Chemical Toolbox for Deciphering the Molecular Origins of Selectivity in Human Extracellular Sulfatases

有了这个奖项，化学部门的生命过程化学项目资助了加州大学圣地亚哥分校的卡米尔·戈多拉博士的研究，以研究两种人类酶的影响，即硫酸和硫酸。这些酶作用于细胞表面，通过识别和修饰糖聚合物上的负电荷基团的模式，糖聚合物是细胞外涂层的一部分。在这样做的过程中，它们控制了参与关键细胞信号传导和发育事件的蛋白质的活性。尽管是调节重要细胞功能的关键因素，但这些酶的结构和它们作用的糖仍然知之甚少。此外，它们在细胞环境中的精确行为需要更好的理解。本研究项目在化学和生物学的界面上开发了一套新的工具和技术，以填补对这些酶的生物学功能的基本认识的空白。这些活动使研究生能够获得化学生物学跨学科领域的培训，重点是碳水化合物化学和生物学。学生们也对碳水化合物在生物学中的各种作用有了更深入的了解。该奖项所提供的教育和技术资源允许将源于该研究项目的知识、工具和技术传播给更广泛的研究界和公众。本研究开发了新的方法来生产人类硫酸酯酶，使结构和机制的详细研究。设想新的化学糖组学工具来捕获和结构表征这些酶的多糖底物。此外，生物化学和基于细胞的分析正在研究它们在胚胎干细胞分化过程中的功能。硫酸酯酶被认为对其硫酸多糖底物的独特带负电区域具有特异性，以补充底物结合域中带正电残基的组织。然而，关于酶的结构和它们首选底物的组成的信息很少。由合成和生物来源的结构明确的硫酸酸化多糖组成的阵列被用于确定硫酸酸化酶同型的首选底物，并描绘酶从多糖中去除硫酸盐如何影响它们与生长因子的相互作用。化学工具箱包括基于底物的荧光探针，用于在生物环境中成像硫酸盐酶活性，以及用于底物化学指纹的活性分子，用于通过质谱分析进行结构表征。结合使用这些工具，可以绘制出硫酸盐酶如何在小鼠胚胎干细胞的神经和间充质分化过程中控制生长因子信号。这些工具是通用的，适用于研究细胞外硫酸酯酶发挥重要调节功能的其他生物系统。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding the research of Dr. Kamil Godula at the University of California – San Diego, to study the effects of two human enzymes known as Sulf1 and Sulf2. These enzymes act on the exterior of cell surfaces by recognizing and modifying the patterns of negatively charged groups on sugar polymers that are part of the outside coating of cells. In doing so they control the activity of proteins involved in critical cell signaling and development events. Despite being a key element in regulating important cellular functions, the structures of these enzymes and the sugars they act on are still poorly understood. Additionally, their precise actions in a cellular context need better understanding. This research project develops a set of new tools and techniques at the interface of chemistry and biology to fill the gaps in the basic understanding of the biological functions of these enzymes. These activities allow graduate students to obtain training in the interdisciplinary area of chemical biology focusing on carbohydrate chemistry and biology. The students also gain a deeper understanding of the varied roles of carbohydrates in biology. The educational and technical resources enabled by this award allow for the dissemination of the knowledge, tools, and techniques stemming from this research project to the broader research community and the general public. This research develops novel methods to produce human sulfatase enzymes enabling detailed investigations of structure and mechanism. New chemical glycomics tools are envisioned to capture and structurally characterize the polysaccharide substrates for these enzymes. Further, biochemical and cell-based assays are under development to investigate their function during embryonic stem cell differentiation. The sulfatase enzymes are thought to have specificity for unique negatively charged regions of their sulfated polysaccharide substrates complementing the organization of positively charged residues in their substrate-binding domains. However, little information exists regarding the structure of the enzymes and the composition of their preferred substrates. Arrays comprising structurally defined sulfated polysaccharides from synthetic and biological sources are in use to identify preferred substrates for the sulfatase enzyme isoforms and to delineate how the enzymatic removal of sulfates from the polysaccharides impacts their interactions with growth factors. The chemical toolbox includes substrate-based fluorogenic probes for imaging sulfatase activity in biological environments and reactive molecules for chemical fingerprinting of substrates for structural characterization by mass spectrometry analysis. In combination, these tools are deployed to map how sulfatase enzymes control growth factor signaling during neural and mesenchymal differentiation of murine embryonic stem cells. The tools are general and suitable for the investigation of other biological systems where extracellular sulfatases exert important regulatory functions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.