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01.12.2016 | Research article | Ausgabe 1/2016 Open Access

Arthritis Research & Therapy 1/2016

Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase

Arthritis Research & Therapy > Ausgabe 1/2016
Dres Damgaard, Mads Emil Bjørn, Maria A. Steffensen, Ger J. M. Pruijn, Claus H. Nielsen
Wichtige Hinweise

Competing interests

DD and CHN are inventors for two patents concerning therapeutic use of monoclonal antibodies against PADs. MEB, MAS and GJMP have no competing interests related to the work described in this article.

Authors’ contributions

DD designed the study, carried out experiments and drafted the manuscript. MEB assisted with study design, experiments and helped to revise the manuscript critically. MAS assisted with experiments and helped to revise the manuscript critically. GJMP expressed, purified and tested recombinant proteins used for the experiments and helped to revise the manuscript critically. CHN designed the study and drafted the manuscript. All authors read and approved the final manuscript.



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. 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.


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 using an in-house PAD activity assay detecting citrullination of fibrinogen.


No 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.


Catalytic 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.
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