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: 2745512
• 负责人: STEVEN D ROSEN
• 金额: $ 22.96万
• 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/2745512
• 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到高 在淋巴结中的内皮静脉（HEV）。 淋巴细胞再循环。 它还在白细胞 - 白细胞和 贩运期间发生的白细胞 - 内皮相互作用 在急性和慢性环境中，白细胞进入炎症部位。 L-选择素在识别离散时充当凝集素样受体 包括Glycam-1，CD34，Podocalyxin，SGP200和 madcam-1。这些配体是硫化，诱导的和溶解的，并且 所有这三个修改都是最佳识别所必需的 由L-选择蛋白。另外，这些糖蛋白被 单克隆抗体（MECA 79和G72），与硫酸盐结合 对其配体活动至关重要的子结构。 详细的 糖水1的碳水化合物分析表明，特异性硫酸盐 发现siAllyl路易斯X的修改：GAL-6-硫酸和N- 乙酰葡萄糖6-硫酸盐。该提议针对 鉴定磺胺转移酶，这催化了这些特异性 HEV相关配体的硫酸化修饰。 少数 修饰碳水化合物链的克隆的磺胺转移酶是小鸡 软骨素6/Keratan硫酸盐硫酸盐硫酸盐转移酶（C6ST/KSST）。这种酶 能够将硫酸盐添加到6位的6位 gal在简单的受体结构中。 我们最近给了克隆3人 与鸡同源的cDNA（GST 1、2和3） C6ST/KSST。 其中之一（GST 3）在 HEV的内皮细胞。 本提案的具体目的 是：1）确定表达的蛋白是否对应于 新克隆的GST（尤其是GST 3）催化适当的添加 硫酸盐部分以建模碳水化合物受体和 Glycam-1的碳水化合物链； 2）识别硫化的 MECA 79认可的碳水化合物表位； 3）获得cDNA 通过使用表达克隆技术来编码HEC磺胺转移酶 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潜在的机制 控制L-选择素的功能配体的表达，并且 从而控制白细胞运输到淋巴机构和 炎症部位。