Autocitrullination and Changes in the Activity of Peptidylarginine Deiminase 3 Induced by High Ca2+ Concentrations

Mizuki Sawata; Hiroki Shima; Kazutaka Murayama; Toshitaka Matsui; Kazuhiko Igarashi; Kazumasa Funabashi; Kenji Ite; Kenji Kizawa; Hidenari Takahara; Masaki Unno

doi:10.1021/acsomega.2c02972

Autocitrullination and Changes in the Activity of Peptidylarginine Deiminase 3 Induced by High Ca²⁺ Concentrations

ACS Omega. 2022 Aug 8;7(32):28378-28387. doi: 10.1021/acsomega.2c02972. eCollection 2022 Aug 16.

Authors

Affiliations

¹ Graduate School of Science and Engineering, Ibaraki University, Hitachi 316-8511, Japan.
² Department of Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan.
³ Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8579, Japan.
⁴ Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
⁵ Kao Corporation, Biological Science Research Laboratory, Odawara 250-0002, Japan.
⁶ College of Agriculture, Ibaraki University, Ami, Inashiki 300-0393, Japan.
⁷ Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Naka 319-1106, Japan.

Abstract

Peptidylarginine deiminases (PADs) are enzymes that catalyze the Ca²⁺-dependent conversion of arginine residues into proteins to citrulline residues. Five PAD isozymes have been identified in mammals. Several studies have shown that the active-site pockets of these isozymes are formed when Ca²⁺ ions are properly bound. We previously characterized the structures of PAD3 in six states. Among these, we identified a "nonproductive" form of PAD3 in which the active site was disordered even though five Ca²⁺ ions were bound. This strange structure was probably obtained as a result of either high Ca²⁺ concentration (∼260 mM)-induced denaturation during the crystallization process or high Ca²⁺-concentration-induced autocitrullination. While autocitrullination has been reported in PAD2 and PAD4 for some time, only a single report on PAD3 has been published recently. In this study, we investigated whether PAD3 catalyzes the autocitrullination reaction and identified autocitrullination sites. In addition to the capacity of PAD3 for autocitrullination, the autocitrullination sites increased depending on the Ca²⁺ concentration and reaction time. These findings suggest that some of the arginine residues in the "nonproductive" form of PAD3 would be autocitrullinated. Furthermore, most of the autocitrullinated sites in PAD3 were located near the substrate-binding site. Given the high Ca²⁺ concentration in the crystallization condition, it is likely that Arg372 was citrullinated in the "nonproductive" PAD3 structure, the structure was slightly altered from the active form by citrulline residues, and probably inhibited Ca²⁺-ion binding at the proper position. Following Arg372 citrullination, PAD3 enters an inactive form; however, the Arg372-citrullinated PAD3 are considered minor components in autocitrullinated PAD3 (CitPAD3), and CitPAD3 does not significantly decrease the enzyme activity. Autocitrullination of PAD3 could not be confirmed at the low Ca²⁺ concentrations seen in vivo. Future experiments using cells and animals are needed to verify the effect of Ca²⁺ on the PAD3 structure and functions in vivo.