Shotgun Glycomics: Linking Glycan Structure and Function

鸟枪糖组学：连接聚糖结构和功能

• 批准号: 7901199
• 负责人: David Fletcher Smith
• 金额: $ 41.08万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7901199
• 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

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）：人类糖的多样性和大小尚不清楚。最近的研究表明，人类基因组编码了数千种糖蛋白，并且糖基化可能是翻译后修饰的最常见类型。但是有多少种不同的聚糖？他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都具有一项功能，例如先天免疫和对感染的抵抗力？聚糖表达如何反应年龄和疾病？几十年来，不同的细胞和组织会产生不同的聚糖结构，而分析聚糖结构的方法的最新进展已导致现代糖果，这主要致力于识别聚糖结构。不幸的是，尽管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知识。不同的细胞形成不同的聚糖结构。尽管所有聚糖结构的知识可以解决化学难题，但它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GlyComic方法，将聚糖结构链接到使用“ Glyco-Chemistry”中的新发展，使我们能够荧光标记从特定细胞或组织糖缀合物中释放出的无甘氨酸。这些新颖的双功能，荧光标签使我们能够检测到原本无法检测到的聚糖的亚微图量，并通过色谱方法净化它们，以获取组织和/或细胞特定标记的 - 聚糖库（TGLS）。标记的Glycans保留了功能组，以使这些库的共价“打印”作为微阵列。随后可以通过聚糖结合蛋白（GBP），病原体和细胞来审查此类微阵列，以识别功能识别和生物学意义的聚糖，然后将其在结构上定义。这种新颖的方法避免了在糖果中定义所有聚糖结构的艰辛过程，无论其功能或识别如何，并允许我们将结构分析集中在潜在的生物学相关聚糖上。开发糖基型TGL类似于定义人基因组的“ shot弹枪”方法，但允许我们靶向分析以将聚糖结构链接到功能。 定义人类糖的所有聚糖结构类似于对人基因组进行测序。人类血液的复杂性和缺乏自动化的聚糖测序方法使定义糖不切实际的“蛮力”方法。在这里，我们提出了一种范式转移策略，该策略是“ shot弹枪”和目标方法的组合。我们将从特定的细胞或组织中组装氨基功能化的，荧光标记的聚糖库，将其打印为聚糖微阵列，鉴定通过聚糖结合蛋白识别的潜在具有生物学相关的聚糖，并在结构上定义了公认的聚糖。生产的稳定，库和聚糖微阵列将作为对聚糖结构和功能的未来分析的切实资源。