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.

血液和血液中 N 连接糖链的唾液酸 (Sia) 残基 血管蛋白通常以α2-6或α2-3附着 连接，并且一些分子在 9-位被选择性地 O-乙酰化 位置。这种多样性会影响某些人对 Sia 的认可 体外血管凝集素。我们将阐明体内生物学 通过操纵某些基因来研究这些发现的重要性 鼠标。唾液酸 O-乙酰转移酶一直很困难 纯化或克隆。我们分离了 cDNA 和基因组克隆 小鼠酯酶 (Lse)，选择性去除 9-O-乙酰基 西亚斯。 Lse 的表达受到高度调控，例如在小鼠中 造血发育。 9-O-乙酰化也会发生 优先选择序列 Siaalpha2-6Galbeta1-4GlcNAc，其 表达由 alpha2-6 唾液酸转移酶基因决定 （ST6Gal I）。该基因受不同作用的调节 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，活性的改变 ST6Gal I 和/或竞争性 alpha2-3 唾液酸转移酶 (ST3Gal III 和 IV) 也可能改变 9-O-乙酰化。为了阐明体内 9-O-乙酰化的调节并追求特定的假设 关于它的作用，我们会做以下工作： 1. 消除表达 Lse 基因在细胞类型中系统地和/或选择性地变化 上面列出的。 2. 改变 alpha2-6 和 alpha2-3 Sia 的比例 破坏 alpha2-6 和 alpha2-3 唾液酸转移酶的表达 （与项目 1 合作）并通过“敲入”cDNA ST3Gal III 进入内源性 ST6Gal I 基因，破坏后者。 在后者小鼠中，α2-6 和 α2-3 Sia 连锁比应 N-聚糖上被反转。 3. 阐明9-O-的变化 这些小鼠中发生的乙酰化以及结构和功能 这些基因操作的后果。变化将是 使用特定探针通过免疫组织学原位检测 唾液酸化和O-乙酰化，并通过化学直接证明 分析。将特别关注肝细胞， 内皮细胞、红细胞和淋巴细胞。 生物效应 涉及 Sia 结合凝集素 H 蛋白、CD22 的相互作用， 将研究 CD33 和 Sialoadhesin（其中一些与 项目 1 和 3）。红细胞对补体的敏感性 红细胞和血浆蛋白的周转率也将被评估。 血液凝固、造血功能的某些方面 还将检查淋巴细胞的分布和功能。 这些研究将有助于确定体内调节和 Sia O-乙酰化在血液和血管中的生物学作用 系统。