Epitope-Based Chicken-Derived Novel Anti-PAD2 Monoclonal Antibodies Inhibit Citrullination.

Masayoshi Aosasa,Takanari Shigemitsu,Keita Koga,Mujo Kim,Motonori Yamaguchi,Tomoko Sakata,Yuji Shoya,Md Saddam Hossain,Kenji Saito

doi:10.1155/2021/6659960

Abstract

The aberrant upregulation of protein arginine deiminase 2- (PAD2-) catalyzed citrullination is reported in various autoimmune diseases (rheumatoid arthritis and multiple sclerosis) and several cancers. Currently, there are no anti-PAD2 monoclonal antibodies (mAbs) that can inhibit the citrullination reaction. Here, an epitope 341YLNRGDRWIQDEIEFGY357 was examined as an antigenic site of PAD2. Chickens were immunized with this epitope, and the generated mAbs were screened for 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 in vitro 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 6–75 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.

Highlights

Citrullination is a type of posttranslational modification that involves the production of citrulline, a noncoding amino acid, through the deimination of arginine
The epitope, 341YLNRGDRWIQDEIEFGY357, was identified (Figure 1). Blastp analysis of this epitope sequence with the chicken protein sequence revealed that the epitope exhibited marked similarity with chicken PAD2 with only two varying residues
A phage scFv display library constructed using chicken immunized with a selective epitope of PAD2 was panned against Recombinant human PAD2 (rhPAD2) for five cycles

Summary

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]. The PAD family comprises five calcium-dependent isozymes (PAD1–4 and 6) [6]. Calcium induces conformational changes and activates the enzyme [7]. PAD2 and PAD4 isotypes, which are mainly expressed in the immune cells, brain, bone marrow, and joints, have piqued the interest of the scientific community for drug discovery [6, 8]

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