title: Generation of monoclonal antibodies against mitocryptide-2: toward a new strategy to investigate the biological roles of cryptides.
abstract: 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 Gi2proteins 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.
authors.affiliation: Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Hattori
authors.affiliation: Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Takenori
authors.initials: T
authors.lastname: Yamada
authors.affiliation: Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Morikawa
authors.affiliation: Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Marutani
authors.affiliation: Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Tsutsumi
authors.affiliation: Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Kodai
authors.initials: K
authors.lastname: Nishino
authors.affiliation: Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: Laboratory of Protein Engineering, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Yoshisuke
authors.initials: Y
authors.lastname: Nishi
authors.affiliation: Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kiso
authors.affiliation: Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
authors.forename: Hidehito
authors.identifier: http://orcid.org/0000-0002-6802-1700
authors.initials: H
authors.lastname: Mukai
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D045303:Cytochromes b / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D024101:Mitochondrial Proteins / Q000276:immunology* / Q000378:metabolism; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D013552:Swine
chemical_list: D000911:Antibodies, Monoclonal; D024101:Mitochondrial Proteins; D010455:Peptides; D045303:Cytochromes b
keywords: cryptide; formyl peptide; mitocryptide; monoclonal antibody; neutrophil