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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 糖磷脂和神经脂参与了不同的生物学过程，它们的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-FTM用于检测不稳定物种，然后通过Sori-Cad和IRMPD有效地分裂。 GM1和GD1A神经毒剂分别用作霍乱毒素和相关LTIIB毒素的运输受体。我们假设人类肠细胞的GD1A神经节因其神经酰胺的结构变异而与脂质筏无关，从而阻止了LTIIB-GD1A复合物的内吞作用。评估毒素受体的神经节结构作为该功能的主持人，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，我们使用了先前开发的TLC板与VC-Maldi-FTM的直接耦合方法。这允许直接TLC-Maldi-FTM，而不会通过TLC板的表面不规则性不利地影响FT高分辨率和质量准确性。碰撞冷却对于稳定和检测完整的神经毒剂是必需的。我们的早期报告描述了从偏振肠上皮细胞系T-84和猴肾细胞中的神经节纯化以及有关毒素生物学机制的功能研究。将样品直接从TLC板板表面直接使用MALDI索引，并采样〜0.2 mm采样步骤。随后由Sori-CAD和IRMPD进行碎片。观察到溶解和高度葡萄糖基化的糖磷脂脂。释放脂质和聚糖的GC/MS分析与这些观察结果一致。现在正在研究未在TLC上迁移的蛋白质片段的馏分，因为它也与毒素结合。准备两个手稿。