title: Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase.
abstract: 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.
authors.affiliation: Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. dres.damgaard@regionh.dk.|Section for Periodontology, Microbiology and Community Dentistry, Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. dres.damgaard@regionh.dk.
authors.forename: Dres
authors.initials: D
authors.lastname: Damgaard
authors.affiliation: Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.|Department of Haematology, Roskilde Hospital, Roskilde, Denmark.
authors.forename: Mads Emil
authors.initials: ME
authors.lastname: Bjørn
authors.affiliation: Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Steffensen
authors.affiliation: Department of Biomolecular Chemistry, Institute for Molecules and Materials, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Pruijn
authors.affiliation: Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.|Section for Periodontology, Microbiology and Community Dentistry, Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Claus H
authors.initials: CH
authors.lastname: Nielsen
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D002956:Citrulline / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D013582:Synovial Fluid / Q000378:metabolism
chemical_list: D002956:Citrulline; D006867:Hydrolases; C000620352:PADI2 protein, human; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D005978:Glutathione
keywords: Citrullination; Peptidylarginine deiminase; Reduced glutathione; Rheumatoid arthritis; Synovial fluid