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: 7371661
• 负责人: STEVEN D ROSEN
• 金额: $ 23.21万
• 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/7371661
• 关键词: Airway Resistance; Allergic; Animals; Anti-Tumor Necrosis Factor Therapy; 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; N-acetylglucosamine-6-O-sulfotransferase; 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; galactose 6-sulfate; 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，淋巴细胞从血液中回家为淋巴结。由淋巴结高的内皮静脉（HEV）上的淋巴细胞滚动来引发归巢。该步骤是由L-选择素（一种C型凝集素）介导的，该凝集素识别HEVS上的一组基于碳水化合物的配体。一个称为MECA-79的功能阻滞mAb识别相同的复合物，称为PNAD。配体是通过GlcNAC-6-硫酸盐，siAllyl Lewis X和Gal-6-硫酸盐修饰的唾液蛋白。我们研究了双基因敲除（DKO）小鼠，其中HEV中存在的两只GlcNAC-6-O-硫代转移酶已经失活了。这些小鼠的淋巴结降低了75％，并且总消除了HEV的PNAD（完全损失MECA-79染色）。我们将调查GAL-6-硫酸盐修饰有助于DKO小鼠中HEV的“残留”配体活性，以及​​GlcNAC-6-O-6-O-硫代转移酶完好无损的小鼠中的可能性（AIM 1）。 PNAD+血管共表达GLCNAC-6-O-硫代转移酶，存在于类风湿关节炎（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械固定模型中概括了。我们表征了一种称为克隆40的新抗体，具有与MECA-79非常相似的结合特性，但更适合用于动物研究。我们将在关节炎的小鼠模型中利用DKO小鼠以及克隆40，以研究硫代转移酶及其硫酸产物在疾病发病机理中的作用（AIM 2）。由于某些鼠模型非常可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导致RA治疗的新方法。我们还将研究一种哮喘的绵羊模型，其中L-选择素似乎起着高度新颖的作用（AIM 3）。我们的实验表明，气道中的奢华白细胞可以利用L-选择素与在发炎的气道中表达的硫酸化粘蛋白配体反应。这种相互作用导致白细胞的激活，这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-选择素结合并引起支气管活性物质的分泌来激活中性粒细胞。最后，我们发现肺中的气道粘蛋白是Siglec-8的配体，Siglec-8是一种已知结合硫化糖和脱脂糖的受体。该siglec存在于嗜酸性粒细胞上，当通过抗体交联时，可能会诱导凋亡。我们将确定这些粘蛋白是否是SIGLEC-8的天然配体，用于在嗜酸性粒细胞上交叉链接Siglec-8，从而触发这些白细胞的凋亡（AIM 4）。这将提供一种稳态控制机制，用于去除在过敏性疾病中积累的嗜酸性粒细胞，例如在哮喘气道中。了解这种机制可能会导致新的药理方法来抑制嗜酸性粒细胞反应。