Recognition and Deconstruction of Uronic Acid-Containing Polysaccharides

除了公认的碳水化合物作为营养来源的作用外，以多糖、蛋白多糖和糖脂形式存在的复杂碳水化合物可以被认为是“细胞的语言”，因为它们调节许多完整的生物事件。为了破译一类碳水化合物--含有糖醛酸的多糖(UCP)在这些过程中所起的作用，我将继续研究与碳水化合物识别和新陈代谢有关的蛋白质的结构和功能特征。UCP的组成相似，因为它们是由重复的元素组成的，这些元素包括β-D-葡萄糖醛酸、α-D-半乳糖醛酸、α-D-4-O-甲基葡萄糖醛酸或它们沿链长的衍生物。然而，尽管成分相似，UCP介导了许多不同的生物相互作用和细胞信号事件。例如：UCP在细胞和组织表面提供独特的机械、粘弹性和渗透势，它们参与整个细胞外基质的配体、营养和细胞运动，并与各种效应分子相互作用，调节细胞内的相互作用。虽然我的研究的长期目标将总体上解决UCPs，但最初的重点将是两种碳水化合物，特别是透明质酸和果胶(高半乳糖醛酸)。这一研究的重点是基于这样一个假设：针对UCP代谢的微生物系统的结构和功能的重建将弥合UCP组成的细微差异与其不同生物学功能之间的知识鸿沟。 尽管透明质酸和果胶作为无毒化合物具有生物学意义和工业相关性，但它们的代谢在很大程度上是糖生物学中一个未被研究的分支。例如，在过去的三年里，一种新型的果胶降解酶被报道，我自己的工作已经鉴定出第一个微生物透明质酸结合蛋白。因此，我应用了一种基于发现的生物信息学方法来识别一系列与透明质酸和果胶相关的新基因，这些基因将成为我们拟议的研究计划的重点。该计划的长期目标是通过三维结构测定来研究透明质酸和果胶识别和代谢蛋白质因子，并使用支持性动力学和热力学技术来探索分子相互作用和作用机制。具体地说，我和我的学员将使用X射线结晶学和小角X射线散射来表征结构，并使用各种互补的分子表征技术，包括生物信息学、糖生物学、酶学、量热法和蛋白质生物化学，以将结构和功能联系起来。这种分子表征和功能分析的统一方法将使我们能够从多个角度解决重要的生物过程，并增强我们研究计划的总体目标的影响，即丰富我们对透明质酸和果胶代谢的理解，这两种主要多糖分别对细胞外基质和植物细胞壁中的生物事件的协调做出相当大的贡献。

Beyond the universally recognized role for carbohydrates as nutrient sources, complex carbohydrates in the form of polysaccharides, proteoglycans, and glycolipids may be considered to be the “the language of the cell” in that they mediate many integral biological events. In an effort to decipher the role that a class of carbohydrates, the uronic-acid containing polysaccharides (UCPs), play in these processes, I will pursue the structural and functional characterization of the proteins involved in carbohydrate recognition and metabolism. UCPs are compositionally similar in that they are made up of repeating elements that include either beta-D-glucuronic acid, alpha-D-galacturonic acid, alpha-D-4-O-methylglucuronic acid, or their derivatives along the chain lengths. However, despite this compositional similarity, UCPs mediate many diverse biological interactions and cell-signaling events. For example: UCPs contribute unique mechanical, viscoelastic, and osmotic potential at cell and tissue surfaces they participate in ligand, nutrient, and cellular motility throughout the extracellular matrix, and they interact with a variety of effector molecules to mediate intracellular interactions. While the long-term objective of my research will address UCPs in general, the initial focus will be on two carbohydrates in particular, hyaluronan and pectin (homogalacturonate). This research focus is based on the hypothesis: that structural and functional reconstruction of microbial systems for UCP metabolism will bridge the knowledge gap between subtle differences in UCP composition and their diverse biological functions. Despite their biological significance and industrial relevance as non-toxic composites, hyaluronan and pectin metabolism is a largely underexplored branch of glycobiology. For example, a novel class of pectin-degrading enzymes has been reported within the past three years and my own work has identified the first microbial hyaluronan binding protein. I have therefore applied a discovery-based bioinformatics approach to identify a series of novel hyaluronan and pectin-related genes that will be the focus of our proposed research program. The long-term objective of the program is to study hyaluronan and pectin recognition and metabolizing protein factors by 3-D structure determination, and to use supportive kinetic and thermodynamic techniques to probe molecular interactions and mechanisms of action. Specifically, my trainees and I will use X-ray crystallography and Small Angle X-ray Scattering for structural characterization and various complementary molecular characterization techniques, including bioinformatics, glycobiology, enzymology, calorimetry and protein biochemistry, to correlate structure and function. This unified approach to molecular characterization and functional analyses will allow us to address important biological processes from multiple perspectives and enhances the impact of the overarching objective of our research program, which is to enrich our understanding of hyaluronan and pectin metabolism; two prominent polysaccharides that make considerable contributions to the coordination of biological events in the extracellular matrix and plant cell walls, respectively.