Hattori Tatsuya, Yamada Takenori, Morikawa Hiroki, Marutani Takayuki, Tsutsumi Koki, Nishino Kodai, Shimizu Toshihiro, Nishi Yoshisuke, Kiso Yoshiaki, Mukai Hidehito
Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
Laboratory of Protein Engineering, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
J Pept Sci. 2017 Jul;23(7-8):610-617. doi: 10.1002/psc.3000. Epub 2017 Mar 29.
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 G 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.
我们最近鉴定出了一个新的中性粒细胞激活肽家族，其中包括线粒体隐肽 -1和线粒体隐肽 -2（MCT -2），它们是由各种线粒体蛋白内源性产生的。其中，MCT -2是一种源自线粒体细胞色素b的N -甲酰化十五肽，被发现能有效促进中性粒细胞迁移和吞噬作用。已在细胞水平研究了MCT -2激活中性粒细胞的信号传导机制，并且已证明MCT -2是甲酰肽受体 -2（也称为甲酰肽受体样1）的内源性特异性配体。还发现MCT -2通过依次激活G蛋白和ERK1/2磷酸化来促进中性粒细胞功能。然而，MCT -2的生理产生、分布和功能尚未阐明。在此，为了研究MCT -2在体内的作用，我们制备了针对人MCT -2（hMCT -2）的单克隆抗体（mAb），这些单克隆抗体具有两种不同特性。一种单克隆抗体NhM2A1不仅与hMCT -2的第10 - 15位区域结合，还识别其C末端切割位点，该位点可能是在细胞色素b的酶促水解时产生的，这表明NhM2A1与hMCT -2特异性相互作用，而不与其亲本蛋白相互作用。此外，我们成功获得了一种特异性中和单克隆抗体NhM2A5，它可阻断hMCT -2的生物活性。具体而言，NhM2A5通过与hMCT -2的第5 - 12位区域结合，抑制hMCT -2诱导的嗜中性/粒细胞分化的HL - 60细胞中β - 己糖胺酶的释放。使用获得的特异性识别hMCT -2而不识别其亲本蛋白细胞色素b且中和hMCT -2生物活性的单克隆抗体进行功能分析，有望揭示目前很难研究的MCT -2的生理作用。版权所有© 2017欧洲肽学会和约翰·威利父子有限公司。
