Glycan engineering via exoplasmic Golgi shuttle of glycosylation building blocks and modulators

项目摘要 多糖在生物学的各个方面都扮演着关键的角色。同时蕴藏着尚未开发的潜力，成为生物医学的目标 研究表明，糖糖在组成和功能方面的特性仍然很差。在缺少 用于细胞工程的多糖组装、遗传、酶和代谢方法的分子模板 表面多聚糖展示有助于建立我们目前对生理学的理解 以及多聚糖的病理生理功能。虽然功能强大，但目前的葡聚糖工程工具尚未 产生了对可以安装在活细胞上的多糖的组成和结构的完全控制。一个 在多糖工程中的重大挑战是递送核苷酸糖构建块的多聚糖和 不同的糖基化调节剂(例如，糖基化酶抑制剂)进入细胞器，其中 细胞的糖基化机制是局部的(即内质网和高尔基体)。 这些化学试剂通常是极性的或带电的，不能穿过多个细胞膜。 将细胞外空间与分泌室分开。 本提案描述了一种细胞不透性核苷酸糖输送方法的发展。 和糖基化抑制剂直接从培养液进入高尔基体，绕过胞浆 车厢。这一新方法利用了细胞内脂质在血浆之间的传递。 细胞膜和各种细胞器。该方案确定了血浆外层叶的脂类。 膜作为潜在的载体将货物运送到高尔基隔室的管腔中。拟议中的工作 将建立1)最大限度地传递化学物质的脂质修饰的结构-活性关系 从细胞表面进入高尔基体腔的货物，2)加载的最佳生物偶联化学 核苷酸和糖基转移酶抑制剂及其在高尔基体腔环境中的释放，以及3) 高负载大分子支架，用于将糖基化调节剂输送和释放到高尔基体中。 提议的方法的一个关键特征是能够改变细胞表面的糖链的组成 不依赖于内源生物合成和回收途径来产生核苷酸糖，如 以及它们从胞浆转运到高尔基体所需的转运体。因此，建议的 该方法将克服目前使用代谢低聚糖可获得的多糖结构的限制 工程学。它还准备为先天性疾病的治疗提供一个通用的机制 由核苷酸糖生物合成和运输缺陷引起的糖基化，一种解决方法 与异常糖基化相关的各种病理生理机制和一种改进的调整方法 非人类生物体中产生的生物制品和组织替代物的糖基化图谱。

Project Summary Glycans play key roles in all aspects of biology. While harboring untapped potential as a target for biomedical research, the glycome is still poorly characterized with respect to composition and function. In the absence of a molecular template for glycan assembly, genetic, enzymatic and metabolic methods for the engineering of cell surface glycan displays have been instrumental in establishing our current understanding of the physiological and pathophysiological functions of glycans. While powerful, the current glycan engineering tools have not yet yielded full control over the composition and structure of glycans that can be installed on living cells. A significant challenge in glycan engineering is the delivery of the nucleotide sugar building blocks of glycans and various modulators of glycosylation (e.g., inhibitors of glycosylation enzymes) into the organelles, where the glycosylation machinery of cells is localized (i.e., the Endoplasmic Reticulum and the Golgi compartment). These chemical agents are often polar or charged, and are unable to cross the multiple cellular membranes separating the extracellular space from the secretory compartments. This proposal describes the development of a method for delivery of cell-impermeable nucleotide sugars and glycosylation inhibitors directly from the culture medium into the Golgi, bypassing the cytosolic compartment. The new method capitalizes on the intracellular trafficking of lipids between the plasma membrane and various cellular organelles. The proposal identifies lipids at the outer leaflet of the plasma membrane as potential carriers to shuttle cargo into the lumen of the Golgi compartment. The proposed work will establish 1) structure-activity relationships for lipid modifications that maximize the delivery of chemical cargo from the cell surface into the Golgi lumen, 2) optimal bioconjugation chemistries for the loading of nucleotide sugars and glycosyl transferase inhibitors and their release in the Golgi lumen environment, and 3) high-payload macromolecular scaffolds for the delivery and release of glycosylation modulators into the Golgi. A key feature of the proposed method will be the ability to alter the composition of cell surface glycans without relying on endogenous biosynthetic and salvage pathways for the generation of nucleotide sugars as well as transporters required for their translocation from the cytosol into the Golgi. Therefore, the proposed method will overcome current limits on glycan structures accessible using metabolic oligosaccharide engineering. It is also poised to offer a general mechanism for the treatment of congenital disorders of glycosylation caused by defects in nucleotide sugar biosynthesis and transport, an approach for addressing various pathophysiologies associated with aberrant glycosylation, and an improved method for tuning glycosylation profiles of biologics and tissue replacements produced in non-human organisms.