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

说明(申请人提供)：乙酰肝素硫酸盐(HS)是细胞外空间丰富的、分子复杂的糖。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或调节微生物与人类细胞之间的相互作用。由于糖的复杂修饰，HS功能是由结构寡糖基序介导的。因此，了解复杂HS糖分子的结构功能关系可能开辟新的干预途径，例如通过干扰恶性肿瘤或传染病中的生长因子信号转导。过去HS功能研究的一个主要问题是无法可视化和分析活体动物中的HS功能基序，以及无法在体内特定的细胞环境中将HS结构与功能直接关联。这一应用程序通过开发使用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，在活体动物中可视化已定义的HS结构。为此，将创造转基因表达HS特异性抗体：：GFP的动物。这将首次允许分析HS模式，即HS在后生动物中的“糖景”和时间和空间的动态。这种活体标记方法将为整个动物的“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做出反应的基因。第二个目标是通过结合使用生化和转基因方法，将HS的结构与功能直接联系起来。为此，标记的蛋白多糖转基因将用于纯化HS核心蛋白，并随后从特定的细胞环境中分离出附着糖。通过细胞特异性表达转基因，就有可能利用已建立的生化方法分离和鉴定细胞特异性、核心蛋白特异性和附着部位特异性HS糖。同时，通过使用这些转基因来检测HS糖依赖细胞迁移表型的挽救，将有可能直接将HS结构与体内功能联系起来。综上所述，这一提议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。 与公共健康相关：具有复杂修饰模式的糖类，如硫酸乙酰肝素，在细胞外空间中含量丰富，并参与调节许多与肿瘤形成有关的细胞-细胞信号通路。此外，肝素硫酸盐对于艾滋病毒和疱疹病毒进入细胞以及一系列微生物与人类宿主细胞之间的细胞间相互作用也很重要。我们建议开发新的工具，使我们能够研究特定的硫酸肝素结构的生物合成，以及在活的生物体中进行详细的硫酸肝素结构功能分析。了解硫酸乙酰肝素的结构功能关系可能最终允许在恶性肿瘤或宿主-微生物相互作用过程中干扰去调节的细胞-细胞信号。

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.