Milne J C, Blanke S R, Hanna P C, Collier R J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Microbiol. 1995 Feb;15(4):661-6. doi: 10.1111/j.1365-2958.1995.tb02375.x.
The edema factor (EF) and lethal factor (LF) components of anthrax toxin require anthrax protective antigen (PA) for binding and entry into mammalian cells. After internalization by receptor-mediated endocytosis, PA facilitates the translocation of EF and LF across the membrane of an acidic intracellular compartment. To characterize the translocation process, we generated chimeric proteins composed of the PA recognition domain of LF (LFN; residues 1-255) fused to either the amino-terminus or the carboxy-terminus of the catalytic chain of diphtheria toxin (DTA). The purified fusion proteins retained ADP-ribosyltransferase activity and reacted with antisera against LF and diphtheria toxin. Both fusion proteins strongly inhibited protein synthesis in CHO-K1 cells in the presence of PA, but not in its absence, and they showed similar levels of activity. This activity could be inhibited by adding LF or the LFN fragment (which blocked the interaction of the fusion proteins with PA), by adding inhibitors of endosome acidification known to block entry of EF and LF into cells, or by introducing mutations that attenuated the ADP-ribosylation activity of the DTA moiety. The results demonstrate that LFN fused to either the amino-terminus or the carboxy-terminus of a heterologous protein retains its ability to complement PA in mediating translocation of the protein to the cytoplasm. Besides its importance in understanding translocation, this finding provides the basis for constructing a translocation vector that mediates entry of a variety of heterologous proteins, which may require a free amino- or carboxy-terminus for biological activity, into the cytoplasm of mammalian cells.
炭疽毒素的水肿因子（EF）和致死因子（LF）成分需要炭疽保护性抗原（PA）才能结合并进入哺乳动物细胞。通过受体介导的内吞作用内化后，PA促进EF和LF穿过酸性细胞内区室的膜进行转运。为了表征转运过程，我们构建了嵌合蛋白，该蛋白由LF的PA识别域（LFN；第1至255位氨基酸）与白喉毒素（DTA）催化链的氨基末端或羧基末端融合而成。纯化的融合蛋白保留了ADP-核糖基转移酶活性，并与抗LF和白喉毒素的抗血清发生反应。在有PA存在的情况下，两种融合蛋白均强烈抑制CHO-K1细胞中的蛋白质合成，但在无PA时则无此作用，并且它们表现出相似的活性水平。添加LF或LFN片段（可阻断融合蛋白与PA的相互作用）、添加已知可阻断EF和LF进入细胞的内体酸化抑制剂或引入减弱DTA部分ADP-核糖基化活性的突变，均可抑制这种活性。结果表明，与异源蛋白的氨基末端或羧基末端融合的LFN在介导该蛋白转运至细胞质中时仍保留其补充PA的能力。除了在理解转运过程中的重要性外，这一发现还为构建一种转运载体提供了基础，该载体可介导多种可能需要游离氨基末端或羧基末端才能具有生物活性的异源蛋白进入哺乳动物细胞的细胞质。
