These findings suggest that both isoforms exhibit variable enzymatic activity [23]

These findings suggest that both isoforms exhibit variable enzymatic activity [23]. its reactivity against the full-length PAD2. Enzyme-linked immunosorbent assay revealed that six mAbs, which were screened from the phage display library, crossreacted with mouse PAD2. Kinetic analysis revealed that mAbs are bound to PAD2 in the nanomolar range, which indicated a strong binding. Results of the citrullination inhibition assay revealed that the half-maximal effective concentration values of mAbs for the inhibition of histone or benzoyl-L-arginine ethyl ester citrullination were in the range of 6C75?nM which supports strong inhibition capabilities. Alanine scanning of epitope revealed that the peptide fragment 344RGDRWIQDEIEF355 was responsible for generating strong antibody responses that inhibit the PAD2-catalyzed citrullination reaction. These antibodies can aid in understanding the extracellular PAD2 function and treating diseases associated with aberrant citrullination. 1. Introduction Citrullination is a type of posttranslational modification that involves the production of citrulline, a noncoding amino acid, through the deimination of arginine. This reaction is catalyzed by the peptidyl-arginine deiminase (PAD) family of enzymes. PADs regulate various cellular processes, including transcriptional regulation of gene expression [1], skin keratinization [2], and the maintenance of myelin sheath insulation [3]. Additionally, citrullination promotes the formation of neutrophil extracellular traps (NETs), a mechanism through which neutrophils capture and eliminate pathogens [4, 5]. In humans, the PAD family comprises five calcium-dependent isozymes (PAD1C4 and 6) [6]. Calcium induces conformational changes and consequently activates the enzyme [7]. Recently, PAD2 Rabbit Polyclonal to NRIP2 and PAD4 isotypes, which are mainly expressed in the immune cells, brain, bone marrow, and joints, have piqued the interest of T0070907 the scientific community for drug discovery [6, 8]. PADs are cytoplasmic or nuclear proteins that lack the transmembrane regions or secretory signal sequences. Hence, the expression and function of PADs are restricted to the cytoplasm [9]. However, recent studies have demonstrated that the expression of PADs is upregulated during inflammation, which results in the upregulation of citrullination, the activation of NETosis, and, consequently, the release of PADs from the damaged cells [10, 11]. Extracellular PADs led to excessive citrullination of proteins, and aberrantly upregulated citrullination are reported in several autoimmune and inflammatory diseases [12], especially rheumatoid arthritis (RA). In T0070907 the synovial fluid (SF) of patients with RA, more than 100 citrullinated proteins have been identified, including several neutrophil-associated intracellular proteins, extracellular matrix proteins, and serum proteins, such as albumin, fibrinogen, and immunoglobulin [13, 14]. Hence, deiminated proteins function as neoantigens and promote the production of anti-citrullinated protein antibodies (ACPAs), which mediate the local inflammatory response and exacerbate the severity of RA [15]. ACPAs are found in approximately 70% of patients with RA. Additionally, the activity of PADs is detected in the SF of patients with RA [16, 17]. Spengler et al. [10] detected both T0070907 PAD2 and PAD4 proteins in the cell-free SF of patients with RA although their expression levels varied in different patients. Interestingly, an study by T0070907 Zhou et al. [18] reported that live neutrophils can inherently express catalytically active PAD4 on the cell surface and that active PAD2 is released spontaneously into the culture media from neutrophils without stimulation. In addition to its involvement in RA progression, PAD2 is involved in the onset and progression of multiple sclerosis (MS) [19], endotoxin-induced lethality [20], and breast cancer [21]. Currently, there are no specific drugs to inhibit T0070907 PAD2. The roles of intracellular or extracellular citrullination in the initiation or progression of RA pathogenesis are unclear. Therefore, the inhibition of extracellular PAD2 using a specific monoclonal antibody (mAb) will aid the elucidation of the biological roles of this isozyme and the treatment of specific diseases associated with dysregulated PAD2 activity. In this study, we aimed to develop a novel PAD2-specific mAb that could inhibit the citrullination activity of PAD2. 2. Materials and Methods 2.1. Materials Keyhole limpet hemocyanin- (KLH-) modified peptide antigen (epitope) was purchased from SCRUM Inc. (Tokyo, Japan). Freund’s adjuvant (complete or incomplete) was procured from WAKO Pure Chemical Industries, Ltd. (Osaka, Japan). The RNA isolation reagent TRIzol and the high pure RNA isolation kit were purchased from Life Technologies (California, USA) and Roche Diagnostics (Basel, Switzerland), respectively. Ficoll-Paque PLUS was purchased from Cytiva (Marlborough, USA). The reagents used in the DNA manipulation procedures were purchased from Takara (Kusatsu, Japan). XL1-Blue electrocompetent cells and VCS-M13 were procured from Stratagene (California, USA). The H-chain.