Selectin mediated cell adhesion under hydrodynamic shear

描述（由申请人提供）：选择物是属于c型凝集素家族的粘附分子。它们在血液白细胞表面和活化的内皮细胞和血小板上组成性表达。这些选择蛋白显著结合o链聚糖，这些聚糖与各种糖蛋白支架上的丝氨酸/苏氨酸残基结合。选择素和o -聚糖之间的这种结合启动了一系列步骤，最终导致白细胞在炎症部位粘附到血管壁上。这种生物分子相互作用也调节正常的免疫反应，并在某些类型的心血管疾病和癌症转移过程中发挥重要作用。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能会导致对抗各种血管疾病的新疗法。因此，在本研究中，我们开发了代谢策略，通过设计在自然选择配体上表达的聚糖来控制白细胞-内皮相互作用。目的1：评价单糖类似物改变多糖结构和白细胞粘附特性的可行性。在这里，我们测试了天然存在的单糖的类似物GalNAc和also focus可以被喂入细胞，以修饰作为选择天然配体的聚糖的核心或末端结构的可能性。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：明确调节白细胞对L-、E-和p -选择素粘附的1（2,3）唾液基转移酶。在这里，我们要么在白细胞中过度表达属于ST3Gal家族的特异性唾液转移酶，要么在这些细胞中同时稳定地沉默一个或多个ST3Gal。这种“系统扰动”对选择配体聚糖结构和白细胞在流体剪切下的粘附功能的影响进行了评估。目的3：确定可逆性唾液酰化在调节白细胞选择配体结构中的作用。在这里，我们量化了人类st3gal催化容易可逆反应的程度，这种性质被称为“可逆唾液化”。然后，我们评估“可逆唾液化”是否在选择配体的生物合成过程中起重要作用。这一目标的成功将证明，除了驱动活细胞中糖缀合物合成的酶速率常数外，由可逆酶活性控制的平衡过程也可能控制聚糖的生物合成。为达到上述目的，采用了多种实验方法。这些研究包括在受控流动条件下的细胞粘附研究，小鼠炎症模型的体内实验，以及在人类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。

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.