Novel Approaches to Structure/Function Analyses of Heparan Sulfate in vivo

描述（由申请人提供）：硫酸肝素（HS）是细胞外空间丰富的分子复杂糖。它们参与调节细胞间的相互作用，例如通过调节生长因子信号传导或调节微生物与人类细胞之间的相互作用。HS功能是由结构寡糖基序介导的，是糖的复杂修饰的结果。因此，了解复杂HS糖分子的结构功能关系可能会开辟新的干预途径，例如通过干扰恶性肿瘤或传染病中的生长因子信号。过去研究HS功能的一个主要问题是无法可视化和分析活体动物的功能性HS基序，以及无法直接将HS结构与体内特定细胞环境中的功能联系起来。该应用程序通过开发使用秀丽隐杆线虫的新工具来解决这些挑战。第一个目标是开发一种新的转基因方法来可视化动物体内定义的HS结构。为此，将创建转基因表达HS特异性抗体：：GFP融合物的动物。这将首次允许分析HS模式，即HS“糖景观”和后生动物的时间和空间动态。这种体内标记方法将为在整个动物中进行“糖景观”的遗传分析打开大门，并将有助于识别调节HS表达和对HS作出反应的基因。第二个目标是通过结合生物化学和转基因方法直接将HS结构与功能联系起来。为此，标记的蛋白多糖转基因将用于纯化HS核心蛋白，随后从定义的细胞环境中纯化附着的糖。通过细胞特异性表达转基因，将有可能使用既定的生化方法分离和表征细胞特异性、核心蛋白特异性和附着位点特异性的HS糖。通过同样使用这些转基因来检测syndecan零突变体的HS糖依赖细胞迁移表型的拯救，将有可能直接将HS结构与体内功能联系起来。总之，本提案将开发新的工具来可视化和分析已定义的HS结构基序在体内的功能。

DESCRIPTION (provided by applicant): Heparan sulfates (HS) are abundant and molecularly complex sugars of the extracellular space. They are involved in regulating cell-cell interactions, for example by modulating growth factor signaling or regulating the interaction between microbes and the human cells. HS functions are mediated by structural oligosaccharide motifs as a result of complex modifications of the sugars. Thus, understanding structure function relationships of the complex HS sugar molecules may open up new avenues of intervention, e.g. by interfering with growth factor signaling in malignancies or, in infectious diseases. A major problem in the study of HS function in the past has been the inability to visualize and analyze functional HS motifs in living animals and, to directly correlate HS structure with function in a defined cellular context in vivo. This application addresses these challenges by developing novel tools using C. elegans. The first aim will develop a novel transgenic approach to visualize defined HS structures in living animals. To this end, animals transgenically expressing HS specific antibody::GFP fusions will be created. This will for the first time allow the analysis of HS patterns, i.e. the HS 'sugar landscape' and dynamics in time and space in a metazoan. This in vivo labeling approach will open the door for genetic analyses of the 'sugar landscape' in a whole animal and, will aid in identifying the genes that regulate HS expression and that respond to HS. The second aim is designed to directly correlate HS structure with function by using a combination of biochemical and transgenic approaches. To this end, tagged proteoglycan transgenes will be used to purify the HS core protein and, subsequently the attached sugars from defined cellular contexts. By cell-specific expressing the transgenes it will be possible to isolate and characterize cell-specific, core protein specific, and attachment site-specific HS sugars using established biochemical methods. By at the same using these transgenes to assay for rescue of an HS sugar dependent cell migration phenotype of a syndecan null mutant, it will be possible to directly correlate HS structure with function in vivo. In summary, this proposal will develop novel tools to visualize and analyze the function of defined HS structural motifs in vivo. PUBLIC HEALTH RELEVANCE: Sugars with complex modification patterns such as heparan sulfates are abundant in the extracellular space and involved in regulating many cell-cell signaling pathways involved in tumor formation. In addition, heparan sulfates are important for entry of HIV and Herpes viruses into cells and, cell-cell interactions between a range of microbes and the cells of the human host. We propose here to develop novel tools that allow us to study the biosynthesis of specific heparan sulfate structures as well as conduct detailed structure function analyses of heparan sulfate in a living organism. Understanding heparan sulfate structure function relationships may ultimately allow to interfere with deregulated cell-cell signaling during malignancies or host- microbe interactions.