Chemical tools for studying membrane protein glycosylation

 描述(申请人提供)：真核细胞中的蛋白质糖基化对许多细胞过程非常重要，包括蛋白质折叠、溶酶体靶向、受体信号传递和细胞-细胞黏附。因此，糖蛋白参与了大多数生理过程，在几乎所有人类主要疾病中都观察到了异常的蛋白质糖基化。尽管糖蛋白具有重要的病理生理意义，但绝大多数糖蛋白还没有在分子水平上得到表征，其糖蛋白的功能也鲜为人知。细胞表面糖基化是动态的，在癌症进展过程中，糖链结构的变化伴随着细胞的转化。然而，由于目前分子工具的局限性，目前还不可能以高空间和时间分辨率跟踪细胞表面多糖的动态变化。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自然产生的多糖的变化。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工具开发和应用的基础，以破译多糖在人类疾病中的功能作用。在目标1中，我们将发展基于铜催化叠氮烯环加成反应(CuAAC)的标记方法，将小分子荧光探针引入到细胞表面的糖偶联物上。这些方法将与改进的频闪成像、时间推移成像相结合，以实现快速形式的定位显微镜，用于生物系统中的糖偶联物的亚衍射极限成像。将开发能够对特定膜蛋白上的葡聚糖进行单分子跟踪的方法。研究表明，肿瘤细胞中的糖基化异常通过调节黏附、受体信号和蛋白表达与肿瘤进展和恶性肿瘤有关。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糖蛋白。我们将应用这种方法对具有不同转移潜力的癌细胞的唾液酸化蛋白质组进行比较分析。我们假设肿瘤细胞中糖基化异常会影响膜蛋白的动态行为，从而影响黏附和肿瘤转移。从Aim 2中鉴定出的具有独特表达模式的糖蛋白将被选作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来改变这些蛋白质在TH细胞表面的糖基化状态。将量化所选蛋白质动态变化对膜的影响，并将评估由此产生的对细胞黏附和迁移的影响(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).