Generation of monoclonal antibodies against mitocryptide ‐2: toward a new strategy to investigate the biological roles of cryptides

We recently identified a novel family of neutrophil‐activating peptides including mitocryptide‐1 and mitocryptide‐2 (MCT‐2) that are endogenously produced from various mitochondrial proteins. Among them, MCT‐2 is an N‐formylated pentadecapeptide derived from mitochondrial cytochrome b and is found to promote neutrophilic migration and phagocytosis efficiently. Signaling mechanisms of neutrophil activation by MCT‐2 have been investigated at the cellular level, and MCT‐2 has been demonstrated to be an endogenous specific ligand for formyl peptide receptor‐2 (also referred to as formyl peptide receptor‐like 1). It was also found that MCT‐2 promoted neutrophilic functions via the activation of Gi2 proteins and phosphorylation of ERK1/2 consecutively. However, the physiological production, distribution, and functions of MCT‐2 are not yet elucidated. Here, to investigate the roles of MCT‐2 in vivo, we generated monoclonal antibodies (mAbs) against human MCT‐2 (hMCT‐2) that have two different characteristics. One mAb, NhM2A1, not only bound to the region of positions 10–15 of hMCT‐2 but also recognized its C‐terminal cleavage site that is presumably produced upon enzymatic hydrolysis of cytochrome b, indicating that NhM2A1 specifically interacts with hMCT‐2 but not its parent protein. Moreover, we succeeded in acquiring a specific neutralizing mAb, NhM2A5, which blocks the bioactivities of hMCT‐2. Specifically, NhM2A5 inhibited hMCT‐2‐indu...

Source: Journal of Peptide Science - March 1, 2017 Category: Biochemistry Authors: Tatsuya Hattori, Takenori Yamada, Hiroki Morikawa, Takayuki Marutani, Koki Tsutsumi, Kodai Nishino, Toshihiro Shimizu, Yoshisuke Nishi, Yoshiaki Kiso, Hidehito Mukai Tags: Special Issue Article Source Type: research