GLYCOLIPIDS AND HEMOLYTIC UREMIC SYNDROME

描述(摘自申请者摘要)：溶血性尿毒症综合征 (HUS)的特点是维罗毒素介导的内皮细胞损伤 这会导致溶血性贫血、血小板减少和多系统疾病。 并发症包括肾功能衰竭。在几乎所有的情况下，维罗毒素 肠出血性大肠杆菌释放的志贺样毒素结合于 内皮细胞上的糖脂，然后被送到内质 从而使28S核糖体失活，停止蛋白质 综合。尽管VT需要糖脂受体 易感性，易感性的程度不直接相关 与受体的数量有关。三者的临床相关性 据观察，儿童和成人在易感性上存在差异 HUS，尽管VT受体和糖脂的表达相似 肾细胞。因此，首席调查员假设 与Vero毒素受体或不同途径对接的差异 VT及其糖脂受体在细胞内转运的调控 这是这些差异的基础。在初步研究中，对VT敏感的Vero 将编码Forssman合成酶(FS)的基因导入细胞。 转染FS的细胞对VT高度耐药，但仍显示 毒素结合。配基印迹显示两种糖脂的存在 受体(R1和R2)，而只有R2，一种新的受体 经鉴定，该基因存在于FS转基因细胞中。在具体目标1中， 首席研究员将鉴定糖脂受体R2，纯化这个 从VT抗性细胞提取的受体，通过质量确认其身份 光谱，并将纯化的R2糖脂外源添加到其他类型的细胞中 对维罗毒素有先天抵抗力。与受体R1的串联实验 将作为一种控制。在具体目标2中，首席调查员 将分析维罗毒素的内化和细胞内转运 霍乱毒素受体糖脂在FS转基因细胞和野生型中的表达 细胞。将使用免疫金电子显微镜来追踪 标记维罗毒素在FS和WT-细胞中的细胞内去向。 霍乱毒素的细胞内运输，它完全结合了 不同的糖脂(GM1)将在这个相同的系统中进行检测。一个 用可诱导启动子滴定FS的表达以了解 是否需要最少量的酶来抵抗室上性心动过速。在……里面 具体目标3，首席调查员将使用他新开发的 转基因小鼠过表达FS以鉴定其基因的表达 该酶的表达及糖脂表达的变化 各种纸巾。HUS的小鼠模型，它未能准确地模拟 VT的作用是因为小鼠表达的糖脂光谱不同于 在人类身上，将使用FS转基因小鼠和窝鼠进行适应 确定糖脂表达改变是否会导致室性心动过速 体内抗药性。

DESCRIPTION (Adapted from applicant's abstract): Hemolytic uremic syndrome (HUS) is characterized by verotoxin-mediated damage to endothelial cells that results in hemolytic anemia, throbocytopenia, and multisystemic complications including renal failure. In almost all cases, verotoxins (shiga-like toxins) released by enterohemorrhagic E. coli bind to glycolipids on the endothelial cells, are then routed to the endoplasmic reticulum, and thereby inactivate the 28S ribosomes and halt protein synthesis. Although glycolipid receptors are required for VT susceptibility, the degree of susceptibility does not correlate directly with the quantity of receptor. The clinical correlative of these observations is that children and adults differ in their susceptibility to HUS despite expressing similar quantities of VT-receptor and glycolipids in renal cells. The principal investigator therefore hypothesizes that differences in docking to verotoxin receptors or distinct pathways controlling intracellular trafficking of VT and its glycolipid receptors underlie these differences. In preliminary studies VT-susceptible Vero cells were transfected with the cDNA encoding Forssman synthetase (FS). FS-transfected cells were highly resistant to VT, yet still demonstrated toxin binding. Ligand blotting demonstrated the presence of two glycolipid receptors (R1 and R2) whereas only R2, a novel receptor not previously identified, was present in FS-transfected cells. In Specific Aim #1, the principal investigator will identify glycolipid receptor R2, purify this receptor from VT-resistant cell extracts, confirm its identity by mass spectroscopy, and add purified R2 glycolipid exogenously to other cell types with a priori resistance to verotoxin. Tandem experiments with receptor R1 will serve as a control. In Specific Aim #2, the principal investigator will analyze internalization and intracellular trafficking of verotoxin and cholera toxin receptor glycolipids in FS-transfected cells and wild-type cells. Immunogold electron microscopy will be used to track the intracellular fate of labeled verotoxin in FS-transfected and WT-cells. Intracellular trafficking of cholera toxin, which binds an altogether different glycolipid (GM1) will be examined in this same system. An inducible promoter will be used to titrate expression of FS to understand whether a minimal amount of the enzyme is required for VT-resistance. In Specific Aim #3, the principal investigator will use his newly developed transgenic mouse model for overexpression of FS to characterize mRNA expression of this enzyme and alterations in glycolipid expression in various tissues. A murine model for HUS, which fails to mimic precisely the effects of VT because mice express a spectrum of glycolipids different from that in humans, will be adapted using FS transgenic mice and littermate controls to determine whether altered glycolipid expression results in VT resistance in vivo.