Expression of peptidylarginine deiminase from Porphyromonas gingivalis in Escherichia coli: Enzyme purification and characterization

Porphyromonas gingivalis peptidylarginine deiminase (PAD) catalyzes the deimination of peptidylarginine residues of various peptides to produce peptidylcitrulline and ammonia. P. gingivalis is associated with adult-onset periodontitis and cardiovascular disease, and its proliferation depends on secretion of PAD. We have expressed two recombinant forms of the P. gingivalis PAD in Escherichia coli, a truncated form with a 43-amino acid N-terminal deletion and the full-length form of PAD as predicted from the DNA sequence. Both forms contain a poly-His tag and Xpress epitope at the N-terminus to aid in detection and purification. The activities and stabilities of these two forms have been evaluated. PAD is cold sensitive; it aggregates within 30 min at 4 °C, and optimal storage conditions are at 25 °C in the presence of a reducing agent. PAD is not a metalloenzyme and does not need a cofactor for catalysis or stability. Multiple l-arginine analogs, various arginine-containing peptides, and free l-arginine were used to evaluate substrate specificity and determine kinetic parameters.     

Peptidylarginine deiminases in citrullination,gene regulation,health and pathogenesis

Peptidylarginine deiminases are a family of enzymes that mediate post-translational modifications of protein arginine residues by deimination or demethylimination to produce citrulline. In vitro, the activity of PADs is dependent on calcium and reductive reagents carrying a free sulfhydryl group. The discovery that PAD4 can target both arginine and methyl-arginine for citrullination about 10 years ago renewed our interest in studying this family of enzymes in gene regulation and their physiological functions. The deregulation of PADs is involved in the etiology of multiple human diseases, including cancers and autoimmune disorders. There is a growing effort to develop isoform specific PAD inhibitors for disease treatment. However, the regulation of the activity of PADs in vivo remains largely elusive, and we expect that much will be learned about the role of these enzymes in a normal life cycle and under pathology conditions.     

Identification of citrullinated eukaryotic translation initiation factor 4G1 as novel autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis. We previously reported that functional variants of the gene encoding peptidylarginine deiminase type 4 were closely associated with RA. The purpose of this study was to investigate the citrullinated autoantigens recognized by serum samples from patients with RA. The human chondrocyte cDNA expression library was citrullinated by PADI4 and was immunoscreened with anti-modified citrulline antibodies and sera from patients with rheumatoid arthritis. One immunoreactive cDNA clone containing a 2480-base pair insert was isolated and sequence analysis revealed that the cDNA included a part of the eukaryotic translation initiation factor 4G1. Immunoreactivity against a recombinant citrullinated eIF4G1 fragment was observed with high specificity in 50.0% of RA patients. The levels of antibodies against citrullinated eIF4G1 were correlated with those of anti-CCP antibodies. Citrullinated eIF4G1 was identified as a candidate citrullinated autoantigen in RA patients. Citrullination of eIF4G1 may thus be involved in the pathogenesis of RA.