title: Porphyromonas gingivalis peptidylarginine deiminase substrate specificity.
abstract: While a group of oral commensals have been implicated in the aetiology of chronic periodontitis; the asaccharolytic Gram negative anaerobe Porphyromonas gingivalis is most commonly reported to be associated with severe forms of the disease. Although a variety of human tissues can produce a number of peptidylarginine deiminase (PAD), enzymes that convert peptide bound arginine residues to citrulline, P. gingivalis is one of the few prokaryotes known to express PAD. Protein and peptide citrullination are important in the development of rheumatoid arthritis and in recent years a number of authors have suggested a possible link between periodontitis and rheumatoid arthritis (RA). Indeed, some have linked P. gingivalis directly to RA via the action of PAD. Accordingly, the prime purpose of this study was to further characterise PAD in P. gingivalis cells particular emphasis on substrate specificity, using arginine containing peptides and RA relevant proteins.
  
  
  METHODS
  P. ginigvalis W50 was anaerobically cultured in BHI broth, cells harvested and resuspended in assay buffer. A colourimetric assay was developed to measure citrulline and employed to determine enzyme activity using the substrate BAEE. The assay was employed to investigate the effects of environmental pH and temperature on activity. Citrullination of BAEE by sonicated cells allowed the proportion of intracellular enzyme to be estimated. Enzyme specificity and substrate preference were investigated by using various arginine containing peptides, proteins and arginine analogues, as substrates and measuring the rate of citrullination. The influence of gingipains on citrullination was assessed by measuring the rates of citrullination of bovine serum albumin in the presence of protease inhibitors.
  
  
  RESULTS
  Enzyme activity decreased by 13% following exposure of cells to 60 °C for 10 min. A comparison of intact and disrupted cells indicated that 90% of PAD activity is cell surface associated and the remainder cytoplasmic. Optimal pH for enzyme activity was between pH 7.5 and 8. All small arginine-containing peptides were citrullinated with reaction rates faster than that for free arginine with rates that varied with arginine residue position and number. Arginine analogues exhibited minimal effect and influence when tested as either substrates or competitive inhibitors. Cells were able to citrullinate yeast enolase, human vimentin and fibrin at varying rates. All proteins were modified at slower rates than those for peptide substrates. Inhibition of gingipains had no influence on the rate of protein citrullination.
  
  
  CONCLUSIONS
  P. gingivalis PAD is a primarily cell surface associated, heat stable, enzyme that exhibits optimal activity under alkaline conditions similar to those present in the inflammatory environment. The enzyme displays high specificity for arginine residues in peptides and modified arginine in all positions and the gingipains did not influence the rate of protein citrullination. The ability of the enzyme to convert arginine residues in all proteins tested would indicate that its presence in inflamed tissue may promote autoimmune reactions by creation of altered host epitopes.
authors.affiliation: School of Dentistry, Faculty of Health Sciences, University of Adelaide, Frome Road, Adelaide, South Australia 5000, Australia; Fakulti Pergigian, Universiti Sains Islam Malaysia, Persiaran MPAJ Pandan Indah, Kuala Lumpur 55100, Malaysia. Electronic address: najmi@usim.edu.my.
authors.forename: Syatirah-Najmi
authors.initials: SN
authors.lastname: Abdullah
authors.forename: Elizabeth-Anne
authors.initials: EA
authors.lastname: Farmer
authors.forename: Llewellyn
authors.initials: L
authors.lastname: Spargo
authors.forename: Richard
authors.initials: R
authors.lastname: Logan
authors.forename: Neville
authors.initials: N
authors.lastname: Gully
mesh_terms: D001120:Arginine / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D000076342:Protein-Arginine Deiminases; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D002956:Citrulline; D001120:Arginine; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases
keywords: Citrullination; Peptidylarginine deiminase; Periodontitis; Porphyromonas gingivalis; Rheumatoid arthritis