CLONING & EXPRESSION OF NOVEL GLYCOSYLTRANSFERASES

克隆

• 批准号: 6653567
• 负责人: STEVEN B LEVERY
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6653567
• 关键词: animal tissue; biological products; biomedical resource; carbohydrates; health care; infection; intestines; lipids; model design /development; nuclear magnetic resonance spectroscopy

Enterotoxigenic E. coli expressing the K88 fimbrial adhesin causes severe intestinal infections in newborn and weaned piglets. Recognition of receptors on the surface of intestinal epithelial cells by K88 fimbrial adhesins, which prevents the removal of bacteria by intestinal peristalsis, is an early step in diarrheic pathogenesis. The three variants of the K88 adhesin (K88ab, K88ac, K88ad) appear to have different receptor specificities. It has been established that some pigs lack intestinal receptors for K88 adhesins and that these animals are resistant to infection by K88+ E. coli. Both glycoproteins and glycolipids have been implicated as K88 adhesin receptors. The aim of the current study was to identify and characterize glycoconjugate receptors for the K88ad adhesin. A neutral glycosphingolipid reactive with the K88 adhesin was isolated and purified from porcine intestinal brush border membranes. Preliminary assessment using anti-carbohydrate monoclonal ant ibodies suggested the presence of a terminal Lewis X epitope on the glycan. A very small aliquot (~ 7 (g) of the glycosphingolipid was submitted first for 1H-NMR analysis, but the results were inconclusive, due to the presence of multiple components in mixture. The sample was analyzed for monosaccharide composition by preparing the TMS derivatives of the methylglycosides followed by GC/MS analysis. TMS methylglycosides were prepared from the sample by methanolysis in 1 N HCl in MeOH, followed by N-acetylation with pyridine and acetic anhydride. The sample was then treated with Tri-Sil. GC/MS analysis was performed using a Hewlett-Packard 5890 GC/5970 MSD with a DB-5 column. Monosaccharides were identified by their retention times and electrospray ionization mass spectra in comparison to standards. The presence of sugars characteristic for neolacto-series glycosphingolipids was detected along with a small amount of fucose, but it was again concluded that the sample was a mixture, with the Lewis X structure belonging to a minor component. In an alternative approach, a set of purified standard glycosphingolipids of confirmed structure (from a library of natural and semi-synthetic compounds available in our laboratory at the CCRC) were sent to the collaborators for direct testing of the binding specificity of the K88 adhesin by HPTLC overlay staining. It was found that neolacto-tetra-and hexaglycosylceramides (nLc4Cer and nLc6Cer) bound the adhesin with a high degree of specificity, although some cross-reaction was observed with the isomeric lacto-tetraosylceramide (Lc4Cer). Binding was not observed with glycosphingolipids bearing Lewis X, Lewis a or histo-blood group H determinants. A manuscript describing this work has been submitted for publication. Currently, another crude preparation of glycosphingolipids from porcine intestinal brush border membranes is being fractionated at the CCRC in order to determine if the putative ligand can indeed be isolated from this natural source.

表达K88纤维蛋白引起的肠毒素大肠杆菌 新生儿和断奶小猪中的严重肠道感染。 识别肠上皮细胞表面的受体 由K88纤维化粘附素，可防止通过 肠蠕动是腹泻发病机理的早期一步。 K88粘合剂（K88AB，K88AC，K88AD）的三种变体似乎 具有不同的受体特异性。 已经确定了 有些猪缺乏K88粘附素的肠受体，而这些 动物对K88+大肠杆菌的感染具有抗性。 两个都 糖蛋白和糖脂已被认为是K88粘附素 受体。 本研究的目的是识别和 表征K88AD粘附素的糖缀合物受体。 一个 分离用K88粘附素反应性中性糖叠醇反应性 并从猪肠道边界膜上纯化。 使用抗碳水化合物单克隆蚂蚁ibodies进行初步评估 建议在聚糖上存在末端路易斯X表位。 一个 提交了非常小的等分试样（〜7（g） 首先进行1H-NMR分析，但由于 混合物中有多个成分的存在。 样本是 通过制备TMS分析单糖组成 甲基糖苷的衍生物，然后进行GC/MS分析。 TMS 通过1 N中的甲烷解析从样品中制备甲基糖苷 HCl在MeOH中，然后用吡啶和乙酸乙酰基化 酐。 然后将样品用Tri-Sil处理。 GC/MS分析 使用DB-5使用惠普5890 GC/5970 MSD进行 柱子。 单糖通过其保留时间和 与标准标准相比，电喷雾电离质谱。 这 糖的存在特征是新作品系列的特征 检测到糖磷脂以及少量的Fucose， 但是再次得出结论，样品是一种混合物，与 属于次要组件的路易斯X结构。 另一种 方法，一组确认的纯化标准糖脂 结构（来自自然和半合成化合物的库 在我们的CCRC实验室中可用）已发送到 直接测试K88的绑定特异性的合作者 HPTLC覆盖染色的粘合剂。 发现 neolacto-tetra and Hexaglycosylceramides（NLC4Cer和NLC6Cer）结合 具有高度特异性的粘附蛋白，尽管有些 与异构体乳酸 - 四糖基酰胺观察到交叉反应 （LC4Cer）。 用糖磷脂的轴承观察到结合 刘易斯X，刘易斯A或Histo-Blood组H决定因素。 手稿 描述这项工作已提交出版。 现在， 猪糖脂果脂的另一种粗制制剂 肠道边界膜在CCRC上被分馏 为了确定是否确实可以与假定的配体隔离 这个自然来源。