Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins.
Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins.
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DOI：
10.1016/j.jbc.2022.101742
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发表时间：
2022-04
期刊：
影响因子：
--
通讯作者：
Mantis NJ
中科院分区：
文献类型：
--
作者：
Czajka TF;Vance DJ;Davis S;Rudolph MJ;Mantis NJ
在哺乳动物细胞的蓖麻毒素中毒过程中，蓖麻毒素的酶(RTA)和结合(RTB)亚基在内质网中解离。然后RTA被转移到细胞质中，在那里，由于它能够清除28S rRNA的sarcin-ricin环(SRL)中的保守残基，它作为核糖体失活蛋白发挥作用。已有研究提出，核糖体P-茎蛋白促进RTA向SRL的募集，其C-末端结构域与RTA上通常被RTB掩盖的空腔相互作用；然而，缺乏证据表明这种相互作用对细胞内的RTA活性至关重要。在这里，我们描述了一组单域抗体(VHH)，它们的表位与RTA上的P-柄结合口袋重叠。与RTA形成的三个VHH(V9E1、V9F9和V9B2)的晶体结构表明，它们不仅封闭了核糖体P-茎结合口袋，而且通过互补决定区3对C-末端多肽的结构模拟。体外实验证实，这些VHs阻断了RTA-P-茎肽的相互作用，保护核糖体不被脱嘌呤。此外，当表达为“体内”时，这些VHH使细胞对蓖麻毒素中毒具有抵抗力。一株VHH(V9F6)在结构上被确定为与P-茎结合口袋紧邻，在体外不能中和细胞内的蓖麻毒素或保护核糖体免受RTA的影响。这些发现与核糖体P-茎蛋白将RTA募集到SRL是蓖麻毒素诱导的核糖体失活所必需的事件相一致。
During ricin intoxication in mammalian cells, ricin's enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin–ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (VHHs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such VHHs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these VHHs block RTA–P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as “intrabodies,” these VHHs rendered cells resistant to ricin intoxication. One VHH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation.
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