Sulfotransferases in the Synthesis of L-Selectin Ligands

L-选择素配体合成中的磺基转移酶

• 批准号: 7681487
• 负责人: STEVEN D ROSEN
• 金额: $ 34.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7681487
• 关键词: Airway Resistance; Allergic; Animals; Antibodies; Apoptosis; Arthritis; Asthma; Binding; Blood; Blood Vessels; C-Type Lectins; Carbohydrates; Cell surface; Complex; Disease; Esters; Exhibits; High Endothelial Venule; Home environment; Homing; Human; Inflammatory; Inorganic Sulfates; Joints; Knock-out; Knockout Mice; L-Selectin; Lead; Leukocyte Trafficking; Leukocytes; Ligands; Lung; Lymphocyte; Mass Spectrum Analysis; Mediating; Modeling; Modification; Mucins; Mus; PNAd; Pathogenesis; Pathologic; Patients; Play; Polysaccharides; Process; Property; Residual state; Rheumatoid Arthritis; Role; Sheep; Staining method; Stains; Techniques; Therapeutic; Tissues; Unspecified or Sulfate Ion Sulfates; Work; asthmatic airway; base; crosslink; eosinophil; human SIGLEC8 protein; keratan sulfate Gal-6-sulfotransferase; leukocyte activation; lymph nodes; mouse model; neutrophil; novel; novel strategies; receptor; research study; response; sialic acid binding Ig-like lectin; sialomucin; sialomucins; sialyl-2-3-(6' -sulfo)galactosyl-1-4-(fucopyranosyl-1-3)-N-acetylglucosamine; sugar; sulfated glycoprotein p50; sulfation; sulfotransferase

DESCRIPTION (provided by applicant): Lymphocytes home from the blood into lymph nodes during the process of lymphocyte recirculation. Homing is initiated by the rolling of lymphocytes on high endothelial venules (HEVs) within the lymph node. This step is mediated by L-selectin, a C-type lectin, which recognizes a set of carbohydrate-based ligands on HEVs. A function-blocking mAb called MECA-79 recognizes the same complex, referred to as PNAd. The ligands are sialomucins modified by GlcNAc-6-sulfate, sialyl Lewis x, and Gal-6-sulfate. We have studied double knockout (DKO) mice in which two GlcNAc-6-O-sulfotransferases that are present in HEVs have been inactivated. These mice exhibit a 75% reduction in homing to lymph nodes and the total elimination of PNAd (complete loss of MECA-79 staining) from HEVs. We will investigate the possibility that Gal-6-sulfate modifications contribute to the "residual" ligand activity on HEVs in DKO mice, as well in mice in which GlcNAc-6-O-sulfotransferases are intact (Aim 1). PNAd+ blood vessels, which co-express GlcNAc-6-O-sulfotransferase, are present in joint tissues of rheumatoid arthritis (RA) patients. This finding is recapitulated in three mechanistically-distinct models of inflammatory arthritis in mouse. We have characterized a new antibody, called Clone 40, with very similar binding properties as MECA-79 but more suitable for use in animal studies. We will employ the DKO mice, together with Clone 40, in the mouse models of arthritis to study the role of the sulfotransferases and their sulfated products in disease pathogenesis (Aim 2). Since some murine models have been very predictive of efficacious human therapeutics (e.g., anti TNF-1 therapy), our work may lead to new approaches for the treatment of RA. We will also investigate a sheep model of asthma in which L-selectin appears to play a highly novel role (Aim 3). Our experiments suggest that extravasated leukocytes in the airways can utilize L-selectin to react with sulfated mucin ligands, which are expressed in inflamed airways. This interaction leads to activation of the leukocytes, which results in an increase in airway resistance and airway responsiveness, two hallmarks of asthma. To evaluate this hypothesis, we will determine whether isolated airway mucins can activate neutrophils through binding to L-selectin and cause the secretion of broncho-active substances. Finally, we have discovered that airway mucins in lungs are ligands for Siglec-8, a receptor known to bind sulfated and sialylated sugars. This Siglec is present on eosinophils and can induce apoptosis when artificially cross-linked by antibodies. We will determine whether these mucins are natural ligands for Siglec-8, serving to crosslink Siglec-8 on eosinophils and thus triggering apoptosis of these leukocytes (Aim 4). This would provide a homeostatic control mechanism for removing eosinophils that accumulate in allergic diseases, such as in asthmatic airways. Understanding this mechanism may lead to new pharmacologic approaches for dampening eosinophil responses.

描述（由申请人提供）：在淋巴细胞再循环过程中，淋巴细胞从血液回到淋巴结。归巢是由淋巴结内高内皮微静脉 (HEV) 上的淋巴细胞滚动启动的。该步骤由 L-选择素（一种 C 型凝集素）介导，它识别 HEV 上的一组基于碳水化合物的配体。一种名为 MECA-79 的功能阻断单克隆抗体可识别相同的复合物，称为 PNAd。配体是由 GlcNAc-6-硫酸盐、唾液酸路易斯 x 和 Gal-6-硫酸盐修饰的唾液酸粘蛋白。我们研究了双基因敲除 (DKO) 小鼠，其中存在于 HEV 中的两种 GlcNAc-6-O-磺基转移酶已被灭活。这些小鼠的淋巴结归巢能力减少了 75%，并且 HEV 中 PNAd 完全消除（MECA-79 染色完全丧失）。我们将研究 Gal-6-硫酸盐修饰是否有助于 DKO 小鼠以及 GlcNAc-6-O-磺基转移酶完整的小鼠中 HEV 的“残留”配体活性（目标 1）。 PNAd+ 血管共表达 GlcNAc-6-O-磺基转移酶，存在于类风湿性关节炎 (RA) 患者的关节组织中。这一发现在小鼠炎症性关节炎的三种机制不同的模型中得到了重现。我们鉴定了一种名为 Clone 40 的新抗体，其结合特性与 MECA-79 非常相似，但更适合在动物研究中使用。我们将在关节炎小鼠模型中使用 DKO 小鼠和 Clone 40 来研究磺基转移酶及其硫酸化产物在疾病发病机制中的作用（目标 2）。由于一些小鼠模型非常能预测有效的人类疗法（例如抗 TNF-1 疗法），因此我们的工作可能会带来治疗 RA 的新方法。我们还将研究绵羊哮喘模型，其中 L-选择素似乎发挥着高度新颖的作用（目标 3）。我们的实验表明，气道中外渗的白细胞可以利用 L-选择素与硫酸化粘蛋白配体反应，硫酸化粘蛋白配体在发炎的气道中表达。这种相互作用导致白细胞活化，从而导致气道阻力和气道反应性增加，这是哮喘的两个标志。为了评估这一假设，我们将确定分离的气道粘蛋白是否可以通过与 L-选择素结合来激活中性粒细胞并引起支气管活性物质的分泌。最后，我们发现肺部的气道粘蛋白是 Siglec-8 的配体，Siglec-8 是一种已知能结合硫酸化和唾液酸化糖的受体。这种 Siglec 存在于嗜酸性粒细胞上，当通过抗体人工交联时可以诱导细胞凋亡。我们将确定这些粘蛋白是否是 Siglec-8 的天然配体，用于交联嗜酸性粒细胞上的 Siglec-8，从而引发这些白细胞的凋亡（目标 4）。这将为去除在过敏性疾病（例如哮喘气道）中积聚的嗜酸性粒细胞提供一种稳态控制机制。了解这一机制可能会带来抑制嗜酸性粒细胞反应的新药理学方法。