SULFOTRANSFERASE 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: 6138654
• 负责人: STEVEN D ROSEN
• 金额: $ 21.73万
• 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 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6138654
• 关键词: carbohydrate receptor; chondroitin sulfates; complementary DNA; enzyme mechanism; epitope mapping; high endothelial venule; human tissue; keratan sulfate; ligands; molecular cloning; monoclonal antibody; selectins; sulfotransferase

L-selectin mediates the initial adhesion of lymphocytes to high endothelial venules (HEV) in lymph nodes during the process of lymphocyte recirculation. It also functions in leukocyte-leukocyte and leukocyte-endothelial interactions that occur during trafficking of leukocytes into inflammatory sites in both acute and chronic settings. L-selectin functions as a lectin-like receptor in recognizing a discrete set of HEV-ligands including GlyCAM-1, CD34, podocalyxin, Sgp200 and MAdCAM-1. These ligands are sulfated, fucosylated and sialylated, and all three of these modifications are required for optimal recognition by L-selectin. In addition, these glycoproteins are recognized by the monoclonal antibodies (MECA 79 and G72), which bind to sulfated substructures that are essential for their ligand activity. A detailed carbohydrate analysis of GlyCAM-1 has revealed that specific sulfation modifications of sialyl Lewis X are found: Gal-6-sulfation and N- acetylglucosamine-6-sulfation. This proposal is directed at the identification of the sulfotransferases, which catalyze these specific sulfation modifications of the HEV-associated ligands. One of the few cloned sulfotransferases that modifies carbohydrate chains is the chick chondroitin 6/keratan sulfate sulfotransferase (C6ST/KSST). This enzyme is capable of catalyzing the addition of sulfate to the 6-position of Gal in simple acceptor structures. We gave recently cloned 3 human cDNAs (GST 1, 2, and 3) which are highly homologous to the chicken C6ST/KSST. One of these (GST 3) is selectively expressed in the endothelial cells of HEV. The specific aims of the present proposal are: 1)To determine whether expressed proteins corresponding to the newly cloned GSTs (especially GST 3) catalyze the appropriate addition of sulfate moieties to model carbohydrate acceptors and to the carbohydrate chains of GlyCAM-1; 2) To identify the sulfated carbohydrate epitope recognized by MECA 79; and 3) To obtain cDNAs encoding HEC sulfotransferases by expression cloning techniques using the sulfation-dependent mAbs (G72 and MECA 79) and L-selectin. Understanding these enzymes has important biomedical implications, because the regulation of these enzymes provides a potential mechanism to control the expression of functional ligands for L-selectin, and thereby to control leukocyte trafficking into lymphoid organs and inflammatory sites.

L-选择素介导淋巴细胞的初始粘附， 淋巴结中的内皮微静脉（HEV）在 淋巴细胞再循环 它还在白细胞-白细胞中起作用， 白细胞-内皮细胞相互作用发生在运输过程中， 白细胞进入急性和慢性环境中的炎症部位。 L-选择素作为一种凝集素样受体， 一组HEV配体，包括GlyCAM-1、CD 34、足糖萼蛋白、Sgp 200和 MAdCAM-1。这些配体被硫酸化、岩藻糖基化和唾液酸化， 这三种修饰都是最佳识别所必需的 L-选择素此外，这些糖蛋白被 单克隆抗体（MECA 79和G72），其结合硫酸化的 其配体活性所必需的亚结构。 详细 GlyCAM-1的碳水化合物分析显示， 发现唾液酸刘易斯X的修饰：半乳糖基-6-硫酸化和N- 乙酰葡糖胺-6-硫酸化。这项建议是针对 鉴定催化这些特定酶的磺基转移酶 HEV相关配体的硫酸化修饰。 为数不多 鸡克隆了修饰碳水化合物链的磺基转移酶 软骨素6/硫酸角质素磺基转移酶（C6 ST/KSST）。这种酶 能够催化硫酸根加成到6-位， 简单受体结构中的半乳糖。 我们给了最近克隆的3个人类 与鸡高度同源的cDNA（GST 1、2和3 C6ST/KSST。 其中之一（GST 3）选择性地表达于 内皮细胞 本提案的具体目标 1）确定是否表达对应于所述蛋白质的蛋白质， 新克隆的GST（特别是GST 3）催化适当的加成 的硫酸酯部分，以模拟碳水化合物受体和 GlyCAM-1的碳水化合物链; 2）为了鉴定GlyCAM-1的硫酸化糖链， MECA 79识别的碳水化合物表位;和3）获得cDNA 通过表达克隆技术编码HEC磺基转移酶 硫酸化依赖性mAb（G72和MECA 79）和L-选择素。 了解这些酶具有重要的生物医学意义， 因为这些酶的调节提供了一种潜在的机制， 控制L-选择素功能性配体的表达，和 从而控制白细胞向淋巴器官的运输， 炎症部位。