Generation of monoclonal antibodies against mitocryptide-2: toward a new strategy to investigate the biological roles of cryptides
Generation of monoclonal antibodies against mitocryptide-2: toward a new strategy to investigate the biological roles of cryptides
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针对 mitocryptide-2 的单克隆抗体的生成：研究 cryptides 生物学作用的新策略
DOI：
10.1002/psc.3000
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发表时间：
2017
影响因子：
2.1
通讯作者：
Hidehito Mukai
中科院分区：
文献类型：
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作者：
Tatsuya Hattori;Takenori Yamada;Hiroki Morikawa;Takayuki Marutani;Koki Tsutsumi;Kodai Nishino;Toshihiro Shimizu;Yoshisuke Nishi;Yoshiaki Kiso;Hidehito Mukai
我们最近发现了一个新的中性粒细胞激活肽家族，包括线粒体肽- 1和线粒体肽- 2 (MCT - 2)，它们是由各种线粒体蛋白内源性产生的。其中，MCT‐2是来源于线粒体细胞色素带的anN‐甲酰基化的五肽，被发现能有效促进嗜中性粒细胞迁移和吞噬。MCT‐2激活中性粒细胞的信号机制已经在细胞水平上进行了研究，MCT‐2已被证明是甲酰基肽受体‐2（也称为甲酰基肽受体样1）的内源性特异性配体。研究还发现，MCT‐2通过连续激活gi2蛋白和磷酸化ERK1/2来促进嗜中性粒细胞功能。然而，MCT‐2的生理产生、分布和功能尚未阐明。为了研究MCT‐2在体内的作用，我们制备了具有两种不同特征的抗人MCT‐2 （hMCT‐2）的单克隆抗体（mab）。其中一个单抗NhM2A1不仅结合到hMCT‐2的10-15位区域，而且还识别出其C端裂解位点，这可能是酶解细胞染色质时产生的，这表明NhM2A1特异性地与hMCT‐2相互作用，而不是与其亲本蛋白相互作用。此外，我们成功地获得了一种特异性中和单抗NhM2A5，它可以阻断hMCT‐2的生物活性。具体来说，NhM2A5通过结合hMCT - 2的5-12位区域，抑制hMCT - 2诱导的中性粒细胞分化HL - 60细胞中β -己糖氨酸酶的释放。利用获得的单抗进行功能分析，这些单抗能够特异性识别hMCT‐2，但不能识别其亲本蛋白，细胞色素，并能中和hMCT‐2的生物活性，有望揭示MCT‐2的生理作用，这是目前很难研究的。版权所有©2017欧洲多肽协会和约翰威利父子有限公司
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 anN‐formylated pentadecapeptide derived from mitochondrial cytochromeband 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‐2in 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 cytochromeb, 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‐inducedβ‐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, cytochromeb, 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.