Abstract
IntroductionMembers of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients.MethodsAn enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined.ResultsApproximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur.ConclusionsWe present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients.
Highlights
Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline
Approximately 150 times lower levels of recombinant human PAD2 than of rhPAD4 were required for citrullination of fibrinogen
We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from rheumatoid arthritis (RA) patients
Summary
Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients. Posttranslational modifications, such as citrullination, methylation or glycosylation, are common alterations that modify protein structure and possibly stability, functionality and antigenicity of proteins. The peptidylarginine deiminases (PADs) are a family of enzymes capable of converting arginine residues in (poly)peptides to citrullines, a process known as citrullination. In RA, PADs play a crucial role in the generation of the citrullinated proteins targeted by anticitrullinated protein antibodies (ACPAs) [7] and citrullinated peptides recognized by autoreactive T cells [8]. Increased levels of PADs and hypercitrullination of proteins have been linked
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