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

通过糖基化构件和调节剂的外质高尔基体穿梭进行聚糖工程

• 批准号: 9809104
• 负责人: Kamil Godula
• 金额: $ 18.02万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9809104
• 关键词: Address; Anabolism; Biological; Biology; Biomedical Research; Bypass; Cell membrane; Cell surface; Cells; Cellular Membrane; Ceramides; Charge; Chemical Agents; Chemicals; Chemistry; Chinese Hamster Ovary Cell; Congenital disorders of glycosylation; Cytidine Monophosphate N-Acetylneuraminic Acid; Cytosol; DNA; Data; Defect; Destinations; Development; Disease; Endoplasmic Reticulum; Engineering; Environment; Enzyme Inhibitor Drugs; Enzymes; Extracellular Space; Functional disorder; Generations; Genetic; Glycobiology; Glycoconjugates; Golgi Apparatus; Golgi Targeting; Impairment; Life; Link; Lipids; Mainstreaming; Medical; Membrane; Membrane Glycoproteins; Metabolic; Methods; Modification; Molecular; Monosaccharides; Oligosaccharides; Organelles; Organism; Pathway interactions; Physiological; Plasma; Play; Polysaccharides; Problem Solving; Process; Proteins; Recycling; Sialic Acids; Site; Sphingolipids; Stimulus; Structure; Structure-Activity Relationship; Surface; Therapeutic; Tissues; Transferase; Work; analog; base; biomacromolecule; design; glycosylation; glycosyltransferase; human disease; improved; inhibitor/antagonist; lipid transport; metabolic engineering; retrograde transport; scaffold; sialylation; stem; sugar nucleotide; synthetic nucleotide; tool; trafficking; uptake

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.

项目概要 聚糖在生物学的各个方面都发挥着关键作用。作为生物医学目标，具有尚未开发的潜力 研究表明，糖组在组成和功能方面仍知之甚少。在没有一个 聚糖组装的分子模板，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 和聚糖的病理生理功能。目前的聚糖工程工具虽然功能强大，但尚未 完全控制了可以安装在活细胞上的聚糖的组成和结构。一个 聚糖工程的重大挑战是聚糖的核苷酸糖构建块的传递和 各种糖基化调节剂（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区室）。 这些化学试剂通常是极性的或带电荷的，并且无法穿过多个细胞膜 将细胞外空间与分泌室分开。 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，绕过胞质 隔间。新方法利用了血浆之间脂质的细胞内运输 膜和各种细胞器。该提案鉴定了血浆外层的脂质 膜作为潜在载体将货物运送到高尔基室的腔内。拟议的工作 将建立 1) 脂质修饰的结构-活性关系，从而最大限度地释放化学物质 货物从细胞表面进入高尔基体腔，2) 用于装载的最佳生物共轭化学 核苷酸糖和糖基转移酶抑制剂及其在高尔基腔环境中的释放，以及3) 用于将糖基化调节剂递送和释放到高尔基体中的高有效负载大分子支架。 该方法的一个关键特征是能够改变细胞表面聚糖的组成 不依赖于内源生物合成和补救途径来产生核苷酸糖 以及从细胞质转运到高尔基体所需的转运蛋白。因此，建议 该方法将克服目前使用代谢寡糖可访问的聚糖结构的限制 工程。它还有望提供治疗先天性疾病的通用机制 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学，以及改进的调节方法 非人类生物体中产生的生物制剂和组织替代品的糖基化谱。