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(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。这一步骤是由L-选择素介导的，它是一种C型凝集素，识别HEV上一组基于碳水化合物的配体。一种名为MECA-79的功能阻断单抗识别相同的复合体，称为PNAD。配体是由GlcNAc-6-硫酸盐、唾液酸基Lewis x和Gal-6-硫酸盐修饰的唾液粘蛋白。我们研究了双基因敲除(DKO)小鼠，在DKO小鼠中，HEV中存在的两个GlcNAc-6-O-磺基转移酶已经失活。这些小鼠表现出75%的归巢到淋巴结和完全消除PNAD(完全失去MECA-79染色)从HEV。我们将研究Gal-6-硫酸盐修饰在DKO小鼠以及GlcNAc-6-O-磺基转移酶完整的小鼠中有助于HEV上的“残留”配体活性的可能性(目标1)。类风湿关节炎(RA)患者关节组织中存在共表达GlcNAc-6-O-磺基转移酶的PNAD+血管。这一发现在三种不同机制的小鼠炎性关节炎模型中得到了概括。我们已经鉴定了一种名为Clone 40的新抗体，它具有与MECA-79非常相似的结合特性，但更适合用于动物研究。我们将利用DKO小鼠和Clone 40在小鼠关节炎模型中研究磺基转移酶及其硫酸盐产物在疾病发病机制中的作用(目标2)。由于一些小鼠模型对有效的人类疗法(如抗肿瘤坏死因子-1疗法)具有很强的预测性，我们的工作可能会为类风湿关节炎的治疗带来新的方法。我们还将研究一个绵羊哮喘模型，在该模型中，L-选择素似乎扮演了一个非常新的角色(目标3)。我们的实验表明，呼吸道中渗出的白细胞可以利用L-选择素与硫化的粘蛋白配体反应，这些配体在炎症的呼吸道中表达。这种相互作用导致白细胞的激活，从而导致哮喘的两个特征--气道阻力和气道反应性的增加。为了评估这一假说，我们将确定分离的呼吸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，并导致支气管活性物质的分泌。最后，我们发现肺中的呼吸道粘蛋白是Siglec-8的配体，Siglec-8是一种已知与硫酸和唾液酸糖结合的受体。这种Siglec存在于嗜酸性粒细胞上，当抗体人工交联时，它可以诱导细胞凋亡。我们将确定这些粘蛋白是否是Siglec-8的天然配体，用于将Siglec-8交联于嗜酸性粒细胞，从而触发这些白细胞的凋亡(目标4)。这将为清除过敏性疾病中积累的嗜酸性粒细胞提供一种动态平衡控制机制，例如在哮喘的呼吸道中。了解这一机制可能会导致抑制嗜酸性粒细胞反应的新的药理学方法。