Chemical tools for studying membrane protein glycosylation

 描述（由申请人提供）：真核细胞中的蛋白质糖基化对于许多细胞过程很重要，包括蛋白质折叠、溶酶体靶向、受体信号传导和细胞-细胞粘附。因此，糖蛋白参与大多数生理过程，并且在几乎所有主要的人类疾病中观察到异常的蛋白质糖基化。尽管糖蛋白的病理生理意义，他们中的绝大多数还没有在分子水平上的特点，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伴随癌症进展中的细胞转化。然而，由于目前的限制，在分子工具，它还不可能遵循动态变化的细胞表面聚糖与高时空分辨率。该项目的长期目标是开发新的成像和蛋白质组学工具，以分析疾病相关过程中天然存在的聚糖的变化。我们的中心假设是，具有快速动力学、高特异性和生物相容性的生物正交点击反应可以作为开发和应用工具来破译聚糖在人类疾病中的功能作用的基础。 在目标1中，我们将开发基于铜催化的叠氮化物-炔环加成（CuAAC）的标记方法，以将小分子荧光探针结合到细胞表面的糖缀合物上。这些方法将结合修改，频闪，延时成像，以实现快速形式的本地化显微镜的子衍射极限成像的糖缀合物在生活系统中。将开发方法，使单分子跟踪聚糖的特定膜蛋白。 研究表明，肿瘤细胞中糖基化异常通过调节粘附、受体信号传导和蛋白质表达与癌症进展和恶性程度相关。末端唾液酸化和岩藻糖基化是这些过程的关键贡献者。在目标2中，我们将开发一种通用的糖蛋白组学方法来鉴定癌细胞中的唾液酸化糖蛋白。我们将应用这种方法比较分析具有不同转移潜力的癌细胞的唾液酸化蛋白质组。 我们推测肿瘤细胞中糖基化的异常会影响膜蛋白的动力学行为，从而影响粘附和肿瘤转移。从Aim 2中鉴定出的具有独特表达模式的糖蛋白将被选择用于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在细胞表面改变这些蛋白质的糖基化状态。将量化所选蛋白质对膜的动力学改变的影响，并评价对细胞粘附和迁移的影响（如果有）（目标3）。

 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).