SIALIC ACID O-ACETYLATION IN BLOOD CELL INTERACTIONS

血细胞相互作用中的唾液酸 O-乙酰化

• 批准号: 6504150
• 负责人: AJIT P VARKI
• 金额: $ 13.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6504150
• 关键词: CD antigens; CD22 molecule; acetylation; cell cell interaction; chemical binding; complement; developmental genetics; erythrocytes; gene expression; genetic regulatory element; genetically modified animals; hematopoiesis; in situ hybridization; laboratory mouse; lectin; lymphocyte; sialate; sialyltransferases

Sialic acid (Sia) residues of N-linked sugar chains on blood and vascular proteins are usually attached in alpha2-6 or alpha2-3 linkages, and some molecules are selectively O-acetylated at the 9- position. This diversity can affect recognition of Sia by certain vascular lectins in vitro. We will elucidate the in vivo biological significance of these findings by manipulation of certain genes in the mouse. Sialic acid O-acetyltransferase(s) have been difficult to purify or clone. We have isolated cDNA and genomic clones of a mouse esterase (Lse) that selectively removes 9-O-acetyl groups from Sias. Expression of Lse is highly regulated e.g., in murine hematopoietic development. 9-O-acetylation also occurs preferentially on the sequence Siaalpha2-6Galbeta1-4GlcNAc, whose expression is determined by the alpha2-6 sialyltransferase gene (ST6Gal I). This gene is regulated by differential action of multiple promoters in cells such as hepatocytes, endothelial cells, erythrocytes and lymphocytes. Thus, alterations in activity of ST6Gal I and/or the competing alpha2-3 sialyltransferases (ST3Gal III & IV) may also alter 9-O-acetylation. To elucidate the in vivo regulation of 9-O-acetylation and to pursue specific hypotheses about its roles,we will do the following: 1. Eliminate expression of the Lse gene systemically, and/or selectively in the cell types listed above. 2. Alter ratios of alpha2-6 and alpha2-3 Sia by disrupting expression of alpha2-6 and alpha2-3 sialyltransferases (Collaboration with Project 1) and by "knocking in" the cDNA for ST3Gal III into the endogenous ST6Gal I gene, disrupting the latter. In the latter mice, alpha2-6 and alpha2-3 Sia linkage ratios should be inverted on N-glycans. 3. Elucidate the changes in 9-O- acetylation occurring in these mice, and structural and functional consequences of these genetic manipulations. Changes will be detected in situ by immunohistology using specific probes for sialylation and O-acetylation, and directly demonstrated by chemical analyses. Particular attention will be directed towards hepatocytes, endothelial cells, erythrocytes and lymphocytes. Biological effects upon interactions involving the Sia-binding lectins H protein, CD22, CD33 and Sialoadhesin will be studied (some in collaboration with Projects 1 and 3). The sensitivity of red cells to complement and the turnover of red cells and plasma proteins will also be assessed. Selected aspects of blood coagulation, hematopoiesis, and the distribution and function of lymphocytes will also be examined. These studies will help to define the in vivo regulation and biological roles of Sia O-acetylation in the blood and vascular systems.

N连锁糖链在血液和 血管蛋白通常附着在alpha2-6或alpha2-3中 链接和某些分子在9-处有选择性地被o-乙酰化。 位置。这种多样性会影响某些人的识别 体外血管凝集素。我们将阐明体内生物学 通过操纵某些基因中这些发现的意义 鼠标。唾液酸O-乙酰转移酶（S）很困难 净化或克隆。我们有A分离的cDNA和基因组克隆 小鼠酯酶（LSE）有选择地去除9-O-乙酰基 SIA。 LSE的表达高度调节，例如在鼠中 造血发育。还会发生9-O-乙酰化 优先在Siaalpha2-6galbeta1-4glcnac的序列上 表达由Alpha2-6 siaLlyltransferase基因确定 （ST6GAL I）。该基因受到差异作用的调节 肝细胞，内皮细胞等细胞中的多个启动子， 红细胞和淋巴细胞。因此，活动的改变 ST6GAL I和/或竞争的Alpha2-3 siAllyltransferases（ST3Gal III和IV）也可能改变9-O-乙酰化。阐明体内 调节9-O-乙酰化并追求特定的假设 关于它的角色，我们将执行以下操作：1。消除表达 在细胞类型中，LSE基因和/或有选择性的 上面列出。 2。alpha2-6和alpha2-3 SIA的变更比率 破坏Alpha2-6和alpha2-3的表达 （与项目1的合作）和“敲” cDNA ST3GAL III进入内源性ST6GAL I基因，破坏了后者。 在后一种小鼠中，alpha2-6和alpha2-3 SIA连锁比应该 倒在n-glycans上。 3。阐明9-O-的变化 这些小鼠发生的乙酰化以及结构和功能 这些遗传操作的后果。更改将是 使用特定的探针对原位检测到原位 溶解和O-乙酰化，并直接通过化学 分析。特别关注的是肝细胞， 内皮细胞，红细胞和淋巴细胞。 生物学效应 在涉及SIA结合凝集素H蛋白CD22的相互作用后 将研究CD33和唾液粘附素（有些与 项目1和3）。红细胞对补体的敏感性和 红细胞和血浆蛋白的营业额也将被评估。 血液凝血，造血和 还将检查淋巴细胞的分布和功能。 这些研究将有助于定义体内调节和 Sia o-乙酰化在血液和血管中的生物学作用 系统。