SULFOTRANSFERASE IN THE SYNTHESIS OF L-SELECTIN LIGANDS

L-选择素介导淋巴细胞对高密度脂蛋白的初始黏附 淋巴结内皮微静脉(HEV)的表达 淋巴细胞再循环。它也作用于白细胞--白细胞和 在运输过程中发生的白细胞与内皮细胞的相互作用 在急性和慢性环境中，白细胞均可进入炎症部位。 L-选择素作为凝集素样受体识别离散的 HEV的一组配体，包括GlyCAM-1、CD34、podocalyin、Sgp200和 MAdCAM-1。这些配体是硫酸盐化、岩藻糖化和唾液酸化的，并且 所有这三个修改都是最佳识别所必需的 作者L-选择素。此外，这些糖蛋白是由 与硫酸盐结合的单抗(MECA 79和G72) 对它们的配基活性至关重要的亚结构。一个详细的 GlyCAM-1的碳水化合物分析表明，特定的硫酸盐化 唾液酸基Lewis X的修饰是：半乳糖-6-硫化和N- 乙酰氨基葡萄糖-6-硫酸酯。这项建议是针对 催化这些特异性的磺基转移酶的鉴定 HEV相关配体的硫酸盐化修饰。为数不多的 克隆的修饰碳水化合物链的磺基转移酶是雏鸡 软骨素6/硫酸角蛋白磺酸转移酶(C6ST/KSST)。这种酶 能够催化硫酸盐加成到6-位。 Gal在简单的受体结构中。我们给最近克隆的3个人 与鸡高度同源的cDNA(GST 1、2和3) C6ST/KSST。其中一个(GST 3)选择性地在 戊型肝炎病毒内皮细胞。本提案的具体目标 是：1)确定表达的蛋白质是否与 新克隆的GSTs(特别是GST3)催化适当的加成反应 硫酸盐部分以模拟碳水化合物受体和 GlyCAM-1的糖链；2)鉴定硫酸盐 MECA 79识别的碳水化合物表位；以及3)获得cDNA 利用表达克隆技术编码HEC磺基转移酶 硫酸依赖单抗(G72和MECA79)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 从而控制白细胞向淋巴器官的转移和 炎症部位。

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.