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‐induced β‐hexosaminidase release in neutrophilic/granulocytic differentiated HL‐60 cells by binding to the region of positions 5–12 of hMCT‐2. Functional analysis using obtained mAbs that specifically recognize hMCT‐2 but not its parent protein, cytochrome b, and that neutralize bioactivities of hMCT‐2 is expected to reveal the physiological roles of MCT‐2, which are presently very difficult to investigate. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. We generated two specific monoclonal antibodies against mitocryptide‐2 that is an endogenous neutrophil‐activating peptide derived from cytochrome b, i.e., NhM2A1 antibody that recognized C‐terminal cleavage site of mitocryptide‐2 and NhM2A5 antibody neutralizing its bioactivities. These antibodies make us possible to investigate physiological production, distribution, and functions of mitocryptide‐2.