A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking.
A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking.
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DOI：
10.1371/journal.pone.0156893
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Mantis NJ
中科院分区：
文献类型：
--
作者：
Herrera C;Klokk TI;Cole R;Sandvig K;Mantis NJ
JJX12 是一种针对蓖麻毒素的工程化双特异性抗体，蓖麻毒素是医学上重要的 A-B 毒素家族的成员，利用逆行运输作为进入靶细胞胞浆的手段。 JJX12 由 RTA-D10 组成，RTA-D10 是一种骆驼单可变域 (VHH) 抗体，针对蓖麻毒素酶亚基 (RTA) 上的表位，通过 15 聚体肽与 RTB-B7 连接，RTB-B7 是针对蓖麻毒素二价半乳糖结合亚基 (RTB) 的 VHH。我们之前报道过，JJX12（而非 RTA-D10 和 RTB-B7 单体的等摩尔混合物）能够被动保护小鼠免受致死剂量蓖麻毒素的攻击，这表明 RTB-B7 和 RTA-D10 的物理连接对于体内毒素中和活性至关重要。我们还报道，JJX12 可能通过分子间交联的形成促进溶液中蓖麻毒素的聚集。在当前的研究中，我们提供了 JJX12 影响人类上皮细胞中蓖麻毒素摄取和运输动态的证据。共聚焦显微镜以及与内吞作用途径特异性抑制剂相结合的活细胞成像表明，JJX12-毒素复合物在哺乳动物细胞表面形成，并通过对阿米洛利（一种已知的巨胞饮抑制剂）敏感的途径内化。此外，在JJX12存在的情况下，蓖麻毒素向跨高尔基体网络的逆行转运显着减少，而毒素在晚期内体中的积累显着增强。总之，我们认为 JJX12 凭借其交联蓖麻毒素的能力，改变了细胞内毒素摄取和运输的途径。
JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin’s enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin’s bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells.