ROLE OF ALPHA2,3 SIALYATED STRUCTURES IN MYELOPOIESIS

ALPHA2,3 唾液酸化结构在骨髓生成中的作用

• 批准号: 6504149
• 负责人: LELAND D POWELL
• 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/6504149
• 关键词: cell adhesion; cell migration; developmental immunology; embryonic stem cell; gene mutation; genetically modified animals; granuloma; hematopoiesis; lectin; ligands; sialate

This Project will examine the distribution and biosynthesis of ligands for the two lectins CD33 and Sialoadhesin (Sn), the distribution of the lectins themselves, and identify functional roles for these lectins in myelopoiesis and immune function. CD33 and Sn are two I-type lectins with considerable similarity including a measurable binding affinity for alpha2-3 sialylated oligosaccharides. Sn is found on subsets of mature macrophages and CD33 on both early myeloid precursors and mature macrophages. While both lectins bind to cells expressing alpha2-3 linked Sia residues, not all cells react to the same extent, indicating that these lectins recognize specific and distinct alpha2-3 sialylated ligands. This observation is of considerable interest, as alpha2-3 Sia residues are the product of a family of alpha2-3 sialyltransferases. Thus, these lectins are potentially capable of discriminating between the products of these different sialyltransferases. The objectives of this Project will be accomplished by pursuing four aims: (1) Determine the expression of ligands for Sn and CD33 in wild type mice and in mice with null mutations in four different alpha2-3 sialyltransferase genes (ST3Gal I, ST3Gal II, ST3Gal III, and ST3Gal IV; from Project 1). Similar studies will be done in mice with a null mutation in sialate:9-O-acetylesterase (from Project 4) which are anticipated to over express 9-O-acetylated Sia residues which can block lectin binding. These studies will establish which tissues express potential ligands, and the relative contributions of different enzymes to their synthesis. (2) Establish the cellular distribution of CD33 in mice. (3) Employ ES cell technology to establish CD33 null or Sn null mice. Tissue specific deletion, using the Cre-loxP approach, will be utilized to circumvent potential embryonic lethality. (4) Examine these mice for specific defects in myelomonocytic cell function. This will be approached by testing specific hypotheses for CD33 and Sn involvement in cell adhesion, by examining the following: hematopoiesis, monocyte trafficking and margination, monocyte-dependent lymphocyte responses, clearance of pathogenic organisms, and granuloma formation. Functional defects demonstrated will likewise be probed for in mice with null alleles of alpha2-3 sialyltransferases or sialate:9-O-acetylesterase, to corroborate a role of lectin-sialic acid recognition in the observed phenotype. These studies will establish the functional roles of specific Sia structures in normal myelopoiesis and relate their function to human disease.

这项计划将研究分布和生物合成 两种凝集素CD33和唾液酸粘附素的配体， 凝集素本身的分布，并确定功能 这些凝集素在骨髓生成和免疫功能中的作用。CD33 和锡是两个具有相当相似性的I型凝集素，包括 唾液酸化α2-3的可测结合亲和力 低聚糖。SN存在于成熟巨噬细胞亚群和 CD33在早期髓系前体细胞和成熟巨噬细胞上的表达。而当 这两种凝集素都与表达α2-3连接的Sia残基的细胞结合， 并不是所有的细胞都有相同的反应，这表明这些细胞 凝集素识别特定和独特的α2-3唾液酸化配体。 这一观察结果是相当有意义的，因为阿尔法2-3Sia 残基是α2-3唾液酸基转移酶家族的产物。 因此，这些凝集素有可能区分 这些不同唾液酸基转移酶的产物之间。 本项目的目标将通过以下四个方面来实现 目的：(1)测定细胞内锡和CD33配体的表达。 野生型小鼠和在小鼠中存在四种不同的零突变 α2-3唾液酸基转移酶基因(ST3GalI、ST3GalII、ST3GalIII、 和ST3Gal IV；来自项目1)。类似的研究将在小鼠身上进行。 唾液酸零突变：9-O-乙酰酯酶(来自项目4) 它们有望过度表达9-O-乙酰化的SiA残基 它可以阻断凝集素的结合。这些研究将确定 组织表达潜在的配体，以及相对的贡献 不同的酶对其合成的影响。(2)建立蜂窝网络 CD33在小鼠体内的分布。(3)采用ES细胞技术 建立CD33缺失或Sn1缺失小鼠。组织特异性删除，使用 将利用CRE-loxP方法来规避潜在的 胚胎致死。(4)检查这些小鼠是否存在特定缺陷 骨髓单核细胞功能。这将通过测试来实现 CD33和锡参与细胞黏附的特定假说 通过检查以下几个方面：造血、单核细胞贩运和 边集作用，单核细胞依赖的淋巴细胞反应，清除 病原体和肉芽肿的形成。功能缺陷 在等位基因为零的小鼠身上也同样会被发现 α2-3唾液酸基转移酶或唾液酸：9-O-乙酰酯酶 证实了凝集素-唾液酸识别在观察到的 表型。这些研究将确定细胞的功能 正常骨髓生成中SIA的特异性结构及其相关关系 对人类疾病的作用。