Anthrax toxin fusion proteins for intracellular delivery of macromolecules
Anthrax toxin fusion proteins for intracellular delivery of macromolecules
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DOI：
10.1046/j.1365-2672.1999.00890.x
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发表时间：
1999-08-01
影响因子：
4
通讯作者：
Varughese, M
中科院分区：
文献类型：
--
作者：
Leppla, SH;Arora, N;Varughese, M
炭疽毒素在炭疽杆菌致病过程中的主导作用表明，炭疽毒素已进化成为一个有效的系统，将其两种催化蛋白组分--水肿因子和致死因子（LF）--递送到宿主细胞的胞质溶胶中。该系统涉及保护性抗原（PA）毒素组分与普遍存在的（且仍未鉴定的）受体的结合、细胞表面的蛋白水解活化、通过内吞作用的内化和通过早期内体膜易位至胞质溶胶（ Leppla 1995）。我们和同事表明，该系统可用于将异源多肽递送到胞质溶胶（ Aroraet al. 1992 ; Milneet等人 1995 ).这项工作使用了其他毒素的催化结构域，这些毒素通常是跨膜转运的（ 对细胞内病原体的免疫性取决于抗原的胞质加工以产生呈递在细胞表面上的与MHC I类分子结合的肽。炭疽毒素传递系统提供了一种模拟这一过程的方法。我们制备了一种融合蛋白，含有LF的（非催化）氨基末端结构域和HIV-1的gp 120包膜糖蛋白。将该重组蛋白与PA沿着施用至抗原呈递细胞使其对gp 120肽特异性细胞毒性T细胞的细胞溶解敏感（Goletzet al. 1997 炭疽毒素系统作为细胞靶向试剂的进一步开发将通过实现细胞类型特异性来促进。PA结构的最新测定（Petosaet al. 1997 ）允许合理的工程改造以用特异性配体结构修饰或替换受体结合结构域。通过将c-Myc肽融合至PA的羧基末端以靶向表达针对该肽的细胞表面抗体的杂交瘤细胞来产生模型系统。通过与肽和PA的无毒突变体的竞争，显示杂交瘤细胞的杀伤是特异性的（ Varugheseet al. 1998 ).
The dominant role played by the anthrax toxin inBacillus anthracispathogenesis shows that the toxin has evolved to be an efficient system for delivering its two catalytic protein components, oedema factor and lethal factor (LF), into the cytosol of host cells. This system involves binding of the protective antigen (PA) toxin component to a ubiquitous (and still unidentified) receptor, proteolytic activation at the cell surface, internalization by endocytosis and translocation through an early endosome membrane to the cytosol ( Leppla 1995). We and colleagues showed that the system can be exploited to deliver heterologous polypeptides to the cytosol ( Aroraet al. 1992 ; Milneet al. 1995 ). This work used the catalytic domains of other toxins which are normally translocated across membranes ( Arora & Leppla 1994) .Immunity to intracellular pathogens depends on the cytosolic processing of antigens to produce peptides that are presented on the cell surface bound to MHC Class I molecules. The anthrax toxin delivery system provides a way to mimic this process. We made a fusion protein containing the (non‐catalytic) amino terminal domain of LF and the gp120 envelope glycoprotein of HIV‐1. Administration of this recombinant protein along with PA to antigen‐presenting cells sensitized them to cytolysis by cytotoxic T‐cells specific to gp120 peptides ( Goletzet al. 1997 ).Further exploitation of the anthrax toxin system as a cell‐targeting reagent would be facilitated by achieving cell type specificity. The recent determination of the PA structure ( Petosaet al. 1997 ) allows rational engineering to modify or replace the receptor‐binding domain with specific ligand structures. A model system was produced by fusing a c‐Myc peptide to the carboxyl terminus of PA so as to target hybridoma cells expressing cell surface antibodies to this peptide. Killing of the hybridoma cells was shown to be specific by competition with the peptide and with non‐toxic mutants of PA ( Varugheseet al. 1998 ).