Activation of peptidylarginine deiminase by reduced glutathione
Peptidylarginine deiminases (PADs) catalyzing the conversion of amino acid arginine to amino acid citrulline (known as citrullination) plays a significant part in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and numerous other inflammatory diseases. Reducing agents like dithiothreitol (DTT) are normally used to define the in vitro activity of PADs.
Recently, a group of investigators, worked hard to evaluate the capacity of reduced glutathione (GSH), the most profuse intracellular small-molecule thiol in vivo, to activate PADs.
Using an in-house PAD activity assay detecting citrullination of fibrinogen, activity 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.
Without addition of an exogenous reducing agent, there was no activity of rhPAD2, rhPAD4 or PADs within SF. In the presence of 1 mM DTT or 10–15 mM GSH, activity of both recombinant and SF PAD was seen. Succeeding 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. Conforming supernatants showed no PAD activity, but addition of exogenous GSH reinstated activity.
The findings from the study recommended reducing conditions as a prerequisite for catalytic activity of PAD. GSH meets this requirement at concentrations comparable with those found within cells. PMA-stimulated granulocytes release active PAD which is reduced by GSH but becomes inactive in the extracellular space.