A Metabolic Engineering Strategy to Map Sialyltransferase Glycosites

项目概要/摘要 标题：映射唾液酸转移酶糖位点的代谢工程策略 糖蛋白唾液酸化在多种生理和疾病相关的过程中起着重要的作用 在病毒感染、B细胞发育和癌症转移等领域的过程。这种翻译后 发生修饰主要是为了终止生长的聚糖，并且可以调节与以下相关的细胞-细胞信号传导： 免疫激活、受体定位以及细胞表面的整体物理性质。唾液酸转移酶， 负责安装这种聚糖终止唾液酸单元的酶家族已经获得了显著的 最近，由于其在多种癌症亚型中的频繁失调，引起了人们的关注。更具体地说， 鉴定特定唾液酸转移酶的蛋白质靶点， 酶活性与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一级蛋白质序列来预测， 通过三维结构和当前糖基化状态的组合。由于这些原因， 开发一种用于询问唾液酸转移酶糖位点的工具将进一步深入了解 唾液酸化是疾病发生和正常生理功能的基础。 唾液酸转移酶ST 6 Gal I（β-半乳糖苷α-2，6-唾液酸转移酶I）催化α-2，6- 连接之间的聚糖终止半乳糖单位和N-乙酰神经氨酸，并牵连在多个 癌症进展的机制。此外，这种酶最近引起了新的关注， 其可溶性循环形式在细胞外唾液酸化中的作用。建议后一项职能为 对调节炎症很重要。目前的建议旨在开发代谢报告系统， 通过凹凸酶和底物工程鉴定唾液酸转移酶糖位点。这种方法将是 应用于研究ST 6 Gal I的蛋白质靶点，但将注意确保该策略可以被应用于 推广到唾液酸转移酶家族的其余成员。在体外优化a 小分子/酶配对，基于质谱的糖蛋白组学分析将用于鉴定ST 6 Gal 转染细胞中的I糖基。最后，将使用气道炎症的小鼠模型来探测 在体内环境中，ST 6 Gal I的细胞外糖位点。

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.