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: 8134629
• 负责人: SRIRAM NEELAMEGHAM
• 金额: $ 30.77万
• 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 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8134629
• 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. PUBLIC HEALTH RELEVANCE: This proposal lies at the interface of bioengineering, biochemistry and medicine. It studies protein-carbohydrate binding interactions that regulate the adhesion of blood leukocytes (white blood cells) to endothelial cells that line blood vessel walls. The goal is to develop novel glycan/carbohydrate engineering approaches to modulate this cell adhesion event, since this can lead to new strategies to ameliorate inflammatory ailments.

描述（由申请人提供）：选择素是属于C型凝集素家族的粘附分子。它们组成型表达于血液白细胞表面和活化的内皮细胞和血小板上。选择素显著地结合与多种糖蛋白支架上的Ser/Thr残基结合的0-连接的聚糖。选择素和O-聚糖之间的这种结合启动了一系列步骤，最终导致白细胞粘附到炎症部位的血管壁。这种生物分子相互作用也调节正常的免疫反应，并且在某些类型的心血管疾病和癌症转移过程中起重要作用。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的速率可以导致对抗各种血管疾病的新疗法。因此，在这个提议中，我们开发了代谢策略，通过工程化在天然选择素配体上表达的聚糖来控制白细胞-内皮细胞相互作用。具体目标是：目标1：评估使用单糖类似物改变聚糖结构和白细胞粘附特性的可行性。在这里，我们测试的可能性，天然存在的单糖，GalNAc和岩藻糖的类似物，可以喂给细胞，以修改作为选择素的天然配体的聚糖的核心或末端结构。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。目的2：确定1（2，3）唾液酸转移酶在调节人白细胞与L-、E-和P-选择素粘附中的作用。在此，我们或者在白细胞中过表达属于ST 3Gal家族的特异性唾液酸转移酶，或者在这些细胞中同时稳定沉默一种或多种ST 3Gal。这种“系统扰动”对选择素-配体聚糖结构和白细胞粘附功能的流体剪切下的效果进行评价。目的3：探讨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ST 3Gals催化容易可逆反应的程度，这种性质称为“可逆唾液酸化”。然后，我们评估，如果“可逆唾液酸化”选择素配体的生物合成过程中起着重要的作用。这一目标的成功将证明，除了在活细胞中驱动糖缀合物合成的酶促速率常数之外，由可逆酶活性控制的平衡过程也可以控制聚糖生物合成。为实现上述目标，采用了多种实验方法。这些包括在受控流动条件下的细胞粘附研究，在炎症小鼠模型中的体内实验，以及在人白细胞和造血祖细胞/干细胞中沉默或过表达特定基因的慢病毒策略。从长远来看，我们预计，新的策略，拮抗选择素配体结合的相互作用将确定从这项工作，可能有助于未来的药物设计。 公共卫生相关性：该提案涉及生物工程、生物化学和医学的界面。它研究调节血液白细胞（白色血细胞）与血管壁内皮细胞粘附的蛋白质-碳水化合物结合相互作用。我们的目标是开发新的聚糖/碳水化合物工程方法来调节这种细胞粘附事件，因为这可以导致改善炎症性疾病的新策略。