Shotgun Glycomics: Linking Glycan Structure and Function

描述(申请人提供)：人类糖类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，糖基化可能是最常见的翻译后修饰类型。但到底有多少种不同的糖链呢？他们的相对表达水平是什么？每个糖链结构是否在人类生物学或疾病过程中都有功能，例如先天免疫和抵抗感染？糖链的表达如何随着年龄和疾病的变化而变化？几十年来，人们已经知道不同的细胞和组织形成不同的糖链结构，最近在分析糖链结构方法上的进展导致了现代糖组学的发展，主要致力于鉴定糖链结构。不幸的是，虽然了解化学结构很重要，但从某种意义上说，它只是证实了我们已经知道的东西；不同的细胞产生不同的糖链结构。虽然所有多糖结构的知识可能会解决一个化学难题，但它并不能为多糖的功能提供生物解决方案。利用我们在“糖化学”方面的新进展，我们提出了一种新的糖学方法，将糖结构与功能/识别联系起来，使我们能够荧光标记特定细胞或组织糖结合物释放的游离糖。这些新颖的、双功能的荧光标记使我们能够检测到原本无法检测到的亚微克量的多糖，并通过层析方法对它们进行纯化，以获得组织和/或细胞特异性的标记聚糖库(TGL)。标记的糖链保留一个官能团，以允许将这些文库作为微阵列共价“打印”。这样的微阵列随后可以用糖结合蛋白(GBP)、病原体和细胞进行询问，以识别功能上被识别的和生物学上有意义的糖，然后对其进行结构定义。这种新颖的方法避免了在糖糖中定义所有糖链结构的繁琐过程，无论它们的功能或识别如何，并允许我们将结构分析集中在潜在的生物相关的糖链上。开发糖链TGL类似于定义人类基因组的“猎枪”方法，但允许我们有针对性地进行分析，将多糖结构与功能联系起来。 定义人类糖链的所有糖链结构类似于对人类基因组进行测序。人类糖链的复杂性和缺乏自动化的糖链测序方法使得用“蛮力”方法来定义糖链是不切实际的。在这里，我们提出了一种范式转换战略，即“猎枪”和有针对性的方法相结合。我们将从特定的细胞或组织中组装氨基功能化的、荧光标记的葡聚糖库，将它们打印为葡聚糖微阵列，识别与生物相关的潜在的糖聚糖结合蛋白，并从结构上定义识别的葡聚糖。所生产的稳定、文库和葡聚糖微阵列将继续作为未来分析葡聚糖结构和功能的有形资源。

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.