Biochemical and Biophysical Research Communications
Comparison of enzymatic properties between hPADI2 and hPADI4☆
Section snippets
Materials and methods
Chemicals.l-Citrulline, Bz-Arg-OEt, and Bz-Arg-OMe were from Sigma. Bz-Arg-NH2 was from Tokyo Kasei Kogyo. Tos-Arg-OMe was from Wako Pure Chemicals Industries.
Cloning of human PADI2 and PADI4, and human filaggrin. We obtained full-length hPADI2 cDNA from ResGen (Invitrogen), and we obtained hPADI4 and filaggrin cDNA by PCR using human bone marrow cDNA [2] and human skin cDNA [28] as the templates, respectively. The hPADI2 cDNA was cloned into a pDEST10 vector (Invitrogen) for expression in SF9
Immunohistochemical localization of hPADI2 and hPADI4 in rheumatoid arthritis synovium
To determine whether hPADI2 and hPADI4 were distributed differentially in the synovial tissue of patients with RA synovitis, fresh-frozen synovial tissues were obtained and immunohistochemical analysis on cryostat tissue sections was performed with an antibody to hPADI2 and hPADI4 (Fig. 1). hPADI2 was expressed in both the lining layer and sublining region (Fig. 1A), while hPADI4 was expressed mainly in the sublining region (Fig. 1C). hPADI2 was expressed in the cytoplasm (Fig. 1B), while
Discussion
Citrullination of peptidylariginine to peptidylcitrulline by PADI is considered to play an important pathogenic role in RA because anti-citrullinated peptide antibody is highly specific in RA [18] and also because the PADI4 gene has an RA-susceptible variant [2]. Among the 5 known human PADI isozymes, hPADI2 and/or hPADI4 are considered to be responsible for the pathogenesis of RA because of their tissue distribution [2], [26], [27]. Although both PADI2 and PADI4 are detected in RA synovial
Acknowledgments
We thank Dr. Akihito Ishigami (Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology) for providing us with hPADI2 vector and for useful technical advice. We thank Dr. Tetsuji Sawada (Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo), Dr. Xiaotian Chang (Laboratory for Rheumatic Diseases, SNP Research Center, The Institute of Physical and Chemical Research), Dr. Takafumi Kohama and Ms. Yoshiko Kagoshima (Sankyo Co., Ltd.), Ms.
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Abbreviations: BAEE, benzoyl-l-arginine ethyl ester; BCEE, benzoyl-l-citrulline ethyl ester; Bz, benzoyl; DTT, dithiothreitol; OEt, O-ethylester; OMe, O-methylester; Tos, tosyl.
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These authors contributed equally to this work.