GLYCOLIPIDS AND HEMOLYTIC UREMIC SYNDROME

描述（改编自申请人摘要）：溶血性尿毒症综合征 (HUS)其特征在于维罗毒素介导的内皮细胞损伤 导致溶血性贫血，血小板减少， 并发症包括肾衰竭。 在几乎所有情况下， （志贺样毒素）由肠出血性E.大肠杆菌结合 糖脂的内皮细胞，然后路由到内质网 从而使28S核糖体和halt蛋白结合 合成. 尽管VT需要糖脂受体 易感性，易感性的程度不直接相关 与受体数量有关。 这些临床相关因素 儿童和成年人在他们的易感性不同， 尽管HUS表达相似数量的VT受体和糖脂， 肾细胞 因此，主要研究者假设， 与verotoxin受体或不同途径对接的差异 控制VT及其糖脂受体的细胞内运输 这些差异的基础。 在初步研究中，VT敏感Vero 用编码Forssman合成酶（FS）的cDNA转染细胞。 FS转染的细胞对VT具有高度抗性，但仍表现出 毒素结合 配体印迹法证实了两种糖脂的存在 受体（R1和R2），而只有R2，一种新的受体，以前没有 经鉴定，存在于FS转染的细胞中。 具体目标#1， 首席研究员将鉴定糖脂受体R2， 从VT抗性细胞提取物中提取受体，通过质量确认其身份 光谱法，并将纯化的R2糖脂外源地添加到其他细胞类型中 对维罗毒素有先天抵抗力 受体R1的串联实验 将作为对照。 在具体目标#2中，首席研究员 将分析verotoxin的内化和细胞内运输， FS转染细胞和野生型中的霍乱毒素受体糖脂 细胞 免疫金电子显微镜将用于跟踪 标记的verotoxin在FS转染的和WT细胞中的细胞内命运。 霍乱毒素的细胞内运输， 不同的糖脂（GM 1）将在同一系统中进行检查。 一个 诱导型启动子将用于滴定FS的表达以理解 是否需要最小量的酶来抵抗VT。 在 具体目标#3，主要研究者将使用他新开发的 用于表征mRNA的FS过表达的转基因小鼠模型 这种酶的表达和糖脂表达的改变， 各种组织。 一种HUS的小鼠模型，它不能精确地模拟 VT的影响，因为小鼠表达的糖脂谱不同于 在人类中，将使用FS转基因小鼠和同窝仔进行适应 确定糖脂表达改变是否导致VT的对照 体内抗性。

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.