Protein citrullination is a post-translational modification of arginine residues that converts peptidyl arginine into peptidyl citrulline. It is an important process implicated in gene regulation, immune function and skin and hair growth. Patients with rheumatoid arthritis (RA) have been shown to possess abnormally elevated levels of auto-antibodies in their synovial fluid that specifically target citrullinated proteins.1
Although abnormal protein citrullination has been implicated in a pathogenic role in RA, that precise role is unclear. It is unknown whether citrullination reflects ongoing inflammation or whether it is involved in the onset of pathogenesis in RA.2 A study by Romero et al. (2014) was able to demonstrate that the cellular hypercitrullination present in synovial fluid (SF) cells in RA may be the result of immune-mediated membranolytic pathways.3 The researchers report that these activate a family of calcium-dependent peptidyl arginine deiminases (PADs) in inflammatory cells within the joint.
Perforin and the membrane attack complex (MAC) induce the citrullination of numerous auto-antigens previously described in RA. The investigators used anti-modified citrulline immunoblotting to identify cellular citrullination. They then defined the perforin- and MAC-induced “citrullinome” by mass spectrometry (MS) and compared it with the citrullinome found in RA SF cells.
Samples from RA SF and neutrophils were incubated overnight at 37°C and centrifuged before analysis using an Easy-nLC 1000 on-line with an Orbitrap Elite coupled to a Nano-Flex ion source (Thermo Scientific). The subsequent data output was searched against the Human International Protein Index database version 3.80.
Previous studies have identified 16 intracellular citrullinated proteins that are confirmed targets of RA auto-antibodies. Of these, 13 were identified in RA SF cells, 14 in perforin-treated cells, and 14 in MAC-treated cells. Citrullinated auto-antigens found in RA SF cells overlapped completely with those found in perforin-treated cells and with 12 auto-antigens in the MAC samples. Cellular hypercitrullination and citrullinated auto-antigen production are therefore common features that unify RA SF cells and PAD activation induced by immune-mediated pore-forming pathways.
Citrullinated cells isolated from an RA joint contained a broad spectrum of citrullination across a range of molecular weights, which the researchers termed cellular hypercitrullination. This was in contrast to previously described patterns of citrullination in neutrophils in response to stimuli—such as cytokines, toll-like receptor ligands, and N-formylated peptides—and during several forms of cell death where histone citrullination is prominent.
Using MS, the authors were able to demonstrate that perforin and MAC pathways are active in the RA joint. The perforin and MAC pathways activate intracellular PADs and induce the pattern of hypercitrullination present in the RA target tissue. This pattern of cellular hypercitrullination activated through two immune-mediated membranolytic pathways is consistent with a preferred model of RA that suggests that infectious or inflammatory events contribute to initiation of immune responses to citrullinated proteins.
Furthermore, neutrophil extracellular traps, or NETs, have been thought to play a role in the pathogenesis of inflammation; these are typically released during a form of pathogen-induced cell death called NETosis. Histone citrullination contributes to the antimicrobial response that allows NETs to form. The authors were able to show, via MS, that NETs are not a source of cellular hypercitrullination found in the RA joint. When NETs were examined by MS, there was no citrullinated protein cargo.
Combined with existing knowledge, this study indicates that there are connections between immune-mediated membranolytic pathways and the activation of the PAD enzymes in RA. The authors suggest that these interactions may be novel targets for treatment of RA.
1. Masson-Bessiere, C., et al. (2001) “The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- and beta-chains of fibrin,” Journal of Immunology, 166(6) (pp. 4177–84).
2. Pongratz, G., and Fleck, M. (2012) “Anti citrullinated protein antibodies and mechanism of action of common disease modifying drugs–Insights in pathomechanisms of autoimmunity,” Current Pharmaceutical Design, 18(29) (pp. 4526–36).
3. Romero, V., et al. (2014) “Immune-Mediated Pore-Forming Pathways Induce Cellular Hypercitrullination and Generate Citrullinated Autoantigens in Rheumatoid Arthritis,” Science Translational Medicine, 5(209) (p. 209ra150), doi: 10.1126/scitranslmed.3006869.
Post Author: Miriam Pollak. Miriam specialised in neuroscience as an undergraduate but traded in lab work for a post graduate degree in science communication.
She has since had a career that has spanned science communication, science education and communications management.
However, Miriam has found her bliss balancing her love of writing and disseminating medical research with managing a multimillion dollar research budget for a childhood cancer charity in Australia.