Abstract
BackgroundCitrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs.MethodsActivity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen.ResultsNo activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10–15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity.ConclusionsCatalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space.
Highlights
Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases
No citrullination was observed when a pool of synovial fluid (SF) from four RA patients was used, we have previously shown that SF contained PAD2 and 1.5–2.5 mM calcium [12, 23]
The addition of rhPAD2 and rhPAD4 to SF did not result in detectable fibrinogen citrullination, except when DTT was present
Summary
Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs. Citrullination refers to the post-translational conversion of protein arginine residues into citrulline residues, a process catalysed by peptidylarginine deiminases (PADs) 1–4 and PAD6 [1]. Citrullination plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) [2, 3] and, possibly, in a number of other autoimmune diseases, Most studies on PAD activity and functional studies of citrullinated proteins have been based on in vitro citrullination using the reducing agent dithiothreitol (DTT) and exogenously added calcium. GSH has been demonstrated in cytosol and in organelles of virtually all cells of the body at the low millimolar range [15], whereas extracellular levels are two to three orders of magnitude lower [16, 17]
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