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

项目摘要 聚糖在生物学的各个方面都起着关键作用。虽然作为生物医学的目标具有未开发的潜力， 尽管在研究中，糖组在组成和功能方面的特征仍然很差。在没有 用于聚糖组装的分子模板，用于细胞工程的遗传、酶促和代谢方法 表面聚糖展示有助于建立我们目前对生理学的理解， 以及聚糖的病理生理功能。虽然功能强大，但目前的聚糖工程工具还没有 完全控制了可以安装在活细胞上的聚糖的组成和结构。一 聚糖工程中的重大挑战是聚糖的核苷酸糖结构单元的递送 各种糖基化调节剂（例如，糖基化酶的抑制剂）进入细胞器，其中 细胞的糖基化机制被定位（即，内质网和高尔基体）。 这些化学试剂通常是极性或带电的，并且不能穿过多个细胞膜 将细胞外空间与分泌隔室分开。 该提案描述了一种用于递送细胞不可渗透的核苷酸糖的方法的开发 和糖基化抑制剂直接从培养基进入高尔基体，绕过细胞溶质 车厢这种新方法利用了脂质在细胞内的运输， 膜和各种细胞器。该提案确定了血浆外叶处的脂质 膜作为潜在的载体穿梭货物进入腔的高尔基室。拟议工作 将建立1）脂质修饰的结构-活性关系， 货物从细胞表面进入高尔基体腔，2）最佳的生物缀合化学用于装载 核苷酸糖和糖基转移酶抑制剂及其在高尔基体腔环境中的释放，和3） 用于将糖基化调节剂递送和释放到高尔基体中的高负载大分子支架。 所提出的方法的一个关键特征将是改变细胞表面聚糖组成的能力 而不依赖于内源性生物合成和补救途径来产生核苷酸糖， 以及它们从胞质溶胶转运到高尔基体所需的转运蛋白。因此，建议 该方法将克服目前对使用代谢寡糖可获得的聚糖结构的限制 工程.它还准备提供一种治疗先天性疾病的一般机制， 糖基化引起的核苷酸糖的生物合成和运输的缺陷，一种方法，解决 与异常糖基化相关的各种病理生理学以及用于调节 在非人类生物体中产生的生物制剂和组织替代物的糖基化谱。

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.