Novel Technologies for Quantitative O-glycomics and Amplification/Preparation of Cellular O-Glycans

定量 O-糖组学和细胞 O-聚糖扩增/制备的新技术

• 批准号: 9167145
• 负责人: Tongzhong Ju
• 金额: $ 64.46万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-13 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9167145
• 关键词: Accounting; Acetylgalactosamine; Address; Affect; Amines; Anabolism; Asparagine; Binding; Biological; Biological Process; Biology; Carbohydrates; Cell Communication; Cell Line; Cell surface; Cells; Chemicals; Cleaved cell; Collection; Complex; Cultured Cells; Cytosol; DNA; Development; Embryonic Development; Enzymes; Fucose; Galactose; Genes; Glycocalyx; Glycoconjugates; Glycolipids; Glycoproteins; Glycosaminoglycans; Glycoside Hydrolases; Glycosphingolipids; Health; High Pressure Liquid Chromatography; Homeostasis; Human; Human Biology; Human Genome; Immunity; Label; Life; Link; Mammalian Cell; Mammals; Mannose; Mass Spectrum Analysis; Metabolic; Methods; Microarray Analysis; Monosaccharides; Mucins; Nature; Nuclear; Oligosaccharides; Organ; Pathway interactions; Play; Polysaccharides; Preparation; Process; Property; Proteins; RNA; Reporter; Reproducibility; Role; Serine; Signal Transduction; Source; Structure; Technology; Threonine; Tissues; abstracting; angiogenesis; base; biological systems; cell type; comparative; extracellular; feeding; glycosyltransferase; innovation; meetings; new technology; novel; success; sugar; tool

Abstract Glycans linked to Asparagine (Asn) (N-glycans, N-glycome) and Serine/Threonine (Ser/Thr) (O-glycans, O- glycome), play critical roles in the molecular interactions that govern many important biological systems. Furthermore, alterations in these glycomes, often associated with pathological states, and can significantly affect cellular properties and functions. In order to fully understand the roles of glycans in biology, functional glycomics, information on specific cellular glycomes is necessary. Glycan analysis, however, often requires release of glycoconjugates prior to structural analysis by modern technologies, such as Mass Spectrometry (MS). While N-glycan analyses are facilitated by the availability of endo-glycosidases that affect their release, no such comparable method exists for O-glycans. Instead, release of O-glycans from glycoproteins often relies on β-elimination, which has many limitations, such as non-specificity, and low efficiency, and the requirement for large amounts of material. The technology for quantitative analysis of glycans, also known as quantitive glycomics, is even more limited. Similarly, the preparation of complex glycans for biological studies is extremely challenging, since only limited numbers and amounts of glycans can be chemically or chemoenzymatically synthesized or isolated from natural sources. To overcome these issues, we are developing a novel technology termed Cell O-glycome Reporter/Amplification(CORA)- a simple, sensitive, and versatile method for profiling mucin type O-glycan structures and amplifying/preparing complex O-glycans from cultured cells fed with peracetylated Benzyl-α-N-acetylgalactosamine [Bn-α-GalNAc(AC)3]. The products, Bn-α- O-Glycans synthesized by cells are secreted into the media and easily isolated for analysis by MS. Our CORA technology has demonstrated high sensitivity, reproducibility, accuracy, and feasibility as well as amplification nature. In this application, we propose to advance our current CORA technology into the Quantitative-CORA and Preparative-CORA for quantitative O-glycomics and amplification/preparation of cellular O-glycans by a study through 4 following Aims. Aim 1: To establish and validate Quantitative-CORA by developing “metabolic Stable Isotopic Labeling of O-glycomes of cultured Cells (SILOC)” with [13C6]Bn-α-GalNAc(Ac)3 for comparative quantitive O-glycomics; Aim 2: To develop Preparative-CORA using 2-PA-5-O-α-GalNAc(Ac)3 which contains a fluorescent tag posing an active Amine group for immobilizing; Aim 3: To validate the Preparative-CORA technology by amplifying and preparing cellular O-glycomes for 20 cell lines using cells as “O-Glycan factories”; Aim 4: To prepare individually purified complex O-glycans from amplified cellular O-glycomes for biological function studies by combining Preparative-CORA, 2D-HPLC Separation and Glycan Microarray technologies. These Quantitative-CORA and Preparative-CORA technologies will meet the critical demands in the field and directly facilitate both cellular O-glycomics and functional glycomics studies.

摘要 与天冬酰胺（Asn）（N-聚糖，N-糖组）和丝氨酸/苏氨酸（Ser/Thr）（O-聚糖，O- 糖组），在控制许多重要生物系统的分子相互作用中起关键作用。 此外，这些糖组的改变，通常与病理状态有关， 影响细胞特性和功能。为了充分了解聚糖在生物学中的作用， 糖组学，关于特定细胞糖组的信息是必要的。然而，聚糖分析通常需要 在通过现代技术（如质谱法）进行结构分析之前释放糖缀合物 （MS）。虽然影响N-聚糖释放的内切糖苷酶的可用性有助于N-聚糖分析， 对于O-聚糖不存在这样的可比方法。相反，从糖蛋白释放O-聚糖通常 依赖于β-消除，其具有许多局限性，例如非特异性和低效率， 需要大量的材料。用于聚糖定量分析的技术，也称为 定量糖组学，甚至更有限。同样，用于生物学研究的复合聚糖的制备 是极具挑战性的，因为只有有限的数量和量的聚糖可以化学地或 化学酶促合成的或从天然来源分离的。为了解决这些问题，我们 开发一种称为细胞O-糖组报告/扩增（CORA）的新技术-一种简单，灵敏， 用于分析粘蛋白型O-聚糖结构和从其扩增/制备复合O-聚糖的通用方法 用全乙酰化苄基-α-N-乙酰半乳糖胺[Bn-α-GalNAc（AC）3]喂养培养的细胞。产物Bn-α- 由细胞合成的O-聚糖分泌到培养基中，并且容易分离用于MS分析。 技术已经证明了高灵敏度，重现性，准确性和可行性以及放大 自然在这个应用中，我们建议将我们目前的CORA技术推进到定量CORA 和用于定量O-糖组学和细胞O-聚糖的扩增/制备的特异性-CORA， 通过以下四个目标进行研究。目的1：通过建立代谢性的CORA模型，建立和验证定量CORA模型 用[13 C6]Bn-α-GalNAc（Ac）3稳定同位素标记培养细胞的O-糖组（SIBR）“用于比较 目的2：利用2-PA-5-O-α-GalNAc（Ac）3（含2-PA-5-O-α-GalNAc（Ac）3 一个荧光标签，构成一个活性胺基团用于固定;目的3：验证的替代CORA 使用细胞作为“O-聚糖”为20种细胞系扩增和制备细胞O-聚糖的技术 目的4：从扩增的细胞O-聚糖制备单独纯化的复合O-聚糖，用于 生物学功能的研究--结合CORA、2D-HPLC分离和聚糖微阵列 技术.这些定量CORA和定量CORA技术将满足以下关键需求： 直接促进细胞O-糖组学和功能糖组学研究。