Ratts Ryan, Trujillo Carolina, Bharti Ajit, vanderSpek Johanna, Harrison Robert, Murphy John R
Department of Medicine, Section of Molecular Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15635-40. doi: 10.1073/pnas.0504937102. Epub 2005 Oct 17.
A 10-aa motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema factor, anthrax lethal factor, and botulinum neurotoxin serotypes A, C, and D was identified by blast, clustal w, and meme computational analysis. Using the diphtheria toxin-related fusion protein toxin DAB(389)IL-2, we demonstrate that introduction of the L221E mutation into a highly conserved residue within this motif results in a nontoxic catalytic domain translocation deficient phenotype. To further probe the function of this motif in the process by which the catalytic domain is delivered from the lumen of early endosomes to the cytosol, we constructed a gene encoding a portion of diphtheria toxin transmembrane helix 1, T1, which carries the motif and is expressed from a CMV promoter. We then isolated stable transfectants of Hut102/6TG cells that express the T1 peptide, Hut102/6TG-T1. In contrast to the parental cell line, Hut102/6TG-T1 cells are ca. 10(4)-fold more resistant to the fusion protein toxin. This resistance is completely reversed by coexpression of small interfering RNA directed against the gene encoding the T1 peptide in Hut102/6TG-T1 cells. We further demonstrate by GST-DT140-271 pull-down experiments in the presence and absence of synthetic T1 peptides the specific binding of coatomer protein complex subunit beta to this region of the diphtheria toxin transmembrane domain.
通过Blast、Clustal W和Meme计算分析，在白喉毒素跨膜螺旋1中发现了一个10个氨基酸的基序，该基序在炭疽水肿因子、炭疽致死因子以及A型、C型和D型肉毒杆菌神经毒素中保守。使用与白喉毒素相关的融合蛋白毒素DAB(389)IL-2，我们证明将L221E突变引入该基序内的一个高度保守残基会导致无毒的催化结构域易位缺陷表型。为了进一步探究该基序在催化结构域从早期内体腔递送至细胞质的过程中的功能，我们构建了一个编码白喉毒素跨膜螺旋1一部分（T1）的基因，该部分携带该基序并由CMV启动子表达。然后我们分离出表达T1肽的Hut102/6TG细胞的稳定转染子，即Hut102/6TG-T1。与亲本细胞系相比，Hut102/6TG-T1细胞对融合蛋白毒素的抗性约高10^4倍。在Hut102/6TG-T1细胞中，针对编码T1肽的基因共表达小干扰RNA可完全逆转这种抗性。我们还通过在有和没有合成T1肽的情况下进行的GST-DT140-271下拉实验，证明了外套膜蛋白复合物亚基β与白喉毒素跨膜结构域的该区域特异性结合。
