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: 6202556
• 负责人: LELAND D POWELL
• 金额: $ 14.89万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202556
• 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.

该项目将研究以下物质的分布和生物合成 两种凝集素 CD33 和唾液酸粘附素 (Sn) 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 Sn 是两种具有相当相似性的 I 型凝集素，包括 对 alpha2-3 唾液酸化的可测量的结合亲和力 寡糖。 Sn 存在于成熟巨噬细胞的亚群中 早期骨髓前体细胞和成熟巨噬细胞上的 CD33。尽管 两种凝集素均与表达 alpha2-3 连接的 Sia 残基的细胞结合， 并非所有细胞的反应程度都相同，这表明这些细胞 凝集素识别特定且独特的 α2-3 唾液酸化配体。 这一观察结果非常有趣，因为 alpha2-3 Sia 残基是α2-3唾液酸转移酶家族的产物。 因此，这些凝集素可能能够区分 这些不同唾液酸转移酶的产物之间。 该项目的目标将通过追求四个目标来实现 目的： (1) 确定 Sn 和 CD33 配体的表达 野生型小鼠和四种不同的无突变小鼠 α2-3 唾液酸转移酶基因（ST3Gal I、ST3Gal II、ST3Gal III、 和 ST3Gal IV；来自项目 1)。类似的研究将在小鼠身上进行 唾液酸：9-O-乙酰酯酶中存在无效突变（来自项目 4） 预计会过度表达 9-O-乙酰化 Sia 残基 它可以阻止凝集素结合。这些研究将确定哪些 组织表达潜在的配体，以及相对贡献 不同酶的合成。 (2) 建立蜂窝 CD33在小鼠体内的分布。 (3)利用ES细胞技术 建立 CD33 缺失或 Sn 缺失小鼠。组织特异性删除，使用 Cre-loxP 方法将用于规避潜在的 胚胎致死率。 (4) 检查这些小鼠的特定缺陷 骨髓单核细胞功能。这将通过测试来解决 CD33 和 Sn 参与细胞粘附的具体假设， 通过检查以下内容：造血、单核细胞运输和 边缘化、单核细胞依赖性淋巴细胞反应、清除 病原微生物和肉芽肿形成。功能缺陷 已证实将同样在具有无效等位基因的小鼠中进行探测 α2-3唾液酸转移酶或唾液酸:9-O-乙酰酯酶， 证实了凝集素-唾液酸识别在观察到的 表型。这些研究将确定以下功能的作用： 正常骨髓细胞生成中的特定 Sia 结构及其相关性 对人类疾病的作用。