Protein citrullination and production of ACPAs are hallmarks of rheumatoidal arthritis (RA). Proteomic analysis of citrullinome of patients with RA revealed complement proteins as targets for PADs. The complement system (CS), essential element of the innate immunity is responsible for protection against microbes, effective clearance of necrotic and apoptotic cells, regulation of inflammation and control of the interplay between the innate and adaptive branches of immune system. The disruption of CS homeostasis leads to pathological processes like: destruction of self-cells and tissues in case of excessive activation, while impaired activation results in reduced efficiency of the clearance of the dead cells leading to the development of the autoimmune diseases (like RA).
The aim of our study was to investigate susceptibility of major CS proteins for deimination by PAD4 in vitro and to check how this modification influences their function.
We confirmed efficient modification and mapped citrullination sites in major CS proteins using phenylglyoxal probes and MS. Citrullination of human serum showed decrease of total CS activity in both classical and alternative pathway, evaluated with hemolytic assays CH50 and AP50 respectively. Using CH50 assay and sera depleted for certain CS components we showed that functionally, among the most vulnerable CS proteins were C3, C4, C1q and C5, which lost around 50-70% of activity upon modification. Western Blot analysis of the activation process for C3 and C5 showed different effects of modification for those proteins, as citrullination decreased rate of C5 proteolytic activation, while decrease in C3 activity is related to impaired interaction of CitC3 with other proteins. Citrullination of major anaphylatoxins was confirmed with RP-HPLC and MS, showing citrullination of crucial C-terminal arginines. C5aCit showed impaired activity on U937-C5aR cells in calcium influx assay.