Abstract
Protein citrullination catalyzed by peptidyl arginine deiminase (PADs) is involved in autoimmune disease pathogenesis, especially in rheumatoid arthritis. Calcium is a key regulator of PAD activity, but under normal physiological conditions it remains uncertain how intracellular calcium levels can be raised to sufficiently high levels to activate these enzymes. In pursuit of trying to identify other factors that influence PAD activity, we identified bicarbonate as a potential regulator of PAD activity. We demonstrate that physiological levels of bicarbonate upregulate citrullination by recombinant PAD2/4 and endogenous PADs in neutrophils. The impact of bicarbonate is independent of calcium and pH. Adding bicarbonate to commercial PAD activity kits could increase assay performance and biological relevance. These results suggest that citrullination activity is regulated by multiple factors including calcium and bicarbonate. We also provide commentary on the current understanding of PAD regulation and future perspective of research in this area.
Highlights
Protein citrullination is the conversion of peptidylarginine to citrulline
Bicarbonate significantly increased fibrinogen citrullination by PAD2 and PAD4 in a dose-dependent manner (Figure 1D). These results demonstrate that the impact of bicarbonate on peptidyl arginine deiminase (PADs) activity was independent of substrates or detection methods and provide additional supportive evidence that bicarbonate regulates recombinant PAD activity
The only essential factor known for PAD enzyme activity is calcium [1], a reducing environment is required to prevent oxidation of cysteine in the catalytic domain [36]
Summary
Protein citrullination (or deimination) is the conversion of peptidylarginine to citrulline. To further confirm the pH-independent effect of bicarbonate and explore the effect of pH on PAD activity, we performed histone H3 citrullination with recombinant enzyme under different pH levels adjusted by HCl and NaOH Both PAD2 and PAD4 showed higher activity at pH of 6.4 compared to 7.2 and 8.0 (Figure 1C). Bicarbonate significantly increased fibrinogen citrullination by PAD2 and PAD4 in a dose-dependent manner (Figure 1D) These results demonstrate that the impact of bicarbonate on PAD activity was independent of substrates or detection methods and provide additional supportive evidence that bicarbonate regulates recombinant PAD activity. PH was adjusted to 7.2 with HEPES in DPBS and DMEM used These results demonstrate that bicarbonate promotes intracellular citrullination at sub-physiological and Figure 2 | Bicarbonate level impacts neutrophil citrullination. There is a requirement for calcium for citrullination, bicarbonate independently increases PAD activity
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