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.

产肠毒素大肠表达K88菌毛粘附素的大肠杆菌引起 新生仔猪和断奶仔猪严重肠道感染。 肠上皮细胞表面受体的识别 通过K88菌毛粘附素，其通过以下方式阻止细菌的去除： 肠梗阻是肠梗阻发病机制的早期步骤。 K88粘附素的三种变体（K88 ab、K88 ac、K88 ad）似乎 具有不同的受体特异性。 已经确定 一些猪缺乏K88粘附素的肠道受体， 动物对K88+ E的感染具有抗性。杆菌 两 糖蛋白和糖脂与K88粘附素有关 受体。 本研究的目的是确定和 表征K88 ad粘附素的糖缀合物受体。 一 分离出与K88粘附素反应的中性鞘糖脂 并从猪肠刷状缘膜中纯化。 使用抗碳水化合物单克隆抗体的初步评估 提示聚糖上存在末端刘易斯X表位。 一 提交了非常小的等分试样（约7 g鞘糖脂 首先进行1H-NMR分析，但结果是不确定的，由于 混合物中存在多种成分。 将样品 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 通过在1 N甲醇中的甲醇分解从样品制备甲基糖苷 HCl的MeOH溶液，然后用吡啶和乙酸乙酯进行N-乙酰化 酐。 然后用Tri-Sil处理样品。 GC/MS分析 使用带有DB-5的Hewlett-Packard 5890 GC/5970 MSD进行 柱 单糖通过其保留时间鉴定， 电喷雾电离质谱与标准品比较。 的 存在新乳糖系列的特征性糖 检测到鞘糖脂沿着少量岩藻糖， 但再次得出结论，该样本是混合物，其中 刘易斯X结构属于微量组分。 在替代方案中 方法，一套纯化的标准鞘糖脂的确认 结构（来自天然和半合成化合物库 我们在CCRC的实验室中提供）被发送到 合作者直接测试K88的结合特异性 粘附素通过HPTLC覆盖染色。 结果发现 新乳糖-四糖基和六糖基神经酰胺（nLc 4Cer和nLc 6Cer）结合 具有高度特异性粘附素，尽管有些 与同分异构的乳糖四糖神经酰胺发生交叉反应 （Lc4Cer）。 结合没有观察到与糖鞘脂轴承 刘易斯X、刘易斯a或组织血型H决定簇。 一份手稿 描述这一工作已提交出版。 目前， 另一种来自猪的鞘糖脂粗品 肠刷状缘膜在CCRC被分离， 为了确定假定的配体是否确实可以从 这个自然的源泉。