From the Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
From the Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
J Biol Chem. 2018 Feb 2;293(5):1810-1819. doi: 10.1074/jbc.M117.815373. Epub 2017 Dec 15.
TcdB (2366 amino acid residues) is an intracellular bacterial toxin that binds to cells and enters the cytosol where it glucosylates small GTPases. In the current study, we examined a putative cell entry region of TcdB (amino acid residues 1753-1851) for short sequences that function as cell-penetrating peptides (CPPs). To screen for TcdB-derived CPPs, a panel of synthetic peptides was tested for the ability to enhance transferrin (Tf) association with cells. Four candidate CPPs were discovered, and further study on one peptide (PepB2) pinpointed an asparagine residue necessary for CPP activity. PepB2 mediated the cell entry of a wide variety of molecules including dextran, streptavidin, microspheres, and lentivirus particles. Of note, this uptake was dramatically reduced in the presence of the Na/H exchange blocker and micropinocytosis inhibitor amiloride, suggesting that PepB2 invokes macropinocytosis. Moreover, we found that PepB2 had more efficient cell-penetrating activity than several other well-known CPPs (TAT, penetratin, Pep-1, and TP10). Finally, Tf assay-based screening of peptides derived from two other large clostridial toxins, TcdA and TcsL, uncovered two new TcdA-derived CPPs. In conclusion, we have identified six CPPs from large clostridial toxins and have demonstrated the ability of PepB2 to promote cell association and entry of several molecules through a putative fluid-phase macropinocytotic mechanism.
TcdB（2366 个氨基酸残基）是一种细胞内细菌毒素，它与细胞结合并进入细胞质，在细胞质中使小分子 GTP 酶糖基化。在当前的研究中，我们研究了 TcdB 的一个假定的细胞进入区域（氨基酸残基 1753-1851），寻找短序列作为细胞穿透肽（CPP）。为了筛选 TcdB 衍生的 CPP，我们测试了一组合成肽增强转铁蛋白（Tf）与细胞结合的能力。发现了四个候选 CPP，对其中一个肽（PepB2）的进一步研究确定了一个天冬酰胺残基是 CPP 活性所必需的。PepB2 介导了包括葡聚糖、链霉亲和素、微球和慢病毒颗粒在内的各种分子的细胞进入。值得注意的是，这种摄取在 Na/H 交换阻滞剂和胞饮抑制剂阿米洛利存在的情况下显著减少，表明 PepB2 引发了巨胞饮。此外，我们发现 PepB2 比其他几种已知的 CPP（TAT、penetratin、Pep-1 和 TP10）具有更高的细胞穿透活性。最后，基于 Tf 测定的筛选来自两种其他大型梭状芽孢杆菌毒素 TcdA 和 TcsL 的肽，发现了两个新的 TcdA 衍生 CPP。总之，我们从大型梭状芽孢杆菌毒素中鉴定出了六个 CPP，并证明了 PepB2 通过一种假定的流体相巨胞饮机制促进了几种分子的细胞结合和进入。
