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.

项目摘要 Glycans在生物学的各个方面都起着关键作用。同时拥有未开发的潜力作为生物医学的目标 研究，在组成和功能方面的糖果表征仍然很差。在没有 用于聚糖组装，遗传，酶促和代谢方法的分子模板用于细胞的工程 表面聚糖显示器有助于建立我们当前对生理的理解 和聚糖的病理生理功能。尽管功能强大，但目前尚未达到聚糖工程工具 可以完全控制可以安装在活细胞上的聚糖的组成和结构。一个 聚糖工程中的重大挑战是聚糖和聚糖核苷酸糖的构建块的挑战 糖基化的各种调节剂（例如，糖基化酶的抑制剂）进入细胞器，其中 细胞的糖基化机制是局部的（即内质网和高尔基体室）。 这些化学剂通常是极性或充电的，并且无法越过多个细胞膜 将细胞外空间与分泌室分开。 该提案描述了开发一种递送细胞可侵蚀核苷酸糖的方法 直接从培养基到高尔基的糖基化抑制剂，绕过胞质 车厢。新方法利用了血浆之间脂质的细胞内贩运 膜和各种细胞细胞器。该提案确定了等离子体外叶的脂质 膜作为潜在的载体，可将货物驶入高尔基体腔腔。拟议的工作 将建立1）脂质修饰的结构活性关系，以最大化化学物质的递送 从细胞表面进入高尔基管的货物，2）用于加载的最佳生物缀合化学 核苷酸糖和糖基转移酶抑制剂及其在高尔基体腔环境中释放，3） 高付费大分子支架，用于将糖基化调节剂传递和释放到高尔基体中。 该方法的关键特征将是改变细胞表面聚糖组成的能力 不依赖内源性生物合成和打捞途径，以生成核苷酸糖作为 以及转运蛋白将其从细胞质转移到高尔基体所需。因此，提议 方法将克服使用代谢寡糖访问的聚糖结构的当前限制 工程。它还准备提供一种一般机制来治疗先天性疾病 由核苷酸糖生物合成和转运的缺陷引起的糖基化，一种解决方法 与异常糖基化相关的各种病理生理和一种改进的调整方法 非人类生物产生的生物制剂和组织替代物的糖基化谱。