly glycosylated MCP-1/CCL2 produced by mammalian cells is protected against degradation during efficient citrullination. Site-directed mutagenesis of the potential glycosylation site at the asparagine-14 residue within human MCP-1 revealed lower expression levels in mammalian expression systems. The glycosylation-mediated recombinant chemokine stabilization allows the production of citrullinated MCP-1/CCL2, which can be effectively used to calibrate crucial assays, such as modified ELISAs.
- Published
- 2023