STRUCTURAL CHARACTERIZATION OF TOXIN-BINDING GANGLIOSIDES BY TLC/VC-FTMS

通过 TLC/VC-FTMS 表征毒素结合神经节苷脂的结构

• 批准号: 8365529
• 负责人: WAYNE I LENCER
• 金额: $ 0.38万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365529
• 关键词: Affect; Binding; Biological; Biological Process; Biology; Cell Line; Cells; Ceramides; Cholesterol; Complex; Coupled; Coupling; Detection; Electrospray Ionization; Endocytosis; Epithelial Cells; Fatty Acids; Funding; GD1a ganglioside; Gangliosides; Glycosphingolipids; Grant; High Pressure Liquid Chromatography; Human; Hydroxylation; Intestines; Kidney; Label; Length; Lipids; Mass Spectrum Analysis; Medicine; Membrane; Membrane Microdomains; Methods; Monkeys; National Center for Research Resources; Oligosaccharides; Polysaccharides; Principal Investigator; Proteins; Pulsar; Reporting; Research; Research Infrastructure; Resolution; Resources; Role; Sampling; Sorting - Cell Movement; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Structure; Surface; Time; Toxin; United States National Institutes of Health; Variant; Vero Cells; base; choleragen receptor; cost; flotillin; ganglioside receptor; prevent; research study; trafficking

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glycosphingolipids and gangliosides participate in diverse biological processes, and their biological roles are dependent on the structures of both the oligosaccharide and the ceramide portions. We have used vibrationally cooled (VC)MALDI-FTMS for the detection of labile species followed by their efficient fragmentation by SORI-CAD and IRMPD. GM1 and GD1a gangliosides serve as trafficking receptors for cholera toxin and the related LTIIb toxin, respectively. We assume that GD1a ganglioside of human intestinal cells is not associated with lipid rafts due to its ceramide structural variation, which prevents endocytosis of the LTIIb-GD1a complex. The toxin receptors' ganglioside structures were evaluated as a moderator of this function, including ceramide chain length, level of saturation and hydroxylation, as well as glycan composition. To analyze these molecules, our previously developed method of direct coupling of TLC plates with VC-MALDI-FTMS was used. This allows direct TLC-MALDI-FTMS without adversely affecting the FT high resolution and mass accuracy by the surface irregularity of the TLC plate. Our earlier reports have described ganglioside purification from polarized intestinal epithelial cell line T-84 and monkey kidney Vero cells and functional studies on the mechanism of toxin biology. The samples were MALDI-desorbed directly off TLC plate surfaces with ~0.2 mm sampling steps. Fragmentation was subsequently performed by SORI-CAD and IRMPD. Both sialylated and highly fucosylated glycosphingolipids were observed. GC/MS analysis of released lipids and glycans were consistent with these observations. In recent experiments, GM1 has been modified with a fluorescent label and its traffic within the cells has been followed. Lencer's group has now determined that only GM1 with unsaturated acyl chains sorts efficiently from PM to the ER. Toxin binding was required, but other membrane components were also required, including cholesterol and the lipid raft protein flotillin. In the BUSM Mass Spectrometry Resource, the difference based on N-acyl fatty acid chains, degree of saturations, etc. of GM1 or fluorescent labeled GM1 is being investigated by high performance liquid chromatography coupled to an electrospray ionization (ESI) QStar Pulsar i time-of-flight (TOF) MS which combines, speed, sensitivity, high mass accuracy and high resolution. MALDI and ESI MS are also being used to verify that the fluorescent label is stable under the cellular conditions.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 鞘糖脂和神经节苷脂参与多种生物过程，其生物学作用取决于寡糖和神经酰胺部分的结构。我们使用振动冷却 (VC)MALDI-FTMS 来检测不稳定物质，然后通过 SORI-CAD 和 IMPD 对其进行有效破碎。 GM1 和 GD1a 神经节苷脂分别充当霍乱毒素和相关 LTIIb 毒素的运输受体。我们假设人肠细胞的 GD1a 神经节苷脂由于其神经酰胺结构变异而与脂筏无关，从而阻止了 LTIIb-GD1a 复合物的内吞作用。毒素受体的神经节苷脂结构被评估为该功能的调节剂，包括神经酰胺链长度、饱和度和羟基化水平以及聚糖组成。为了分析这些分子，我们使用了我们之前开发的 TLC 板与 VC-MALDI-FTMS 直接耦合的方法。这允许直接进行 TLC-MALDI-FTMS，而不会因 TLC 板的表面不规则性而对 FT 高分辨率和质量精度产生不利影响。我们早期的报告描述了从极化肠上皮细胞系 T-84 和猴肾 Vero 细胞中纯化神经节苷脂以及毒素生物学机制的功能研究。样品直接从 TLC 板表面进行 MALDI 解吸，采样步长约为 0.2 mm。随后由 SORI-CAD 和 IRMPD 进行破碎。观察到唾液酸化和高度岩藻糖基化的鞘糖脂。释放的脂质和聚糖的 GC/MS 分析与这些观察结果一致。在最近的实验中，GM1 已被荧光标记修饰，并对其在细胞内的运输进行了追踪。 Lencer 的研究小组现已确定，只有具有不饱和酰基链的 GM1 才能有效地从 PM 到 ER 进行分类。需要毒素结合，但还需要其他膜成分，包括胆固醇和脂筏蛋白弗洛林蛋白。在 BUSM 质谱资源中，基于 GM1 或荧光标记 GM1 的 N-酰基脂肪酸链、饱和度等的差异正在通过与电喷雾电离 (ESI) QStar Pulsar i time 联用的高效液相色谱进行研究飞行 (TOF) MS 结合了速度、灵敏度、高质量精度和高分辨率。 MALDI 和 ESI MS 也用于验证荧光标记在细胞条件下是否稳定。