ROLE OF ALPHA2,3 SIALYATED STRUCTURES IN MYELOPOIESIS

本项目将研究的分布和生物合成的 两种凝集素CD 33和唾液酸粘附素（Sn）的配体， 分布的凝集素本身，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是两种具有相当大相似性的I型凝集素， 对唾液酸化的α 2 -3的可测量的结合亲和力 低聚糖。Sn存在于成熟巨噬细胞的亚群上， 早期髓样前体细胞和成熟巨噬细胞上的CD 33。而 两种凝集素都与表达α 2 -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 凝集素识别特异性和不同的α 2 -3唾液酸化配体。 这一观察结果相当有趣，因为α 2 -3 Sia 残基是α 2 -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 这些不同的唾液酸转移酶的产物之间。 该项目的目标将通过以下四个方面来实现： 目的：（1）检测人肝癌细胞中Sn配体和CD 33配体的表达情况。 野生型小鼠和在四种不同的 α 2 -3唾液酸转移酶基因（ST 3Gal I，ST 3Gal II，ST 3Gal III， 和ST 3Gal IV;来自项目1）。类似的研究也将在老鼠身上进行 唾液酸：9-O-乙酰酯酶无效突变（来自项目4） 预期其过表达9-O-乙酰化Sia残基 其可以阻断凝集素结合。这些研究将确定哪些 组织表达潜在的配体， 不同的酶来合成它们。(2)建立细胞 CD 33在小鼠体内的分布。(3)采用ES细胞技术， 建立CD 33缺失或Sn缺失小鼠。组织特异性缺失，使用 Cre-loxP方法将用于规避潜在风险 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实现 CD 33和Sn参与细胞粘附的特定假设， 通过检查以下内容：造血、单核细胞运输和 边集，单核细胞依赖性淋巴细胞反应， 病原微生物和肉芽肿形成。功能缺陷 将同样在具有无效等位基因的小鼠中进行探测 α 2 -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方面的职能： 正常骨髓细胞中特异性Sia结构及其相关性 对人类疾病的作用。

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.