Comparison of enzymatic properties between hPADI2 and hPADI4

doi:10.1016/j.bbrc.2004.11.152

Biochemical and Biophysical Research Communications

Volume 327, Issue 1, 4 February 2005, Pages 192-200

https://doi.org/10.1016/j.bbrc.2004.11.152 Get rights and content

Abstract

In the sera of rheumatoid arthritis (RA) patients, autoantibodies directed to citrullinated proteins are found with high specificity for RA. Peptidylarginine deiminases (PADIs) are enzymes responsible for protein citrullination. Among many isoforms of PADIs, only PADI4 has been identified as an RA-susceptibility gene. To understand the mechanisms of the initiation and progression of RA, we compared the properties of two PADIs, human PADI2 and human PADI4, which are present in the synovial tissues of RA patients. We confirmed their precise distribution in the RA synovium and compared the stability, Ca²⁺ dependency, optimal pH range, and substrate specificity. Small but significant differences were found in the above-mentioned properties between hPADI2 and hPADI4. Using LC/MS/MS analysis, we identified the sequences in human fibrinogen indicating that hPADI2 and hPADI4 citrullinate in different manners. Our results indicate that hPADI2 and hPADI4 have different roles under physiological and pathological conditions. Further studies are needed for the better understanding of the role of hPADIs in the initiation and progression of RA.

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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Serum C-Fbg levels were significantly higher in bacteremia patients than in HC (p < 0.001) and positively correlated with WBC and neutrophil count. Further, C-Fbg levels were significantly higher in phases III and IV of bacteremia than in HC (p < 0.001). MLR analysis indicated that log C-Fbg had a stronger relationship with log neutrophil counts than other certain inflammatory markers (p < 0.01).
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Citrullination is the post-translational conversion of arginine into citrulline in proteins. The reaction is catalyzed by peptidylarginine deiminase (PAD), of which five isoforms exist. Fibrinogen is a substrate for PAD2 and PAD4, and citrullinated fibrinogen (cFBG) has been detected in patients with inflammatory diseases. In purified systems, cFBG is known to inhibit the release of fibrinopeptide A (FPA) and B (FPB) and impairs fibrin polymerization. However, the effect of cFBG on fibrin structure and fibrinolysis in a plasma environment remains unclear. We hypothesized that citrullination of fibrinogen impairs fibrin properties.
Fibrinogen was citrullinated by recombinant PAD2 and PAD4. The impact of cFBG on fibrin structure was investigated by turbidity measurements in fibrinogen-deficient plasma spiked with cFBG or native fibrinogen.
Citrullination of fibrinogen by PAD2 dose-dependently reduced the rate of fibrin polymerization, as well as the overall hemostasis potential of fibrin, the maximum velocity of fibrin formation, the fibrin mass/length ratio, and the lysis of fibrin clots.
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Citrullination is a post-translational protein modification, which is associated with inflammation in general and is thought to play an important pathogenic role in rheumatoid arthritis (RA). Here a mass spectrometry-based proteomics approach was applied to identify citrullination sites in synovial fluid fibrinogen from four RA patients. In general, high disease activity correlated with increased number of identified citrullination sites and higher relative citrulline occupancy. Altogether, 23 sites were identified, of which 9 have not been previously reported to be citrullinated in vivo. Citrullination at site α84, α123, α129, α547, α573, α591, β334 and γ134 was identified in more than one patient, and these positions were therefore regarded as hotspots. Following citrullination of fibrinogen in vitro using human recombinant peptidylarginine deiminase 2 (PAD2), a total of 46 citrullination sites were identified, including 6 hitherto unreported in vitro citrullination sites. Twenty-two out of the 23 citrullination sites identified in vivo were also detected in vitro, supporting the validity of the identifications.
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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.

¹: These authors contributed equally to this work.

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Comparison of enzymatic properties between hPADI2 and hPADI4☆

Abstract

Section snippets

Materials and methods

Immunohistochemical localization of hPADI2 and hPADI4 in rheumatoid arthritis synovium

Discussion

Acknowledgments

J. Biol. Chem.

J. Invest. Dermatol.

J. Biol. Chem.

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Neuroimmunol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Invest. Dermatol.

Anal. Biochem.

Anal. Biochem.

Cell

Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis

Nat. Genet.

Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils

J. Leukoc. Biol.

Molecular cloning of cDNAs of mouse peptidylarginine deiminase type I, type III and type IV, and the expression pattern of type I in mouse

Eur. J. Biochem.

cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I

Biochem. J.