Shotgun Glycomics: Linking Glycan Structure and Function

鸟枪糖组学：连接聚糖结构和功能

• 批准号: 7515462
• 负责人: David Fletcher Smith
• 金额: $ 29.45万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7515462
• 关键词: Age; Binding Proteins; Biological; Cells; Chemical Structure; Chemicals; Chemistry; Development; Disease; Future; Glycoconjugates; Glycoproteins; Human; Human Biology; Human Genome; Knowledge; Libraries; Link; Methods; Natural Immunity; Polysaccharides; Post-Translational Protein Processing; Printing; Process; Relative (related person); Resistance to infection; Resources; Shotguns; Solutions; Structure; Tissues; functional group; glycosylation; novel; novel strategies; pathogen; response; size

DESCRIPTION (provided by applicant): The diversity and size of the human glycome is unknown. Recent studies have revealed that the human genome encodes thousands of glycoproteins, and that glycosylation is likely the most common type of post-translational modification. But how many different glycans are there? What are their relative levels of expression? Does each glycan structure have a function, either in human biology or in disease processes, such as innate immunity and resistance to infection? How does glycan expression change in response to age and disease? It has been known for decades that different cells and tissues make different glycan structures, and recent advances in methods for analysis of glycan structures has led to modern glycomics, which is mainly devoted to identifying glycan structures. Unfortunately, while knowing chemical structures is important, in a sense it only confirms what we already know; different cells make different glycan structures. While knowledge of all glycan structures may solve a chemical puzzle, it does not provide a biological solution to glycan function. We propose a new glycomic approach to link glycan structure to function/recognition using our new developments in "glyco-chemistry" that allow us to fluorescently tag free glycans released from specific cell or tissue glycoconjugates. These novel, bifunctional, fluorescent tags allow us to detect sub-microgram quantities of glycans that are otherwise undetectable, and purify them by chromatographic methods to obtain tissue- and/or cell- specific tagged-glycan libraries (TGLs). The tagged glycans retain a functional group to allow covalent "printing" of these libraries as microarrays. Such microarrays can subsequently be interrogated with glycan binding proteins (GBPs), pathogens, and cells to identify functionally recognized and biologically significant glycans, which will then be structurally defined. This novel approach avoids the laborious process of defining all glycan structures in a glycome regardless of their functional or recognition, and allows us to focus structural analyses on potentially biological relevant glycans. Developing glycomic TGLs is analogous to the "Shotgun" approach to defining the human genome, but allows us to target our analyses to link glycan structure to function. Defining all of the glycan structures of the human glycome is analogous to sequencing the human genome. The complexity of the human glycome and the lack of automated glycan sequencing methods make a "brute-force" approach to defining the glycome impractical. Here we propose a paradigm- shifting strategy, which is a combination of "shotgun" and targeted approaches. We will assemble amino functionalized, fluorescent-tagged glycan libraries from specific cells or tissues, print them as glycan microarrays, identify potentially biologically relevant glycans that are recognized by glycan binding proteins, and structurally define the recognized glycans. The stable, libraries and glycan microarrays produced will remain as a tangible resource for future analyses of both glycan structure and function.

描述（由申请人提供）：人类糖组的多样性和大小未知。最近的研究表明，人类基因组编码数千种糖蛋白，而糖基化可能是最常见的翻译后修饰类型。但是有多少种不同的聚糖呢？它们的相对表达水平是多少？每个聚糖结构在人类生物学或疾病过程中是否都有功能，例如先天免疫和抗感染能力？聚糖表达如何随年龄和疾病而变化？几十年来，人们已经知道不同的细胞和组织会产生不同的聚糖结构，而聚糖结构分析方法的最新进展催生了现代糖组学，主要致力于识别聚糖结构。不幸的是，虽然了解化学结构很重要，但从某种意义上说，它只能证实我们已经知道的东西；不同的细胞产生不同的聚糖结构。虽然了解所有聚糖结构可以解决化学难题，但它并不能为聚糖功能提供生物学解决方案。我们提出了一种新的糖组学方法，利用我们在“糖化学”方面的新进展将聚糖结构与功能/识别联系起来，该方法使我们能够荧光标记从特定细胞或组织糖缀合物释放的游离聚糖。这些新颖的双功能荧光标签使我们能够检测否则无法检测到的亚微克量的聚糖，并通过色谱方法纯化它们以获得组织和/或细胞特异性标记聚糖库（TGL）。标记的聚糖保留了一个官能团，以允许将这些文库共价“打印”为微阵列。随后可以用聚糖结合蛋白（GBP）、病原体和细胞来检测此类微阵列，以识别功能上可识别且具有生物学意义的聚糖，然后对其进行结构定义。这种新颖的方法避免了定义糖组中所有聚糖结构（无论其功能或识别如何）的费力过程，并使我们能够将结构分析重点放在潜在的生物学相关聚糖上。开发糖组 TGL 类似于定义人类基因组的“鸟枪”方法，但允许我们有针对性地进行分析，将聚糖结构与功能联系起来。 定义人类糖组的所有聚糖结构类似于对人类基因组进行测序。人类糖组的复杂性和自动化聚糖测序方法的缺乏使得“强力”方法来定义糖组不切实际。在这里，我们提出了一种范式转变策略，它是“霰弹枪”和有针对性的方法的结合。我们将从特定细胞或组织组装氨基功能化、荧光标记的聚糖文库，将其打印为聚糖微阵列，识别聚糖结合蛋白识别的潜在生物学相关聚糖，并在结构上定义所识别的聚糖。所产生的稳定的文库和聚糖微阵列将继续作为未来分析聚糖结构和功能的有形资源。