A Metabolic Engineering Strategy to Map Sialyltransferase Glycosites

绘制唾液酸转移酶糖位图的代谢工程策略

• 批准号: 10468652
• 负责人: Nicholas Alexander Till
• 金额: $ 6.72万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-26 至 2024-07-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10468652
• 关键词: Active Sites; Acute; Amino Acid Sequence; Area; Attention; Automobile Driving; Azides; B-Cell Development; Biological Assay; Cell Line; Cell model; Cell surface; Cells; Colorectal Cancer; Development; Disease; Disease Progression; Engineering; Ensure; Enzyme Kinetics; Enzymes; Family; Fluorescence; Galactose; Galactosides; Glycoproteins; In Vitro; Inflammation; Injections; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Metabolic; Modification; Molecular; Mus; N-Acetylneuraminic Acid; Neoplasm Metastasis; Physiological; Play; Polysaccharides; Post-Translational Protein Processing; Process; Proteins; Recombinants; Reporter; Role; ST6Gal I; Sialic Acids; Sialyltransferases; System; Therapeutic; Virus Diseases; Work; airway inflammation; analog; base; cancer subtypes; colon cancer cell line; enzyme substrate; extracellular; glycoproteomics; glycosylation; immune activation; immune function; in vivo; insight; intercellular communication; member; metabolic engineering; mouse model; mutant; patient prognosis; physical property; receptor; response; sialylation; small molecule; sugar; three dimensional structure; tool; tool development; tumor progression

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.

项目摘要/摘要 标题：绘制siallyltransfrassy gllycosites的代谢工程策略 糖蛋白溶解酶在广泛的生理和疾病有关 病毒感染，B细胞发育和癌症转移等地区的过程。这种翻译后 修饰主要是为了终止增长的聚糖，并且可以调节与 免疫激活，受体定位以及细胞表面的大量物理特性。 siallyltransferases， 负责安装这种聚糖终止唾液酸单元的酶家族已获得显着 最近注意，部分原因是它们在多种癌症亚型中经常失调。更具体地说， 识别给定硝基转移酶的蛋白质靶标已导致对分子的了解增加 酶活性与肿瘤进展之间的联系。不幸的是，由于事实，这是一项挑战任务 溶酶糖材并非仅基于原发性蛋白质序列而预测，而是定义的 通过三维结构和当前糖基化状态的组合。由于这些原因， 开发用于询问乙烯基转移酶糖材料的工具将为分子提供进一步的见解 在驱动疾病和正常生理功能方面的基础基础。 SiAlllltransferase st6gal I（β-半乳糖苷α-2,6-溶解酶I）催化α-2,6--的形成 聚糖末端的半乳糖单元和N-乙酰神经氨酸酸之间的联系，并暗示多个 癌症进展的机制。此外，该酶最近吸引了基于重新关注的酶 在其可溶性的作用上，循环形式可能在细胞外裂囊化中具有。建议以后的功能为 调节炎症很重要。当前的建议旨在开发代谢记者系统 通过凸起/孔酶和底物工程识别乙烯基转移酶糖材料。这种方法将是 应用于研究ST6GAL I的蛋白质靶标，但将注意确保该策略可以是 将其概括为辅酶酶家族的其余成员。在体外优化A之后 小分子/酶对，基于质谱的糖蛋白质组学分析将用于识别ST6GAL 我在翻译细胞中的糖材料。最后，将使用气道注入的鼠标模型探测 在体内环境中，ST6GAL I的细胞外糖材料。