HEPARANOMICS: STRUCTURE-FUNCTION RELATIONSHIPS

说明(申请人提供)：硫酸乙酰肝素(HS)在细胞表面和细胞外基质中以蛋白多糖的形式大量表达，具有不同的硫酸盐化模式、链长度和链数量，从而导致巨大的结构特征。每条链同时与许多蛋白质相互作用，包括各种信号分子、形态因子、受体等，并协调各种生物学功能。对它们生物合成途径的调控已经开始揭示它们在斑马鱼、线虫、果蝇等模式生物中的作用。然而，缺乏有效的合成方法，加上从动物模型中分离出少量的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。这一建议的中心假设是，具有保守临界基团的多个寡糖序列可以阐明相同的生物反应，或者给定的具有一组临界基团的寡糖序列可以阐明不止一种生物反应。确定这些关键基团及其空间距离将足以确定支配HS-蛋白质相互作用的规则。开发一种通用的酶方法来快速组装各种大小的HS应该能让我们检验我们的假设，并描绘出支配HS-蛋白质相互作用的规则。我们建议构建寡糖库，并筛选它们与各种生长因子和受体形成信号复合体的能力，这些生长因子和受体在心、肺和脑的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属于生物功能的关键基团。在合成ATIII结合位点中获得的知识将被用于设计不同大小的HS文库，这应该是定义所有相互作用蛋白质和阐明发生这种相互作用的化学规则的关键。这项拟议的研究应该为蛋白多糖的糖生物学奠定坚实的基础。

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.