A Metabolic Engineering Strategy to Map Sialyltransferase Glycosites

项目摘要/摘要 新陈代谢工程策略定位唾液酸转移酶糖基 糖蛋白唾液酸化在广泛的生理和疾病相关领域发挥着重要作用。 在病毒感染、B细胞发育和癌症转移等方面的过程。这一翻译后 修饰主要是为了终止生长中的多糖，并可以调节与以下相关的细胞-细胞信号 免疫激活、受体定位以及细胞表面的大量物理性质。唾液酸转移酶， 负责安装这种终止糖链的唾液酸单位的酶家族已经获得了显著的 最近受到关注，部分原因是它们在多种癌症亚型中频繁的失调。更确切地说， 识别特定唾液酸基转移酶的蛋白质靶标有助于加深对该分子的理解 酶活性和肿瘤进展之间的联系。不幸的是，这是一项具有挑战性的任务，因为 唾液酸化糖基不能仅仅基于初级蛋白质序列来预测，而是被定义 由三维结构和当前糖基化状态相结合。出于这些原因， 开发一种询问唾液酸基转移酶糖基的工具将提供对分子的进一步了解 唾液酸化在驾驶疾病中的基础和正常的生理功能。 唾液酸转移酶ST6GalI(β-半乳糖苷α-2，6-唾液酸基转移酶I)催化α-2，6-唾液酸基转移酶的形成。 糖链末端的半乳糖单位和N-乙酰神经氨酸之间的联系，并与 癌症进展的机制。此外，这种酶最近引起了新的关注，基于 关于其可溶的循环形式在细胞外唾液酸化中的作用。后一项职能建议为 对调节炎症很重要。目前的提案旨在开发代谢报告系统，以 通过凸起/空穴酶和底物工程鉴定唾液酸基转移酶糖位。这种方法将是 应用于研究ST6Gal I的蛋白质靶点，但将注意确保该策略可以 泛指唾液酸基转移酶家族的其余成员。经过体外优化后的一种 小分子/酶对、基于质谱学的糖蛋白质组分析将用于鉴定ST6Gal I在转基因细胞中的糖基座。最后，将使用小鼠的呼吸道炎症模型来探索 体内环境下ST6Gal I的胞外糖位。

PROJECT SUMMARY/ABSTRACT Title: A Metabolic Engineering Strategy to Map Sialyltransferase Glycosites Glycoprotein sialylation plays an important role in a wide range of physiological and disease-related processes in areas such as viral infection, B cell development, and cancer metastasis. This posttranslational modification occurs primarily to terminate a growing glycan, and can modulate cell-cell signaling relevant to immune activation, receptor localization, as well as bulk physical properties of the cell surface. Sialyltransferases, the family of enzymes responsible for installing this glycan-terminating sialic acid unit, have garnered significant attention recently, due in part to their frequent dysregulation in multiple cancer subtypes. More specifically, identifying the protein targets of a given sialyltransferase has led to increased understanding of the molecular link between enzymatic activity and tumor progression. Unfortunately, this is a challenging task, due to the fact that sialylation glycosites are not predictable based solely on primary protein sequence, but instead are defined by a combination of three-dimensional structure and current glycosylation state. For these reasons, the development of a tool for interrogating sialyltransferase glycosites would provide further insight into the molecular basis of sialylation in driving disease and normal physiological function. The sialyltransferase ST6Gal I (β-galactoside α-2,6-sialyltransferase I) catalyzes the formation of α-2,6- linkages between a glycan-terminating galactose unit and N-acetylneuraminic acid, and is implicated in multiple mechanisms for cancer progression. Additionally, this enzyme has recently attracted renewed attention based on the role its soluble, circulating form may have in extracellular sialylation. This latter function is proposed to be important to modulating inflammation. The current proposal seeks to develop a metabolic reporter system to identify sialyltransferase glycosites through bump/hole enzyme and substrate engineering. This approach will be applied to studying the protein targets of ST6Gal I, but attention will be paid to ensuring the strategy can be generalized to the remaining members of the sialyltransferase enzyme family. After in vitro optimization of a small molecule/enzyme pair, mass spectrometry-based glycoproteomic analysis will be used to identify ST6Gal I glycosites in transfected cells. Finally, a mouse model of airway inflammation will be used to probe the extracellular glycosites of ST6Gal I in an in vivo setting.