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: 8170895
• 负责人: WAYNE I LENCER
• 金额: $ 0.46万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170895
• 关键词: Affect; Binding; Biological; Biological Process; Biology; Cell Line; Cells; Ceramides; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupling; Detection; Endocytosis; Epithelial Cells; Funding; GD1a ganglioside; Gangliosides; Glycosphingolipids; Grant; Human; Hydroxylation; Institution; Intestines; Kidney; Length; Lipids; Manuscripts; Membrane Microdomains; Methods; Monkeys; Oligosaccharides; Polysaccharides; Preparation; Protein Fragment; Reporting; Research; Research Personnel; Resolution; Resources; Role; Sampling; Source; Structure; Surface; Toxin; United States National Institutes of Health; Variant; Vero Cells; choleragen receptor; ganglioside receptor; prevent; trafficking

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. Here, vibrationally cooled (VC)MALDI-FTMS was used 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. Collisional cooling is necessary for stabilization and detection of intact gangliosides. 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. A fraction, which could be a protein fragment, that does not migrate on TLC is now being studied, since it also binds with the toxin. Two manuscripts are in preparation.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 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。在这里，振动冷却 (VC)MALDI-FTMS 用于检测不稳定物质，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。碰撞冷却对于完整神经节苷脂的稳定和检测是必要的。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。目前正在研究一种在薄层色谱法上不迁移的片段，可能是蛋白质片段，因为它也与毒素结合。两份手稿正在准备中。