HEPARANOMICS: STRUCTURE-FUNCTION RELATIONSHIPS

描述（由申请人提供）：硫酸乙酰肝素（HS）在细胞表面和细胞外基质中作为蛋白聚糖大量表达，具有不同的硫酸模式、链长和链数，导致巨大的结构特征。每条链同时与多种蛋白质相互作用，包括各种信号分子、形态因子、受体等，并协调各种生物功能。它们的生物合成途径的调节已经开始揭示它们在斑马鱼、秀丽隐杆线虫、果蝇等模式生物中的作用。然而，缺乏有效的合成方法以及从动物模型中分离的少量HS链限制了我们对这些分子如何在分子水平上调节各种生理和病理过程的了解。该建议的中心假设是，具有保守关键基团的多个低聚糖序列可能阐明相同的生物反应，或者具有一组关键基团的给定低聚糖序列可能阐明多种生物反应。确定这些关键基团及其空间距离将足以确定支配hs -蛋白相互作用的规则。开发一种通用的酶促方法来快速组装各种大小的HS，应该允许我们测试我们的假设，并描绘出决定HS-蛋白相互作用的规则。我们建议构建寡糖文库，并筛选它们与各种生长因子和受体形成信号复合物的能力，这些生长因子和受体在心脏、肺和大脑发育中发挥核心作用。在稳定同位素的帮助下，我们将开发新的结构生物学方法来阐明赋予生物功能的关键群体。在合成ATIII结合位点中获得的知识将用于设计各种基于大小的HS文库，这些文库应该是定义所有相互作用蛋白质和阐明这种相互作用发生的化学规则的关键。本研究为蛋白多糖的糖生物学研究奠定了坚实的基础。

DESCRIPTION (provided by applicant): Heparan sulfates (HS) are abundantly expressed on the cell surface and in the extracellular matrix as proteoglycans with differential sulfation pattern, chain length and number of chains leading to vast structural features. Each chain interacts simultaneously with numerous proteins including a variety of signaling molecules, morphogens, receptors, etc and orchestrates various biological functions. Modulation of their biosynthetic pathways has begun to shed light on their role in such model organisms as zebra fish, c. elegans, drosophila, etc. However, the lack of efficient synthetic approaches coupled with a small amount of HS chains isolated from animal models has limited our knowledge of how these molecules regulate various physiological and pathological processes at the molecular level. The central hypothesis of this proposal is that multiple oligosaccharide sequences with conserved critical groups may elucidate the same biological response or that a given oligosaccharide sequence with a set of critical groups may elucidate more than one biological response. Determining those critical groups and their spatial distance would be sufficient to determine the rules that dictate HS-protein interactions. Development of a general enzymatic approach for the rapid assembly of HS of various sizes should allow us to test our hypothesis and to delineate the rules that dictate HS-protein interactions. We propose to construct the oligosaccharide libraries and screen their ability to form signaling complex with various growth factors and receptors that play a central role in heart, lung and brain development. With the aid of stable isotopes, we will then develop novel structural biology approaches to elucidate the critical groups that attribute a biological function. The knowledge gained in synthesizing ATIII binding sites will be used in designing diverse size-based HS libraries that should be the key to defining all interacting proteins and elucidating the chemical rules by which such interactions take place. This proposed study should establish a firm foundation for the glycobiology of proteoglycans.