Identification of host cell factors required for intoxication through use of modified cholera toxin.
Identification of host cell factors required for intoxication through use of modified cholera toxin.
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DOI：
10.1083/jcb.201108103
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发表时间：
2011-11-28
期刊：
影响因子：
--
通讯作者：
Ploegh HL
中科院分区：
文献类型：
--
作者：
Guimaraes CP;Carette JE;Varadarajan M;Antos J;Popp MW;Spooner E;Brummelkamp TR;Ploegh HL
一种新的标记策略应用于霍乱毒素A1亚基的背景下，允许跟踪其细胞内运输途径和识别参与细胞中毒的宿主蛋白质的预组装的全毒素。我们描述了一种新的标记策略，以位点特异性地将荧光团，生物素和蛋白质连接到霍乱毒素（CTx）的A1亚基（CTA 1）的C末端，否则正确组装和活性CTx复合物。使用生物素化的N-连接的糖基化报告肽连接到CTA 1，我们提供了直接的证据，约12%的内化CTA 1池到达ER。我们还探索了分选酶标记的方法，将白喉毒素的催化亚基作为毒性弹头连接到CTA 1上，从而将CTx转化为细胞致死毒素。这种新的毒素缀合物使我们能够在人类细胞中进行遗传筛选，其鉴定出ST 3GAL 5、SLC 35 A2、B3 GALT 4、UGCG和ELF 4为CTx中毒所必需的基因。前四个编码参与神经节苷脂合成的蛋白质，神经节苷脂是已知的CTx受体。ST 3GAL 5和SLC 35 A2突变克隆细胞的鉴定和分离揭示了神经节苷脂对CTx中毒的先前未被认识到的差异贡献。
A novel labeling strategy is applied to cholera toxin subunit A1 in the context of a pre-assembled holotoxin allowing tracking of its intracellular trafficking pathway and identification of host proteins involved in cell intoxication. We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ∼12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.
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DOI：
10.1083/jcb.131.4.951
发表时间：
1995-11
期刊：
The Journal of cell biology
影响因子：
--
作者：
Lencer WI;Constable C;Moe S;Jobling MG;Webb HM;Ruston S;Madara JL;Hirst TR;Holmes RK
通讯作者：
Holmes RK
DOI：
10.1073/pnas.97.2.745
发表时间：
2000-01-18
影响因子：
11.1
作者：
Arunachalam, B;Phan, UT;Cresswell, P
通讯作者：
Cresswell, P
影响因子：
48
作者：
Chen, I;Howarth, M;Ting, AY
通讯作者：
Ting, AY
影响因子：
3.3
作者：
Bernardi, Kaleena M.;Forster, Michele L.;Tsai, Billy
通讯作者：
Tsai, Billy
影响因子：
3.2
作者：
Dixit, Garima;Mikoryak, Carole;Draper, Rockford K.
通讯作者：
Draper, Rockford K.