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

基本信息

批准号：
10313364
负责人：
Nicholas Alexander Till
金额：
$ 6.56万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/10313364
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 Patients Physiological Play Polysaccharides Post-Translational Protein Processing Process Prognosis 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 physical property receptor response sialylation small molecule sugar three dimensional structure tool tool development tumor progression

项目摘要

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.

项目概要/摘要标题：绘制唾液酸转移酶糖基图谱的代谢工程策略糖蛋白唾液酸化在广泛的生理和疾病相关中发挥着重要作用病毒感染、B 细胞发育和癌症转移等领域的过程。修饰主要是为了终止生长的聚糖，并且可以调节与相关的细胞间信号传导免疫激活、受体定位以及细胞表面的大量物理特性。负责安装这种聚糖末端唾液酸单元的酶家族已吸引了大量的研究人员最近引起关注，部分原因是它们在多种癌症亚型中频繁出现失调。识别给定唾液酸转移酶的蛋白质靶标可以增加对分子的了解不幸的是，由于事实上，这是一项具有挑战性的任务。唾液酸化糖基化位点不能仅根据初级蛋白质序列来预测，而是可以定义由于这些原因，通过三维结构和当前糖基化状态的组合。开发一种用于检测唾液酸转移酶糖位点的工具将提供对分子机制的进一步了解。唾液酸化驱动疾病和正常生理功能的基础。唾液酸转移酶 ST6Gal I（β-半乳糖苷 α-2,6-唾液酸转移酶 I）催化 α-2,6- 的形成聚糖末端半乳糖单元和 N-乙酰神经氨酸之间的连接，并且涉及多种此外，这种酶最近引起了人们的重新关注。其可溶性循环形式在细胞外唾液酸化中可能发挥的作用。对调节炎症很重要当前的提案旨在开发一种代谢报告系统通过凹凸/孔酶和底物工程鉴定唾液酸转移酶糖位点。应用于研究 ST6Gal I 的蛋白质靶点，但要注意确保该策略可以经过体外优化后推广到唾液酸转移酶家族的其余成员。小分子/酶对，基于质谱的糖蛋白组分析将用于鉴定 ST6Gal 最后，将使用气道炎症的小鼠模型来探测转染细胞中的糖基化位点。 ST6Gal I 在体内环境中的胞外糖位点。