Toxin Potency is Linked to Holotoxin Disassembly by Protein Disulfide Isomerase

 描述（由申请方提供）：来自霍乱弧菌的霍乱毒素（CT）和来自产肠毒素大肠杆菌的不耐热毒素（LT）是高度相关的AB 5型蛋白毒素，含有催化A1亚基、A2接头和细胞结合同型五聚体B亚基。CT和LT作为完整的AB 5毒素从细胞表面行进到中毒细胞的内质网（ER）。在ER中，催化A1亚基通过蛋白质二硫键异构酶（PDI）的作用与毒素的其余部分解离。游离的A1亚基然后穿过ER膜并进入胞质溶胶，在那里它引起细胞病变效应。CT和LT在A和B亚基上共有约80%的序列同一性。这些毒素与它们共有的GM 1神经节苷脂表面受体表现出高亲和力的相互作用，并对它们共有的Gsa靶标显示出相似的体外ADP-核糖基化活性。然而，CT在细胞培养中比LT更有效：它比LT产生更大的细胞病变效应，并且在比LT更短的时间内产生这种效应。毒性的差异先前已被映射到非催化A2亚基：嵌合LTA 1/CTA 2/LTB 5毒素在体内比野生型LT更有效并且与野生型CT一样有效，而嵌合CTA 1/LTA 2/CTB 5毒素表现出的体内毒性不如野生型CT，但与野生型LT相当。A2亚基影响毒性的机制仍然未知。我们假设A2亚基，通过其在全毒内A1亚基的定位，负责建立PDI-全毒结合的亲和力 以及从全毒素中置换A1的效率。这些事件影响A1亚基传递到胞质溶胶的程度，从而影响中毒。我们已经注意到CTA 1和LTA 1亚基在它们的全毒素中的位置不同，因为它们各自的A2接头的方向不同。此外，我们已经发现，PDI介导的LT的拆卸比PDI介导的CT的拆卸效率低。因此，我们预测与CT相比，LT的有限体内效力是由于PDI对LT全毒素的无效分解。该项目将使用野生型毒素以及嵌合CTA 1/LTA 2/CTB 5和LTA 1/CTA 2/LTB 5毒素来测试我们的模型。这些毒素将用于涉及以下的研究中：（i）表面等离子体共振（SPR），以计算PDI-全毒素结合的结合速率;（ii）SPR，以真实的时间监测PDI介导的全毒素分解;和（iii）基于细胞的测定，以监测A1亚基递送至胞质溶胶。含有LTA 2亚基的毒素被预测表现出， 与含有CTA 2亚基的毒素相比，（i）对PDI的亲和力较低;（ii）通过PDI的txin分解效率较低;和（iii）胞质A1亚基水平降低。细胞生物学和生物物理学中的其他方法将用于排除对差异体内毒素活性的其他可能贡献。该项目将产生大量关于宿主-LT相互作用的新信息，并将提供详细的分子模型，解释A2亚基如何最终负责CT和LT的不同体内活性。

 DESCRIPTION (provided by applicant): Cholera toxin (CT) from Vibrio cholerae and heat-labile toxin (LT) from enterotoxigenic Escherichia coli are highly related AB5-type protein toxins that contain a catalytic A1 subunit, an A2 linker, and a cell-binding homopentameric B subunit. CT and LT travel as intact AB5 toxins from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit is dissociated from the rest of the toxin y the action of protein disulfide isomerase (PDI). The free A1 subunit then crosses the ER membrane and enters the cytosol where it elicits a cytopathic effect. CT and LT share ~80% sequence identity across both the A and B subunits. The toxins exhibit high affinity interactions with their shared GM1 ganglioside surface receptor and display similar in vitro ADP-ribosylation activities against their shared Gsa target. Yet CT is much more potent than LT in cell culture: it elicits a greater cytopathic effect than LT, and it does so in a shorter time frame than LT. The difference in toxicity has previously been mapped to the non-catalytic A2 subunit: a chimeric LTA1/CTA2/LTB5 toxin is more potent than wild-type LT and as potent as wild-type CT in vivo, while a chimeric CTA1/LTA2/CTB5 toxin exhibited in vivo toxicity that was less potent than wild-type CT but comparable to wild-type LT. The mechanism by which the A2 subunit influences toxicity remains unknown. We hypothesize the A2 subunit, through its positioning of the A1 subunit within the holotoxin, is responsible for establishing the affinity of PDI-holotoxin binding and the efficiency of A1 displacement from the holotoxin. These events affect the extent of A1 subunit delivery to the cytosol and, thus, intoxication. We have noted the CTA1 and LTA1 subunits are positioned differently within their holotoxins because of the orientation of their respective A2 linkers. Furthermore, we have found the PDI-mediated disassembly of LT is less efficient than the PDI-mediated disassembly of CT. We accordingly predict the limited in vivo potency of LT, in comparison to CT, is due to inefficient disassembly of the LT holotoxin by PDI. This project will use wild-type toxins as well as chimeric CTA1/LTA2/CTB5 and LTA1/CTA2/LTB5 toxins to test our model. These toxins will be used in studies involving (i) surface plasmon resonance (SPR) to calculate the on-rate for PDI-holotoxin binding; (ii) SPR to monitor PDI-mediated holotoxin disassembly in real time; and (iii) cell-based assays to monitor A1 subunit delivery to the cytosol. Toxins containing the LTA2 subunit are predicted to exhibit, in comparison to toxins containing the CTA2 subunit, (i) lower affinity for PDI; (ii) less efficient txin disassembly by PDI; and (iii) reduced levels of the cytosolic A1 subunit. Additional methods in cell biology and biophysics will be used to discount other possible contributions to differential i vivo toxin activity. This project will generate a substantial body of new information on host-LT interactions and will provide a detailed molecular model explaining how the A2 subunit is ultimately responsible for the different in vivo activities of CT and LT.