Selectin mediated cell adhesion under hydrodynamic shear

选择素介导的流体动力剪切下的细胞粘附

• 批准号: 8466353
• 负责人: SRIRAM NEELAMEGHAM
• 金额: $ 25.82万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8466353
• 关键词: Address; Adhesions; Anabolism; Binding; Biochemistry; Biomedical Engineering; Blood; Blood Platelets; Blood Vessels; Buffers; C-Type Lectins; Carbohydrates; Cardiovascular Diseases; Cell Adhesion; Cell Adhesion Molecules; Cell Line; Cell Surface Receptors; Cells; Chemicals; Data; Disease; Drug Design; Drug Kinetics; Endothelial Cells; Engineering; Enzymes; Equilibrium; Event; Exhibits; Family; Family member; Fucose; Future; Galactose; Gene Expression; Genes; Glycoconjugates; Glycolipids; Glycoproteins; Glycosaminoglycans; Goals; Golgi Apparatus; Half-Life; Hematopoietic; Human; Immune response; Indium; Infiltration; Inflammation; Inflammatory; Lead; Leukocytes; Life; Ligand Binding; Ligands; Link; Liquid substance; MMP11 gene; Malignant Neoplasms; Mediating; Medicine; Membrane Glycoproteins; Metabolic; Methods; Monosaccharides; Mus; Organ; P-Selectin; P-selectin ligand protein; Pathology; Pathway interactions; Pharmacodynamics; Physiological; Plasma; Play; Polysaccharides; Process; Property; Proteins; RNA Interference; Reaction; Reporting; Role; ST3Gal II; ST3Gal IV; Selectins; Series; Serum; Sialyltransferases; Site; Stem cells; Structure; Subfamily lentivirinae; Surface; System; Testing; Time; Tissues; Trisaccharides; Ursidae Family; Vascular Endothelial Cell; Work; analog; base; beta-galactoside alpha-2,3-sialyltransferase; carbohydrate receptor; carbohydrate structure; combat; enzyme activity; feeding; glycosylation; glycosyltransferase; in vivo; inhibitor/antagonist; insight; interest; knock-down; metastatic process; migration; mouse model; novel; novel strategies; overexpression; premature; prevent; progenitor; research study; scaffold; sialylation; small molecule; stem; success; tool

DESCRIPTION (provided by applicant): The selectins are adhesion molecules belonging to the C-type lectin family. They are expressed constitutively on the surface of blood leukocytes and on activated endothelial cells and platelets. The selectins prominently bind O-linked glycans that are bound to Ser/Thr residues on a variety of glycoprotein scaffolds. Such binding between selectins and O-glycans initiate a series of steps that eventually results in the adhesion of leukocytes to blood vessel walls at sites of inflammation. This biomolecular interaction also conditions normal immune response, and it plays an important role in certain types of cardiovascular disorders and cancer metastatic processes. It is widely believed that controlling the rate of leukocyte adhesion by antagonizing selectin-ligand interactions can lead to new therapies to combat a variety of vascular ailments. Thus, in this proposal, we develop metabolic strategies to control leukocyte-endothelial interactions by engineering the glycans that are expressed on natural selectin-ligands. The specific aims are: Aim 1: To evaluate the feasibility of using monosaccharide analogs to alter glycan structures and leukocyte cell adhesion properties. Here, we test the possibility that analogs of the naturally occurring monosaccharide, GalNAc and also Fucose, can be fed to cells in order to modify either the core or terminal structures of glycans that function as the natural ligands for the selectins. We evaluate the ability and mechanism by which these chemical inhibitors permeate cells, engage and modify glycan biosynthetic pathways, and inhibit cell adhesion. Aim 2: To define the precise 1(2,3) sialyltransferase(s) regulating leukocyte adhesion to L-, E- and P-selectin in humans. Here, we either over- express specific sialyltransferases belonging to the ST3Gal family in leukocytes, or stably silence one or more ST3Gals at the same time in these cells. The effect of this "system perturbation" on selectin-ligand glycan structure and leukocyte cell adhesion function under fluid shear is evaluated. Aim 3: To determine the role for reversible sialylation in regulating leukocyte selectin-ligand structure. Here, we quantify the extent to which human ST3Gals catalyze readily reversible reactions, a property called 'reversible sialylation'. We then evaluate if 'reversible sialylation' plays an important role during the biosynthesis of selectin-ligands. Success in this aim will demonstrate that besides enzymatic rate constants that drive glycoconjugate synthesis in living cells, equilibrium processes governed by reversible enzymatic activity may also control glycan biosynthesis. Diverse experimental methods are applied to accomplish the above aims. These include cell adhesion studies under controlled flow conditions, in vivo experiments in a mouse model of inflammation, and lentiviral strategies to silence or overexpress specific genes in human leukocytes and hematopoietic progenitor/stem cells. In the long run, we anticipate that novel strategies to antagonize selectin-ligand binding interactions will be identified from this work that may aid future drug design.

描述(申请人提供)：选择素是属于C型凝集素家族的黏附分子。它们结构性地表达于血白细胞表面以及活化的内皮细胞和血小板上。选择素主要结合O-连接的多聚糖，这些多聚糖结合在各种糖蛋白支架上的丝氨酸/苏氨酸残基上。选择素和O-葡聚糖之间的这种结合启动了一系列步骤，最终导致白细胞与炎症部位的血管壁粘连。这种生物分子相互作用也制约着正常的免疫反应，在某些类型的心血管疾病和癌症转移过程中发挥着重要作用。人们普遍认为，通过拮抗选择素-配体相互作用来控制白细胞粘附率可以带来对抗各种血管疾病的新疗法。因此，在这项建议中，我们开发代谢策略来控制白细胞和内皮细胞的相互作用，方法是设计表达在天然选择素配体上的多糖。具体目的是：目的1：评价单糖类似物改变糖链结构和白细胞黏附特性的可行性。在这里，我们测试了自然产生的单糖的类似物GalNAc和岩藻糖可以喂给细胞的可能性，以便改变作为选择素的天然配体的多糖的核心或末端结构。我们评估了这些化学抑制物渗透细胞、参与和修饰多糖生物合成途径以及抑制细胞黏附的能力和机制。目的：确定调节人白细胞与L、E和P选择素黏附的精确1(2，3)唾液酸转移酶(S)。在这里，我们要么在白细胞中过度表达属于ST3Gal家族的特定唾液酸基转移酶，要么在这些细胞中稳定地沉默一个或多个ST3Gal。评估了这种“系统扰动”对流体剪切力作用下选择素-配体糖链结构和白细胞黏附功能的影响。目的：确定可逆唾液酸化在调节白细胞选择素-配体结构中的作用。在这里，我们量化了人类ST3Gals催化可逆反应的程度，这种性质被称为“可逆唾液酸化”。然后，我们评估“可逆唾液酸化”是否在选择素配体的生物合成过程中发挥重要作用。这一目标的成功将证明，除了在活细胞中驱动糖共轭合成的酶速率常数之外，可逆酶活性控制的平衡过程也可能控制糖的生物合成。为了达到上述目的，我们采用了多种实验方法。这些措施包括在受控流动条件下的细胞黏附研究，在小鼠炎症模型中的体内实验，以及在人类白细胞和造血祖/干细胞中沉默或过度表达特定基因的慢病毒策略。从长远来看，我们预计将从这项工作中确定对抗选择素-配体结合相互作用的新策略，这可能有助于未来的药物设计。