SELECTIN MEDIATED CELL ADHESION UNDER HYDRODYNAMIC SHEAR

选择流体力学剪切下介导的细胞粘附

• 批准号: 7035175
• 负责人: SRIRAM NEELAMEGHAM
• 金额: $ 25.37万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7035175
• 关键词: carbohydrate biosynthesis; carbohydrate structure; cell adhesion; chemical structure function; chemical synthesis; clinical research; disaccharides; flow cytometry; glycoproteins; glycosylation; human subject; inflammation; inhibitor /antagonist; laboratory mouse; leukocyte adhesion molecules; ligands; liquid chromatography; mathematical model; model design /development; monosaccharides; oligosaccharides; polysaccharides; protein structure function; selectins; shear stress; small molecule; surface plasmon resonance; western blottings

DESCRIPTION (provided by applicant): This research project examines the function of carbohydrate chains that are O-linked to leukocyte cell- surface glycoproteins. By acting as the natural ligands of the selectin family of adhesion molecules, these glycoproteins control the rates of leukocyte adhesion in the human vasculature during normal immune response, inflammatory diseases and certain types of cardiovascular disorders. It is widely believed that controlling the rate of leukocyte adhesion in vascular disorders can lead to new therapies to combat these ailments. Thus, in the current proposal, we evaluate two mechanisms for controlling selectin-ligand binding. In Aim 1, we develop and test the ability of unique molecules based on an unusual disaccharide carbohydrate structure (GalNAc(31,3GalNAca-O-Methyl) to competitively inhibit selectin binding interactions with its ligand. Our preliminary data suggests that this disaccharide alone can bind P- selectin. We also demonstrate that appropriate modification of this unit can dramatically enhance the binding affinity of the resulting carbohydrate for selectins, when compared with the prototypic selectin ligand sialyl Lewis-X. In Aim 2, we test an approach where small-molecule metabolic inhibitors are designed based on the structure of monosaccharides that compose natural selectin ligands. These modified monosaccharides are fed to cells in order to interfere with the biosynthesis of specific carbohydrate epitopes on the glycoprotein ligands of selectins. More specifically, these molecules are directed to alter either the core or terminal residues of glycans expressed by an important leukocyte selectin-ligand called PSGL-1 (P-selectin glycoprotein ligand- 1). We evaluate the ability and mechanism by which these chemical inhibitors permeate cells, engage and modify glycan biosynthetic pathways and inhibit cell adhesion. In Aim 3, to complement the experimental work above, a Systems Biology based mathematical model is developed to simulate biochemical networks that regulate O-glycan biosynthesis in leukocytes. Many of the assumptions in this mathematical model are experimentally validated. Diverse experimental methods are applied to accomplish the above three aims. These include cell adhesion studies under controlled flow, in vivo experiments in a mouse model of acute inflammation, western blot analysis, molecular biology based approaches, flow cytometry, surface plasmon resonance and liquid chromatography. In the long run, we anticipate that small-molecule selectin-antagonists will be identified from this work that may aid future drug design. Mathematical models developed will enhance the application of metabolic engineering principles in the area of biological chemistry. Such analysis can also provide the rationale for the chemical synthesis of new inhibitors and for interpretation of experimental observations.

描述（由申请人提供）：本研究项目检查与白细胞表面糖蛋白O-连接的碳水化合物链的功能。通过充当粘附分子的选择素家族的天然配体，这些糖蛋白在正常免疫应答、炎性疾病和某些类型的心血管疾病期间控制人脉管系统中白细胞粘附的速率。人们普遍认为，控制血管疾病中白细胞粘附的速度可以带来对抗这些疾病的新疗法。因此，在目前的建议，我们评估两种机制控制选择素配体结合。在目标1中，我们开发并测试了基于不寻常的二糖碳水化合物结构（GalNAc（31，3GalNH 4-O-甲基））的独特分子竞争性抑制选择素与其配体结合相互作用的能力。我们的初步数据表明，这种二糖单独可以结合P-选择素。我们还表明，适当的修改，这个单位可以显着提高所得到的碳水化合物的选择素的结合亲和力，当与原型选择素配体唾液酸化路易斯-X相比。在目标2中，我们测试了一种基于组成天然选择素配体的单糖结构设计小分子代谢抑制剂的方法。将这些修饰的单糖供给细胞以干扰选择素的糖蛋白配体上的特异性碳水化合物表位的生物合成。更具体地说，这些分子是针对改变由称为PSGL-1（P-选择素糖蛋白配体-1）的重要白细胞选择素-配体表达的聚糖的核心或末端残基。我们评估了这些化学抑制剂渗透细胞、参与和修饰聚糖生物合成途径以及抑制细胞粘附的能力和机制。在目标3中，为了补充上述实验工作，开发了基于系统生物学的数学模型来模拟调节白细胞中O-聚糖生物合成的生化网络。该数学模型中的许多假设都得到了实验验证。为了实现上述三个目标，采用了多种实验方法。这些包括在受控流动下的细胞粘附研究、急性炎症小鼠模型中的体内实验、蛋白质印迹分析、基于分子生物学的方法、流式细胞术、表面等离子体共振和液相色谱。从长远来看，我们预计，小分子选择素拮抗剂将确定从这项工作，可能有助于未来的药物设计。所开发的数学模型将加强代谢工程原理在生物化学领域的应用。这种分析也可以为新抑制剂的化学合成和实验观察的解释提供理论基础。