Systems Biology of Glycosylation

描述(申请人提供)：糖基化是蛋白质和脂质的一种重要的翻译后修饰。这一过程控制着调节人类发育、免疫和疾病的细胞识别和信号传递过程。目前的提案旨在开发基于系统生物学的计算和实验方法，以增强我们对细胞糖基化途径的理解。特别是，我们的重点是更好地了解调节人类白细胞上O-连接的多糖形成的特征。通过结合属于选择性家族的黏附分子，这些O-葡聚糖在调节白细胞与血管内皮细胞的黏附中发挥关键作用，血管内皮细胞排列在炎症和心血管疾病部位的血管壁上。我们的总体假设是，在糖基化反应网络的电子模拟中，可以识别控制人类白细胞上选择素配体形成的限速步骤。这些限速步骤的明确和特定的扰动可以减少炎症期间体内白细胞-内皮细胞的黏附/迁移。其具体目标是：1)开发计算模型来预测控制细胞糖基化的限速步骤。2)定量研究糖基转移酶和多肽骨架在调节O-连接糖基化和白细胞选择结合功能中的作用。3)检测沉默糖基转移酶对白细胞在骨髓中的滞留和细胞向炎症部位迁移的影响。该项目涉及在基于系统生物学的建模、定量生物工程实验、蛋白质组学、糖生物学、免疫学和动物模型方面具有专业知识的研究人员之间的合作。从基因到蛋白质/酶，再到碳水化合物结构和细胞黏附功能，体外和体内的实验研究跨越了多个尺度。计算机模拟整合了这些信息，以确定系统扰动对多糖结构和功能的影响。预期的项目成果包括：i)定义用于描述糖基化反应网络的称为GlycoML的新标准。Ii)结合实验和理论，揭示糖基化的潜在细胞内/代谢靶点，可以定量和明确地改变选择素配体结构。3)精确的α(2，3)唾液酸基转移酶(S)和α(1，3)岩藻糖基转移酶(S)的定义，它们调节人白细胞中选择素配体的生物合成。IV)更好地理解了肽骨架在调节O-糖链的起始、延伸和终止中的作用。V)在炎症、腹膜炎和慢性阻塞性肺疾病(COPD)的动物模型中进行验证，这是使用计算机模拟和体外实验产生的关键假说。

DESCRIPTION (provided by applicant): Glycosylation is an important post-translational modification of proteins and lipids. This process controls cell recognition and signaling processes that regulate human development, immunity and disease. The current proposal aims to develop Systems Biology based computational and experimental methodologies to enhance our understanding of cellular glycosylation pathways. In particular, our focus is on better understanding the features that regulate the formation of O-linked glycans on human leukocytes. By binding adhesion molecules belonging to the selecting family, these O-glycans play a critical role in regulating leukocyte adhesion to vascular endothelial cells that line blood vessel walls at sites of inflammation and cardiovascular disease. Our overall hypothesis is that "In silico modeling of glycosylation reaction networks can identify rate limiting steps that control the formation of selectin-ligands on human leukocytes. Defined and specific perturbation of these rate-limiting steps can reduce leukocyte-endothelial cell adhesion/migration in vivo during inflammation." The specific aims are: 1) to develop computational models to predict the rate-limiting steps that control cellular glycosylation. 2) To quantify the role of selected glycosyltransferases and the peptide backbone in regulating O-linked glycosylation and leukocyte selecting-binding function. 3) To test the effect of silencing glycosyltransferases on leukocyte retention in the bone marrow, and cell migration to sites of inflammation. The project involves collaboration between investigators with expertise in Systems Biology based modeling, quantitative bioengineering experimentation, proteomics, glycobiology, immunology and animal models. Experimental studies span multiple scales from genes, to proteins/enzymes, to carbohydrate structure and cell adhesion function, both in vitro and in vivo. The computer modeling integrates this information to determine the effect of system perturbation on glycan structure and function. Expected project outcomes include: I) Definition of a new standard called GlycoML for the description of glycosylation reaction networks. ii) Combined use of experiment and theory to reveal potential intra-cellular/metabolic targets of glycosylation that can quantitatively and definitively alter selectin-ligand structures. iii) Definition of the precise a (2, 3)sialyltransferase(s) and a (1, 3) fucosyltransferases(s) that regulate selectin-ligand biosynthesis in human leukocytes. iv) Improved understanding of the role of the peptide backbone in regulating O-glycosylation chain initiation, extension and termination. v) Validation in animal models of inflammation, peritonitis and COPD (chronic obstructive pulmonary disease), key hypothesis generated using computer simulation and ex vivo experimentation.