Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen.

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 amino-terminal domain of LF (LFN; 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: (LFN > Lys8 > Lys6 > Lys3). Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive. Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA. 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 LFN blocked the action of LFNDTA, but not of Lys6DTA. 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.

炭疽毒素的致死因子（lethal factor, LF）与水肿因子（edema factor, EF）可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（protective antigen, PA）结合，并被转位至细胞质基质，进而作用于胞内靶标。已有研究证实，将LF的氨基末端结构域（LFN；氨基酸残基1-255）与特定异源蛋白进行基因融合，可增强这些蛋白经PA依赖途径转位至细胞质基质的能力。本研究发现，短链赖氨酸、精氨酸或组氨酸残基序列，同样可增强蛋白经PA依赖途径完成胞质递送的效率。将这类多聚阳离子短序列融合至白喉毒素酶活性A链（diphtheria toxin A chain, DTA；氨基酸残基1-193）的氨基末端后，PA可介导该融合蛋白转位至细胞质基质，并抑制细胞蛋白质合成。转位效率与短序列长度呈正相关：LFN > 八聚赖氨酸（Lys8）> 六聚赖氨酸（Lys6）> 三聚赖氨酸（Lys3）。六聚赖氨酸的活性约为六聚精氨酸（Arg6）或六聚组氨酸（His6）的100倍，而六聚谷氨酸（Glu6）与（丝氨酸-丝氨酸-甘氨酸）₂序列则无活性。六聚精氨酸融合的DTA在细胞培养体系中会发生部分降解，这或可解释其活性相较于六聚赖氨酸融合DTA偏低的原因。这类多聚阳离子短序列可能结合于细胞表面的阴离子位点（可能位于PA上），使融合蛋白可与PA共同被内吞，并被递送至内体，随后转位进入细胞质基质。过量游离的LFN可阻断LFN-DTA的作用，但无法抑制六聚赖氨酸-DTA的活性。这一结果表明，结合LF/EF结合位点并非转位过程的必需步骤，同时提示多聚阳离子标签可通过结合另一不同位点发挥作用。本研究不仅阐明了炭疽毒素的转位机制，同时可为开发将异源蛋白与肽段递送至哺乳动物细胞胞质的递送系统提供理论参考。