Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen
Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen
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DOI：
10.1073/pnas.93.16.8437
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发表时间：
1996-08-06
影响因子：
11.1
通讯作者：
Collier, RJ
中科院分区：
文献类型：
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作者：
Blanke, SR;Milne, JC;Collier, RJ
炭疽毒素的致死因子(LF)和水肿因子(EF)通过其氨基末端结构域与毒素敏感细胞表面的保护性抗原(PA)结合，并被转移到胞浆，在那里它们作用于细胞内靶点，将LF(LF(N)；残基1-255)的氨基末端结构域与某些异源蛋白基因融合，从而增强这些蛋白的PA依赖递送到胞浆的能力。我们在这里报道，短片段的赖氨酸、精氨酸或组氨酸残基也可以增强蛋白质的这种依赖PA的递送，这些多阳离子束与白喉毒素酶A链(DTA；残基1-193)的氨基末端融合使其能够被PA移位到胞浆中，并抑制蛋白质的合成。易位效率依赖于肌束长度：(Lf(N)&Gt；Lys(8)&Gt；Lys(6)&Gt；Lys(3))。Lys(6)的活性是Arg(6)或His(6)的近100倍，而Glu(6)和(SerSerGly)(2)则没有活性。Arg(6)DTA在细胞培养中被部分降解，这可能是其活性低于Lys(6)DTA的原因。聚阳离子束可能结合到细胞表面的阴离子位点(可能在PA上)，使融合蛋白与PA共吞并传递到内体，在那里发生转位到胞浆，过量的游离LF(N)阻止了LF(N)DTA的作用，但不阻止Lys(6)DTA的作用，这意味着与LF/EF位点的结合不是转位的必需步骤，并表明聚阳离子标签结合到不同的位置，除了阐明炭疽毒素的转位过程外，这些发现可能有助于开发将异源蛋白和多肽输送到哺乳动物细胞细胞质的系统。
The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets, Genetically fusing the aminoterminal domain of LF (LF(N); residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol. We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery, Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis. The efficiency of translocation was dependent on tract length: (LF(N) > Lys(8) > Lys(6) > Lys(3)). Lys(6) was approximate to 100-fold more active than Arg(6) or His(6), whereas Glu(6) and (SerSerGly)(2) were inactive. Arg(6)DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys(6)DTA. The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs, Excess free LF(N) blocked the action of LF(N)DTA, but not of Lys(6)DTA, This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site, Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells.