PROTECTIVE ANTIGEN-BINDING DOMAIN OF ANTHRAX LETHAL FACTOR MEDIATES TRANSLOCATION OF A HETEROLOGOUS PROTEIN FUSED TO ITS AMINO-TERMINUS OR CARBOXY-TERMINUS
PROTECTIVE ANTIGEN-BINDING DOMAIN OF ANTHRAX LETHAL FACTOR MEDIATES TRANSLOCATION OF A HETEROLOGOUS PROTEIN FUSED TO ITS AMINO-TERMINUS OR CARBOXY-TERMINUS
复制标题
DOI：
10.1111/j.1365-2958.1995.tb02375.x
复制
发表时间：
1995-02-01
影响因子：
3.6
通讯作者：
COLLIER, RJ
中科院分区：
文献类型：
--
作者：
MILNE, JC;BLANKE, SR;COLLIER, RJ
炭疽毒素的水肿因子（EF）和致死因子（LF）组分需要炭疽保护性抗原（PA）结合并进入哺乳动物细胞。在通过受体介导的内吞作用内化后，PA促进EF和LF穿过酸性细胞内室的膜的易位。为了表征易位过程，我们产生了由LF的PA识别结构域（LF（N）;残基1-255）融合到白喉毒素（DTA）催化链的氨基末端或羧基末端，纯化的融合蛋白保留ADP-核糖基转移酶活性，并与抗LF和白喉毒素的抗血清反应。两种融合蛋白在PA存在下强烈抑制CHO-K1细胞中的蛋白质合成，但在PA不存在下不抑制，并且它们显示出相似的活性水平。这种活性可以通过添加LF或LF（N）片段（其阻断融合蛋白与PA的相互作用）、通过添加已知阻断EF和LF进入细胞的内体酸化抑制剂、或通过引入减弱DTA部分的ADP-核糖基化活性的突变来抑制。结果表明，LF（N）融合到一个异源蛋白的氨基端或羧基端保留其能力，以补充PA介导的蛋白质易位到细胞质。除了其在理解易位中的重要性之外，这一发现为构建易位载体提供了基础，该载体介导多种异源蛋白质进入哺乳动物细胞的细胞质，这些异源蛋白质可能需要游离的氨基或羧基末端以获得生物活性。
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 (LF(N); 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 LF(N) 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 LF(N) 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.