SULFOTRANSFERASE IN THE SYNTHESIS OF L-SELECTIN LIGANDS

L-选择素配体合成中的磺基转移酶

基本信息

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
项目类别：
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

项目摘要

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对高浓度的初始粘附淋巴结内皮小静脉（HEV）淋巴细胞再循环。它还在白细胞-白细胞和运输过程中发生的白细胞-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-选择素作为凝集素样受体来识别离散的一组 HEV 配体，包括 GlyCAM-1、CD34、podocalyxin、Sgp200 和 MAdCAM-1。这些配体被硫酸化、岩藻糖基化和唾液酸化，并且所有这三个修改都是最佳识别所必需的通过L-选择素。此外，这些糖蛋白被单克隆抗体（MECA 79 和 G72），可与硫酸化物结合对其配体活性至关重要的子结构。详细的 GlyCAM-1 的碳水化合物分析表明，特定的硫酸化作用发现了唾液酸路易斯 X 的修饰：Gal-6-硫酸化和 N- 乙酰氨基葡萄糖-6-硫酸化。该提案针对的是磺基转移酶的鉴定，其催化这些特定的 HEV 相关配体的硫酸化修饰。少数之一克隆的修饰碳水化合物链的磺基转移酶是小鸡软骨素 6/硫酸角质素磺基转移酶 (C6ST/KSST)。这种酶能够催化硫酸盐加成到 6 位简单受体结构中的 Gal。我们最近克隆了 3 个人与鸡高度同源的 cDNA（GST 1、2 和 3） C6ST/KSST。其中之一 (GST 3) 选择性表达于 HEV的内皮细胞。本提案的具体目标是： 1）确定表达的蛋白是否对应新克隆的 GST（尤其是 GST 3）催化适当的添加硫酸盐部分来模拟碳水化合物受体和 GlyCAM-1 的碳水化合物链； 2) 硫酸盐的鉴别 MECA 79 识别的碳水化合物表位； 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单克隆抗体（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-选择素功能配体的表达，以及从而控制白细胞转运至淋巴器官炎症部位。