Shotgun Glycomics: Linking Glycan Structure and Function

描述（由申请人提供）：人类糖的多样性和大小是未知的。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。但是一共有多少种聚糖呢？它们的相对表达水平是什么？每个聚糖结构是否在人类生物学或疾病过程（如先天免疫和抗感染）中都有功能？聚糖表达如何随年龄和疾病变化？几十年来，人们已经知道不同的细胞和组织产生不同的聚糖结构，近年来聚糖结构分析方法的进展导致了现代糖组学，主要致力于鉴定聚糖结构。不幸的是，虽然知道化学结构很重要，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聚糖结构。虽然对所有聚糖结构的了解可能会解决化学难题，但它并没有提供聚糖功能的生物学解决方案。我们提出了一种新的糖偶联方法，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，使我们能够荧光标记从特定细胞或组织糖偶联物释放的游离聚糖。这些新颖的双功能荧光标记使我们能够检测到亚微克量的聚糖，否则无法检测到，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文库（TGLs）。标记的聚糖保留一个官能团，允许这些文库作为微阵列共价“打印”。这样的微阵列随后可以用聚糖结合蛋白（GBPs）、病原体和细胞进行询问，以识别功能上可识别的和生物学上重要的聚糖，然后对其进行结构定义。这种新颖的方法避免了在不考虑其功能或识别的情况下定义糖类中所有聚糖结构的费力过程，并使我们能够将结构分析集中在潜在的生物学相关聚糖上。开发糖基化TGLs类似于定义人类基因组的“散弹枪”方法，但允许我们将分析目标定位于将糖基结构与功能联系起来。

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.