Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

Abstract

Introduction: Members 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.

Methods: An 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.

Results: Approximately 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.

Conclusions: We 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.

Figure 1

In situ citrullination of human fibrinogen. (A) Fibrinogen (Fib) or citrullinated fibrinogen (cFib) was immobilized to enzyme-linked immunosorbent assay (ELISA) plates at a concentration of 1 μg/ml. Increasing concentrations of the anti-cFib monoclonal antibody (mAb; clone 20B2) were added, followed by addition of enzyme-conjugated rabbit anti-mouse immunoglobulin antibodies and o-phenylenediamine substrate. All data points represent means and ranges of duplicate measurements of optical density at 490 nm. (B) ELISA plates were coated with 1.0 μg/ml human fibrinogen and incubated with citrullination buffer including recombinant human peptidylarginine deiminase (rhPAD) enzyme, ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), MQPAD4 (49 mU/ml) or rhPAD4 (1,500 ng/ml). Shown is the activity as a percentage of the maximal activity obtained for each enzyme, corresponding to the activity at 4 hours. Anti-cFib mAb was used at a concentration of 0.5 μg/ml, as determined from the graph shown in (A). Symbols and error bars represent means and ranges of duplicate measurements.

Figure 2

Comparison between enzymatic activities of peptidylarginine deiminases 2 and 4. Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen. (A) Recombinant human peptidylarginine deiminase 2 (rhPAD2) or rhPAD4 raised in-house were used for citrullination at mass concentrations ranging from 0.1 ng/ml to 15 μg/ml. (B) Commercially available ModiQuest (MQ) PAD2 or MQPAD4 enzymes were used in units ranging from 0.1 mU/ml to 100 mU/ml. Following a 3-hour citrullination period, the anti-cFib mAb (0.5 μg/ml) was used for detection of citrullinated fibrinogen. Duplicate measurements of optical density (OD) at 490 nm are shown as mean and range.

Figure 3

Calcium dependency of peptidylarginine deiminases. Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml). Dithiothreitol was added at a concentration of 1.0 mM, and CaCl₂ was added at concentrations ranging from 50 μM to 10 mM. Following 3 hours of incubation, citrullination was measured using mouse anti-cFib monoclonal antibody (0.5 μg/ml). Shown is the activity as a percentage of maximal activity for each enzyme. Symbols and error bars represent means and ranges of duplicate measurements.

Figure 4

Peptidylarginine deiminases activity in synovial fluid samples from rheumatoid arthritis patients. (A) Synovial fluid (SF) samples from five rheumatoid arthritis patients were applied on enzyme-linked immunosorbent assay plates coated with fibrinogen (1 μg/ml) after 1:3 dilution into Tris buffer with 1 mM dithiothreitol (DTT; black columns), Tris buffer containing 1 mM DTT and 10 mM CaCl₂ (grey columns) or Tris buffer containing 1 mM DTT and 10 mM ethylenediaminetetraacetic acid (EDTA; white columns). Following a 3-hour incubation period, the monoclonal mouse anti-citrullinated fibrinogen (anti-cFib) (0.5 μg/ml) was used for detection of citrullinated fibrinogen. Peptidylarginine deiminase (PAD) activity is presented as optical density (OD) at 490 nm of duplicated measurements. (B) Association between PAD2 concentration and PAD activity in the five SF samples diluted 1:3 in citrullination buffer containing 10 mM CaCl₂ (grey circles, solid line) or no additive calcium (black circles, dotted line). Pearson’s correlation coefficients (r) and levels of significance are shown.