Chemical tools for studying membrane protein glycosylation

研究膜蛋白糖基化的化学工具

• 批准号: 9196522
• 负责人: Ben Ovryn
• 金额: $ 25.15万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9196522
• 关键词: Chemical; tools; studying; membrane; protein

 DESCRIPTION (provided by applicant): Protein glycosylation in eukaryotic cells is important for numerous cellular processes, including protein folding, lysosomal targeting, receptor signaling and cell-cell adhesion. Glycoproteins, therefore, are involved in most physiological processes, and aberrant protein glycosylation is observed in almost all major human diseases. Despite the pathophysiological significance of glycoproteins, the vast majority of them have not been characterized at the molecular level, and the functions of their glycans are poorly understood. Cell surface glycosylation is dynamic and changes in glycan structure accompany cell transformation in cancer progression. However, because of current limitations in molecular tools it is not yet possible to follow the dynamic changes of cell-surface glycans with high spatia and temporal resolution. The long-term objective of this project is to develop new imaging and proteomic tools to analyze changes in naturally- occurring glycans in disease-related processes. Our central hypothesis is that bioorthogonal click reactions with fast kinetics, high specificity ad biocompatibility can serve as the foundation upon which tools can be developed and applied to decipher the functional roles of glycans in human disease. In Aim 1, we will develop labeling methods based on copper-catalyzed azide-alkyne cycloaddition (CuAAC) to incorporate small-molecule fluorescent probes on to cell-surface glycoconjugates. These methods will be combined with modified, stroboscopic, time-lapse imaging to achieve a fast form of localization microscopy for sub-diffraction-limit imaging of glycoconjugates in living systems. Methods will be developed to enable single molecule tracking of glycans on a specific membrane protein. Studies have shown that abnormal glycosylation in tumor cells is associated with cancer progression and malignancy by regulating adhesion, receptor signaling and protein expression. Terminal sialylation and fucosylation are key contributors to these processes. In Aim 2, we will develop a general glycoproteomic approach to identify sialylated glycoproteins in cancer cells. We will apply this approach for comparative analysis of the sialylated proteomes of cancer cells with distinct metastatic potentials. We hypothesize that abnormal glycosylation in tumor cells will affect the dynamic behaviors of membrane proteins, and thus adhesion and cancer metastasis. Glycoproteins identified from Aim 2 with unique expression patterns will be selected for further biological studies. We will alter glycosylation status of these proteins directly on th cell surface by in situ glycosylation reactions and by glycosidase treatment. The impact of the altered dynamics of the selected proteins on the membrane will be quantified, and the resulting influence, if any, upon cell adhesion and migration will be evaluated (Aim 3).

 描述（由适用提供）：真核细胞中的蛋白质糖基化对于许多细胞过程，包括蛋白质折叠，溶酶体靶向，受体信号传导和细胞细胞粘合剂很重要。因此，糖蛋白参与了大多数物理过程，并且在几乎所有主要的人类疾病中都观察到异常的蛋白质糖基化。尽管糖蛋白具有病理生理意义，但绝大多数在分子水平上尚未表征，其聚糖的功能知之甚少。细胞表面糖基化是动态的，并且聚糖结构的变化癌症进展中的细胞转化。但是，由于分子工具的当前局限性，因此无法遵循具有高海峰和临时分辨率的细胞表面聚糖的动态变化。该项目的长期目标是开发新的成像和蛋白质组学工具，以分析与疾病相关的过程中天然发生聚糖的变化。我们的中心假设是，具有快速动力学的生物正交点击反应，高特异性AD生物相容性可以作为可以开发和应用工具来破坏聚糖在人类疾病中的功能作用的基础。 In Aim 1, we will develop labeling methods based on copper-catalyzed azide-alkyne cycloaddition (CuAAC) to incorporate small-molecule These methods will be combined with modified, stroboscopic, time-lapse imaging to achieve a fast form of localization microscopy for sub-diffraction-limit imaging of glycoconjugates in living systems.将开发方法以实现特定膜蛋白上的聚糖的单分子跟踪。研究表明，通过调节粘附，受体信号传导和蛋白质表达，肿瘤细胞中糖基化异常与癌症进展和恶性有关。终末溶性和诱导化是这些过程的关键因素。在AIM 2中，我们将开发一种一般的糖蛋白质组学方法，以鉴定癌细胞中的脱糖糖蛋白。我们将采用这种方法来比较具有不同转移电位的癌细胞的溶解蛋白。我们假设肿瘤细胞异常的糖基化将影响膜蛋白的动态行为，从而影响粘合剂和癌症转移。从AIM 2鉴定出具有独特表达模式的糖蛋白将被选择进行进一步的生物学研究。我们将通过原位糖基化反应和糖苷酶处理直接改变这些蛋白质的糖基化状态。将评估所选蛋白质改变动力学对膜的影响，并将评估所得的影响（如果有的话）对细胞粘合剂和迁移的影响（AIM 3）。